Introduction: Neurological manifestations are emerging as relatively frequent complications of corona virus disease 2019 (COVID-19), including stroke and encephalopathy. Clinical characteristics of the latter are heterogeneous and not yet fully elucidated, while the pathogenesis appears related to neuroinflammation in a subset of patients.Case: A middle-aged man presented with acute language disturbance at the emergency department. Examination revealed expressive aphasia, mild ideomotor slowing, and severe hypocapnic hypoxemia. Multimodal CT assessment and electroencephalogram (EEG) did not reveal any abnormalities. COVID-19 was diagnosed based on chest CT findings and positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse transcription PCR (RT-PCR) on nasopharyngeal swab. The following day, neurological symptoms progressed to agitated delirium and respiratory status worsened, requiring admission to the ICU and mechanical ventilation. Brain MRI and cerebrospinal fluid (CSF) studies were unremarkable. RT-PCR for SARS-CoV-2 on CSF was negative. He received supportive treatment and intravenous low-dose steroids. His neurological and respiratory status resolved completely within 2 weeks.Conclusions: We report a patient with reversible COVID-19-related encephalopathy presenting as acute aphasia, mimicking stroke or status epilepticus, eventually evolving into delirium. Although large-vessel stroke is frequently encountered in COVID-19, our case suggests that focal neurological deficits may occur as the earliest feature of encephalopathy. Neurological status reversibility and the absence of abnormalities on brain MRI are consistent with a functional rather than a structural neuronal network impairment. Coronavirus disease 2019 (COVID-19) is characterized predominantly by respiratory symptoms, although neurological manifestations are increasingly described, including encephalopathy and stroke . CoaguloA hypertensive, dyslipidemic, right-handed 54-years-old man with a 1-week history of sore throat, dysgeusia, and hyposmia presented with acute language disturbance developed within 1 h at the emergency department.2/FiO2 = 252), elevated D-dimer level (3.32 mg/L), lymphopenia, and elevated inflammatory markers  involvement. Encephalopathy in COVID-19 has been shown to have various manifestations with ranging severities, characterized by subacute onset and fluctuations or progressive course, eventually reversible . AlthougWe report a patient with COVID-19-related encephalopathy presenting with acute aphasia, mimicking stroke. Although large-vessel stroke is frequently encountered in COVID-19, our case suggests that COVID-19-related encephalopathy may present with acute language disturbances as the earliest feature. Neurologists should be aware of this clinical presentation, as it may have an impact on the differential diagnosis and management of these patients.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the patient for the publication of any potentially identifiable images or data included in this article.UP and LM drafted the manuscript. All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


MicroRNAs (miRNA) are a class of non-protein-coding RNAs that have significant biological and pathological functions. The importance of miRNAs as potential cancer diagnostic biomarkers is gaining attention due to their influence in the regulation of cellular processes such as cell differentiation, proliferation and apoptosis. The aim of this study was to identify significant miRNAs from saliva as potential diagnostic biomarkers in the early diagnosis and prognosis of head and neck squamous cell carcinoma (HNSCC).Five differentially expressed miRNAs  were selected from Next Generation Sequencing (NGS) miRNA data generated from our previous study using saliva of 12 HNSCC patients and 12 healthy controls. Their differential expressed miRNAs were subsequently validated by RT-qPCR using saliva samples from healthy controls (n = 80) and HNSCC patients (n = 150). Total RNA was isolated from 150 saliva samples of HNSCC patients and was transcripted into cDNA by TaqMan MicroRNA Reverse Transcription Kit. Using quantitative RT-PCR analysis, salivary miRNAs were identified in HNSCC patients (n = 150) and healthy controlled cases (n = 80). T-tests were used to compare the differences among the various clinical variants.On average 160 ng/μl was isolated from 500 μl of saliva. Overall, a good correlation observed between the HNSCC and some of miRNAs expression levels. Salivary miR-let-7a-5p (P<0.0001) and miR-3928 (P< 0.01) were significantly down regulated in saliva of HNSCC patients relative to age and sex-matched healthy controls. A number of salivary miRNAs (miR-let-7a-5p and miR-3928) were correlated with lymph node metastasis  and tumour size , respectively. However, our preliminary analysis showed no significant differences in salivary miR-1470, miR-345-5p or miR-7703 expression between patients and healthy controls. Most notably, our analysis showed that salivary miR-let-7a-5p and miR-3928 expression levels have significant sensitivity and specificity to distinguish between patients with HNSCC and healthy controls.This study concluded that salivary miR-let-7a-5p and miR-3928 has the potential to be novel non-invasive biomarkers for early detection and prognosis of HNSCC. Head and neck squamous cell carcinoma (HNSCC) is the fifth most frequent carcinoma in men worldwide and includes tumours at various sites in the oral and upper aerodigestive tract with annSalivary biomarkers are ideal non-invasive diagnostic tool for diseases including cancer , 7–10. TSince its discovery in 1993, miRNAs have been considered a novel regulator biomarker for gene expression . MiRNA imiR-let-7a-5p was found up-regulated in human prostate cancers and this miRNA accompanied the prostate oncogenic process . HoweverUnstimulated whole saliva was collected before surgery from patients attended the Gold Coast University Hospital (GCUH) in Gold Coast, Queensland with HNSCC (n = 150) at different stages of the disease, from different sites . Similarly, whole saliva samples were collected from healthy controls (n = 80) without a history of any malignancy, and who had good oral hygiene. In addition to verbal, written informed consent obtained from all participants. Samples collected from a period of December 1, 2015, to May 30, 2018, from the GCUH. HNSCC were classified according to the extent or size of tumour (T), spread to the regional lymph nodes (N) and the presence or absence of metastasis (M) according to TNM classification (American Joint Committee on Cancer) - [Ct (miR-16)], and the relative miRNA expression level were calculated as 2^-(ΔCt) which is commonly used in miRNAs detection studies. The p-value less than 0.05 was considered as significant.In total, there were 150 saliva samples from HNSCC patients admitted to the GCUH and 80 saliva samples were collected from healthy matching controls, students and staff of Griffith University. The tumour localizations, histopathological features, treatment options, and demographic properties of the HNSCC patients are listed in Based on previous studies performed on profiling miRNAs from different samples and their potential as targets and functions on oral cancer, five salivary miRNAs  were selected for further confirmation using the RT-qPCR technique. We investigated these five validated neoplasm-associated miRNAs in supernatant saliva from both patients with HNSCC and controls groups. Two validated miRNAs  were significantly down regulated . MoreoveReceiver Operator Curves (ROC) were created to evaluate the discriminatory power of miR- let-7a-5p and miR- 3928 for their ability to distinguish between control and disease groups. These miRNAs were revealed to provide a good discriminative ability with AUC values of 0.85and 0.74, respectively. There are no saliva-based biomarkers available in the early detection or as a prognostic indicator for HNSCC. We are reporting two novel biomarkers (miR- let-7a-5p and miR- 3928) in saliva that we had identified to be a reliable with a potential to aid in the diagnosis and prognosis of HNSCC. This adds credibility to the existing knowledge on low level expression of miR-let-7a and miR-3928 as well as the identified list of targeted oncogenes or suppressor genes , 34. ThiHowever, cell proliferation has been inhibited by the increased level of miR-let-7a in the cell line of pancreatic cancer . SeveralWe report significant down-regulation of miR-3928 in the supernatant saliva samples from HNSCC patients. Tumour inhibition role of miR-3928 has been demonstrated in osteosarcoma . It has in vitro and xenograft tumour model in vivo using hepatocellular carcinoma cell lines [Recent reports confirmed that, HBX induces miR-3928v through NF-κB/EGR1 signalling pathway and accelerated hepatocellular carcinoma by decreasing the expression of suppressor gene VDAC3 using calorimetry, colony formation, migration/invasion, vascular mimicry assays ll lines Our results concluded that the expression levels of the miR-let-7a-5p and miR-3928 were significantly decreased in supernatant saliva samples of patients with HNSCC compared with the healthy matched controls. Furthermore, ROC curve analysis revealed that slivery miR-let-7a-5p and miR-3928 expression levels have significant sensitivity and specificity to differentiate between HNSCC patients and healthy controls. This study suggests that the downregulation of miR-let-7a-5p and miR-3928 in patients with HNSCC could be useful tool as an early diagnostic aid and prognostic indicator for HNSCC. Further studies using a larger number of patients, different sites and stages of HNSCC are necessary before elucidating the full potential of the above two miRNAs as salivary biomarkers for HNSCC.


Conventional magnetic resonance imaging (MRI) sequences have known limitations in target delineation for radiation treatment (RT) planning of cerebral gliomas. Advanced physiology-based MRI techniques and radionuclide imaging techniques, including positron emission tomography (PET) with amino acid radiopharmaceuticals, may increase the specificity for glioma tissue characterization. Our work aims to provide a comprehensive review of the advanced MRI and PET imaging modalities that can complement conventional MRI for RT planning of gliomas. A detailed overview of their basic principles and clinical results is given based on the most updated literature.The accuracy of target delineation in radiation treatment (RT) planning of cerebral gliomas is crucial to achieve high tumor control, while minimizing treatment-related toxicity. Conventional magnetic resonance imaging (MRI), including contrast-enhanced T1-weighted and fluid-attenuated inversion recovery (FLAIR) sequences, represents the current standard imaging modality for target volume delineation of gliomas. However, conventional sequences have limited capability to discriminate treatment-related changes from viable tumors, owing to the low specificity of increased blood-brain barrier permeability and peritumoral edema. Advanced physiology-based MRI techniques, such as MR spectroscopy, diffusion MRI and perfusion MRI, have been developed for the biological characterization of gliomas and may circumvent these limitations, providing additional metabolic, structural, and hemodynamic information for treatment planning and monitoring. Radionuclide imaging techniques, such as positron emission tomography (PET) with amino acid radiopharmaceuticals, are also increasingly used in the workup of primary brain tumors, and their integration in RT planning is being evaluated in specialized centers. This review focuses on the basic principles and clinical results of advanced MRI and PET imaging techniques that have promise as a complement to RT planning of gliomas. The mainstay of treatment for patients with either low-grade or high-grade gliomas (HGG) is surgical resection followed by a combination of radio and chemotherapy ,2. In paModern RT techniques, including intensity-modulated radiotherapy (IMRT), stereotactic RT (SRT), and radiosurgery (SRS), allow better conformality of dose to the target with a subsequent decrease in treatment-related complications . HoweverIn this regard, an accurate delineation of tumor volumes and organs at risk (OARs) is critical to ensure maximum target dose and sparing of the surrounding normal brain structures to maintain high tumor control, while minimizing treatment-related toxicity. Most radiation treatment centers are equipped with dedicated computed tomography (CT) scanners that provide precise geometric information of anatomical structures, as well as electron density information for accurate dose calculation in treatment planning systems. Magnetic Resonance Imaging (MRI), using postoperative contrast-enhanced T1-weighted and T2-weighted sequences, has progressively replaced CT imaging because of its excellent soft-tissue contrast, high spatial resolution, and widespread availability. Based on these characteristics, MRI represents the current standard imaging modality for glioma target volume delineation; however, conventional imaging does not provide biological information, such as regional blood volume and microstructural architecture. Limitations of conventional MRI sequences include: (1) Limited capability in differentiating between treatment-related changes and disease progression in previously treated gliomas ,12,13; , diffusion MRI (dMRI), and perfusion MRI (PWI), providing relevant metabolic, structural, and hemodynamic information for treatment planning and monitoring ,19,20. RIn this article, we provide a comprehensive overview of the basic principles and clinical results of the advanced MRI and PET imaging modalities that can complement conventional MRI for RT planning of gliomas.MRI, using pre- and postcontrast T1-weighted and T2-weighted sequences, typically fluid-attenuated inversion recovery (FLAIR) sequences, is mandatory for precise radiation treatment of gliomas . For patFor grade 3 glioma, the typical total dose is 59.4 Gy at 1.8 Gy per fraction . The GTVHowever, it should be noted that the use of variable GTV-to-CTV margins of 10–20 mm represents an oversimplification of the disease biology and recurrence risk pattern . A largeFor recurrent gliomas undergoing reirradiation, the GTV is still defined as the visible lesion on MRI contrast-enhanced T1-weighted sequences; however, limited total doses and small GTV-to-CTV margins of 0-5 mm are usually utilized during reirradiation, either SRS or fractionated SRT, to limit the higher risk of radiation-induced brain necrosis associated with a second course of RT . In this1H-MRS) has been largely used to detect and quantify several endogenous cellular metabolites in brain gliomas in vivo, including choline (Cho), N-acetyl-aspartate (NAA), creatine (Cr), lactate (Lac), and lipid (Lip) Fluorodeoxyglucose (FDG) is the workhorse of PET. Its intracellular transport is primarily mediated by the sodium-independent glucose transporter 1 (GLUT-1), which is constitutively expressed by the endothelial cells forming the BBB and by the glial cells methyl-l-methionine (MET), O-(2-[18F]fluoroethyl)-l-tyrosine (FET) and 3,4-dihydroxy-6-[18F]fluoro-l-phenylalanine (F-DOPA), are now being successfully used in the management of patients with primary or secondary brain tumors fluoro-1-(2-nitro-1-imidazolyl)-2-propanol (FMISO) and [18F]fluoroazomycin arabinoside (FAZA) [2 < 10 mmHg), are progressively reduced, leading to the production of reactive radicals that bind covalently and irreversibly, to intracellular molecules. Conversely, in the presence of molecular oxygen, these tracers are oxidized back and exit the cell. An additional mechanism of tracer accumulation might be related to glutathione conjugation [4-methylthiosemicarbazone) (ATSM) was studied in more detail [2-ATSM and free Cu(I). By contrast, in normoxic cells, Cu(I)-ATSM is reoxidized and exits the cells. 64Cu-ATSM presents potential advantages for RT planning, including its half-life (12.7 h) and higher TBR than nitroimidazole-based tracers [Tumor hypoxia is characterized by an oxygen concentration below critical Onditions . Severalnditions ,147. Thee (FAZA) . Nitroimjugation . Comparejugation . Amongste detail . Cu-ATSM tracers ,147. How tracers ,153.It has been shown that Hypoxia PET imaging may aid RT planning in patients with lung and head and neck cancer by safely guiding dose escalation to tumor regions with higher tracer uptake ,155,156.In summary, several hypoxia-targeting radiopharmaceuticals are available for clinical use and have shown prognostic and therapeutic relevance for solid tumors. Nevertheless, the application of hypoxia-PET in RT planning of glioma has been limited so far. Well-designed trials should evaluate whether hypoxia PET-based RT planning is feasible and advantageous for patients with glioma.There is evidence that MRI techniques may contribute to hypoxia imaging, in addition to radionuclide techniques ,166. The2). Low-grade gliomas exhibit higher OEF and lower CMRO2, while HGG showed lower OEF, higher CMRO2, and higher neovascularization markers [2) of HGG, that drives neo-angiogenesis, supplying oxygen without an OEF, due to the abnormal and inefficient neoangiogenetic network [Quantitative Blood-oxygen-level-dependent (qBOLD) MR imaging exploits the intrinsic paramagnetic properties of deoxy-hemoglobin to infer data regarding blood oxygenation . This ap markers . This ha network . In two  network ,170. By  network . By quan network , and tenIn recurrent gliomas, standard procedures have not yet been established. However, the aim is to limit the re-irradiated volume to the absolutely necessary, i.e., to the active tumor region, to enhance efficacy and decrease possible neurotoxicity. The delineation of tumor volume should, therefore, be restricted to the contrast-enhancing T1 abnormality. Unfortunately, anatomical contrast-enhanced T1-weighted MRI is not suited for differentiating tumor progression from radiation necrosis and therapy-related changes ,171,172.MRSI-derived Cho/NAA abnormality may be relevant for defining the target volume for Gamma Knife radiosurgery (GK-SRS) or other re-irradiation forms in recurrent gliomas. Early retrospective studies demonstrated that patients with recurrent GBM showing areas of elevated Cho/NAA ratio outside of the target volume for GK-SRS had worse OS survival than patients for whom the Cho/NAA abnormality was fully treated ,174. In As for dMRI integration in the re-irradiation setting, additional RT dose painting or boost target based on ADC lowest values has been proposed by Orlandi et al. in a simulation study in recurrent GBM . The feaPWI has been proposed in the re-irradiation setting based on of early studies showing that quantitative DSC-derived metric of rCBV correlates with histological tumor fraction and OS in recurrent GBM . Wang et123I-methyl-tyrosine for RT planning [p = 0.03) [Amino acid radiopharmaceuticals have also been used in the re-irradiation setting. Grosu et al. included 44 patients with HGG  in a study using MET-PET or planning . Treatme = 0.03) . Miwa et = 0.03) . Followi = 0.03) , they us = 0.03) .More recently, Møller et al. performed a Phase I dose-volume escalation trial integrating FET-based BTV (>1.6 TBR threshold) into the RT planning of 31 patients with recurrent HGG . The PTVPotential advantages and current limitations of advanced MRI techniques and amino acid PET in the re-irradiation setting of glioma have been summarized in Several studies compared the information provided by amino acid PET and MRSI ,187, dMRSo far, there are very few available studies directly comparing advanced MRI techniques with amino acid PET in the setting of RT planning. Dissaux et al. compared FET PET with standard MRI sequences and DSC PWI in 30 patients with newly diagnosed HGG scheduled for concomitant chemoradiotherapy. Overlap volumes and spatial similarity between contrast-enhanced MRI-based GTV and FET-based GTV were low, with more than 5 mL of FET-based GTV falling outside the MRI-based CTV (GTV + 20 mm). In contrast, there was a larger agreement between MRI-based GTV and rCBV tumor volume. The authors concluded that MRI underestimates the metabolically active tumor volume and that FET PET is expected to improve the accuracy of target volume delineation, although follow-up clinical information and patterns of recurrence were not provided .low) showed poor overlap with PET-based (median non-overlapping volume = 69.5%) or MRI-based GTV (median non-overlapping volume = 72.2%). The recurrent pattern analysis showed a significantly smaller overlap between recurrent tumor volume and GTV-ADClow compared with PET-based or conventional MRI-based GTV [Another study, mentioned earlier in this review, compared dMRI with contrast-enhanced MRI and FET PET for RT planning of GBM at recurrence . The tumased GTV .Advanced physiology-based MRI techniques and amino acid PET demonstrate higher specificity with respect to conventional MRI for characterizing the biological attitude of cerebral gliomas and are being increasingly used in RT planning in specialized centers and in prospective single- and multi-institutional trials. Amongst the advanced MRI techniques, MRS allows the most accurate depiction of tumor metabolism, thus appearing as a promising imaging method for RT planning integration, especially by using MRSI. However, its widespread application outside specialized centers is still limited by significant technical challenges. On the other hand, dMRI has been proven to be a sensitive, but indirect, method for characterization of glioma tissue microstructure and cellularity, easy to be implemented on clinical scanners. DTI and MR tractography may be useful complements in RT planning as they can help define the anisotropic tumor expansion and invasion of normal white matter structures. The information provided by PWI is relevant for characterization and quantification of tumor neoangiogenesis, but notwithstanding its undoubtful value in predicting glioma prognosis and monitoring of treatments, its impact on RT planning deserves confirmation in larger prospective studies and requires standardization of PWI methods within and across sites. Taking advantage of the wide availability of MRI and its ability to obtain different biological information within a single exam, the combination of multiple advanced MRI techniques may enhance their implementation in the RT planning setting. Amino acid PET is an extremely robust imaging technique that depicts the actual tumor extent with better accuracy than conventional MRI. The inclusion of amino acid PET in RT planning can change the planned treatment volumes in a significant number of patients, and the most recent studies showed a promising correlation between PET-positive tumor volume and patterns of relapse. Larger, prospective studies are warranted to correlate the changes of RT targets according to the information derived from PET and MRI with biological and molecular tumor changes, and to assess the possible clinical impact on treatment efficacy.


Early diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection prevents widespread transmission, manages outbreak control, improves poor prognosis, and reduces mortality of the Coronavirus disease 2019 (COVID-19). Policymakers, public health authorities, healthcare providers, and physicians have deployed COVID-19 control intervention and have made crucial decisions in choosing the appropriate COVID-19 diagnostic tests.Antigen detection tests are diagnostic tests that quickly detect viral components or the virus directly without the need for a laboratory by testing samples collected from nasopharyngeal swabs and urine, and only reveal the active viral infection, not the recovery situation . They arBMC Medicine greater healthcare cost-saving and health outcomes of antigen detection rapid diagnostic tests compared to nucleic acid tests for symptomatic persons [Ricks et al. now report in  persons . The maiThe cost-effectiveness of SARS-CoV-2 diagnostic tests is a major global concern as well as the speed, accuracy, and simplicity of testing. Cost-effectiveness analysis facilitates evidence-based solutions for policymakers to prevent COVID-19 transmission. Silent transmission is also an important public health issue to control COVID-19. Approximately 50% of transmissions occur from asymptomatic or presymptomatic persons . In the Ricks et al.’s research strongly suggests that policymakers could choose COVID-19 antigen detection tests to control COVID-19 instead of nucleic acid tests for symptomatic persons, based on evidence, even with some limitations in the infectivity and transmission dynamics of SARS-CoV2 in the modelling analysis . To make


Neutrophils, the most abundant leukocytes in human blood, are essential fighter immune cells against microbial infection. Based on the finding that neutrophils can either restrict or promote cancer progression, tumor-associated neutrophils (TAN) are classified into anti-tumor N1 and pro-tumor N2 subsets. One of the major mechanisms underlying the tumor-promoting function of N2-TANs is suppression of adaptive immune cells, in particular, cytotoxic T lymphocytes. Currently, no established methodologies are available that can unequivocally distinguish immunosuppressive TANs and granulocytic/polymorphonuclear myeloid-derived suppressor cells (G/PMN-MDSC). In view of the critical role of PMN-MDSCs in immune evasion and resistance to cancer immunotherapy, as established from data obtained with diverse cancer models, therapeutic strategies targeting these cells have been actively developed to enhance the efficacy of immunotherapy. Here, we have reviewed the available literature on strategies targeting PMN-MDSCs and summarized the findings into four categories: (1) depletion of existing PMN-MDSCs, (2) blockade of the development of PMN-MDSCs, (3) blockade of PMN-MDSC recruitment, (4) inhibition of immunosuppressive function. Owing to their high mobility to inflamed organs and ability to trespass the blood-brain barrier, neutrophils are outstanding candidate carriers in nanoparticle-based therapies. Another attractive application of neutrophils in cancer therapy is the use of neutrophil membrane-derived nanovesicles as a surrogate of extracellular vesicles for more efficient and scalable drug delivery. In the second part of the review, we have highlighted recent advances in the field of neutrophil-based cancer drug delivery. Overall, we believe that neutrophil-based therapeutics are a rapidly growing area of cancer therapy with significant potential benefits. Neutrophil differentiation and phenotype are profoundly influenced in multiple ways by solid tumors. Considerable evidence obtained from experimental tumor models and cancer patients supports morphological and functional heterogeneity of tumor-associated neutrophils (TANs)9. In a dichotomy similar to M1/M2 macrophages, TANs have been classified as tumor-inhibitory N1 and tumor-promoting N2 based on their functional differences10. Characteristic nuclear morphologies are potentially associated with N1-polarized and N2-polarized TANs. For instance, N1 neutrophils have hypersegmented nuclei whereas N2 neutrophils have banded or ring-like nuclei10. Recently, lectin-type oxidized LDL receptor-1 (LOX1)11 and fatty acid transport protein 2 (FATP2)12 were identified as distinguishing factors for the two populations of neutrophils in cancer patients, representing a significant advance in the classification of functionally distinct neutrophils based on molecular markers. Despite being a convenient model to describe TANs, N1/N2 classification is an oversimplification of neutrophil heterogeneity. Further molecular characterization of TANs based on single cell gene expression analysis is warranted to define the spectrum of neutrophil polarization within the tumor microenvironment.Neutrophils are the most abundant population of white blood cells and often the first responders to the site of infection. Neutrophils are activated to kill bacterial pathogens 7. Both share a cellular lineage origin and several morphological and phenotypic features, and currently, no surface markers are available that can be readily used for distinguishing the two cell types. By definition, the difference is that PMN-MDSCs potently suppress T cells whereas TANs may or may not possess immunosuppressive activity15. Considering N1/N2 classification, PMN-MDSCs and N2 neutrophils appear equivalent or essentially the same population. We believe that PMN-MDSC still presents a valid and helpful definition to describe a specialized neutrophil state that promotes tumor progression mainly through dampening adaptive immunity. Therefore, in this review, we will refer to immunosuppressive neutrophils and PMN-MDSCs interchangeably to indicate the population of TANs with immunosuppressive activity.The distinction between TANs and granulocytic/polymorphonuclear myeloid-derived suppressor cells (G/PMN-MDSCs) is a controversial issue17.In view of the dominant negative regulatory role of PMN-MDSCs in anti-tumor immunity in many experimental cancer models, significant research has focused on understanding and targeting this cell population as a means of sensitizing tumors to current immunotherapies. Meanwhile, a separate yet related line of research has exploited the strong chemotactic property of neutrophils to deliver tumor-killing drugs. In the current review, we focus on two aspects, specifically, immunosuppressive neutrophil targeting and neutrophil-based drug delivery. For details on functions and mechanisms of TANs and PMN-MDSCs, readers may refer to a number of recent outstanding reviewsvia antibodies targeting immune checkpoint molecules, such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) and programmed death 1/programmed death ligand 1 (PD-1/PD-L1), has made the most significant impact19. However, the efficacy of immunotherapy is limited to a subset of patients and specific cancer types, mainly due to de novo or acquired resistance to immunotherapy21, and driven by tumor-intrinsic or tumor-extrinsic factors23. Among the resistance mechanisms, the impact of tumor-infiltrating PMN-MDSCs is one of the central barriers that impairs cytotoxic T lymphocytes (CTL) activity within the tumor bed. Thus, strategies targeting PMN-MDSCs, in combination with immunotherapy, present a promising approach to overcome resistance to immunotherapy. The currently available methods of PMN-MDSC targeting are classified as: (1) depletion of existing PMN-MDSCs, (2) blockade of the development of PMN-MDSCs, (3) blockade of PMN-MDSC recruitment, and (4) inhibition of immunosuppressive function.Immunotherapy has revolutionized cancer treatment and become a new pillar of cancer therapy. Among the various immunotherapy modalities developed to date, immune checkpoint blockade (ICB) 24 demonstrated that gemcitabine is capable of reducing circulating MDSC and TGFβ-1 levels and elevating the effector T cell: Treg ratio in human pancreatic adenocarcinoma. The group showed a significant decrease in granulocytic but not monocytic MDSCs in the peripheral blood of patients with pancreatic adenocarcinoma eight days after gemcitabine treatment, supporting the utility of gemcitabine in depleting PMN-MDSCs and enhancing immunotherapy. Another chemotherapeutic agent, 5-fluorouracil (5FU), causes cell death by preventing synthesis of DNA and RNA25. Vincent and co-workers showed that 5FU induced a decrease in the number of MDSCs in the tumor microenvironment via triggering apoptosis and promoting IFN-γ production by tumor-infiltrated T cells to enhance antitumor immunity in a mouse EL4 cancer model26. Moreover, 5FU showed no preference for killing monocytic over granulocytic MDSCs and exerted no significant effects on T, natural killer, dendritic or B cells. Therefore, both gemcitabine and 5FU are considered promising agents to eliminate PMN-MDSCs in cancer patients. The antitumor effects of these drugs may be mediated, at least in part, by their selective toxicity against PMN-MDSCs [Figure 1A(1)].Chemotherapeutic drugs are reported to adversely affect MDSC viability. One of the typical side-effects of gemcitabine, a chemotherapeutic agent for many cancer types, including pancreatic cancer, is myelotoxicity. A study by Eriksson et al.+ is used in combination with other markers, such as HLA-DR− CD15+, to identify PMN-MDSCs. Therefore, antibodies and their derivatives targeting CD33 are putative candidate agents for PMN-MDSC depletion. AMV564 is a novel CD33/CD3 tetravalent bispecific antibody currently in early phase clinical trials for relapsed or refractory acute myeloid leukemia (AML) (NCT03144245). Cheng and co-workers27 reported that AMV564 treatment efficiently decreased the number of MDSCs and increased the CD4+ and CD8+ T cell numbers in primary bone marrow microenvironments from patients with myelodysplastic syndromes. The CD33-targeted antibody drug conjugate, gemtuzumab ozogamicin (GO), was reapproved in 2017 for treatment of AML28. To improve treatment options, it is important to determine whether agents, such as AMV564 and GO, demonstrate anti-MDSC activity in patients with solid tumors and show combinatorial synergy with immunotherapy [Figure 1A(2)].Targeting of surface markers presents an attractive depletion approach. In mouse models, anti-Gr1 or anti-Ly6G antibody is often used to deplete PMN-MDSCs. However, Gr1 or Ly6G (part of the Gr1 antigen) is absent in humans, restricting this approach to experimental models. In cancer patients, CD3329 showed that apolipoprotein E (ApoE), which is transcriptionally activated by liver X nuclear receptor (LXR), reduces the survival levels of both tumoral and circulating granulocytic and monocytic MDSCs by triggering apoptosis in mouse and human cancer models [Figure 1A(3)]. LXRβ agonists, such as GW3965 and RGX-104, induced depletion of immunosuppressive MDSCs (both granulocytic and monocytic) and impairment of tumors. Through depletion of MDSCs, LXRβ agonists augmented the efficacy of anti-PD-1 immunotherapy in melanoma and lung cancer models. Currently, the efficacy of RGX-104 as monotherapy and in combination with ICB immunotherapeutics, such as Nivolumab or Ipilimumab, is being evaluated in a phase I clinical trial for patients with malignant solid tumors and lymphoma (NCT02922764).Novel strategies to stimulate apoptosis of MDSCs are under development. For example, Tavazoie et al.Taken together, the literature suggests that PMN-MDSCs can be efficiently depleted using chemotherapeutic or biological agents and targeted therapeutics. When combined with immunotherapeutic strategies, these treatments significantly enhance the effectiveness of immunotherapy and prolong survival of cancer patients.30. These cytokines are capable of promoting accumulation of MDSCs originating from bone marrow and blocking their differentiation into mature myeloid cells, such as granulocytes, dendritic cells, and macrophages32. Thus, inhibition of these growth factors, their receptors or implicated downstream pathways may impair the PMN-MDSC population. For example, reduction in the number and activity of PMN-MDSCs by all-trans retinoic acid (ATRA)33, IL-1234, and anti-G-CSF antibody35 have been reported [Figure 1B(1)].An alternative strategy to reduce PMN-MDSC number is to redirect their developmental pathway, either by inhibiting the conversion of immature myeloid cells into PMN-MDSCs or inducing differentiation of PMN-MDSCs into other mature myeloid cells that possess limited immunosuppressive activity. PMN-MDSC formation is induced by tumor-derived cytokines, such as granulocyte macrophage-colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), and vascular endothelial growth factor (VEGF)36 demonstrated that ultra-low doses of paclitaxel, which neither increased MDSC apoptosis nor blocked MDSC generation, could stimulate differentiation of tumor-infiltrating MDSCs into mature dendritic cells in a TLR4-independent manner in a murine melanoma model [Figure 1B(2)]. Immunostimulatory CpG oligonucleotides delivered via intratumoral injection reduced the immunosuppressive activity of MDSCs in the tumor bed by stimulating their differentiation into macrophages with tumoricidal capability37 [Figure 1B(3)].Theoretically, normalizing aberrant differentiation of MDSCs back into mature myeloid cells with diminished immunosuppressive and enhanced antitumor activities presents an ideal strategy with little anticipated toxicity. A number of studies support this possibility. Paclitaxel is a well-known chemotherapy drug for several cancer types. Interestingly, Michels et al.Overall, blockade of the PMN-MDSC development, either by blocking their generation or inducing differentiation into non-suppressive myeloid cells, is an efficient strategy to abolish immunosuppression induced by PMN-MDSCs.via specific chemotactic pathways. Several chemokine signaling pathways are involved in PMN-MDSC recruitment to the tumor microenvironment. Inhibitors of chemokine receptors are therefore promising candidate agents that may aid in preventing PMN-MDSCs from entering the tumor bed and shaping the immunosuppressive microenvironment . Earlier studies using CXCR2 inhibitors (SB225002 and SX-682), a neutralizing antibody and peptide (pepducin) confirmed CXCR2 signaling as the central pathway for recruitment of PMN-MDSCs. Notably, CXCR2 blockade inhibited tumor progression in various tumor models41 and restored the sensitivity of highly refractory tumor models to immunotherapy43, a testimony for a key role of PMN-MDSCs in immunosuppression and resistance to cancer immunotherapy. A fusion protein, mCCR5-Ig, was generated to hinder PMN-MDSC accumulation via blocking CCR5 signaling in melanoma models45. In animal models of breast cancer metastasis, blocking the γδT cell/IL-17/neutrophil axis by neutralization of IL17 or G-CSF prevented neutrophil accumulation and downregulated the T cell-suppressive phenotype46. Ongoing clinical trials are underway to determine whether blocking PMN-MDSC recruitment can effectively enhance cancer immunotherapy. For example, NCT03161431 is an ongoing phase I clinical trial evaluating a combination of the CXCR1/2 inhibitor, SX-682, and ICB antibody, pembrolizumab, in patients with metastatic melanoma.PMN-MDSCs are recruited Based on the above findings, it is reasonable to suggest that blocking recruitment of PMN-MDSCs presents an effective prophylactic approach to inhibit the immunosuppressive activity of the tumor microenvironment, which may improve the efficacy of immunotherapy.Downregulation of the immunosuppressive function of PMN-MDSCs can be achieved by either silencing the signaling that controls immunosuppressive activity or diminishing immunosuppressive products secreted by PMN-MDSCs. Such treatments may help reprogram the tumor microenvironment to have less hostility toward CTLs and improve the chances of success of cancer immunotherapy.Figure 1D(1)]. Patnaik et al.47 observed that inhibiting receptor tyrosine kinase signaling with Cabozantinib in pten-/-p53-/- mouse prostate tumor models promoted infiltration of neutrophils with antitumor activities into tumors, attenuating cancer progression. In this context, CXCL12 and HMGB1 are two tumor-secreted neutrophil chemotactic factors that recruit CXCR4-expressing neutrophils with antitumor properties. Another study by our group demonstrated that other than recruiting N1-like neutrophils, cabozantinib as well as the dual PI3K/mTOR inhibitor, dactolisib, significantly downregulated immunosuppressive genes, such as Arg1, Ncf1 and Ncf4, in tumor-infiltrating PMN-MDSCs through targeting PI3K signaling43. By suppressing PMN-MDSCs, these drugs synergized with ICB to eradicate both primary and metastatic prostate tumors in the PB-Cre+L/L p53L/L Smad4L/LPten mouse model43. Isoform-selective PI3K inhibitors, such as the PI3Kγ inhibitor PI-54949, PI3Kβ inhibitor GSK263677150, PI3Kδ/γ inhibitor IPI-14551 and (-)-4-O-(4-O-β-D-glucopyranosylcaffeoyl) quinic acid52 were shown to suppress tumor-promoting myeloid cells, including PMN-MDSCs, and enhance immunotherapy. Given the importance of STAT3 in MDSC expansion and activation32, silencing of STAT3 in PMN-MDSCs via an antisense oligonucleotide tethered to CpG oligonucleotide (CpG-STAT3ASO) reduced circulating PMN-MDSCs and promoted the CTL to Treg ratio in prostate cancer models53. Tumor-derived COX2-PGE2 signaling plays a key role in promotion of PMN-MDSCs in both animal models and human PBMCs, and COX2 inhibitors, such as celecoxib and SC-236, have been shown to diminish the number and activity of PMN-MDSCs, in part, through inhibition of STAT3 in myeloid cells60. S100A8/A9 is a heterodimeric pro-inflammatory mediator involved in both chronic and acute inflammation through activation of TLR4- or RAGE-mediated inflammatory pathways61. In gastric cancer patients, PMN-MDSCs accumulating in the tumor bed secreted higher levels of S100A8/A9. Inhibition of S100A8/A9 and its receptor (RAGE) impaired the ability of PMN-MDSCs to suppress T cell proliferation and IFN-γ production62. The effects of S100A8/A9 on blood, splenic and tumoral MDSCs were also effectively abolished by a peptide-Fc fusion protein (peptibody), which outperformed anti-Gr1 antibody and completely depleted both granulocytic and monocytic MDSCs in lymphoid organs and tumors in mice without affecting pro-inflammatory immune cell types, such as dendritic cells63. Inhibition of phosphodiesterase-5 (PDE5) by sildenafil  induced downregulation of arginase 1 and nitric oxide synthase-2, thereby reducing activity of intratumoral MDSCs65. Surprisingly, a long noncoding RNA, Pvt1, was shown to regulate the immunosuppressive activity of PMN-MDSCs in the tumor microenvironment, and siRNA-mediated silencing of Pvt1 inhibited intratumoral PMN-MDSC activity in the Lewis lung carcinoma mouse model66 [Figure 1D(1)].Various strategies have been developed to dampen the immunosuppressive activity of PMN-MDSCs [Figure 1D(2)]. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are the main weapons used by PMN-MDSCs to cause tolerance or death of CTLs71 and tumorigenic mutations in epithelial cells72. Another major mechanism used by PMN-MDSCs to suppress T cells is production of Arginase-1 that leads to diminished L-arginine levels, affecting T cell function74. Arginase-1 remains in the inactive form within granules of neutrophils and becomes activated when neutrophils release their granule content76. The agents shown to neutralize or inhibit the production of these molecules include N-hydroxy-nor-l-arginine (nor-NOHA)67, uric acid77, and bardoxolone methyl78. A recent study by our group demonstrated that RNS from intratumoral PMN-MDSCs promoted nitration of Tyr394 of lymphocyte-specific protein tyrosine kinase (LCK) in CTLs and abolished T cell receptor signaling through disruption of the phosphorylation cascade77. Neutralizing RNS with uric acid was a satisfactory strategy that negated the effects of PMN-MDSCs and greatly promoted the efficacy of immunotherapy in both prostate and lung cancer models. However, the optimal approach to neutralize PMN-MDSC-derived RNS in cancer patients remains to be established.The anti-inflammatory factors  produced by PMN-MDSCs additionally present promising targets. Treatments that act by inhibiting these factors are considered the last line of defense against the effects of PMN-MDSCs [8. NETs can immobilize and neutralize pathogens including fungi, bacteria, and viruses79. Recently, NETs have been shown to assist metastasis by trapping circulating tumor cells (CTCs), promoting cancer cell invasion, and awakening cancer cells from dormancy83. Strategies to inhibit NETosis or remove existing NETs could therefore present an effective way to diminish metastasis. The agents exerting anti-NETosis effects in cancer models include DNase I-coated nanoparticles that digest NETs80, antibodies against NET-remodeled laminin81, heparin that sequesters histone from NETs with its negative charges84, and inhibitors of protein-arginine deiminase 4 (PAD4), an enzyme required for NET formation85. Use of these inhibitors should be carefully evaluated in patients, since inhibition of NETs may also compromise their important functions in innate immunity.A particularly interesting strategy to target N2 neutrophils is through blocking neutrophil extracellular traps (NET). NETs are composed of DNA fibers with granule-derived antimicrobial proteins  secreted by neutrophils through a process known as NETosisWe have summarized the four main strategies employed to target PMN-MDSCs, including depletion using apoptosis-induced agents, blockade of the development, blockade of recruitment from the bone marrow, and elimination of immunosuppressive signaling and associated factors. Several important challenges and issues remain in developing selective therapeutics to target this population of neutrophils. First, the methodology used to define PMN-MDSCs in experimental models and clinical samples makes evaluation cumbersome. Due to the lack of definitive markers to distinguish PMN-MDSCs from other types of neutrophils, isolated intratumoral PMN-MDSCs must be subjected to a T cell co-culture assay to evaluate whether the therapeutic agent can diminish immunosuppressive activity. This method is feasible for experimental models but difficult to implement in clinical trials. Second, the signaling mechanisms used by PMN-MDSCs to suppress T cells are often hyperactive in other cancer cells or M2-polarized macrophages in the tumor microenvironment. Therefore, it is often difficult to attribute the therapeutic effect solely to PMN-MDSCs. Third, due to the complexity and redundancy of immunosuppressive mechanisms, blocking one mechanism may be compensated by other mechanisms, leaving T cells still suppressed. Therefore, blockage of PMN-MDSC recruitment may be a favorable strategy because without continuous recruitment from the bone marrow, tumors are expected to exhaust their supply of infiltrating PMN-MDSCs rapidly and become susceptible to killing by CTLs activated by immunotherapy. Fourth, most of the existing knowledge on PMN-MDSCs has been obtained from mouse models. However, the differences between human and mouse immune systems, especially in the context of neutrophils, and their roles in cancer and immunotherapy make translation of findings from mouse models to clinical therapy a long and difficult process. To address these challenges, future research should focus on identifying biomarkers highly specific for PMN-MDSCs and developing more selective therapeutics (antibody-drug conjugate or bispecific antibody based on highly selective PMN-MDSC surface markers). New humanized mouse models incorporating PMN-MDSCs, autologous cancer cells and T cells of human origin should be developed as a tool for elucidation of PMN-MDSC biology and therapeutics.3. Owing to high responsiveness and mobility, neutrophils present an outstanding candidate cellular carrier for various antitumor reagents86. So far, two main strategies have been employed: (1) use of live neutrophils and (2) use of neutrophil membrane-derived nanovesicles as drug delivery tools.As the most abundant immune cells in the circulation, neutrophils play an essential role in immune responses to tissue damage or infection. Upon inflammation, neutrophils are the first immunocytes recruited to the inflammation site87. Limited NP-based delivery strategies have been successful in the clinic and solving the NP delivery problem should accelerate clinical translation of nanomedicine. The remarkable ability of neutrophils to migrate from the circulation to various tissues supports their utility as promising drug carriers to enhance the therapeutic effect91. Recently, Wang et al.92 showed that activated but not resting neutrophils or monocytes were able to take up drug-loaded albumin NPs in a Fcγ receptor-dependent manner. Neutrophils treated with albumin NPs loaded with Syk inhibitor, piceatannol, lost “outside-in” β2 integrin signaling activity and detached from the inflamed vasculature in vascular inflammation and lung injury models. This capability of activated neutrophils to take up NPs is potentially exploitable for cancer treatment.Targeting of solid tumors with nanoparticles (NPs) loaded with antitumor drugs is an attractive therapeutic option. However, the vast majority of administered NPs are not effectively delivered to solid tumors94 demonstrated that neutrophil-mediated delivery of NPs across the blood vessel barrier into tumor tissues after induction of inflammation is a valid approach to precisely deliver therapeutic agents in tumor models. In both investigations, acute stimulation of inflammation, either via a monoclonal antibody or photosensitization, was required for neutrophil recruitment. Neutrophils delivered pyropheophorbide-a loaded NPs or anti-CD11b antibody-linked gold nanorods to the tumor bed and induced a tumoricidal effect .In two studies, Chu et al.96. Xue et al.97 reported that neutrophils carrying paclitaxel (PTX) liposomes could penetrate the brain and suppress recurrence of glioma in mice whose tumors had been resected surgically. The pro-inflammatory factors generated at the site of resection triggered massive infiltration of neutrophils. The high concentration of inflammatory signals in the brain further triggered release of liposomal PTX from neutrophils, facilitating delivery of PTX to residual tumor cells . As a result, neutrophil-mediated delivery of drugs efficiently impeded relapse and improved survival.A particular advantage of using neutrophils as the vehicle for drug delivery is their ability to penetrate the blood brain barrier (BBB) and reach inflamed brain tumors owing to high migratory capacityin situ setting, loading neutrophils with toxic anti-cancer drugs ex vivo, such as paclitaxel, is expected to be a significant challenge, since neutrophils are highly sensitive and have a short lifetime ex vivo. Moreover, chemotherapeutic drugs may significantly impair neutrophil viability and functions, such as cell migration98. Nevertheless, Xue and colleagues97 showed that PTX liposomes exert negligible toxicity to neutrophils compared with free PTX, suggesting that ex vivo loading of neutrophils with carefully designed chemotherapeutic drug-containing NPs is feasible and loaded neutrophils may survive long enough after infusion to effectively target tumors and generate the desired therapeutic effect.In contrast to the delivery of non-toxic agents, such as photosensitizers or gold NPs in an While still an emerging field of research, studies to date have revealed that the use of neutrophils as vesicles for drug delivery can improve the efficacy of immunotherapy and aid in resistance to tumor recurrence. With broader application of immunotherapy in cancer treatment, we expect neutrophil-mediated drug delivery to become a valuable complementary approach.in vivo, leading to compromised therapeutic effects and elevated risk of systemic immune responses100.Lipid-based nanomaterials, such as liposomes, are extensively used as carriers for drug delivery due to several suitable physical and chemical properties. However, the exogenous nature of liposomes may elicit immunogenicity 101. However, clinical application of EVs is currently impeded by low yield, inefficient drug loading and difficulty in scalability102.Extracellular vesicles (EV) are lipid bilayer-delimited particles naturally released from cells that are loaded with proteins, lipids, nucleic acids and sometimes even organelles. These vesicles play important roles in intercellular communication under both physiological and pathological conditions. A variety of cell adhesion molecules are expressed on the EV surface, facilitating their utility as carriers for drug delivery102 reported a strategy to generate EVs using nitrogen cavitation (NC-EVs) that instantly disrupts neutrophils to form nanosized membrane vesicles. NC-EVs are similar to naturally secreted EVs (NS-EVs) by neutrophils but include less nuclear acids and organelles. Due to the high yield of production (16 times higher than NS-EVs), NC-EVs are potentially applicable for clinical use. In the study, NC-EVs loaded with piceatannol (an anti-inflammatory drug) alleviated acute lung inflammation/injury and sepsis induced by lipopolysaccharide in mice . The data suggest that nitrogen cavitation is feasible for efficient manufacture of neutrophil-membrane-derived EVs on a large scale. This approach may therefore be adapted for the efficient production of chemotherapy drug-loaded NC-EVs.Gao et al.103. Compared with uncoated NPs, NM-NPs displayed enhanced cellular association under shear flow in vitro and higher capture efficiency of CTCs in vivo. For therapeutic purposes, NM-NPs loaded with carfilzomib, a second-generation proteasome inhibitor, exhibited impressive activity in depleting CTCs in the circulation and inhibiting the initiation and development of metastasis in animal models . This exciting study reveals the potential of of NP-loaded neutrophil-derived vesicles in treating metastasis, a direction warranting further investigation.A nanosized neutrophil-mimicking drug delivery system (NM-NP) was developed by Kang and co-workers by coating neutrophil membranes on the surface of poly (lactic-co-glycolic acid) NPs without damaging membrane-associated proteins and binding affinitiesWe have reviewed two related topics: (1) how to target immunosuppressive neutrophils and (2) how to use neutrophils to battle cancer. For targeting immunosuppressive neutrophils, four types of strategies have been developed, including depletion, redirection of differentiation, blockage of recruitment, and functional inactivation of PMN-MDSCs. These approaches require a deeper mechanistic understanding of neutrophils in cancer development and immune evasion. Among the many challenges of neutrophil targeting, perhaps the most prominent is molecular discrimination of TANs, especially PMN-MDSCs, from the remaining neutrophil populations required for innate immunity against bacterial and fungal pathogens. We believe that technological advances, especially single cell profiling techniques, such as single cell RNA-seq and singe cell proteomics, will lead to greatly enhanced abilities to classify heterogeneous neutrophil populations, pinpoint specific protein markers in “bad” neutrophils and spare “good” neutrophils to avoid neutropenia as a major side-effect of neutrophil-targeted therapy.Neutrophils are a double-edged sword in cancer development. Strategies should be developed not only to disrupt pro-tumor neutrophils but also reinstate anti-tumor neutrophils. Application of neutrophils and their membranes for better drug delivery and novel therapeutics has already generated impressive results in preclinical models and neutrophil-based therapeutics present a rapidly growing area of cancer therapy. With the accumulating research efforts dedicated to targeting tumor-associated immunosuppressive neutrophils and devising novel neutrophil-based therapies, we expect significantly higher numbers of cancer patients to benefit from immunotherapy and personalized targeted therapies in the next decade.


In vivo two-photon imaging was used to directly visualize MMP9 activity using a FITC-gelatin probe and leakage of intravenous dye during photothrombotically induced capillary ischemia. Results showed that the NOS inhibitor, L-NIL, at concentrations affecting both iNOS and constitutive NOS isoforms, attenuated capillary leakage at pericyte soma-specific locations and substantially reduced FITC-gelatin cleavage. We also found that combined administration of L-NIL and anisomycin, an inhibitor of protein synthesis, led to near complete elimination of FITC-gelatin cleavage and vascular leakage. These results indicate that both nitric oxide synthase and new protein synthesis are involved in the rapid activation of MMP-9 at somata of capillary pericytes during ischemia.Nitric oxide serves essential roles in normal vascular physiology, but paradoxically contributes to vascular pathology in disease. During brain ischemia, aberrant nitric oxide levels can cause cellular injury through induction of nitrosative/oxidative stress and post-translational activation of matrix-metalloproteinase-9 (MMP-9). We recently demonstrated that brain pericyte somata were associated with very early and localized MMP-9 activation along capillaries during cerebral ischemia, leading to focal blood-brain barrier disruption. Here, we tested whether this effect was dependent upon nitric oxide production.  This was coupled with a photothrombotic model of capillary occlusion to induce ischemia during imaging, and then rapidly track its pathological consequence. These studies revealed extensive overlap between the somata of capillary pericytes, FITC-gelatin cleavage and leakage of blood plasma. These findings point to pericytes as a trigger for rapid MMP-9 activation, likely through induction of existing pools of MMP-9 or rapid MMP-9 expression. However, the prior studies did not delineate the upstream signals involved in pericyte-associated MMP-9 activation.During stroke, rapid activation of the proteolytic enzyme, matrix-metalloproteinase-9 (MMP9) leads to degradation of the neurovascular unit and compromise of the blood-brain barrier (BBB) . This prischemia . This waischemia . To studin vivo.Nitric oxide (NO) is a diffusible signaling molecule integral for regulation and maintenance of vascular homeostasis . Nitric in vivo during two-photon imaging, by using focal photothrombotic approach to occlude small regions of capillary bed in the cerebral cortex lysine (L-NIL), together with FITC-gelatin 30 min prior to photothrombosis. Two doses of L-NIL were tested: A lower dose expected to inhibit primarily iNOS (3.8 μM) and a higher dose (19 μM) expected to begin inhibiting constitutive forms, nNOS and eNOS as well. The low-dose of 3.8 μM affected neither the occurrence of vascular leakage nor MMP-9 activation . HoweverWe further asked whether rapid MMP-9 activation was entirely a post-translational process or also involved new protein synthesis. To test this, we administered the protein synthesis inhibitor, anisomycin , either alone or in combination with intra-cortical injection of 19 μM L-NIL . AnisomyWe further compared the effects of anisomycin, alone or in combination with L-NIL on MMP-9 activity at leakage locations . AnisomyWe have previously shown that pericytes are involved in very early BBB damage during ischemia, and that the vascular damage near their somata corresponds to rapid increases MMP-9 activity . The speOur findings add further weight to the notion of pericyte sensitivity in vascular pathology by showing that NO can act on pericyte MMP-9 activation very rapidly after ischemic onset. This is complementary to prior work showing that pericytes are exquisitely sensitive to nitrosative/oxidative stress during brain ischemia and . Yemisciin vivo  at high levels, but also iNOS (Nos2) at lower levels in the normal brain microvasculature . The proin vivo . HoweverIn summary, our results provide a basis for deeper mechanistic studies on pericytes as inducers of BBB disruption in stroke, and potentially other cerebrovascular diseases were MMP-9 has been implicated in microvascular pathology . PericytTdtomato reporter mice (Ai14) on a C57/Bl6 background were purchased from Jackson Labs (Jax #: 007914) . These mSkull-removed, dura-intact craniotomies  were genTwo-photon imaging was performed with a Sutter Moveable Objective Microscope and a Coherent Ultra II Ti:Sapphire laser source. A wavelength of 800 nm was used for excitation of FITC-gelatin and Texas red-dextran. Excitation was tuned to 975 nm to successively capture tdTomato fluorescence at the start of each experiment. Green and red emission was simultaneously collected using ET525/70m and ET605/70m filter sets, respectively (Chroma Corp.). Throughout the duration of imaging, mice were maintained under light isoflurane (0.75%) supplied in medical air . Pulse oximetry  was used to monitor blood oxygen saturation and heart rate to ensure that cardiovascular function was normal during imaging.Procedures for vascular imaging and analysis have been described previously . BrieflyRestricted photothrombotic capillary occlusions have been previous described . Brieflyin vivo using two-photon microscopy were rendered in 3-D using Imaris 7.7 Software (Bitplane). Lateral sampling was 0.41 μm per pixel and axial sampling was 1 μm per pixel. Vascular leakage was quantified from 3-D renderings of the Texas red-dextran labeled channel, and also confirmed in 2-dimensional images by scrolling through z-stacks with Fiji/ImageJ software. This method of quantification was previous described  in PBS, together with the FITC-gelatin, which was then injected intracortically.Anisomycin was administered intraperitoneally at a concentration of 75 mg per kg of mouse body weight. Anisomycin was prepared by adding the minimum amount of 1 M HCL required to bring the drug into solution. Sodium hydroxide (1 M) was then added to bring the solution to a neutral pH. Saline was then added to bring the solution to the appropriate concentration . IntrapeAll statistical analyses were performing using Graphpad PRISM 9 software. Statistical details are provided in the legend of each figure.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher. Source data is provided in The animal study was reviewed and approved by IACUC of the Medical University of South Carolina.RU performed the data collection and analysis. RU and AS wrote the manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this commentary, we discuss possible mechanisms of the COVID-19-induced AKI, indicating potential new approaches for risk stratification of SARS-CoV-2 infected patients that may help clinical decision in this emerging scenario.Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the pathogen responsible of atypical pneumonia that has affected more than 330,000 people and caused death of more than 14,000 patients . Coronavirus spike (S) glycoproteins mediate entry into cells end predominantly lung epithelial alveolar cells precipitating an interstitial pneumonia that evolves through acute respiratory distress syndrome (ARDS) [AKI represents a life-threatening complication in critically ill patients, often leading to increased risk of death. As recently reported by Wilson et al., the onset of moderate-to-severe AKI described a higher-risk subset of ARDS patients, with a significant risk for mortality . ConsideAs described, AKI developed in average 9 days after admission together with secondary infections and acute cardiac damage , 3. Age,Therefore, determining the risk for developing AKI in SARS-CoV-2 infected patients or progressing to severe AKI requiring renal replacement therapies  is an imA positive test suggesting a high risk of developing AKI may require an early nephrology consultation , the cloARDS patients developing AKI are a higher risk for severe outcomes and mortality. In our opinion, it is compelling to establish a prediction model to stratify SARS-CoV-2 infected patients according to AKI severity; this approach might lead to an innovative biomarker-based interventional strategy in this emerging clinical contest leading to a better allocation of hospital resources.


Oncolytic virus (OV) is a new therapeutic strategy for cancer treatment. OVs can selectively infect and destroy cancer cells, and therefore act as an in situ cancer vaccine by releasing tumor-specific antigens. Moreover, they can remodel the tumor microenvironment toward a T cell-inflamed phenotype by stimulating widespread host immune responses against the tumor. Recent evidence suggests several possible applications of OVs against cancer, especially in combination with immune checkpoint inhibitors. In this review, we describe the molecular mechanisms of oncolytic virotherapy and OV-induced immune responses, provide a brief summary of recent preclinical and clinical updates on this rapidly evolving field, and discuss a combinational strategy that is able to overcome the limitations of OV-based monotherapy. Viruses are continuously co-evolving with our immune systems and have developed sophisticated mechanisms to manipulate host immune responses . With scA big breakthrough has been achieved in the field of cancer immunotherapy over the last decade. Immune checkpoint inhibitors (ICIs) targeting cytotoxic T lymphocyte-associated protein 4 (CTLA-4); programmed cell death 1 (PD-1); and PD-1’s main ligand PD-L1, have been introduced for the treatment of more than a dozen types of cancers ,19,20. HIn this review, we will summarize OVs’ mode of action and provide recent preclinical and clinical evidence supporting OV as an ideal platform for combination immunotherapy.Tumor selectivity is an essential prerequisite of OVs to guarantee maximal oncolysis while minimizing off-target effects on normal tissue . Since tThe therapeutic efficacy of OVs depends on two main modes of action . (1) OVsBesides tumor cells and immune cells, other cellular components within the TME also respond to OV therapy ,28,29,30Over the past two decades, OV therapeutics have grown very rapidly with the advancement of molecular biology, virology, immunology, and genetic engineering . Table 1Melanoma is one of the most sensitive types of malignancy for cancer immunotherapy. T-VEC demonstrated its clinical efficacy in patients with melanoma and has inaugurated the era of oncolytic virotherapy. T-VEC is the first FDA-approved oncolytic herpesvirus, genetically modified to selectively replicate within tumor cells and to increase tumor antigen presentation by dendritic cells through granulocyte-macrophage colony-stimulating factor (GM-CSF) transgene expression . HSV-1 iClinicalTrials.gov NCT00769704) was a randomized phase III trial that compared T-VEC and GM-CSF in patients who had histologically confirmed unresectable stage IIIB/C/IV melanoma with at least one cutaneous, subcutaneous, or nodal lesion [OPTiM  have completed phase I trials in glioma patients, and phase II trials with these OVs are now ongoing . The thiPancreatic cancer remains one of those with the poorest prognosis and major causes of cancer-related deaths worldwide . Its pooBreast cancer is the most commonly diagnosed cancer among women. Despite recent advances in molecularly targeted therapies, there are still treatment-resistant cases; thus, OVs have become new therapeutic options against these intractable breast cancers. Various types of OVs have been investigated in patients with breast cancer. The novel oncolytic HSV-encoding interleukin 12 (designated G47Δ-mIL12) showed significant anti-tumor effects in a preclinical model of syngeneic triple-negative breast cancer (4T1) . OncolytBecause OVs as single agents showed limited efficacy in clinical trials , a combiImmune checkpoint blockade has revolutionized the therapeutic landscape of advanced cancer over the last decade. ICIs are monoclonal antibodies that block immune checkpoint proteins, such as PD-1, PD-L1, or CTLA-4, which are natural brakes of the immune system, from interacting with their binding partners ,62. Thisp = 0.022). The median progression-free survival was 13.5 months with combo therapy and 4.5 months with ipi monotherapy. Therefore, the T-VEC plus ipi combo showed remarkable tumor burden reduction and durable activity in patients with melanoma [Recently, the combination of OVs with ICIs has been intensively investigated in many clinical trials . Combinamelanoma .+ (CD8+) T cells, elevated PD-L1 expression, and IFN-γ gene expression after T-VEC treatment. However, objective responses were not associated with baseline CD8+ T cell infiltration or baseline IFN-γ signature. These results indicate that T-VEC may improve the efficacy of pembrolizumab by changing the TME [In a phase Ib trial for patients with advanced melanoma, 21 patients were treated with T-VEC followed by combination therapy with pembrolizumab. This combination therapy was well tolerated and showed no dose-limiting toxicities. The objective response rate and complete response rate were 62% and 33%, respectively. Patients who responded to combination therapy demonstrated increased intratumoral cluster of differentitiation 8 the TME . On the On the basis of tolerable safety for intrahepatic injection of T-VEC , a phaseThe ONCOS-102 is a chimeric oncolytic adenovirus armed with human GM-CSF and Ad5/3 chimeric capsids, and it has shown promising anti-tumor effects through the upregulation of PD-L1 in a phase I clinical trial against solid tumors (NCT01598129) [In a phase Ib trial for patients with metastatic or unresectable renal cell carcinoma (REN026), IV infusion of Pexa-Vec also reported promising results in combination with cemiplimumab (anti-PD-1) in an American Association for Cancer Research (AACR) 2020 meeting . The oveChimeric antigen receptor T (CAR-T) cell therapy implies in vitro design, modification, and amplification of T cells from a patient’s blood to grant them the ability to recognize the surface antigens on tumor cells via the transduced CAR structure on the T cell surface. After amplification in the laboratory, these CAR-T cells are administered intravenously to the patient with cancer. In a mouse neuroblastoma model, oncolytic adenovirus armed with the chemokine RANTES  and the cytokine interleukin-15 (Ad5Δ24) enhanced migration and proliferation of CAR-T cells . TherefoBispecific antibodies are a novel class of anti-tumor agents that simultaneously target two different types of antigens or epitopes by combining two antibodies . BispeciBiTE applications in solid tumors have shown some limitations, such as low tumor penetration and off-target effects . To overRecently, tri-specific killer cell engagers (TriKEs) have also been developed. Eric Vivier et al. generated a new trifunctional antibody that engages two activation receptors (NKp46 and CD16) on natural killer cells and a tumor antigen on cancer cells . They deAlthough OVs can induce anti-tumor immunity through multiple mechanisms and serve as an ideal platform for combination immunotherapy, there are still many issues to be solved to optimize OV-based immunotherapy. These include viral species, delivery platforms, intratumoral viral spread, and dosing strategies ,8,27. InA variety of viral species have been developed as OVs. Because different kinds of viruses have diverse sizes, shapes, genetic materials, and pathogenicity, understanding the unique biological characteristics of OVs is an essential step for developing the most effective anti-tumor oncolytic virotherapy ,27. For OVs can be delivered locally  or systemically (mainly intravenously). Local intratumoral injection is the most common route of administration; intratumoral delivery maximizes the concentration of OVs at target tumor lesions while minimizing systemic toxicity ,82,83. HNew delivery platforms have also improved the therapeutic effects of OVs. Usually, viral particles are rapidly degraded by the host immune system. Various agents such as nanoparticles, liposomes, polyethylene glycol, and polymeric particles have been used to deliver OVs from the systemic circulation to the local TME ,85. MagnThe intratumoral distribution of OV within the TME is also a critical factor that determines its anti-tumor efficacy. Besides the aforementioned virus size and envelope type, a dense ECM within tumors serves as a physical barrier to intratumoral OV infiltration and diffusion ,28,83. TOV clearance through anti-viral immunity can also limit OV-induced anti-tumor efficacy ,82. TherThe immunosuppressive TME is another hurdle against effective oncolytic virotherapy. Tumor cells continuously secrete immunosuppressive chemokines and cytokines such as IL-10, TFG-β, and arginase-1 to reduce the amplitude of OV-induced anti-cancer immune responses ,93,94. TOver the past two decades, OVs have emerged as promising immunotherapeutic agents against advanced cancers. Since the FDA approval of T-VEC as a new cancer therapy for patients with melanoma in 2015, many OVs have demonstrated modest anti-tumor efficacies with tolerable toxicity profiles as a monotherapy when administered through either intratumoral or systemic routes in clinical trials. To improve on this, researchers generated genetically modified OVs and combined them with other therapeutic modalities, most frequently with ICIs. In the future, researchers will develop new combination therapies with other agents, generate newly genetically engineered OVs, and produce new delivery systems. After overcoming current hurdles of oncolytic virotherapy, such as physical barriers, immunosuppressive TME, and host clearing of OVs, OVs will be the most powerful therapeutic strategy in cancer treatment.


PLR was recorded in response to light stimuli of various intensities. DAMGO eliminated PLR evoked by light with intensities below melanopsin activation threshold but not that evoked by bright blue irradiance that activated melanopsin signaling, although in the latter case, DAMGO markedly slowed pupil constriction. CTAP or genetic ablation of MORs in ipRGCs slightly enhanced dim-light-evoked PLR but not that evoked by a bright blue stimulus. Our results suggest that endogenous opioid signaling in the retina contributes to the regulation of PLR. The slowing of bright light-evoked PLR by DAMGO is consistent with the observation that systemically applied opioids accumulate in the vitreous and that patients receiving chronic opioid treatment have slow PLR.Opioid peptides and their receptors are expressed in the mammalian retina; however, little is known about how they might affect visual processing. The melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs), which mediate important non-image-forming visual processes such as the pupillary light reflex (PLR), express β-endorphin-preferring, µ-opioid receptors (MORs). The objective of the present study was to elucidate if opioids, endogenous or exogenous, modulate pupillary light reflex (PLR) via MORs expressed by ipRGCs. MOR-selective agonist [D-Ala Over the past 25 years, the liberalization of laws governing opioid prescription for the treatment of chronic non-cancer pain has led to dramatic increases in opioid use, often referred to as an opioid epidemic in the United States ,2,3. Whis after onset of high irradiance stimuli -enkephalin (DAMGO) or the MOR-selective antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP)  (2 mg/mL each) were administered via unilateral intravitreal injection (2 µL/eye) under isoflurane anesthesia following application of topical 0.5% proparacaine [n” stands for the number of mice tested throughout the paper.MOR-selective agonist [D-Alaaracaine . ControlOur previous multielectrode array data shows that the maximal effect of DAMGO for reducing ipRGC response was reached at ~1 µM. . Intravip < 0.05 considered significant.All data were analyzed using SigmaPlot11 . Specific statistical comparisons are described in-text. Data are presented as mean ± SEM, and Our results indicate that intraocular opioids acting on MORs of ipRGCs are negative modulators of the PLR and are suggestive of a potential increase in endogenous opioid concentrations in the dark-adapted retina. Future studies should investigate the effect of systemic opioid administration on both the static and dynamic PLR, as this may have a significant impact on the interpretation of diagnostic pupillometry and serve as a potential biomarker of systemic opioid effect.


Flaviviridae family of positive-sense RNA viruses contains important pathogens of humans and other animals, including Zika virus, dengue virus, and hepatitis C virus. The Flaviviridae are currently divided into four genera—Hepacivirus, Pegivirus, Pestivirus, and Flavivirus—each with a diverse host range. Members of the genus Hepacivirus are associated with an array of animal species, including humans, non-human primates, other mammalian species, as well as birds and fish, while the closely related pegiviruses have been identified in a variety of mammalian taxa, also including humans. Using a combination of total RNA and whole-genome sequencing we identified four novel hepaci-like viruses and one novel variant of a known hepacivirus in five species of Australian wildlife. The hosts infected comprised native Australian marsupials and birds, as well as a native gecko (Gehyra lauta). From these data we identified a distinct marsupial clade of hepaci-like viruses that also included an engorged Ixodes holocyclus tick collected while feeding on Australian long-nosed bandicoots (Perameles nasuta). Distinct lineages of hepaci-like viruses associated with geckos and birds were also identified. By mining the SRA database we similarly identified three new hepaci-like viruses from avian and primate hosts, as well as two novel pegi-like viruses associated with primates. The phylogenetic history of the hepaci- and pegi-like viruses as a whole, combined with co-phylogenetic analysis, provided support for virus-host co-divergence over the course of vertebrate evolution, although with frequent cross-species virus transmission. Overall, our work highlights the diversity of the Hepacivirus and Pegivirus genera as well as the uncertain phylogenetic distinction between.The  Hepaciviruses and their close phylogenetic relatives (i.e. ‘hepaci-like’ viruses) infect a broad range of vertebrates, including humans, non-human primates  currently contain eleven defined virus species that infect humans and variety of other mammals (Acridotheres tristis) . Human pegivirus (HPgV), previously known as GB virus C, infects humans with an unproven link with clinical illness (cpz) is closely related to HPgV , a native Australian marsupial (Ixodes holocyclus) feeding on another Australian marsupial   . These d nasuta) , althougFlaviviridae, a more diverse set of genome structures, including segmented forms, have been identified in some invertebrate flaviviruses  approach to identify additional novel hepaci-like viruses in Australian wildlife. This approach has previously proven successful in identifying a variety of viruses in Australian wildlife, including native Australian and invasive species ,b,c. To 2.The magpie-lark and pelican were handled under a series of NSW Office of Environment and Heritage Licences to Rehabilitate Injured, Sick, or Orphaned Protected Wildlife (No. MWL000100542). Samples were collected under the Opportunistic Sample Collection Program of the Taronga Animal Ethics Committee, and scientific licence SL100104 issued by the NSW Office of Environment and Heritage. Ticks were removed from the long-nosed bandicoot under the approval of the NSW Office of Environment and Heritage Animal Ethics Committee (No. 000214/05) and scientific licence SL100104.Gehyra lauta) collected at West Leichardt Station, Queensland in 2013 (Pelecanus conspicillatus) collected at Blackwall Bay, New South Wales (NSW) in 2013. The pelican presented with an ongoing syndrome of profound weakness, diarrhoea, dyspnoea, and death, consistently associated with myocardial degeneration to necrosis. Magpie-lark hepaci-like virus (MaHV)  was obtained from brain (RNASeq library VERT14) and heart samples (RNASeq library VERT 15) of an injured juvenile magpie-lark  collected at Warigee, NSW in 2013 (I.holocyclus) collected while feeding on a healthy long-nose bandicoot (P.nasuta) at North Head, NSW in 2016. These individual tick samples were part of a larger pool of ticks  used to identify the partial genome sequence of Collins Beach virus (Phascolarctos cinereus) ‘Pacific Chocolate’ infected with chlamydia . For animal tissues, ∼50 mg pieces were prepared from livers and other organs by dissecting on a sterile and cool surface before homogenisation in 1 ml of lysis buffer (RLT Plus with 0.1% (v/v) β-mercaptoethanol and 0.5% (v/v) reagent DX) using a TissueRuptor  for 5 min at maximum speed. Whole ticks were similarly processed except were pre-washed in cold, sterile 1X PBS before homogenisation in lysis buffer. Lysates were then purified as per the manufacturers protocol before elution in nuclease-free water. RNA concentration and integrity were determined using a NanoDrop spectrophotometer (ThermoFisher) and TapeStation (Agilent). RNA samples were pooled in equal proportions based on animal tissue types and syndrome . Illumina TruSeq stranded RNA libraries were prepared on the pooled samples following rRNA depletion using a RiboZero Gold kit (Epidemiology) at the Australian Genome Research Facility (AGRF), Melbourne. The rRNA depleted libraries were then sequenced on an Illumina HiSeq 2500 system (paired-end 100 or 125 bp reads) to depths of between 13 and 28 million paired reads.de novo assembly using Trinity 2.5.1 . Briefly, Superscript IV VILO master mix was used to generate cDNA from individual RNA samples before RT-PCR screening with Platinum SuperFi following the manufacturers recommended protocol. Following the confirmation of viruses to individual samples, long overlapping PCRs (2–4 kb) were designed from the mapped RNASeq data to amplify the available viral genome  for the hepaciviruses identified here using Platinum SuperFi. Viral amplicons were then pooled, purified, and sequenced using the Nextera XT library prep kit and an Illumina MiSeq (150 nt paired reads). Viral genomes were then assembled using MegaHit v1.1.3 .Homo sapiens), birds  and marsupials. We focused on these taxonomic groups because 1, although HCV is clearly a human virus it is uncertain whether it is present in non-human primates (which may be indicative of virus-host co-divergence), 2, a number of novel hepaci- and pegi-like viruses have recently been identified in avian hosts suggesting that these might be a rich source of novel viruses, and 3, to confirm the presence of a marsupial specific-lineage as suggested by our previous studies. Accordingly, three different sets of single- and paired-end RNA sequencing SRAs were analysed: one included 2,312 avian SRAs  from Germany. These three fragments were concatenated into a single genome, Blue tit hepaci-like virus (BtHV). The three fragments, and their respective coding regions, are shown in Three virus fragments were identified in three runs  from SRA project SRX565268, isolated from Eurasian blue tit (The hepaci- and pegi-like viruses identified in this study were subjected to an online sequence similarity search using NCBI blastx as well as a conserved domain (CDD) search. Genome annotation was performed using Geneious Prime (version 2019.2.1) . SpecifiTo investigate the evolutionary relationships among the hepaci- and pegi-like viruses we collated the amino acid sequences for the complete polyprotein of each genome from the ten novel viruses identified here, and from related viruses taken from the NCBI Refseq database, including both Brushtail possum hepacivirus and Collins Beach hepacivirus  available in the RDP4 package, assuming default parameters . CompletTo determine the relative frequencies of co-divergence and cross-species transmission in the evolutionary history of hepaci- and pegi-like viruses and their vertebrate hosts, we utilised the co-phylogenetic method available in the Jane package (version 4) . To thisWe identified four novel hepaci-like viruses and one new hepacivirus variant from five meta-transcriptomic libraries representing a variety of Australian wildlife samples . The fouPropithecus diadema) sampled in Toamasina province, Madagascar, and termed Sifaka hepaci-like virus (SfHV-mad), 2, a Senegal bushbaby  virus termed Bushbaby hepaci-like virus (BbHV), and 3, three hepaci-like virus fragments recovered from a Eurasian blue tit (C.caeruleus) in Montpellier, France, that were compiled into a viral single genome termed BtHV. Similarly, two pegi-like virus sequences were isolated from a 1, Common marmoset  termed Marmoset pegi-like virus (MHV), and 2, a South African Vervet monkey (Chlorocebus pygerythrus) that were assembled into a single viral genome termed Simian pegi-like virus (SPV-saf). Finally, we identified a partial fragment of KHV in the SRA screen (library SRX501262) that we re-sequenced to obtain a complete viral genome (VERT20). All individual sequence fragments are described in the Three hepaci-like virus sequences and two pegi-like virus sequences were identified through SRA data mining . The hepEach of the novel hepaci-like viruses identified here, as well as CBV1, underwent genome annotation and were compared to the fully annotated Bald eagle hepacivirus as a reference  Krekulo. All of To determine the evolutionary relationships of the novel hepaci- and pegi-like viruses we utilised complete polyprotein amino acid sequences . MaximumP.diadema and share 91 per cent amino acid identity. These two viruses cluster with a group of rodent hepaciviruses. In contrast, BbHV, sampled from a Senegal bushbaby, groups with the equine and CHVs, with HCV falling as a sister-group to this clade. Importantly, however, as this part of the phylogeny has poor bootstrap support, it is uncertain whether BbHV is more closely related to the equine/ CHVs or to HCV.The mammalian hepaci-like virus clade is the largest and best characterised and contains four of the novel viruses identified in this study, including the two primate hepaci-like viruses identified via SRA mining . SfHV-maThe remaining novel hepaciviruses identified in the mammalian clade were associated from marsupials: the Australian koala and a tick bloodmeal from a long-nosed bandicoot in the case of CBV1. The previously identified Brushtail possum hepacivirus and Collins beach hepacivirus fall into this clade. Notably, the partial sequence of Collins beach hepacivirus was found in the same pooled tick libraries as the CBV1 described here and they share 74 and 89 per cent identity at the nucleotide and amino acid levels, respectively. The ticks that made up these libraries were sampled from long-nosed bandicoots in New South Wales, Australia . This maPapio cynocephalus) from Tanzania (Chlorocebus sabaeus) from Gambia, and Red colobus monkey (Piliocolobus tephrosceles) from Uganda (Saguinus labiatus), mystax monkeys (Saguinus mystax) and owl monkeys (Aotus trivirgatus)  .Trachemys scripta elegans) and Wenling moray eel hepacivirus, identified from a moray eel (Gymnothorax reticularis), fall basal to all known pegi-like viruses, even though other fish and turtle viruses group with the hepaci-like viruses. To investigate this issue in more detail we performed an additional phylogenetic analysis of amino acid sequences of the NS2/3 and NS5 regions separately  and Hepacivirus F  —although the putative recombinant region in the latter was extremely short . To confThe existence of a number of taxon-specific clades (e.g. avian-specific hepaci-like viruses) is compatible with virus-host co-divergence, particularly if the hepaci- and pegi-like virus phylogeny is rooted (as here) using the fish-associated hepacivirus that represent the most phylogenetically divergent host taxa  Fig. 3)Fig. 3). To explore possible co-divergence in the hepaci- and pegi-like viruses in more detail, we estimated the relative frequency of four evolutionary ‘events’: co-divergence, duplications, host-switching and extinction. Although this suggested that host-switching (i.e. cross-species virus transmission) was the most common co-phylogenetic process (33–35 estimated events), there was clearly also evidence for relatively commonplace virus-host co-divergence  from Germany , that cluster with turtle-associated hepaci-like viruses. Similarly, as noted above, it is striking that two previously identified hepaci-like viruses, Hepacivirus sp., identified in a Red-eared slider turtle and Wenling moray eel hepacivirus fall basal to all known pegi-like viruses, even though other fish and turtle sequences group with the hepaci-like viruses. Similarly, rather than falling as a sister-group to those hepaci-like viruses sampled from placental mammals, it is notable that the marsupial hepaci-like viruses fall within the placental mammal phylogenetic diversity. Such a phylogenetic pattern clearly highlights the complexity of the current division into Hepacivirus and Pegivirus genera and strongly suggests that a re-classification of these viruses is required.There are, however, some notable exceptions to co-divergence, such as rodent hepacivirus and Hepacivirus J, identified in bank voles  (G. lauta), and the two previously identified GHVs from China. The gecko clade itself clusters with the avian hepaciviruses, which in turn groups with the pegi-like viruses. Importantly, however, there is relatively weak support for the nodes in question, and if this reptile–avian cluster grouped with the hepaci-like viruses then the overall phylogeny would offer better support to virus-host co-divergence.The avian hepaci-like virus clade characterised here comprises BtHV, recovered from the SRA, as well the novel Australian pelican and MaHVs, three variants of DuHV , and theigoides) . SimilarP. cinereus) and wombats (Vombatus ursinus), similarly formed a distinct clade (The novel KHV and CBV1 (identified in ticks feeding on a long-nosed bandicoot) group closely together, along with the single marsupial-infecting hepaci-like virus identified previously, Brushtail possum hepacivirus . Togethect clade . Hence, ct clade . The plaFlaviviridae, and are responsible for more than 17 per cent of total infectious diseases and cause upwards of 700,000 deaths per year  they do not act as ‘biological syringes’ as other arthropods such as mosquitos and biting flies do (Arthropod vectors are known to transmit a wide variety of infectious disease agents, including members of the per year . All theeviously  tentativflies do . As bothveaa064_Supplementary_DataClick here for additional data file.


Oxidative stress is a common feature of tuberculosis (TB), and persons with reduced antioxidants are at more risk of TB. TB patients with relatively severe oxidative stress had also more advanced disease as measured by the Karnofsky performance index. Since adverse effects from anti-TB drugs are also mediated by free radicals, TB patients are prone to side effects, such as hearing loss. In previous articles, researchers appealed for clinical trials aiming at evaluating N-acetyl cysteine (NAC) in attenuating the dreaded hearing loss during multidrug-resistant TB (MDR-TB) treatment. However, before embarking on such trials, considerations of NAC's overall impact on TB treatment are crucial. Unfortunately, such a comprehensive report on NAC is missing in the literature and this manuscript reviews the broader effect of NAC on TB treatment. This paper discusses NAC's effect on mycobacterial clearance, hearing loss, drug-induced liver injury, and its interaction with anti-TB drugs. Based on the evidence accrued to date, NAC appears to have various beneficial effects on TB treatment. However, despite the favorable interaction between NAC and first-line anti-TB drugs, the interaction between the antioxidant and some of the second-line anti-TB drugs needs further investigations. Oxidative stress is a common feature of tuberculosis (TB)  and is eAnti-TB drugs induce several adverse effects in TB patients, and oxidative stress is implicated in mediating these adverse effects. Drug-induced liver injury (DILI)  and hearResearchers previously appealed for clinical trials aiming at evaluating N-acetyl cysteine (NAC) in attenuating hearing loss in multidrug-resistant TB (MDR-TB) patients owing to the gravity of the problem , 8. HoweNAC demonstrated antimycobacterial properties in previous studies of different models –11. NAC NAC is a precursor of GSH , and GSHEscherichia coli, intracellular cysteine-induced reactive oxygen species (ROS) leading to DNA damage [NAC also has a direct antimycobacterial effect  independA damage  and a siA damage . MoreoveA damage . By avoiγ) production [α), cytokines known to exacerbate oxidative stress. NAC also increased the immunological activities of various cells including natural killer (NK) cells and macrophages against mycobacterium [Moreover, NAC modulated immunity against tuberculosis where it increased interleukin-2 (IL-2), IL-12, and interferon gamma , 60 days of oral NAC treatment partially restored red blood cell GSH concentrations and serum total antioxidant capacity. NAC also reduced spleen mycobacterial load as well as lesion burden and the severity in the lungs [NAC demonstrated antimycobacterial effects in animal studies , 26. In he lungs .The difference in the two studies, concerning the reduction in lung mycobacterial load, could be due to species variation, the difference in infection methodology, and the duration of infection. Guinea pigs are very susceptible to infection by MTB and clearing the mycobacteria may be difficult for them while mice are resistant to mycobacteria . The stuNCT03281226, NCT03702738).NAC also demonstrated several beneficial effects including facilitation of mycobacterial clearance in a clinical trial. In a double-blind, randomized clinical trial of 67 newly diagnosed TB patients, NAC as an adjunct to anti-TB drugs improved smear conversions 3 weeks after initiation of DOTs (directly observed therapies) . NAC at in vitro animal studies and a clinical trial which reported antimycobacterial effects of NAC, studies by Khameneh et al. and Vilchèze et al. did not confirm the antimycobacterial effects of the antioxidant in their experiments [Despite numerous eriments , 29  are at a higher risk of ototoxicity . ProlongThe rate of ototoxicity also varies with the particular aminoglycosides used in the MDR-TB treatment. In a study comparing the rate of ototoxicity induced by capreomycin versus amikacin, the risk of ototoxicity in patients treated with amikacin was increased five times compared with those treated with capreomycin . In anotNAC attenuated aminoglycoside-induced ototoxicity in various studies , 50, andAminoglycosides cause ototoxicity through the generation of free radicals and these radicals, in turn, attack the hair cells of the vestibulocochlear nerve . In thisIn animal studies, enhancing the antioxidant systems and/or free radical-scavenging capacity protected against aminoglycoside-induced ototoxicity. Animals overexpressing superoxide dismutase, the enzyme responsible for scavenging ROS, were resistant to kanamycin-induced ototoxicity . NAC alsNAC protected against aminoglycoside-induced hearing loss in clinical trials of non-TB patients , 61, 62 DILI is one of the major adverse effects of treatment with anti-TB drugs. Patients treated with anti-TB drugs may experience DILI ranging from simple hepatic enzyme elevations to severe clinical hepatitis. In the presence of hepatitis symptoms, such as abdominal pain, nausea, vomiting, unexplained fatigue, or jaundice, hepatotoxicity is defined as an elevation of liver enzymes, alanine (ALT) and/or aspartate (AST) transaminase, more than three times the upper limit of the normal range (ULN). But in the absence of symptoms, either of the enzymes must be elevated more than five times ULN , 67. TheDILI could have serious implications on the outcome of TB patient treatment , 70. SomDifferent risk factors are associated with the development of anti-TB DILI. Various studies suggested advanced age as one of the risk factors , 72 evenNAC attenuates anti-TB DILI through scavenging free radicals formed during the metabolism of the drugs as well as through enhancing the synthesis of GSH . Anti-TBMAFK encoding MafK  increased susceptibility to anti-TB DILI [NOS2A, encoding inducible nitric oxide synthase (iNOS) and BACH1, encoding BTB and CNC homology 1 (bach1), increased susceptibility to anti-TB DILI [NOS2A encodes the enzyme nitric oxide synthase while BACH1 encodes Bach1, whose binding with Nrf2 leads to the downregulation of antioxidant enzymes. Additionally, loss of function mutations on HMOX1, the gene encoding for heme oxygenase 1 recognized as phase II antioxidant enzyme, and NQO1, which encodes NAD (P)H: quinone oxidoreductase 1, were also associated with susceptibility to anti-TB DILI [According to studies on genetic polymorphisms of oxidant-antioxidant systems, mutations leading to reduced antioxidant enzymes or increased prooxidant enzymes resulted in increased susceptibility to anti-TB DILI , 84, 85.-TB DILI   cells also demonstrated a protective property of NAC against anti-TB-induced hepato-/cytotoxicity [NAC prevented anti-TB drug-induced hepatotoxicity in animal studies , 83 Tab. A studytoxicity  (Table 3A clinical trial of NAC in newly diagnosed TB patients also showed protective effects against hepatotoxicity  Table 3Table 3. Based on the evidence accrued to date, NAC appears to have various beneficial effects on TB treatment and its evaluation in clinical trials is justifiable. For maximal protection by NAC against anti-TB-induced hearing loss, we need to continue the administration of NAC for a little longer after completion of aminoglycoside administration. Moreover, the interaction between NAC and some of the second-line anti-TB drugs needs further investigations despite favorable interaction between NAC and first-line anti-TB drugs, bedaquiline and clofazimine.