Glioblastoma (GBM) is the most common malignant brain tumor. Although current treatment strategies, including surgery, chemotherapy, and radiotherapy, have achieved clinical effects and prolonged the survival of patients, the gradual development of resistance against current therapies has led to a high recurrence rate and treatment failure. Mechanisms underlying the development of resistance involve multiple factors, including drug efflux, DNA damage repair, glioma stem cells, and a hypoxic tumor environment, which are usually correlative and promote each other. As many potential therapeutic targets have been discovered, combination therapy that regulates multiple resistance-related molecule pathways is considered an attractive strategy. In recent years, nanomedicine has revolutionized cancer therapies with optimized accumulation, penetration, internalization, and controlled release. Blood-brain barrier (BBB) penetration efficiency is also significantly improved through modifying ligands on nanomedicine and interacting with the receptors or transporters on the BBB. Moreover, different drugs for combination therapy usually process different pharmacokinetics and biodistribution, which can be further optimized with drug delivery systems to maximize the therapeutic efficiency of combination therapies. Herein the current achievements in nanomedicine-based combination therapy for GBM are discussed. This review aimed to provide a broader understanding of resistance mechanisms and nanomedicine-based combination therapies for future research on GBM treatment. The standard GBM treatment involves maximal surgical resection, followed by postoperative radiotherapy and adjuvant chemotherapy with temozolomide (TMZ)3, the first-line chemotherapeutic drug for GBM treatment approved by the Food and Drug Administration (FDA). The recurrence rate of GBM, however, is as high as 90% due to incomplete tumor removal and development of resistance4. Although numerous clinical trials have demonstrated that patient survival is significantly prolonged with standard treatment5, the median survival of patients with GBM is < 2 years, and only approximately 5% of patients survive > 5 years6.Glioblastoma (GBM), classified as a grade IV glioma, is the most common and aggressive adult brain tumor, accounting for 50% of all gliomas8. Thus, commonly used chemotherapeutic drugs are not used in GBM treatment due to poor penetration across the BBB. Specifically, epirubicin, a structural analog of the anthracycline, doxorubicin, exhibits prominent cytotoxic against GBM tumor cells in vitro, but cannot serve as a clinical medication due to poor BBB permeability10. Although TMZ exhibits a distinct advantage in crossing the BBB, accumulation of TMZ in GBM is far from satisfactory. TMZ has been reported to be one of the substrates of P-glycoprotein (P-gp), which serves as an efflux pump on the apical membrane side of the endothelial cells forming the BBB12. Only 20% of the dose of TMZ enters the brain13. As a benefit of nanotechnology, chemotherapeutic drugs can be loaded into nanocarriers to improve BBB penetration efficiency through various transcytosis pathways8. GBM, however, is highly heterogeneous and prone to mutate14, thus providing a favorable environment to develop resistance to chemotherapeutic drugs. Considering the complicated signal networks and various compensatory mechanisms involved in the development of drug resistance, combination therapies that synergistically regulate multiple cancer-associated pathways using different treatments or drugs provide a promising strategy for overcoming drug resistance16. Further, nanotechnology optimizes the in vivo pharmacokinetics and biodistribution between multiple types of drugs, including small molecule drugs, therapeutic genes, and proteins, leading to improvement in the therapeutic efficiency and alleviation of side effects17. In this review we discuss mechanisms of drug resistance in patients with GMB and a possible strategy to overcome drug resistance. Then, we discuss the essential role of nanotechnology in combination therapies, outline current nanomedicine-based combination therapies for GBM treatment , and analyze the challenges to offer a reference for future research.Compared to other malignant tumors, the blood-brain barrier (BBB) presents a unique and challenging biological barrier to effective drug delivery for GBM treatment18. In addition, the highly heterogeneous and hypoxic environment of GBM provides favorable conditions for developing drug resistance. This section focuses on common resistance mechanisms and potential strategies to improve chemosensitivity.There are two major approaches by which tumor cells develop drug resistance: 1) impeding the accumulation of chemotherapeutic drugs within tumor cells; and 2) developing DNA repair mechanisms to lower drug sensitivity19. For example, the expression of a series of solute carriers (SLCs) is downregulated. Thus, the uptake of drugs depending on SLCs to effect intracellular transportation is significantly decreased18. To overcome resistance caused by this mechanism, drugs can be loaded into delivery systems and enter tumor cells through alternative transport pathways.Most chemotherapeutic drugs are only active inside tumor cells. Thus, to impair chemotherapeutic efficacy, tumor cells usually develop drug resistance by blocking or limiting access of the drug at the site of action20. In a previous report, nearly 60% of chemotherapeutic drugs were recognized by P-gp, then transported outside the cell21, thus leading to decreased intracellular drug accumulation and diminished therapeutic activity. During GBM treatment, increased P-gp levels have been reported from tissue samples of clinical patients who exhibit TMZ resistance and tumor recurrence22. Knockdown of MDR1, which codes P-gp in GBM cells, results in enhanced TMZ-mediated cell death, thus suggesting that the MDR1 gene has an important role in TMZ resistance23. Moreover, extracellular vesicles (EVs) usually contain mRNAs, miRNAs, and lncRNAs related to drug efflux, which are released from resistant cells and transferred to sensitive cells, further resulting in acquired resistance of surrounding cells within the tumor mass. To overcome resistance caused by this mechanism, small molecular inhibitors, such as P-gp inhibitors and small interfering RNA (siRNA) targeting ABC efflux gene families, have been designed to reduce drug efflux and alleviate TMZ resistance.Drug molecules must remain in the tumor cell at a sufficient concentration rather than pass through the cell to achieve effective treatment. ATP-binding cassette (ABC) transporter family proteins, including P-gp, multidrug resistance proteins (MRPs), and brain cancer resistance proteins (BCRPs), are usually overexpressed on tumor cell membranes and play an important role in drug resistance6 position of guanine residues24; however, under prolonged treatment, cancer cells gradually develop DNA repair mechanisms that reduce chemosensitivity. O6-methylguanine methyl transferase (MGMT), a DNA repair enzyme that eliminates the methyl group in O6-methylguanine and repairs drug-induced DNA damage, is considered to be a major factor in the development of TMZ resistance. Thus, TMZ usually fails to realize the expected therapeutic effect for GBM in patients with high MGMT expression. Moreover, MGMT expression increases with the development and recurrence of GBM25, thus leading to difficulties in the treatment of patients in the mid and late stages, as well as the relapse stage, which significantly affects the therapeutic effect. The use of a high dose of TMZ is the currently recommended strategy to counteract the attenuating effect of MGMT on DNA alkylation26; however, this maneuver does not overcome drug resistance and can even exacerbate drug resistance. To this end, DNA repair inhibitors, such as O6-benzylguanine (BG) and siRNA-targeted MGMT, have been developed to alleviate TMZ resistance based on the DNA damage repair mechanism.The mechanism by which numerous chemotherapeutic drugs cause apoptosis of tumor cells is to induce DNA damage. TMZ, an oral alkylating agent, usually causes DNA damage by alkylating or methylating DNA at the O27, GSCs are a dominant factor in cancer progression, metastasis, and relapse, as well as the development of drug resistance28. Thus, although the initial treatment kills the bulk of tumor cells, a small number of residual GSCs survive and account for recurrence and treatment failure. Moreover, Liu et al.29 reported that the expression of ABC transporters is elevated in patients with recurrent GBM compared to patients with newly diagnosed GBM, which manifests as more severe drug resistance in recurrent GBM and treatment difficulty.Cancer stem cells (CSCs) are a population of undifferentiated and highly tumorigenic cancer cells that exhibit proliferative and self-renewal properties. While GSCs account for as few as 3%–5% of GBM tumors29. Sonic hedgehog (SHH) signaling also has a vital role in regulating GSC self-renewal and tumorigenesis30. Moreover, it has been reported that inhibition of the SHH pathway sensitizes GSCs to TMZ treatment, providing a possible direction for overcoming TMZ resistance31. In addition, the Wnt/β-catenin pathway involves upregulation of MGMT expression, thus inducing the development of TMZ resistance in GBM32. To achieve effective treatment of GBM, novel therapeutic approaches are required to target the tumor bulk, and more importantly, to aim at GSCs to overcome resistance mechanisms.To develop promising therapies for elimination of GSCs, it is essential to understand the intrinsic signaling pathways responsible for the growth, renewal, and development of GBM. For example, Notch signaling is activated in GSCs, thus suppressing differentiation and maintaining stem-like properties, further contributing to tumorigenesis and therapeutic resistance33 reported the interrelationship between hypoxia and TMZ resistance of GBM cells using a basement membrane extract (BME)-based 3D model. Hypoxia-inducible factor-1alpha (HIF-1α) is a crucial transcription factor. HIF-1α is significantly upregulated in the hypoxic microenvironment, which further leads to the activation of several genes associated with the hypoxia-responsive element (HRE), including the MDR1 gene, one of the genes related to drug resistance34.Hypoxia is one of the characteristics of glioblastoma tissues. Hypoxia leads to an increase in genetic mutations, inflammation, the epithelial-mesenchymal transition (EMT), drug resistance, and autophagy. Musah-Eroje and Watson35. For example, acute treatment with TMZ inhibits PI3K/Akt-mTOR and causes the transient induction of autophagy, further leading to TMZ resistance in GBM therapy. To solve this problem, chloroquine (CQ) and CQ analogs, which have been reported to inhibit autophagy, are used in clinical trials preceding treatment with TMZ.Autophagy, an important homeostatic cellular recycling mechanism, has a cytoprotective role by which excessive or unnecessary proteins and injured or aged organelles are degraded. Autophagy is usually activated after chemotherapy or radiotherapy to protect tumor cells from DNA damage, and thus is considered an important factor in the development of drug resistance36. Persano et al.37 reported that silencing HIF-1α expression reduces MGMT-related drug resistance to TMZ, implying a positive relationship between HIF-1α and chemoresistance in GSCs. In addition, autophagy is usually upregulated in a hypoxic tumor environment with a lack of nutrition, further promoting GSC survival and migration, which are closely related to the development of chemoresistance38. Moreover, other potential pathways are still being discovered, which makes resistance mechanisms clear and increase the difficulties in overcoming resistance mechanisms.The mechanisms underlying drug resistance in patients with GBM are complex and interconnected. For example, a hypoxic tumor environment with poor vasculature density is a hotbed for GSCs, which increase the levels of GSC marker expression and promote a cancer stem-like phenotypevia an enhanced permeability and retention (EPR) effect39. Further, the accumulation, penetration, internalization, and controlled release of DDSs can be optimized through modulation of nanoparticle physicochemical and mechanical properties, including size40, shape41, surface chemistry42, and mechanical softness43. Moreover, BBB penetration efficiency has also been optimized with the development of nanotechnology. Through the receptors or transporters on the BBB, DDSs actively target brain tissues with high specificity, selectivity, and affinity. For example, glucose transporters (GLUTs) facilitate the transportation of glucose from the blood to the brain, which could be utilized to penetrate through BBB. Wu et al.44 used maltobionic acid, a glucose derivative, to modify a nanodelivery system, thus achieving enhanced BBB penetration efficiency. Angiopep-2 is a peptide targeting low-density lipoprotein receptor-related protein 1 (LRP1), which is often used to cross the BBB. Zheng et al.45 developed angiopep-2 ligand camouflaged polymeric siRNA nanomedicine, with which BBB penetration and GBM targeting was achieved. In addition, Fan et al.46 developed human H-ferritin (HFn) nanocarriers that successfully crossed the BBB through transferrin receptor 1 (TfR1)-mediated endocytosis, resulting in efficient tumor killing. Thus, introducing suitable ligands on DDS significantly improves BBB penetration efficiency.The rapid development in nanotechnology has provided new opportunities for cancer treatment. Multiple drug delivery systems (DDSs), including liposomes, polymers, and inorganic nanoparticles, have been designed to satisfy the diverse delivery requirements of different drugs. Current DDSs exhibit low immunogenicity, a high loading capacity, and preferentially accumulate in tumor tissues 47 utilized mPEG-PLGA nanoparticles to co-deliver PTX and TMZ for GBM treatment. The best synergistic effect was achieved when the weight ratio of PTX-to-TMZ was 1:5 for U87 cells and 1:100 for C6 cells, demonstrating importance of the drugs ratio. The optimal drug ratio in cancer cells is usually different from the optimal drug ration in vitro due to the complicated tumor environment. To overcome this problem, Zhang et al.48 demonstrated a macrocyclic-amphiphile-based self-assembled nanoparticle (MASN) to precisely load multiple drugs for cancer treatment. Such macrocyclic host-based nanoparticles load drugs via host-guest interactions with a defined stoichiometry (most often 1:1), thus allowing multiple drugs to be precisely loaded with the optimal ratio. Moreover, owing to the sharp decline in the binding affinity as a response to the tumor environment, the MASN releases the loaded drugs simultaneously upon reaching tumors. Thus, with a ratiometric delivery ability of multiple drugs, a MASN has the potential to be an effective multi-drug delivery platform for combination chemotherapies of multiple cancers, including GBM.For most combination chemotherapies, the ratio of the drugs or active ingredients in the combination is a crucial factor. Benefit from nanotechnology, pharmacokinetics, biodistribution, as well as the ratio of different drugs, are optimized to achieve better synergistic therapeutic efficacy. Xu et al.4 first loaded small molecular drugs into carboxylated azocalix arene (CAC4A) via a host-guest interaction, which was further mixed with phenylboronic acid (PBA)-modified polyethyleneimine (PEI-PBA) and pDNA to form a calixarene-embedded polyplex core (denoted as CEPC)49. It is worth noting that CAC4A offers a steric barrier to avoid interference between the molecular drugs and the pDNA loaded in the CENP, thus enhancing the combined therapeutic efficacy and alleviating side effects. Considering the common feature of gene delivery for different cancers, this strategy is also expected to be used for GBM treatment. Therefore, through an elaborate design of DDS, it is possible to maximize the therapeutic efficiency of combination therapies.Combinations of chemotherapy and gene therapy have also been successfully developed for cancer treatment. Small molecular drugs usually achieve anti-tumor effects by binding to cancer cell DNA and inducing DNA damage, which may interfere with the function of therapeutic genes, further leading to a compensation in the anti-tumor efficiency of both drugs and therapeutic genes. To solve this problem, Giese et al.50. Also, the combined treatment of nimotuzumab, a monoclonal antibody against epidermal growth factor receptor (EGFR), and rapamycin, an mTOR inhibitor, achieved more efficient outcomes than TMZ treatment on patient-derived human glioma cells51. These results indicate the feasibility of combination treatment. In this section, we discuss multiple combination therapies, including combination chemotherapy, chemotherapy-gene therapy, chemo-phototherapy, and chemo-immunotherapy, to provide a reference for designing drug combinations and delivery systems for further research .Because GBM resistance is complicated in multiple molecular pathways and the compensation mechanisms, inhibiting a single target is not sufficient to reverse resistance. Thus, combination therapy that targets multiple pathogenic pathways is an attractive strategy to address drug resistance and improve chemosensitivity of GBM. Recent evidence from clinical trials suggests that a combination of lomustine and TMZ improves overall survival when used as a first-line treatment for patients with MGMT methylation52; however, the therapeutic effect of combination multiple chemotherapy drugs is limited by poor BBB penetration, limited tumor-targeting ability, and high systemic toxicity. To solve these issues with the assistance of nanomaterials, Zou et al.53 successfully developed a novel strategy for the co-delivery of TMZ and CDDP with a pH-responsive acetal grated dextran (AcDEX) core and GBM cell membrane . It has been reported that engineered nanoparticles with GBM CCM target homologous cancer cells due to homotypic recognition by the abundant proteins on the surface of CCM54. Moreover, because the GBM cell membrane readily traverses the BBB due to the downregulation of the specific proteins (ZO-1 and claudin-5) in tight junctions55, such biomimetic nanoparticles achieve efficient BBB transportation and GBM targeting. As a result, MNPs@TMZ+CDDP crosses the BBB and delivers both TMZ and CDDP into the tumor. Compared with single drug-loaded nanoparticles, MNPs@TMZ+CDDP extends survival up to 3-fold without noticeable side effects. The EGFR and mesenchymal-epithelial transition factor (MET) have been shown to be involved in TMZ resistance and GBM cell growth56. As two inhibitors, Inherbin3 and cMBP block their corresponding pathways, thus impairing DNA damage repair to inhibit drug resistance. To co-deliver Inherbin3 and cMBP, and regulate the activity of EGFR and MET, Meng et al.57 developed a dual functionalized brain-targeting nanoinhibitor (denoted BIP-MPC-NP) through in situ polymerization based on 2-methacryloyloxy ethyl phosphorylcholine (MPC), followed by the surface conjugation with Inherbin3 and cMBP. As a result, BIP-MPC-NP effectively decreased the activation of EGFR and MET, thereby attenuating DNA damage repair in drug-resistant GBM and enhancing the sensitivity of GBM to TMZ.Combination drug therapy with two or more drugs to disturb multiple pathogenic pathways is the most direct strategy to address the challenge of drug resistance. Co-delivering drugs for reducing MGMT activity is the most common strategy. For example, cisplatin (CDDP), a common clinical anticancer drug for multiple cancers, binds DNA and causes crosslinking of purine bases, thereby destroying the function of DNA and inhibiting cell mitosis. Moreover, CDDP has also exhibited the ability to decrease MGMT activity, suggesting its potential in combination therapies with a DNA-damaging agent. In a phase II trial, the combination of CDDP and TMZ increased patient survival compared with TMZ alone58. Based on ART, Ismail et al.59 employed artesunate-phosphatidylcholine (ARTPC) to encapsulate TMZ and constructed a special ApoE-functionalized liposomal nanoplatform (denoted ApoE-ARTPC@TMZ). Such a nanoplatform enhanced GBM sensitivity to TMZ and achieved great combination chemotherapy for resistant tumors. Similarly, dihydrotanshinone (DHT), another traditional Chinese medicine, induces glioma cell apoptosis by upregulating caspase-3 expression and reduces drug resistance by downregulating MGMT and P-gp expression61. Wang et al.62 utilized glioma cell-derived reassembled exosomes to co-deliver TMZ and DHT (denoted R-EXO-T/D). The results showed that the combination of DHT and TMZ significantly enhanced the anti-glioma effect of TMZ in an orthotopic GL261-bearing mice model by inhibiting drug efflux and DNA repair damage.As a commonly used clinical Chinese medicine, artesunate (ART) involves multiple pathways within tumor cells. Recent research reported that ART interferes with the expression of MGMT and alleviates GBM drug resistance by inhibiting the Wnt/β-catenin pathway63 reported that the potent small molecule inhibitor, JQ1, inhibited the expression of BET bromodomain proteins, further regulating the DNA damage response in multiple cancer cell lines, including U87MG cells. In IC50 analyses of U87MG and GL261 cells, the combinatorial index (C.I.) values of TMZ and JQ1 were 0.95 and 0.94 for U87MG and GL261 cells, respectively, indicating an additive cytotoxic effect. Moreover, increased DNA damage was observed from the groups treated with the combination of JQ1 and TMZ. Further, Lam et al.64 utilized transferrin-functionalized PEGylated NPs (Tf-NPs) to co-deliver JQ1 and TMZ, penetrate the BBB, and target GBM. In vivo experiments demonstrated that JQ1 and TMZ co-loaded Tf-NPs significantly sensitize gliomas to TMZ therapy, resulting in effective tumor growth inhibition in both U87MG- and GL261-bearing mice.Floyd et al.65 designed B19 aptamer-conjugated dendrimer nanoparticles to co-deliver paclitaxel (PTX) and TMZ (denoted Apt-NPs). Owing to the high affinity between B19 aptamer and CD133, a most recognizable biomarker on GSCs, Apt-NPs target U-87 stem cells and induce apoptosis, further reducing the resistance in GBM.In addition, co-delivering the drugs for regulating DNA damage or inhibiting drug efflux, specifically inducing GSCs apoptosis, is another practical approach. Behrooz et al.66. CQ and its derivative [hydroxychloroquine (HCQ)] are lysosomotropic agents that increase lysosomal pH, thereby blocking autophagosome fusion with the lysosome67. Ruan et al.68 designed functional GNPs to co-deliver DOX and HCQ for chemotherapy and autophagic interference. As expected, the released HCQ inhibited the DOX-induced cytoprotective autophagy and re-sensitized glioma cells to DOX, thus providing an effective strategy for overcoming drug resistance.It has also been reported that inhibiting cytoprotective autophagy improves chemosensitivity in GBMIn addition to combination chemotherapy, gene therapy, which directly disturbs disease-causing genes or drug-resistant genes, is another method to overcome tumor drug resistance. Gene therapy refers to a treatment that utilizes gene editing tools to down-regulate or up-regulate, replace, or insert specific genes to alleviate the development of diseases. For example, using siRNA to downregulate the overexpression of MGMT in GBM cells effectively enhances the sensitivity of GBM to TMZ and alleviates the drug resistance of GBM.69 designed an attractive liposome-based hypoxia-radiosensitive nanoparticle nanoplatform denoted RDPP(Met)/TMZ/siMGMT for co-delivery of TMZ and siMGMT. Xie et al.69 encapsulated TMZ into RDPP by hydrophobic interaction and attached siMGMT to the surface of RDPP via an electrostatic interaction. Furthermore, surface modification of RGD facilitated RDPP to transport TMZ and siMGMT across the BBB and targeted GBM. Owing to the hypoxia-triggered degradation of RDPP, TMZ and siMGMT were released in the tumor microenvironment. Because siMGMT significantly decreased the expression of MGMT and restored the sensitivity of GBM to TMZ, the results showed that RDPP(Met)/TMZ/siMGMT effectively inhibited GBM proliferation and prolonged the survival of mice in an U87MG brain tumor mouse model. Similarly, Wang et al.70 developed chitosan-PEG-polyethylenimine (PEI)-coated iron oxide nanoparticles for siMGMT loading, followed by targeting ligand (CTX) conjugation . Such nanoparticles reduced MGMT expression in both GBM cells and GSCs, thus improving TMZ chemosensitivity and prolonging the survival of mice bearing orthotopic GBM6 tumors. In addition, recent research has shown that the signal transmitter and activator of transcription 3 (STAT3) is one of the genes that regulates cell proliferation and anti-apoptosis in various tumor cells. STAT3 induces the expression of MGMT in GBM cells, leading to the development of drug resistance72. Rehman et al.73 employed siRNA to silence the expression of STAT3 in GBM to relieve the resistance of GBM to TMZ. Based on exosomes isolated from bone marrow mesenchymal stem cells (BMSCs), Rehman et al.73 constructed a multifunctional nanocarrier to deliver TMZ or siSTAT3, followed by modification with HMOX1-specific peptide (HSSP), which targets GBM transport (denoted HSSP-BMSCExo). As a result, HSSP-BMSCExo effectively crossed the BBB, silenced STAT3, restored GBM sensitivity to TMZ, and eventually inhibited tumor proliferation in a TMZ-resistant U251 (U251-TR) brain tumor mouse model.The instability of gene editing tools (nucleic acid or protein) in blood circulation and the difficulty of penetrating the BBB and entering GBM cells across amphiphilic cell membranes limit their application in regulating drug resistance of GBM. With the development and advances in nanotechnology, nanomaterials are emerging as ideal non-viral vectors capable of overcoming multiple biological barriers of nucleic acid delivery to GBM. For example, Xie et al.75. Ma et al.76 constructed a biological camouflaged nanosystem based on liposome and macrophage exosomes to co-deliver si-c-Myc, si-mTOR, and TMZ. Through effective downregulation of c-Myc and mTOR expression, the proliferation of GSCs was significantly suppressed, thus attenuating TMZ resistance. Such a dual-targeting inhibition strategy efficiently inhibited tumor growth and prolonged survival in TMZ-resistant GBM-bearing mice. In addition, the Hedgehog (Hh)/Gli1 signaling pathway maintains the proliferation and survival of GBM cells and GSCs77. Downregulation of Gli1 expression with RNAi technology suppresses GSC proliferation and reduces chemoresistance. Melamed et al.78 developed polyethylenimine-wrapped spherical nucleic acid nanoparticles (PEI-SNAs) as a siGli1 delivery carrier. With enhanced cellular uptake and endosomal escape, Gli1 PEI-SNAs effectively silenced Gli1 expression and inhibited the self-renewal capacity of GSCs. Thus, Gli1 PEI-SNAs significantly relieved TMZ resistance and induced effective cell apoptosis under low doses of TMZ.c-Myc is also commonly activated in various CSCs, and the upregulation of mTOR contributes to c-Myc expression79 developed mesoporous silica nanoparticles (MSNPs) to co-deliver miR221-targeting polyarginine-peptide nucleic acid (R8-PNA) and TMZ (denoted PNA-TMZ-MSNPs). The in vitro results showed that PNA-TMZ-MSNPs effectively induced an apoptosis rate up to 70.9% in a TMZ-resistant T98G cell line. It has been reported that overexpression of miR-125b enhances the sensitivity of the DOX-resistant breast cancer cell line, MCF-7/R, to DOX and exhibits a similar potential in other tumors, including GBM. Wang et al.80 encapsulated DOX and miR-125b into an amphiphilic co-polymer (γ-PGA-co-PLA-DPPE) containing poly [gamma-glutamic acid (γ-PGA)], polylactide (PLA), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), and formed co-polymer NPs using a nano-precipitation method. The results showed that miR-125b aggravates cell cycle arrest caused by DNA damage by DOX and activates the AMPK/p53 pathway in U251 cells, thus improving chemosensitivity and promoting tumor cell apoptosis.Recently, more and more reports have suggested that the abnormal expression profile of miRNAs is closely related to cancer cell drug resistance. Downregulation of miR221 expression is beneficial in restoring the sensitivity of GBM cells to TMZ. As a commonly used anti-miR targeting tool, peptide nucleic acids (PNAs) have been shown to effectively target miRNAs. Bertucci et al.81 developed microbubble-modified lipid-polymer hybrid nanoparticles (denoted MB-LPHNs-cRGD) for efficient delivery of dual-gRNA/Cas9 plasmids targeting the MGMT gene (pCas9/MGMT). With focused ultrasound (FUS)-assisted BBB opening, MB-LPHNspCas9/MGMT-cRGD was effectively transported to GBM cells, thus relieving the drug resistance of GBM through MGMT downregulation. Finally, MB-LPHNs-cRGD significantly inhibited tumor growth and prolonged survival of T98G tumor-bearing mice.As a revolutionary gene editing tool, the CRISPR/Cas9 system has also been used to overcome challenges in GBM drug resistance. For example, Yang et al.82. PDT employs photosensitizers (PSs) to absorb light and convert energy to cytotoxic ROS or heat to induce tumor cell apoptosis. In addition to inducing apoptosis, PDT also exhibits the ability to circumvent drug resistance, providing an important strategy to overcome GBM resistance84. Similarly, PTT induces tumor cell apoptosis directly via heat generation85. Thus, combining phototherapy with TMZ is expected to significantly enhance the tumor suppressive effect.Phototherapy, including photodynamic therapy (PDT) and photothermal therapy (PTT), as a non-invasive, highly selective, and controllable strategy, has attracted more and more attention in cancer treatment86 constructed multifunctional pluronic P85/F127 nanoparticles with biotin modification (m-NPs) to co-load TMZ and PS verteporfin (VP) for chemotherapy combined with PDT for GBM treatment. In addition to the excellent drug-loading ability, pluronics reduce drug efflux by inhibiting P-glycoprotein expression. As a result, in vitro experiments showed that VP/TMZ-coloaded mNP is more effective than VP-loaded m-NP or TMZ-loaded m-NP treatment alone, thereby proving that PDT combined with low-dose TMZ effectively suppresses tumor growth and overcomes the resistance by circumventing the drug resistance pathway. Similarly, Zhang et al.87 also constructed an angiopep-2 (Ang) peptide-modified multifunctional nanocarrier (denoted T-TKNPVP) to co-load VP and PTX. Under X-ray irradiation, T-TKNPVP produces cytotoxic ROS and releases PTX. As a result, T-TKNPVP significantly inhibits tumor proliferation and prolongs mouse survival in a U87-MG brain tumor mouse model through combination X-ray-induced PDT and chemotherapy.As an important element in phototherapy, PS converts light into ROS or heat; however, the limited BBB crossing and tumor penetration efficiency in GBM severely restrict the therapeutic effect of phototherapy combined with chemotherapy in overcoming tumor drug resistance. Recently, with the rapid development of nanotechnology, nanomaterials with the characteristics of small particle size, easy surface modification, and strong permeability provide solutions to the problems. For example, Pellosi et al.88 designed TMZ-loaded GNPs with anti-EphA3 modification on the surface (denoted anti-EphA3-TMZ@GNPs) to target GBM cells . Under laser irradiation, anti-EphA3-TMZ@GNPs enhance tumor cell apoptosis through a chemophotothermal synergistic effect. Moreover, such chemophotothermal treatment activates p53 and reduces MGMT expression. As a result, anti-EphA3-TMZ@GNPs effectively inhibit T98G glioma tumor proliferation and reverse GBM drug resistance. Zeng et al.89 designed porous silicon nanoparticles to load TMZ (denoted TMZ/Psi NPs) and achieved chemo-PTT and hyperbaric oxygen (HBO) therapy. Upon exposure to NIR light, Psi generates mild heat for thermotherapy (40-44°C), thus enhancing the drug effects. Moreover, HBO relieves the hypoxic microenvironment in GBM and increases TMZ sensitivity. As a result, TMZ/Psi NPs presents significant antitumor effects in C6 tumor-bearing mice with the combination of chemotherapy, PTT, and HBO.In addition to PDT combined with chemotherapy, PTT has also been combined with chemotherapy to overcome drug resistance in GBM. For example, Yu et al.90 fabricated a multifunctional phototheranostic agent (denoted DTRGD NPs) for chemo/photodynamic/photothermal synergistic therapy. DTRGD NPs were constructed by co-encapsulating PS [dicysteamine-modified hypocrellin derivative (DCHB)] and octadecane-modified TMZ derivative (TMZ-C18) with DSPE-mPEG2000 and DSPE-PEG2000-cRGD. With the BBB crossing ability, DTRGD NPs significantly inhibit GBM tumor growth and alleviate GBM drug resistance by chemo/photodynamic/photothermal synergistic therapy.Moreover, Zhang et al.Cancer immunotherapy, which aims to activate the host immune system to recognize and destroy tumors, has emerged as a potential strategy for cancer treatment after surgery, chemotherapy, and radiotherapy. By targeting the immune system rather than the tumor itself, immunotherapy achieves precise recognition and killing of tumor cells in an antigen-specific manner. In the past few decades, cancer immunotherapies, including immune checkpoint inhibitors (ICIs), adoptive T cell transfer (ATC), and chimeric antigen receptor T-cell (CAR-T), have achieved great success and revolutionized cancer treatment.91 designed a 2-deoxy-D-glucose modified lipid polymer nanoparticle to load TMZ and siPD-L1 (denoted TMZ/siPD-L1@GLPN/dsb) as combination chemotherapy and immunotherapy . Such nanoparticles downregulate the expression of PD-L1 in tumor cells, thus reversing the tumor suppressive microenvironment and reducing the activity of MGMT. With effective BBB penetration, enhanced GBM accumulation, and TMZ sensitivity, TMZ/siPD-L1@GLPN/dsb effectively inhibit GBM tumor proliferation and prolong the rat survival time in an orthotopic C6/TR brain tumor rat model. In another report, MnO2 nanoparticles were utilized to load TMZ, followed by surface modification of poly (ethylene glycol)-poly (β-amino ester) to improve stability (denoted MT@PAE)92. By increasing BBB permeability using ultrasound, MT@PAE and PD-L1 antibodies crossed the BBB, accumulated in GBM, and relieved the tumor immunosuppressive microenvironment. Moreover, MnO2 nanoparticles also relieved the hypoxic microenvironment by consuming excessive H2O2. As a result, the combination of MT@PAE and PD-L1 antibodies overcame GBM drug resistance and inhibited GBM tumor growth in a G422 brain tumor mouse model.Therapeutic antibodies have been used to restore the activity of immune cells in GBM and reshape the tumor immunosuppressive microenvironment, which is closely related to GBM drug resistance. Recently, many clinical trials have utilized immune checkpoint blockade therapy combined with chemotherapy against GBM. Unfortunately, the results of these clinical trials are not satisfactory. Several studies have shown that the inability of therapeutic antibodies to cross the BBB limit their therapeutic efficacy against GBM. Over recent decades, with the rapid development of nanomaterials, various nanocarriers have been developed to overcome this issue. For example, Liu et al.2) and manganese oxide (MnO) nanoparticles have recently been used to modulate tumor hypoxia, which is considered an important factor to induce drug resistance. Tan et al.93 synthesized iRGD-modified polymeric micelles {polyethylene glycol-poly(2-(diisopropyl-amino) ethyl methacrylate, [PEG-PDPA]} to encapsulate TMZ and MnO (denoted iRPPA@TMZ/MnO). The released Mn2+ induced intracellular oxidative stress to cause tumor cell death via Fenton-like activity and the production of O2 further alleviated tumor hypoxia. Thus, such nanoparticles enhanced GBM tumor inhibition efficiency and alleviated GBM drug resistance via synergistic chemodynamic therapy and chemotherapy.Manganese dioxide  has been widely reported to induce the differentiation of CSCs by blocking off signaling pathways. In this study the combination of RA and TMZ-loaded GNP-associated LIUS stimulation exhibited a significant and synergistic effect on promoting CSCs differentiation and further enhancing TMZ sensitivity.Sonodynamic therapy has been commonly used in cancer therapy, which not only enhances cancer cells apoptosis, but also regulates the tumor microenvironment. Moreover, low-intensity ultrasound (LIUS) has exhibited the ability to induce CSC attachment and differentiationTreatment of GBM remains a challenge, and TMZ resistance is one of the major factors in treatment failure. Although this review summarized resistance mechanisms simply as reduced drug uptake, increased drug efflux, DNA damage repair, and heterogeneous tumor microenvironment, more-and-more studies have shown that TMZ resistance is mediated by multiple molecular pathways. For example, the overexpression of EGFR, galectin-1, and Mdm2, as well as the mutation of p53 and phosphatase and tensin homolog (PTEN), have essential roles in drug resistance, providing a number of potential targets for relieving drug resistance and increasing difficulties in overcoming drug resistance. Thus, therapeutic regimens aimed at a single target usually fail to achieve effective GBM treatment. Although combination treatments targeting multiple pathways exhibit significant advantages over single target treatment, GBM is likely to develop compensation mechanisms through unknown pathways owing to intrinsic heterogeneity. As advances in molecular biology research, more innovative therapeutic targets and inherent interrelationships and compensation mechanisms are discovered and identified, laying a foundation for drug discovery and the combination of various drugs or therapies for effective long-term GBM treatment.Nanomedicine has revolutionized GBM therapies. Current delivery methods based on nanotechnology have significantly improved BBB permeability and GBM accumulation of therapeutic agents and reduced systemic toxicity. Moreover, nanoparticles designed to load multiple therapeutics exhibit great potential in achieving effective combination therapy. With increasing knowledge of GBM molecular pathways related to drug resistance and the further development of delivery strategies, nanomedicine can be further optimized to achieve more effective GBM treatment. In addition, most combination treatments in the current studies co-deliver multiple therapeutics without carefully designing and optimizing the synergistic effects between the drugs. Thus, future research should focus on the factors  that affect the efficiency of drug combinations in the design of new treatments for overcoming drug resistance.


Avian paramyxovirus type 1 (APMV-1) is a virus of birds that results in a range of outcomes, from asymptomatic infections to outbreaks of systemic respiratory and neurologic disease, depending on the virus strain and the avian species affected. Humans are rarely affected; those who are predominantly experience mild conjunctivitis. We report a fatal case of neurologic disease in a 2-year-old immunocompromised child in Australia. Metagenomic sequencing and histopathology identified the causative agent as the pigeon variant of APMV-1. This diagnosis should be considered in neurologic conditions of undefined etiologies. Agnostic metagenomic sequencing methods are useful in such settings to direct diagnostic and therapeutic efforts. Orthoavulavirus genus, Paramyxoviridae family  , respiratory, and digestive diseases, depending on tropism of the particular strain (A 2-year-old child with a history of infantile pre-B cell acute lymphoblastic leukemia (ALL) was admitted to hospital with nausea and vomiting after 3 weeks of upper respiratory tract symptoms. As part of the Associazione Italiana di Ematologia Oncologia Pediatrica and the Berlin-Frankfurt-Münster Acute Lymphoblastic Leukemia (AIEOP-BFM ALL) clinical trial, she had been randomized to receive blinatumomab, a bispecific T-cell–engaging antibody targeting CD19-expressing B cells; her last dose was 6 months before admission  were unremarkable. Investigations for autoimmune causes of encephalitis all yielded negative results, including testing for antibodies to myelin oligodendrocyte glycoprotein aquaporin 4, N-methyl-d-aspartate (NMDA) receptor, contactin-associated protein-like 2 (CASPR2), leucine-rich glioma-inactivated 1 (LGI-1), gamma aminobutyric acid-B (GABA-B), dipeptidyl-peptidase-like protein 6 (DPPX), and immunoglobulin-like cell adhesion molecule 5 (IgLON5). Neurotropin was elevated in cerebrospinal fluid (CSF) at 1,752 nmol/L (reference range 6–30 nmol/L), representing significant CNS inflammation. Ultra-rapid exome sequencing performed by Acute Care Genomics, a tool that enables diagnosis of genetic disorders within 5 days, yielded no genetic abnormalities (Haematoxylin and eosin (H&E) staining of brain biopsy tissue performed 20 days after admission demonstrated almost complete cortical necrosis with some subpial sparing. The cortex was replaced by sheets of foamy macrophages with some gliosis and scattered, predominantly interstitial, CD3-positive T cells. Only rare residual small neurons were identified in the superficial cortex with NeuN immunostaining. No microglial nodules, viral inclusions, or viral cytopathic effects were apparent , panel ADespite therapy with broad-spectrum antimicrobial drugs, antiseizure medications, immunomodulators, and a ketogenic diet, the patient’s condition did not improve. Over the course of 2 weeks, serial cerebral MRI showed progressive and widespread inflammatory changes with increasing left frontal and insular T2 signal hyperintensity evolving into laminar necrosis as well as T2 hyperintensity of deep gray-matter structures . Treatmehttps://www.twistbioscience.com/resources/product-sheet/twist-pan-viral-panel) and 281,601 sequences by in-house methods. The only other nonhuman sequences of note included low levels (<230 reads) of human pegivirus (HPgV) sequences identified using both methods, as previously identified in similar studies (data not shown). We conducted comprehensive, hypothesis-free metagenomic testing of the biopsied brain tissue using established panviral hybridization-capture  before phylogenetic analysis with PhyML version 3.3.20180214 (www.atgc-montpellier.fr/phyml) using the Le-Gascuel (LG) protein substitution model  strain . The avion model . An analon model  indicateon model . The comAPMV-1 infection of the brain tissue was confirmed using virus-specific real-time quantitative PCR assays (data not shown) and immunohistochemistry at the Australian Centre for Disease Preparedness. The APMV-1 nucleoprotein identified using immunohistochemistry was present in occasional clusters within cells in the brain tissue. At least some of those small pyramidal cells appeared to be neurons with an axon-like process , panel BThe first documented case of APMV-1 infection in humans was reported in 1942 in Australia , there is no clear association between HPgV and human disease, nor evidence of neurotropism. Because HPgV has a reported seroprevalence of up to 20% in the human population, this finding most likely represents passive transport into CSF (In birds, APMV-1 infects respiratory, neural, lymphoid, and other tissues, causing a range of signs that are partially dependent on the viral strain. In the human case we report, it is likely that the PPMV-1 infection began in the upper respiratory tract before the onset of FIRES and spread to the CNS, given the history of respiratory signs. Although no exposure was identified, it is likely the virus was transmitted inadvertently via direct contact with pigeon feces or infected fluids. The virus is known to remain stable in pigeon feces and can be spread by windborne dust, extending the risk beyond just localized environments (The term FIRES refers to an epileptic encephalopathy characterized by intractable seizures after a febrile illness. It is diagnosed in the absence of infectious encephalitis or a defined trigger and most commonly affects otherwise healthy children and young adults (The use of APMV-1 strains as an oncolytic therapy has been examined in preclinical and clinical trials; the favorable properties of APMV-1 result in an antitumor response (This case demonstrates the valuable role of metagenomics in complex clinical cases and highlights its potential future use in routine diagnostics. Although early diagnosis of PPMV-1 in this case was unlikely to have changed the clinical trajectory, including metagenomics early in similar cases is likely to have a substantial effect on patient outcomes. Compared with traditional methods, metagenomics can detect uncommon and novel pathogens in different disease phenotypes, which is particularly relevant in immunocompromised patients for whom a broad range of opportunistic pathogens must be considered (Incorporating metagenomic sequencing into routine diagnostics has several barriers and challenges. Metagenomic sequencing is expensive. High-throughput sequencing requires highly skilled staff, including bioinformaticians with expertise in data analysis (Additional information for case of fatal human neurologic infection caused by pigeon avian paramyxovirus-1, Australia.


SARS-CoV-2 infection is a pandemic that affects predominantly upper airways and lungs. It may lead to reactivation of known inflammatory rheumatic diseases and/or initiation of various granulomatous disorders. Necrotising sarcoid granulomatosis (NSG) is a rare condition that can be confused with malignancy, granulomatosis with polyangiitis, and sarcoidosis. Herein we reported the development of NSG following a SARS-CoV-2 infection which mimicked granulomatosis with polyangiitis. Extra-pulmonary involvement is found in one third of the cases, with ocular being the most common. At imaging, multiple nodules or a solitary mass are found accompanied by mediastinal lymphadenopathy at one third of the cases. It can be clinically mistaken for GPA or malignancy on radiological examination; therefore, biopsy and histopathological evaluation are necessary. SARS CoV-2 infection is a pandemic characterised with upper airway and lung involvement that in some patients may progress to respiratory and multiorgan failure. The clinical manifestations of disease are heterogeneous, which include an asymptomatic carrier, acute respiratory disease , and pneumonia of varying degrees of severity. SARS-CoV-2 may lead to reactivation and/or initiation of various inflammatory rheumatic and granulomatous diseases such as sarcoidosis and/or GPA.4 There is no data in the literature yet regarding relationship between SARS-CoV-2 infection and NSG. Herein we reported a 27-year-old male patient who developed SARS-CoV-2-induced NSG.Necrotising sarcoid granulomatosis (NSG) is a rare systemic condition characterised by sarcoid-like granulomas, granulomatous vasculitis, and varying degrees of infarct-like necrosis. It was first described in 1973, and it has been unclear since then whether it is a different entity or a spectrum of a known disease such as sarcoidosis or granulomatosis with polyangiitis (GPA).A 27-year-old non-smoker male patient presented to the pulmonology outpatient clinic with complaints of flu-like symptoms and dry cough starting two weeks ago. Patient’s history was obtained; lifelong never smoker and reported no medicine/illicit drug/alcohol use. There was no allergy or systemic illness history.Figure 1). Multiple solid nodular lesions in both lungs, with the largest measuring around 3 cm in the right lung middle lobe lateral segment were found on CT scan . Tests were planned for the differential diagnosis of malignancy, nonspecific infections, tuberculosis, and granulomatosis with polyangiitis, based on the patient’s age and complaint profile. Laboratory tests were found as following: erythrocyte sedimentation rate (ESR): 5 mm/h , C-reactive protein (CRP): 1,2 mg/dL , hydroxy-vitamin-D3: 6,5 ng/mL , angiotensin-converting enzyme (ACE): 47 U/L . Complete blood count, serum calcium levels, liver and kidney function tests, urinalysis were found normal. Rheumatic serologic test was performed: anti-nuclear antibody, rheumatoid factor, anti-cyclic-citrullinated peptide antibody, anti-neutrophilic cytoplasmic antibodies, serum immunoglobulin levels and immunoglobulin subsets, serum complement C3/C4 levels were found to be normal. Interferon-gamma releasing assay for tuberculosis was positive, but microbiological cultures including Löwenstein-Jensen were found negative. 18-Fluorodeoxyglucose Positron emission tomography (FDG-PET) - CT was performed; apart from parenchymal nodular lesion in the lungs , FDG distribution was normal including hilar-mediastinal lymph nodes . The CT component of the PET confirmed that the paranasal sinuses were also normal. Bronchoscopy was scheduled for the lesion in the middle lobe, bronchoalveolar lavage and bronchoscopic transbronchial punch biopsies were performed. Histopathological findings resulted as chronic inflammation of unknown aetiology and transthoracic fine-needle aspiration biopsy (FNAB) was planned. The two consecutive biopsies resulted as benign cytology. A definitive diagnosis could not be reached after bronchoscopy and fine needle aspiration biopsy (FNAB), and video-assisted thoracoscopic surgery (VATS) biopsy was recommended, and pathology revealed between areas of massive fibrinoid necrosis, histological staining revealed clustered sarcoid granulomas and vasculitic involvement, indicating the diagnosis of NSG .Physical examination was unremarkable. The patient works as a translator in our hospital and is at risk for SARS-CoV-2 infection; PCR test was performed and was found positive. The patient was treated only with paracetamol, no additional treatment was required. One month later, the control PCR test was negative, but a lung computed tomography (CT) was planned because he had a dry cough. The patient had a baseline normal lung-X-ray before the infection . Outpatient follow-up of the patient, whose general condition is good, continues.The patient was treated with 1 mg/kg/day methylprednisolone with isoniazid prophylaxis; the dose of corticosteroid then decreased gradually to 8mg/day. At month three the clinical and radiologic findings have nearly completely disappeared .SARS-CoV-2 is an infection characterised with predominantly upper airways and lungs involvement. It is a heterogenous disease which may lead to rapid acute respiratory distress syndrome (ARDS) and pulmonary insufficiency resulting in high morbidity and mortality.9 SARS-CoV-2 acts by binding to angiotensin converting enzyme II (ACE II) receptors in target organs such as lung alveolar type 2 cells, leading to downregulation of the expression of ACE II, which in turn results in excessive production of Angiotensin II. This might lead to an excess of stimulation of angiotensin II receptor (ATR1) in innate immune cells, leading to a pro-inflammatory response that favours T-helper 1 polarization, which is responsible of the elaboration of IFN-γ, IL-2 and TNF-α. In this pro-inflammatory environment including mucosal activated invariant T cells (MAITc) activation, both ACE upregulation and/or Angiotensin II accumulation could therefore stimulate granuloma formation and lead to granulomatous responses.10 Therefore, SARS-CoV-2 might trigger granulomatous diseases via the renin-angiotensin system and activation of innate immune system. Some authors suggest that this MAIT cells might have protective effects from severe or life-threatening infection. These patients have less clinical symptoms and organ involvement.11 Recently, the efficacy of various anti-rheumatic drugs in the treatment of SARS-CoV-2 infection were reported. Cherian JJ et al. review the efficacy and safety baricitinib and tocilizumab among hospitalized patients with SARS-CoV-2 infection.12 They reported that baricitinib and tocilizumab both are recommended interchangeably by various guidelines for the management of SARS-CoV-2 infection considering the mortality data and other comparable efficacy and safety outcomes. Pelechas E et al. reported five patients with RA under treatment with conventional and biological DMARDs which have SARS-CoV-2 infection.13 The authors hypothesise that anti-rheumatic drugs may ameliorate the clinical picture and disease course of COVID-19 in RA patients. The efficacy of various DMARDs with different mechanism of action in the treatment of SARSCoV-2 infection may explain with the fact that immune system dysregulation and release of higher amounts of cytokines are responsible in its pathogenesis. Targeting the MAIT cells could be an interesting field in both SARSCoV-2 and granulomatous diseases treatment strategies. More studies in this regard are needed.The relationship between SARS-CoV-2 infection and development of granulomatous lung diseases are not known yet. One of the hypotheses are the model showing links between SARS-CoV-2 and granulomatous process mechanisms.NSG is a rare disease that can affect anyone at any age and has a wide range of differential diagnoses, the most common of which are malignancy and granulomatosis with polyangiitis. Several triggering factors were reported, and SARS-CoV-2 infection could be one of these.


Acrocephalus arundinaceus individual  by using Illumina MiSeq amplicon sequencing. Single exons, representing MHC class I (MHC-I) and class IIB (MHC-IIB) alleles, were sequenced in the focal individual and mapped to the annotated MHC alleles in the original long-read genome assembly. Eighty-four percent of the annotated MHC-I alleles in the original long-read genome assembly were detected using 55% of the amplicon alleles and likewise, 78% of the annotated MHC-IIB alleles were detected using 61% of the amplicon alleles, indicating an incomplete annotation of MHC genes. In the haploid genome assembly, each MHC-IIB gene should be represented by one allele. The parental origin of the MHC-IIB amplicon alleles in the focal individual was determined by sequencing MHC-IIB in its parents. Two of five larger scaffolds, containing 6–19 MHC-IIB paralogs, had a maternal and paternal origin, respectively, as well as a high nucleotide similarity, which suggests that these scaffolds had been incorrectly assigned as belonging to different loci in the genome rather than as alternate haplotypes of the same locus. Therefore, the number of MHC-IIB paralogs was overestimated in the haploid genome assembly. Based on our findings we propose amplicon sequencing as a suitable complement to long-read sequencing for independent validation of the number of paralogs in general and for haplotype inference in multigene families in particular.Long-read sequencing offers a great improvement in the assembly of complex genomic regions, such as the major histocompatibility complex (MHC) region, which can contain both tandemly duplicated MHC genes  and high repeat content. The MHC genes have expanded in passerine birds, resulting in numerous MHC paralogs, with relatively high sequence similarity, making the assembly of the MHC region challenging even with long-read sequencing. In addition, MHC genes show rather high sequence divergence between alleles, making diploid-aware assemblers incorrectly classify haplotypes from the same locus as sequences originating from different genomic regions. Consequently, the number of MHC paralogs can easily be over- or underestimated in long-read assemblies. We therefore set out to verify the MHC diversity in an original and a haplotype-purged long-read assembly of one great reed warbler  Multigene families contain paralogs (gene copies) that have evolved by repeated gene duplication and share high sequence similarity and similar functions . SeveralAcrocephalus arundinaceus, with 4–21 MHC-I alleles found per haplotype (In passerine birds (Aves: Passeriformes), the number of MHC paralogs have expanded massively leading to high MHC diversity  and then use HTS to genotype these PCR amplicons from large numbers of individuals at a low cost . Thus, it is possible to simultaneously co-amplify all MHC alleles in every individual by PCR , and MHC related genes, i.e., genes expected to be found in the MHC region, such as the antigen peptide transporter genes, TAP1 and TAP2  and in the post-processed haploid genome assembly (Purge Haplotigs). In the Purge Haplotigs assembly alternate haplotypes had been removed to generate an improved haploid representation of the genome, though this procedure is challenging in scaffolds containing MHC genes due their repetitive nature and differences between haplotypes. Using amplicon HTS data from the parents of the focal individual, we overcame this challenge and determined the parental origin of the annotated MHC alleles, verifying the MHC haplotypes in the haploid long-read genome assembly.de novo genome assembly reconstructed from long-read sequencing , short-read sequencing , and optical mapping data  from a female individual, our focal individual  in order to genotype MHC-I and MHC-IIB genes in a great reed warbler family including the focal individual, two siblings as well as both parents. The birds are part of a long-term monitored wild population at Lake Kvismaren in Sweden . The focFor both MHC-I and MHC-IIB, only the amplicons sequenced from the focal individual and its parents were used in the present study. All three samples were run in replicates for all primer pair combinations except for HNalla/HN46. Obtained sequences were trimmed of the adapters and primer sequences using Cutadapt . TrimmedFor the parents and the focal individual, the numbers of filtered reads for both MHC-I primer pairs (PPs) ranged between 19,193 and 34,310 . The maximum percentage of reads for an MHC-I amplicon allele  was 10.8–12.9% and the minimum percentage of reads was 0.12–0.23% for all individuals. In addition, to keep an allele we took into account that (i) it should be found in both replicates of the same individual within PP combination or (ii) it should be found with both PPs in the same individual or (iii) it should be found in at least two individuals in the family independently of the PP combination . Following these rules, we retained two additional MHC-I alleles with low number of reads (0.06%), but which were amplified in the focal individual and its parents.i.e., for which we obtained the largest number of different MHC-IIB amplicon alleles post-trimming (PP1: 93 and PP5: 89). Only sequences of the expected length or that were shorter by a combination of a multiple of 3 bp (considering the length of a full codon) were kept. The two other MHC-IIB PPs gave a low number of unique amplicon alleles in the focal individual (PP2: 74 and PP3: 65) and data from both PPs were discarded  for all MHC-IIB amplicon alleles is lower as many more alleles are amplified simultaneously in a single individual. In the focal individual, the number of unique MHC-I and MHC-IIB amplicon alleles amplified was assessed for each primer pair combination (http://130.235.244.92/apps/seqeqseq.html).For MHC-IIB, we selected data from the two best-performing primer pair combinations (PPs) iscarded . The numiscarded . Consequbination . FinallyIn order to identify paternally and maternally inherited annotated MHC alleles, MHC-I and MHC-IIB amplicon alleles found in the focal individual were sorted into three categories. Amplicon alleles found in the focal individual and the father were identified as paternal (P) whereas amplicon alleles found in the focal individual and the mother were identified as maternal (M). Amplicon alleles found in the focal individual and both parents were identified as unresolved alleles (U).® 2020.1.2 (v. 11.0.6) to the original great reed warbler long-read genome assembly  and to post-processed haploid genome assembly (Purge Haplotigs) which has gone through the post-assembly filtering procedure  in MEGA version 11 .In the original long-read genome assembly of the focal individual, GRW Falcon-2017, 25 MHC-I and 96 MHC-IIB full-length alleles were annotated in primary contigs , hence the primary contigs of GRW Falcon-2017 represent a diploid version of the MHC region. Following the post-assembly procedure (Purge Haplotigs), which aimed to remove sequences that represent alternative haplotypes of other sequences and give a haploid representation of the genome, 18 MHC-I and 66 MHC-IIB full-length genes were included and of these, 15 MHC-I and 56 MHC-IIB genes contained a predicted full-length open reading frame ORF, . In totaTwenty-nine MHC-I alleles and 95 MHC-IIB alleles were amplified in the focal individual using amplicon HTS  of the annotated alleles in GRW Falcon-2017 were successfully detected by amplicon alleles in the focal individual . For MHCSixteen MHC-I exon 3 amplicon alleles mapped to 21 annotated MHC-I genes in GRW Falcon-2017 –S3. FourFifty-eight MHC-IIB exon 2 amplicon alleles mapped to 78 annotated full-length MHC-IIB alleles in GRW Falcon-2017 . Ten of For MHC-I, 89% (16 of the 18 full-length genes) of the annotated MHC genes in Purge Haplotigs were successfully detected by amplicon alleles in the focal individual . The sucThe parental amplicon alleles that mapped to the annotated full-length tandemly duplicated MHC-I and MHC-IIB genes in the Purge Haplotigs assembly show which scaffolds that are dominated by paternal (P) and maternal (M) MHC genes. Thus, since seven MHC-I genes were assigned as paternal and only one was assigned as maternal on scaffold 508 (Acar-UA*508), this scaffold is likely to represent a paternal haplotype .There were 54 MHC-IIB genes in the Purge Haplotigs assembly with information about parental origin  of the tandemly duplicated MHC-IIB genes containing an ORF between the five larger scaffolds to investigate whether any pair-wise comparison among the scaffolds represent putatively complementary haplotypes. The mean nucleotide p-distance between genes in the two scaffolds Aaru-DAB*554 and Aaru-DAB*357 was lower (0.057) than the mean nucleotide p-distances of other scaffold comparisons  was largely recovered using amplicon HTS data . Amplicon alleles (MHC-I exon 3 and MHC-IIB exon 2) frequently mapped to more than one full-length annotated MHC allele in the genome assembly, highlighting the fact that the MHC diversity based on amplicon alleles to some extent underestimates the true MHC diversity. This is because the amplicon alleles only represent a portion of the full-length annotated alleles of MHC genes in the genome and several annotated full-length MHC genes share identical MHC-I exon 3 and MHC-IIB exon 2 sequences in the great reed warbler. However, we think that MHC diversity based on amplicon alleles also may overestimate the true MHC diversity of full-length genes. This is because short amplicons sequences (MHC-I exon 3 and MHC-IIB exon 2) at times are derived from remnants of full-length genes, i.e., annotated full-length alleles, described in the GRW Falcon-2017 assembly was detected using only approximately half of the MHC-I and MHC-IIB amplicon alleles (55% for MHC-I and 61% for MHC-IIB). This was a little unexpected, given that the GRW Falcon-2017 assembly reflects the diploid representation of the MHC region and is a rather high-quality assembly where the MHC scaffolds have been carefully annotated. Therefore, it suggests that the genome assembly does not describe the full MHC diversity. Nevertheless, one additional explanation for the low MHC diversity in the genome assembly compared to the MHC diversity based on amplicon alleles is linked to how MHC genes evolve. The MHC multigene family is believed to evolve according to the “birth and death” model  than maternal (N = 7) MHC-I amplicon alleles in the focal individual, but we find it unlikely that the focal individual only would inherit a single maternal MHC-I allele that contained an ORF as indicated in the GRW Falcon-2017 assembly. Hence, we believe that some of the maternal MHC-I genes failed to be assembled. We envision that the assembly procedure has mixed maternal and paternal haplotypes in the genome interval, and that the use of future long-read sequencing approaches  will be able to yield a more correct haplotype assignment and gene order.The assembler resolved 11 MHC-I paralogs organized in tandem on scaffold Aaru-UA*508 in Purge Haplotigs. Most of the annotated MHC-I genes were paternal, and only one annotated MHC-I gene (Acar-UA*508_8) was maternal. Likewise, there were more paternal  combination .The number of MHC-IIB amplicon alleles that contained stop-codons are stated within brackets for each primer pair (PP) combination . Fewer unique amplicon alleles were found with two MHC-IIB PPs (PP2 and PP3) and data from both PPs were discarded before the trimming and the mapping steps.Click here for additional data file.10.7717/peerj.15480/supp-2Supplemental Information 2Number of MHC-I and MHC-IIB amplicon allele sequences that match to annotated MHC alleles in genome assemblies  of the focal individual. The number of amplicons alleles being mapped has been assessed using the standard Geneious RNA mapper .Click here for additional data file.10.7717/peerj.15480/supp-3Supplemental Information 3® (Geneious RNA mapper). Non-functional genes are indicated with the symbol Ψ .All primary and associated MHC-I scaffolds (Aaru-UA scaffold) included in the GRW Falcon-2017 are presented. Scaffolds indicated with # were discarded after the post-assembly procedure was performed in the Purge Haplotigs assembly. Amplicon alleles were separated into three categories related to their inheritance in the focal individual: paternal alleles , maternal alleles  and unresolved alleles . Annotated alleles not detected with amplicon mapping are highlighted in grey. One amplicon allele was successfully amplified in the focal individual (both replicates) but not found in its parents and have the prefix “Seq”. The mapping procedure was performed in Geneious PrimeClick here for additional data file.10.7717/peerj.15480/supp-4Supplemental Information 4® (Geneious RNA mapper). Non-functional genes are indicated with the symbol Ψ .All primary and associated MHC-IIB scaffolds (Aaru-DAB scaffold) included in the GRW Falcon-2017 are presented . Scaffolds indicated with # were discarded after the post-assembly procedure was performed in the Purge Haplotigs assembly. Amplicon alleles were separated into three categories related to their inheritance in the focal individual: paternal alleles , maternal alleles  and unresolved alleles . Additional amplicon alleles that were corresponding to multiple hits with higher mismatches are indicated with numbers (based on matrix of similarity). Annotated MHC alleles not detected or not assigned with amplicon mapping are highlighted in grey. The mapping procedure was performed in Geneious PrimeClick here for additional data file.10.7717/peerj.15480/supp-5Supplemental Information 5Mean amino-acid pairwise distances  and standard errors  between annotated scaffolds including tandemly duplicated MHC-IIB gene copies in open reading frame (ORF) in the focal individual: Acar-DAB*120_2;4–10;12–14;16–18, Acar-DAB*301_1;2;4;6, Acar-DAB*357_1–4;6–9, Acar-DAB*45_2–9 and Acar-DAB*554_1–9.Click here for additional data file.10.7717/peerj.15480/supp-6Supplemental Information 6i.e., shared; numbers in blue, percentages in brown).Out of 29 MHC-I amplicon alleles 16 were mapping to annotated alleles and out of 95 MHC-IIB amplicon alleles 58 were mapping. The upper panel represents amplicon alleles that mapped to the assembly and are separated into two categories: alleles mapping once  and alleles mapping multiple times . Out of the 25 annotated MHC-I alleles, 21 were detected by amplicon alleles and out of the 100 annotated MHC-IIB alleles, 78 were detected by amplicon alleles.The lower panel describes the annotated MHC alleles in the GRW Falcon-2017 assembly, which were detected by amplicon alleles, and is separated into two categories: alleles detected uniquely  and different alleles detected by the same amplicon allele  and all MHC-IIB gene copies at four scaffolds with tandemly duplicated genes in open reading frame (ORF) in the focal individual: Aaru-DAB*357 (purple), Aaru-DAB*45 (light grey), Aaru-DAB*301 (medium grey) and Aaru-DAB*120 (dark grey).Click here for additional data file.10.7717/peerj.15480/supp-8Supplemental Information 8List of MHC-I (exon 3) amplicon alleles in the focal individual, corresponding sequences obtained with both MHC-I primer pair combinations  and matching MHC-I allele in GeneBank .Click here for additional data file.10.7717/peerj.15480/supp-9Supplemental Information 9List of MHC-IIB (exon 2) amplicon alleles in the focal individual, corresponding nucleotide sequences obtained with both MHC-IIB primer pair combinations (PP1 (trimmed length sequences) and PP5) and matching MHC-IIB allele in GeneBank .Click here for additional data file.


Background: Increased studies on the basis of bulk RNA-sequencing (RNA-seq) data of cancer identify numbers of immune-related genes which may play potential regulatory roles in the tumor microenvironment (TME) without in-depth validation.Methods: In the current study, the immunological correlation and cell subpopulation expression pattern of FMNL1 were analyzed using public data. In addition, the cell subpopulation expression pattern of FMNL1 was also deeply validated using single-cell RNA-sequencing (scRNA-seq) and multiplexed quantitative immunofluorescence (mQIF).Results: Bulk FMNL1 mRNA was related to better prognosis in hepatocellular carcinoma (HCC) and was able to identify immuno-hot tumor in not only HCC but also multiple cancer types. Bulk FMNL1 mRNA also predicted the response to immunotherapy in multiple cancers. Further validation using scRNA-seq and mQIF revealed that FMNL1 was a biomarker for immune cells.Conclusions: FMNL1 is a biomarker for immune cells in not only hepatocellular carcinoma, but also multiple cancer types. Moreover, immune infiltration analysis using the bulk RNA-seq data would be further validated using scRNA-seq and/or mQIF to describe the cell subpopulation expression pattern in tumor tissues for more in-depth and appropriate understanding. Increasing studies on the basis of bulk RNA-sequencing (RNA-seq) data of cancer have been conducted to investigate the features of the tumor microenvironment (TME) and identify numbers of immune-related genes that may play potential regulatory roles in TME, but only a few researchers provided in-depth insights into candidate genes In our previous study, we uncovered that Formin-like gene 1 (FMNL1) was tightly related to immune infiltration in gastric cancer In this research, the bulk RNA-seq data from the Cancer Genome Atlas (TCGA) database was utilized to explore the correlation between FMNL1 and features of TME, and FMNL1 was found to be tightly correlated with features of TME in not only hepatocellular carcinoma (HCC) but also multiple cancer types. However, further validation using single-cell RNA-sequencing (scRNA-seq) and multiplexed quantitative immunofluorescence (mQIF) revealed that, FMNL1 was just a biomarker for immune cells. Thus, bulk RNA-seq analysis of immune-related genes should be further validated using sc-RNA-seq or mQIF to describe the cell subpopulation expression pattern in tumor tissues.http://xenabrowser.net/datapages/). All abbreviations for tumor types were shown in The RNA-seq data and clinical annotation in the Cancer Genome Atlas (TCGA) were obtained from the Xena  in HCC contained immunomodulators, the activities of the cancer immunity cycle, infiltration levels of tumor-infiltrating immune cells (TIICs), and the expressions of inhibitory immune checkpoints. First, the ESTIMATE algorithm was apllied to calculate Tumor Purity, ESTIMATE Score, Immune Score, and Stromal Score The standard workflow of the python-based toolkit Scanpy (version 1.5.1) http://tisch.comp-genomics.org/gallery/) The scRNA-seq database Tumor Immune Single-cell Hub  was provided by Outdo BioTech . The tissue microarray included 88 para-tumor tissues and 92 HCC tissues. Outdo BioTech provided comprehensive clinico-pathological details and follow-up information for the cohorts. In this study, the tissue microarray was used for multiplexed quantitative immunofluorescence (mQIF). The Clinical Research Ethics Committee of Outdo Biotech  gave its approval for the research of tissue microarray.Using a previously established procedure, the mQIF was immediately carried out on the tissue microarray to quantify the levels of FMNL1, CD45, and CK8 in the HCC samples and simultaneously detecting DAPI FMNL1 mRNA level could predict the response to immunotherapy, the GSE100797, GSE126044, MEDI4736, and PRJEB23709 cohorts To check whether the bulk http://www.proteinatlas.org/) dataset is launched to describe all the human proteins in cells, tissues, and organs utilizing the integration of multi-omics The Human Protein Atlas  and progression-free survival (PFS), but had no significant correlation with overall survival (OS) and disease-specific survival (DSS). We also examined the correlation between FMNL1 expression and clinical parameters, and found that FMNL1 was correlated with gender , and CK8 (a biomarker for glandular epithelium and adenocarcinoma cell). The clinical parameters of the in-house cohort were exhibited in Supplementary S Figure .To further explore the role of FMNL1 in formation of the inflamed tumor microenvironment (TME) in HCC patients, we firstly divided the immune subpopulations into FMNL1+ and FMNL1- based on the expressed count of FMNL1. Then, CellPhoneDB software was performed to dissect the interactions among various subpopulations. Results showed that compared with FMNL1- immune cells, FMNL1+ immune cells presented significantly more interactions with other cell subpopulations A-B. NotaWe further identified the significant ligand-receptor interactions among these cell types using CellphoneDB. Results showed that T/NK (FMNL1-) and myeloid (FMNL1-) cells rarely interacted with malignant cells, but T/NK (FMNL1+) and myeloid (FMNL1+) cells presented many unique interactions with malignant cells D-E.For example, T/NK (FMNL1+) cells communicated with malignant cells via CCR6-CCL20 D, which Recently, cancer immuno-correlation analysis of candidates is a boom in bioinformatics via restraining TGF-β1 signaling FMNL1 is a classic member of Formin proteins and mediates the polymerization of F-actin FMNL1 is mostly expressed in immune cells and tissues, including peripheral blood leukocytes, the spleen, and the thymus, while playing a crucial oncogenic role in numerous cancer types + cells predicting prolonged OS verified in this research, we speculated that FMNL1+ immune cells had stronger migration capability, increasing their propensity to act as anti-tumor roles. In addition, FMNL1 was also could be detected in tumor cells, which acted as a critical oncogene in multiple cancers. As a result, we hypothesized that varied prognostic characteristics in diverse malignancies were caused by the balance of FMNL1 expression in tumor and immune cells.In this report, we revealed that higher FMNL1 expression could predict well clinical outcome in HCC. However, prior studies showed that elevated FMNL1 expression was linked to poor prognosis in a number of malignancies, including clear cell renal cell carcinoma Overall, the in-depth analysis beyond bulk RNA-sequencing was prepormed involving the immunological correlation and cell subpopulation transcriptomic pattern of FMNL1. Although FMNL1 was tightly correlated with immune infiltration in HCC, and even in pan-cancer, it was a novel biomarker for immune cells, but might not be a critical regulator in tumor cells to mediate tumor immunity due to the distinctive expression pattern of FMNL1. Thus, blind immune infiltration analysis based on bulk RNA-seq data should be further validated using scRNA-seq or mQIF for more deeply appropriate conclusions.Supplementary figures and tables.Click here for additional data file.


Oral treprostinil has been shown to improve exercise capacity and delay disease progression in patients with pulmonary arterial hypertension (PAH), but its effects on hemodynamics are not well-characterized. The FREEDOM-EV trial was a Phase III, international, placebo-controlled, double-blind, event-driven study in 690 participants with PAH who were taking a single oral PAH therapy. FREEDOM-EV demonstrated a significantly reduced risk for clinical worsening with oral treprostinil taken three times daily and did not uncover new safety signals in PAH patients. Sixty-one participants in the FREEDOM-EV trial volunteered for a hemodynamics sub- study. Pulmonary artery compliance (PAC), a ratio of stroke volume to pulmonary pulse pressure, significantly increased from Baseline to Week 24 in the oral treprostinil group compared with the placebo group . There was a significant increase in cardiac output in the oral treprostinil group compared to the placebo group  and a corresponding significant reduction in pulmonary vascular resistance (PVR)  from Baseline to Week 24. These data suggest that increased compliance contributes to the physiological mechanism by which oral treprostinil improves exercise capacity and delays clinical worsening for patients with PAH. FREEDOM-EV demonstrated a significantly reduced risk for clinical worsening in patients with pulmonary arterial hypertension (PAH) who took oral treprostinil three times daily . Prospec1.NCT01560624) study underwent two RHCs  [Sixty-one participants of the FREEDOM-EV NCT0560624 stSix participants with missing or mismatched CO measurement methodology, Fick (direct or indirect) or thermodilution, at baseline and follow up were not included in CO-based analyses. The treatment groups were compared using analysis of covariance with change from baseline for each log-transformed hemodynamic parameter as the dependent variable, treatment as a fixed effect, and log-transformed baseline hemodynamic parameter as a covariate.2.2.1The sub-study treatment groups were generally balanced , but tho2.2Mean PAC increased 44% at Week 24 (raw mean, Median oral treprostinil doses  at Week 3.This FREEDOM-EV hemodynamic sub-study is the first prospective, placebo-controlled study to examine the effects of oral treprostinil taken three times daily in PAH patients. Treprostinil treatment resulted in a significant increase in PAC and CO with an associated reduction in PVR compared to the placebo group at Week 24. These data can be explained by the known pulmonary vasodilator effect of prostacyclin therapies . CompliaTreatment groups were generally well-balanced at baseline and similar to the parent study . The subPrevious data on the hemodynamic effects of oral treprostinil are limited . An openProspective hemodynamic studies of prostacyclin receptor agonists have had mixed results. In a phase II study of 61 PAH patients on mono- or combination therapy, ralinepag demonstrated significant improvements in PVR, SVR and PAPm compared to placebo at Week 22 . A post-In summary, in patients with PAH, addition of oral treprostinil to approved oral monotherapy improved PAC, PVR, and CO. These improvements combined with the anti-platelet and anti-inflammatory properties of oral treprostinil may have contributed to the significant reduction in risk-adjusted clinical worsening in FREEDOM-EV ,4–6. TheSupplementry Material


Short-term and long-term blood pressure variability (BPV) in hemodialysis (HD) population are risk factors of cardiovascular diseases (CVD) and all-cause mortality. There is no full consensus on the best BPV metric. We compared the prognostic role of intra-dialytic and visit-to-visit BPV metrics for CVD morbidity and all-cause mortality in HD patients.A retrospective cohort of 120 patients on HD was followed up for 44 months. Systolic blood pressure (SBP) and baseline characteristics were collected for 3 months. We calculated intra-dialytic and visit-to-visit BPV metrics, including standard deviation (SD), coefficient of variation (CV), variability independent of the mean (VIM), average real variability (ARV) and residual. The primary outcomes were CVD events and all-cause mortality.In Cox regression analysis, both intra-dialytic and visit-to-visit BPV metrics were associated with increased CVD events , but not associated with increased all-cause mortality . Overall, intra-dialytic BPV showed greater prognostic ability than visit-to-visit BPV for both CVD event  and all-cause mortality .Compared to visit-to-visit BPV, intra-dialytic BPV is a greater predictor of CVD event in HD patients. No obvious priority was found among various BPV metrics.The online version contains supplementary material available at 10.1186/s12882-023-03162-w. Hemodialysis (HD) population is under high risk of morbidity and mortality particularly due to cardiovascular disease (CVD) , which aHD patients have higher BPV than the general population due to the routine removal of fluids and electrolytes during each dialysis session and volume-related progressive BP increase during the interdialytic interval . AccordiAmong all the studies mentioned above, several metrics representing variation were mainly implemented including standard deviation (SD) , 11, coeAlthough multiple studies have already proved the correlation between BPV and cardiovascular morbidity as well as all-cause mortality in HD patients, whether BPV is a modifiable risk factor remains undetermined and there are currently no guidelines for treatment recommend based on BPV . FurtherThis was a retrospective study. The inclusion criteria were as followed: (1) patients with end-stage renal disease (ESRD) from the Dialysis Center at Tsinghua Changgung Hospital between January 1st 2018 and Dec 31st 2018; (2) aged ≥ 18 years old; (3) on maintenance HD for more than 3 months in 2018. Patients were excluded under the following circumstances: (1) had no available BP measurement data more than 3 months in 2018; (2) died or transferred to another dialysis center before Dec 31st 2018. Finally, a total of 120 patients were included in the cohort.Data was extracted from the electronic medical record from Tsinghua Changgung Hospital according to the organization’s standard clinical protocols. HD patients received regular HD thrice-weekly , and each dialysis session generally lasted for 4 h. All BP during each dialysis session in our Center was routinely and automatically measured by HD machine in a consensus manner . The patient was in a resting state and lying position, and the BP on the non-fistula arm was measured. The peri-dialytic SBP during the 3 months exposure period for each patient  was used for the calculation of BPV metrics . Intra-dialytic BPV  and visit-to-visit BPV  were selected as indicators of short-term BPV and long-term BPV, respectively. All 5 SBP measures of dialysis sessions were used for intra-dialytic BPV, while only the pre-dialytic SBP of dialysis sessions were used for visit-to-visit BPV.As for intra-dialytic BPV, five different metrics were analyzed using peri-dialysis SBP: (1) standard deviation (SD); (2) coefficient of variation (CV), calculated as SD divided by the mean and multiplied by 100%; (3) variability independent of mean (VIM), calculated as: ocument} ; (4) aveocument} ; (5) aveocument} . As for ocument} , 14. DetData for covariates were collected from electronic clinical records, including demographic characteristics , personal history , comorbidities , dialysis-related variables  and laboratory parameters . Patients with any of the following conditions  were considered to have CVD history. Laboratory parameters were measured monthly. The average values during the exposure period served as the baseline data.Our primary outcomes were CVD events and all-cause mortality. The follow-up period was from Jan 1st 2019 to Aug 31st 2022. CVD events include heart failure, myocardial infarction, ventricular arrhythmias, cerebral infarction, cerebral hemorrhage, and peripheral artery disease (PAD) required surgical intervention. CVD events were acquired from hospitalization records and reviewed by physicians to ascertain. Deaths were identified from death certificates of hospital records. Participants were censored at the following circumstances: (1) received a kidney transplant; (2) transferred to another dialysis facility; (3) come to the end of the follow-up period.Continuous variables were described by mean and SD, while categorical variables were described by frequency and proportion. Spearman’s correlation test was performed in order to evaluate the association between BPV metrics and covariates.The BP data from each patient were analyzed separately as intradialytic data and visit-to-visit BP data. Patients were divided into two groups based on their intra-dialytic or visit-to-visit CV, and the mean CV were used as cutoff values. The high CV group was defined as greater than or equal to the mean CV. The low CV group was defined as less than the mean CV. Variables were compared between the high and low CV groups using Wilcoxon rank sum test. Kaplan-Meier estimator was used to calculate the cumulative incidence of cardiovascular events and all-cause death. Log-rank test was subject to compare survival between the two groups.To further assess the potential association of BPV with CVD morbidity and all-cause mortality, unadjusted (containing only one of the BPV metrics) and adjusted Cox proportional hazards models were conducted. Covariates plausibly associated with CVD morbidity or all-cause mortality  were selected for the adjustment: age, gender, smoking history, drinking history, diabetes, hyperlipidemia, CVD history, tumor, Kt/V, albumin, hemoglobin and creatinine. Phosphorus and ultrafiltration volume were also adjusted. The process deciding confounders is shown in Table S3 . Receiver operating characteristic (ROC) curves were applied to compare the prognostic ability of different BPV metrics. Bootstrap test or DeLong’s test was used for comparing ROC curves.All analyses were performed using R . The R pThere were 218 patients underwent maintenance HD in Dialysis Center of Tsinghua Changgung Hospital since January 1st 2018. After excluding by less than 3 months of HD (n = 81), death (n = 9) and transfer to another dialysis center (n = 8) in 2018, total 120 adult patients on regular HD were enrolled in the cohort. During the 3 months exposure period, 4386 HD treatments and 21,408 SBP measurements were recorded. Table Table Patients were divided into two groups based on the cutoff of the mean intra-dialytic or visit-to-visit CV separately . Characteristics of high and low CV groups were shown in Table S2 . SBP was not different between the two groups. Hemoglobin and albumin were significantly decreased in the high intra-dialytic CV group  but not in the high visit-to-visit CV group  compared with corresponding low CV group. High intra-dialytic CV group had increased CVD morbidity and all-cause mortality compared with low intra-dialytic CV group , but visit-to-visit CV groups had no such difference .During follow-up, 42 patients encountered CVD event, and overall CVD morbidity was 35%, including 28 cardiac diseases (66.7%), 7 strokes (16.7%), and 7 peripheral vascular disease (16.7%). 35 deaths occurred in 331.5 patient-years of follow-up. The mortality rate was 105.6 deaths/1000 patient-years. 14 deaths (40%) were attributed to cardiovascular causes. Infection and tumor were the other two main causes accounting for 6 (17%) and 7 deaths (20%) respectively.Crude and adjusted hazard ratios (HRs) for CVD event and all-cause mortality were shown in Table Area under curve (AUC) of unadjusted Cox regression model was presented in Table In this study, we recruited total 120 patients on maintenance HD as a retrospective cohort, and calculated the intra-dialytic as well as visit-to-visit BPV metrics using peri-dialytic SBP during the 3 months exposure period. We found both intra-dialytic and visit-to-visit BPV were associated with CVD morbidity but not with all-cause mortality. Overall, intra-dialytic BPV outperformed visit-to-visit BPV in prediction of CVD event. Although without reaching significance, intra-dialytic BPV also showed better performance in prediction of all-cause mortality than visit-to-visit BPV.There are already multiple studies that proved both short-term BPV –10 and lShort-term BPV reflects the various physiologic mechanisms, such as autonomic modulation, arterial stiffness, humoral response, blood viscosity, behavior, emotional and environmental factors, whereas long-term BPV additionally involves compliance with prescribed therapeutic regimen and seasonal climate changes . In HD pGreater prognostic ability of intra-dialytic BPV may suggest a major role of dialysis-related factors causing adverse outcomes. Dialysis could be considered as a shock due to the rapid removal of fluids and electrolytes, which may better reflect the body’s ability to regulate BP under stress, like a cardiac stress test for heart evaluation. The limitation of intra-dialytic BPV is that it can’t reflect influence out of dialysis unit such as fluid accumulation, circadian fluctuation and behavior changes. Moreover, peri-dialytic SBP is not the only choice to evaluate BPV. Ambulatory SBP and home SBP measurement have shown superior risk prediction for adverse outcomes –28. And Our study comprehensively compared the prognostic ability of intra-dialytic and visit-to-visit BPV metrics in predicting CVD events and all-cause mortality, which provides evidence for selecting the most efficient BPV metric. Determining a widely accepted and efficient BPV metric is the first step to build a BPV-based guideline for HD population. Non-pharmacologic treatments, such as restricting sodium intake and dialysis prescription, and antihypertensive medications are main approaches to manage BP and volume in HD patients . NeverthIn conclusion, this study showed that both intra-dialytic and visit-to-visit BPV were associated with risk of CVD events in HD patients. Overall, intra-dialytic BPV performed better prognostic ability than visit-to-visit BPV in prediction of CVD morbidity, suggesting intra-dialytic BPV to be a better predictor in HD population.Below is the link to the electronic supplementary material.Supplementary Material 1


A 48-year-old otherwise healthy woman with recurrent urinary tract infections presented to the emergency department with 1 month of fever and flank tenderness. Three months prior, she was diagnosed with a 3-cm right staghorn stone; interventions were postponed due to the SARS-CoV-2 pandemic. Percutaneous nephrolithotomy was attempted: unexpectedly, dye injected into the ureter filled the bowel on fluoroscopy, suggesting a renal-enteric fistula (a). Computed tomography demonstrated xanthogranulomatous pyelonephritis (XGP), a rare chronic pyelonephritis, with perinephric stranding abutting a thickened duodenum. Endoscopy was performed to plan for intervention, revealing a fistulous opening in the second segment of the duodenum (b). Pyeloduodenal fistula is an extremely rare, late-presenting sequela of XGP (


Pneumococcal conjugate vaccines (PCVs) provide protection against vaccine-type pneumococcal disease in both children and adults. Growing evidence suggests that PCVs also reduce pneumonia and lower respiratory tract infections (LRTIs) more broadly, including protecting against viral-associated respiratory diseases. In this short narrative review, we highlight clinical studies investigating whether PCVs might have a role in reducing coronavirus disease, both those caused by endemic human coronaviruses (HCoVs) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). These studies include two randomized controlled trials assessing HCoV-associated pneumonia, one each in children and older adults, and two observational studies of PCV13 effectiveness against HCoV-associated LRTI and COVID-19 in adults. We discuss possible mechanisms for PCV protection including preventing viral pneumococcal co-infections and the possibility that pneumococci in the upper respiratory tract might modify the host immune response to SARS-CoV-2. Lastly, we identify knowledge gaps and further questions on the potential role of PCVs during the COVID-19 pandemic. Streptococcus pneumoniae (pneumococcus) is a leading bacterial cause of community-acquired pneumonia (CAP) in both children and adults [S. pneumoniae and respiratory viruses, particularly influenza virus, are well-documented [Lower respiratory tract infections (LRTIs) remain a major cause of morbidity and mortality worldwide. LTRIs are caused by a wide range of viral, bacterial, and fungal pathogens, some of which also colonize the upper respiratory tract of healthy individuals. d adults . Synergicumented . Pneumoccumented , 4.Coronaviruses (CoVs) are enveloped RNA viruses. Endemic human coronaviruses (HCoVs) 229E, NL63, OC43, and HKU1 typically cause mild, seasonal upper respiratory tract illness, but can cause LRTIs, especially in older adults and people with co-morbidities . Three eAs part of a comprehensive systematic literature review on PCVs and viral respiratory infections, registered in the PROSPERO database (registration number: CRD42022339625), we identified four clinical studies investigating the efficacy or effectiveness of PCV against HCoVs or COVID-19  of viral-associated pneumonia among hospitalized children . ProtectIn a case–control study, Lewnard et al. evaluated PCV13 effectiveness against virus-associated LRTIs among adults in California, United States during the pre-COVID-19 pandemic period . MatchedLewnard and colleagues also investigated PCV13 effectiveness against COVID-19 outcomes in a cohort of older adults in California during 2020 . To reduIn both children and adults, the limited available evidence raises the possibility that PCVs reduce the risk of HCoV-associated respiratory disease. These results indicate that pneumococci might play a role in the development or severity of HCoV infections. Some of the observed efficacy might be due to prevention of pneumococcal-HCoV co-infection, particularly for the PCV9 trial. Since there are no suitable diagnostics for identifying non-bacteremic pneumococcal pneumonia in children, some of the HCoV-associated pneumonia cases may have been pneumococcal-HCoV co-infections for which only HCoV was detected, and these would be more likely to occur in the children who did not receive PCV. Pneumococcal pneumonia is also underdectected in adults due to limited sensitivity of blood culture and urinary antigen tests, so some of the reductions in coronavirus-associated disease in adults might be due to fewer pneumococcal co-infections , 11. StuIn adults, observational studies have reported reduced risk of COVID-19 after vaccination with other vaccines including influenza vaccines and recombinant zoster vaccine , 15. ProFor PCV13, another plausible mechanism exists: the observed reduction in COVID-19 risk might be due to pneumococcal carriage modifying host immune responses . In a stThe clinical studies included in our summary have their own inherent limitations and biases. The two RCTs were not designed to evaluate coronavirus outcomes, and post-hoc or exploratory analyses should be interpreted with caution. The detection of HCoVs in children with pneumonia in the PCV9 trial is insufficient to demonstrate causality, however the reductions observed in the vaccine arm, plus consistency with the finding that efficacy against vaccine-type invasive pneumococcal disease was greater in HIV-uninfected children, suggest that PCV9 protects against HCoV-pneumococcal co-infection . All obsThe potential impact of PCVs on disease due to coronaviruses, including SARS-CoV-2, remains unknown. PCV9 efficacy against HCoV-associated pneumonia seen in South African children may not apply to SARS-CoV-2, as protection against individual HCoVs varied, and HCoVs primarily affect the upper respiratory tract whilst SARS-CoV-2 tropism is broader . High-quIn adults, evidence of PCV13 effectiveness against COVID-19 stems from a single cohort study conducted prior to COVID-19 vaccine availability . CurrentBecause PCVs in adults are not widely used, the opportunity exists for high-quality observational studies that incorporate design elements to mitigate risk of bias to address some remaining questions . In vitrAlthough available data are limited, there is emerging evidence that PCVs might reduce the risk of respiratory disease caused by coronaviruses including SARS-CoV-2. Future studies can further explore the relationship between pneumococci, coronaviruses, and host immune responses, and the mechanisms by which PCVs might alter these interactions. Understanding the broader impact of PCVs can help optimize vaccination strategies to prevent respiratory infections.


Psychobiotics are a group of probiotics that affect the central nervous system related functions and behaviors mediated by the gut-brain-axis via immune, humoral, neural, and metabolic pathways to improve not only the gastrointestinal function but also the antidepressant and anxiolytic capacity.To assess the efficacy the combination of selective serotonin reuptake inhibitors antidepressants (SSRI) and probiotic supplement containing Lactobacillus Plantarum CECT7485 and Lactobacillus Brevis CECT7480 (PLANTARUM) in patients with mild-to-moderate depression.9 CFU once per day. Thirty patients received SSRI antidepressants only without probiotics intake. The severity of depressive symptoms was assessed using Hamilton Depressive Rating Scale (HDRS) and Patient Health Questionnaire (PHQ-9).Sixty patients with mild-to-moderate depression  were included in an 8-week open label study. Thirty participants received either SSRI antidepressants with PLANTARUM at a dose of 1.0 × 10After 8 weeks intervention, a clinically significant reduction of HDRS total score  was detected in patients with mild-to-moderate depression who received SSRI antidepressants and PLANTARUM , compared with participants who didn’t receive probiotics . A significant reduction of PHQ-9 total score  was identified in patients with mild-to-moderate depression who received SSRI antidepressants and PLANTARUM . However, the participants received SSRI antidepressant only didn’t meet a clinically significant reduction depressive symptoms  by PHQ-9 scale.The combination use of SSRI antidepressants and probiotic supplement PLANTARUM significantly reduced the depressive symptoms.None Declared