Evidence suggest that critically ill COVID-19 patients are at higher risk of developing anxiety symptoms, which may be related to or exacerbated by patients concerns regarding their health status and recovery.To assess anxiety symptoms in critically ill COVID-19 survivors, 1-2 months after hospital discharge and to analyze its association with concerns reported by patients regarding their own health status and recovery.In the framework of MAPA prospective research, this preliminary study included COVID-19 patients admitted in the Intensive Care Medicine Department (ICMD) of a University Hospital. Patients were excluded if they had an ICMD length of stay (LoS) ≤24h, terminal illness, major auditory impairment or inability to communicate at the evaluation time. Participants were assessed at a scheduled telephone follow-up appointment, with Generalized Anxiety Disorder Scale (GAD-7). Additional questions were asked to assess the survivors’ post-discharge concerns regarding discrimination against for COVID-19, infection of a family member, re-infection or sequelae related to COVID-19.Eighty-three patients were included  and 24% had anxiety symptoms. Anxiety scores were higher in survivors who reported being afraid of being discriminated against for COVID-19 , being re-infected  and having sequelae .These findings revealed that anxiety is common in COVID-19 survivors and is associated with post-discharge patients concerns that may limit patient daily living. This study emphasizes the importance of psychological assessment and follow-up of the COVID-19 survivors, in order to support these patients recovery.No significant relationships.


The main causes of anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis are ovarian teratoma and herpes simplex virus (HSV) encephalitis. We present a rare case of suspected anti-NMDAR encephalitis caused by corpus callosotomy (CC). An 18-year-old woman with Lennox-Gastaut syndrome underwent CC. Although left hemiplegic due to cerebral hemorrhage and impaired consciousness due to cerebral venous sinus thrombosis (CVST) appeared postoperatively, anticoagulant therapy quickly improved CVST and impaired consciousness. However, various unexplained symptoms such as insomnia, hallucination, impulsivity, impaired consciousness, and a new type of drug-resistant cluster seizures gradually developed over a 2-month period. Magnetic resonance imaging revealed the gradual extension of a hyperintense area from the right frontal lobe on fluid-attenuated inversion recovery images. Intravenous methylprednisolone pulse was initiated from postoperative day (POD) 74, followed by intravenous immunoglobulin (IVIg) therapy, although white blood cell counts were normal in all three cerebrospinal fluid (CSF) examinations. After IVIg therapy, the above unexplained symptoms promptly improved. On POD 103, antibodies against NMDAR were revealed in both the serum and CSF collected before these immunotherapies. The patient was transferred to a rehabilitation hospital due to residual left hemiplegia. Psychiatric symptoms and a new onset of drug-resistant seizures may be suggestive of postoperative anti-NMDAR encephalitis, even if CSF findings are mild. Approximately 20–30% of patients with epilepsy reportedly cannot achieve a seizure-free state despite optimal medical treatment ,2,3. SomAnti-NMDAR encephalitis is a type of autoimmune encephalitis caused by antibodies against the GluN1 subunit of the NMDAR . PatientAlthough several cases of non-bacterial encephalitis after craniotomy have been reported, most were attributable to herpes simplex virus (HSV) encephalitis ,16,17,18This is the first report of suspected anti-NMDAR encephalitis after CC. The diagnosis was challenging because of a pre-existing intellectual disability, mild CSF findings, and the difficulty of obtaining results for the anti-NMDAR antibody. We suggest the importance of checking the criteria for possible autoimmune encephalitis when a case shows unexplained progressive neurological deterioration after neurosurgery. Cases that meet these criteria should be investigated for neuron surface antibodies, even if CSF and MRI findings are mild.An 18-year-old woman with drug-resistant epilepsy was admitted for CC due to daily seizures that persisted even after vagus nerve stimulation. There was no family history of epilepsy. The seizure types were tonic seizures, spasms, and drop attacks. She had an intellectual disability  and was diagnosed with Lennox-Gastaut syndrome (LGS). Preoperative MRI revealed cortical scarring in the right parietal and left occipital lobes A, althouOn postoperative day (POD) 1, CT revealed a cerebral hemorrhage on the medial side of the right frontal lobe B, and thNew symptoms such as insomnia, hallucinations, delusions, and dysphagia became slowly apparent from a month after CC. Impaired consciousness  and fever of approximately 38 °C relapsed, and a new type of seizure and tachycardia (120 beats/min) were observed from 2 months postoperatively . The seizure type differed from the preoperative seizure types, appearing as daily clusters of 10 or more consecutive focal impaired awareness seizures (FIASs) with conjugate deviation of the eyes to the left followed by elevation of the left arm.The electroencephalogram showed diffuse polymorphic slowing with lower amplitude in the right frontal region and a reduction of generalized slow spike-and-slow-wave complexes found before CC, although extreme delta brush  was not This case met the criteria for possible autoimmune encephalitis  as proposed by Graus et al. , althougAlthough slight disinhibition and impulsivity became apparent as the level of consciousness improved, second-line immunosuppressive therapy with rituximab or cyclophosphamide was not administered because seizure frequency decreased to once or twice a month, and MRI showed a reduction in the area of FLAIR hyperintensity on POD 141 . Due to This report describes a case of suspected postoperative anti-NMDAR encephalitis that sub-acutely developed various symptoms such as psychiatric symptoms, impaired consciousness, and cluster seizures and recovered from these symptoms immediately after starting immunotherapy. We suspected autoimmune encephalitis because these symptoms occurred after CVST resolved, and it met the criteria for possible autoimmune encephalitis. However, this diagnosis was challenging to make and was delayed because only one previous case report has described anti-NMDAR encephalitis after epilepsy surgery, pre-existing intellectual disability concealed psychiatric symptoms, and the CSF and MRI in this case displayed few findings showing autoimmune encephalitis. We emphasize the possibility of autoimmune encephalitis as a complication of neurosurgery even with normal CSF and MRI findings.Clinical symptoms were the key clues to considering autoimmune encephalitis in the present case. Patients with anti-NMDAR encephalitis show a variety of acute or subacute symptoms such as psychiatric symptoms, seizures, movement disorders, impaired consciousness, central hypoventilation, and dysautonomia . The preCSF pleocytosis in autoimmune encephalitis is considered milder than that in virus encephalitis . A recenThe rate of abnormal findings on an MRI is reportedly 35–50% in anti-NMDAR encephalitis, and no or only mildly abnormal MRI findings despite symptom intensity seemed to be a feature of this encephalitis ,32,33. TThe main triggers of anti-NMDAR encephalitis are ovarian teratomas and HSV encephalitis . ApoptosUnfortunately, we cannot prove that this patient developed anti-NMDAR encephalitis after epilepsy surgery because of the possibility of false-positive results and previous anti-NMDAR encephalitis. However, the likelihood of false-positive results in the present case seems very low, because both the serum and CSF showed positive results for anti-NMDAR antibody. Although false-positive or false-negative results for antibodies against NMDAR can occur if only serum is used, the false-positive rate has been reported as only 0.8% when CSF is investigated . RecentlWe documented a rare case of suspected anti-NMDAR encephalitis after CC for LGS. We propose the importance of considering autoimmune encephalitis in cases showing unexplained progressive neurological deficits after neurosurgery, even if CSF and MRI findings are mild.


Despite the initially reported high efficacy of vaccines directed against ancestral SARS-CoV-2, repeated infections in both unvaccinated and vaccinated populations remain a major global health challenge. Because of mutation-mediated immune escape by variants-of-concern (VOC), approved neutralizing antibodies (neutAbs) effective against the original strains have been rendered non-protective. Identification and characterization of mutation-independent pan-neutralizing antibody responses are therefore essential for controlling the pandemic. Here, we characterize and discuss the origins of SARS-CoV-2 neutAbs, arising from either natural infection or following vaccination. In our study, neutAbs in COVID-19 patients were detected using the combination of two lateral flow immunoassay (LFIA) tests, corroborated by plaque reduction neutralization testing (PRNT). A point-of-care neutAb LFIA, NeutraXpress™, was validated using serum samples from historical pre-COVID-19 negative controls, patients infected with other respiratory pathogens, and PCR-confirmed COVID-19 patients. Surprisingly, potent neutAb activity was mainly noted in patients generating both IgM and IgG against the Spike receptor-binding domain (RBD), in contrast to samples possessing anti-RBD IgG alone. We propose that low-affinity, high-avidity, germline-encoded natural IgM and subsequent generation of class-switched IgG may have an underappreciated role in cross-protection, potentially offsetting immune escape by SARS-CoV-2 variants. We suggest Reverse Vaccinology 3.0 to further exploit this innate-like defense mechanism. Our proposition has potential implications for immunogen design, and provides strategies to elicit pan-neutAbs from natural B1-like cells. Refinements in future immunization protocols might further boost long-term cross-protection, even at the mucosal level, against clinical manifestations of COVID-19. As of mid-2022, more than 560 million people globally have been infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for coronavirus disease 2019 (COVID-19). Over 6.3 million people have died of infection-mediated complications. The extraordinarily rapid development of several vaccines in the first year of field deployment has saved close to 20 million lives that would otherwise have been lost to COVID-19 , and mayLikewise, passive transfer of therapeutic neutralizing monoclonal antibodies (mAb) was initially successful in treating COVID-19 before the arrival of the variants , but manTherefore, for neutAb developers, active questions are: 1) Whether it is possible to identify, characterize and isolate pan-neutAbs against the majority of current and future SARS-CoV-2 variants and 2) if this venture is successful, whether developing recombinant pan-neutAbs as prophylactic and therapeutic modalities can stay ahead of evolving variants? These questions also apply to vaccine development to stimulate durable pan-neutralizing antibody responses.The answer to the first question is in the affirmative. By adopting high throughput single cell sequencing of thousands of Spike-enriched memory B cells (MBCs) from the PBMCs of convalescent patients , researcNevertheless, as the S2 regions are conserved among multiple human coronaviruses, blocking the interaction of the highly conserved S2’ site and the fusion peptide by 76E1 can effectively block virus-cell fusion and broadly neutralize seven human coronaviruses, including two α-coronaviruses (HCoV-229E and HCoV-NL63) and five β-coronaviruses  . Thus, ath, 2022, the FDA restricted the emergency use of Bamlanivimab and Etesevimab (co-administered) and REGEN-COV (Casirivimab and Imdevimab) to “patients that are likely to have been infected with or exposed to a variant that is susceptible to these treatments”.The answer to the second question is more complex. Large-scale manufacturing, clinical trials and regulatory approval of the recombinant neutAbs typically take a long time and are hugely expensive. In light of certain neutAbs being “highly unlikely to be active against the Omicron variant, which is circulating at a very high frequency throughout the United States”, on January 24To avoid such restrictions in future therapeutic mAbs, researchers need to reassess if there are fundamental immunological elements missing in our current understanding of such pan-neutAbs and of the durability of vaccine-induced protection. The following sections are devoted to such mechanistic discussions, albeit with a focus on mRNA vaccination.nd dose of either Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) mRNA vaccines in subjects having no prior history of SARS-CoV-2 infection  responses drive the development of two distinct but equally important B cell populations: the high-affinity long-lived plasma cells (LLPCs) , 28 geneFor example, upon infection or immunization with one flavivirus , the low-affinity cross-reactive MBCs generated had a very limited capacity to re-enter secondary GCs, and were excluded from GC-somatic hypermutation. However, these MBCs developed enhanced affinities towards a heterologous flavivirus  by forming extrafollicular plasmablasts . Lineagei.e., whether responses are through creating fundamentally new specificities through secondary GCs or through the selection of pre-existing clones without further affinity maturation. Unlike antigen-specific LLPCs derived from high-affinity naïve B cells, precursors of MBCs encoding low-affinity antibodies with germline sequences largely bypass secondary GCs in recall responses  by SARS-CoV-2 specific antibodies in the early response has also been confirmed by other studies  of SARS-CoV-2-infected subjects, Kreer and colleagues found highly potent neutAbs developed early after infection and exhibited limited ongoing somatic hypermutation . From so studies , 37–45.  studies  and seve studies , 47. The studies , a featuThe B cell compartment can be divided into two developmentally and functionally distinct populations, B-1 and B-2 B cells . B-1 cele.g., defense against microbial pathogens in innate immunity), and B-2 cell antibodies are selected for affinity to a pathogen (via class-switching and low-degree somatic hypermutation) into antibodies with greater breadth and affinity to rising variants. To the contrary, because of the BCR sequence use, the malleability of neutAbs of B-2 ontogeny may be limited and these might be funneled towards high affinity with a LLPC host cell fate, targeting only the original strain. These neutAbs are highly specific to the homologous immunogen and yet are not flexible in their response and are eventually outpaced by a rapidly-mutating pathogen.It is important to understand the origin of neutAbs for SARS-CoV-2. B-1 cell antibodies are selected for function  to detect PRNT90 ≥ 80 neutAb activity  Whether neutAb activities can be detected in the early phase of SARS-CoV-2 infection (<40 days post onset of symptoms); and 2) Whether RBD-specific IgM contributes to the serum neutralizing activity. First, we stratified PCR-confirmed COVID-19 patients based on days following symptom onset and IgM/IgG profiles of anti-RBD antibodies in their serum samples. For that, we used an LFIA rapid test DISCOVID™ to detect the presence of RBD-specific IgM and/or IgG in their preserved serum samples. The specificity (96.6%) and sensitivity (92.7%) of DISCOVID™ have been previously determined with historical samples collected prior to the pandemic and in PCR-confirmed COVID-19 patients 2. ThereP<0.05)  adequate time post symptom onset; and 2) IgG isotype RBD reactivity, indicative of post-GC events. However, in direct comparison to IgM+IgG+ subgroup (66.7% samples neutAb positive), there appears to be much lower neutralization activity in IgM-IgG+ subgroup (30% samples positive) (P<0.05) Figure 1P<0.05) . Such invs. intramuscular inoculation); 2) antigen dose and persistence (weeks vs. days); and 3) antigen form [particulate intact virus vs. cell-surface displayed Spike trimer locked in a stabilized pre-fusion state (de novo expression of the vaccine antigen is similar to the presentation of engulfed soluble antigens by antigen-presenting cells (APCs); following vaccination, full length Spike protein expressed by monocytes, macrophages and dendritic cells (DCs) stimulates conventional B-2 cells in the axillary lymph nodes via the classical MHC class I and class II antigen presentation pathways  route of antigen exposure . For pathogens with strong immune-evasion potential, what could be the ideal strategy to aid the development of pan-protective vaccines against the circulating strains and emerging variants?Widespread danger from SARS-CoV-2 still lingers, due to the evolution of new escape variants impervious to current vaccination strategies. A broader sobering outlook includes the estimation that 58% (218 out of 375) of infectious diseases currently confronted by humanity worldwide may be aggravated by the effects of climate change in the near future . There iin silico using the genomic sequence information of the pathogen without the need for growing the specific microorganism. This strategy directly led to the successful development of the meningococcal B vaccine , and SBIR 75N93019C00014, 75N93020C00042, and 75N93022C00011.We thank Dr. Xuemei Zhong for the insightful discussion on the basic immunological mechanisms involved in this topic.WG is employed by Antagen Diagnostics, Inc., which is the developer of DISCOVID™ and NeutraXpress™. WG is also employed by company Antagen Pharmaceuticals, Inc. SCR is a scientific founder of Purinomia Biotech Inc and consults for eGenesis, AbbVie and SynLogic Inc; his interests are reviewed and managed by HMFP at Beth Israel Deaconess Medical Center in accordance with the conflict-of-interest policies. Authors J-SL and Y-MH are employed by AB Biosciences, Inc. Authors WZ and SH are employed by Shijiazhuang Hipro Biotechnology Co.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The continuum of social threat ranges from anxiety to paranoia. Examining the factors that predict and mediate the relationship between social anxiety and persecutory paranoia will help with the development of interventionist-causal theories that can guide the development of new treatments.To investigate mediators between social anxiety and persecutory paranoia in a prospective cross-cultural analogue sample.A 3-month follow-up online survey included participants aged ≥18-years-old in Thailand and the UK. Recruitment was via advertisements on websites and social media. Participants completed questionnaires at baseline (T1) and 3-month follow-up (T2) measuring social anxiety, paranoia, depression. Mediators were: stigma; internal and external shame; social rank; self-esteem; and safety behaviours. We used linear regression to examine predictors of paranoia and mediation analysis to test indirect effects. Estimating the indirect effects was calculated by 10,000 bootstrapping bias-corrected 95% confidence intervals.At follow-up, 186  Thai and 236  UK respondents completed the survey. Regarding change scores (T2-T1), higher paranoia was significantly predicted by higher social anxiety and external shame controlling for age, gender, depression. A simple mediation model controlling for depression showed significant indirect effects for external shame  and safety behaviours . A multiple mediation model found external shame was a significant mediator .These cross-cultural data suggest that external shame may mediate the prospective relationship between social anxiety and paranoia. These data suggest the potential for treatment of persecutory fears and social anxiety in psychosis by targeting shame-related cognitions.


The rapid development of CRISPR-Cas genome editing tools has greatly changed the way to conduct research and holds tremendous promise for clinical applications. During genome editing, CRISPR-Cas enzymes induce DNA breaks at the target sites and subsequently the DNA repair pathways are recruited to generate diverse editing outcomes. Besides off-target cleavage, unwanted editing outcomes including chromosomal structural variations and exogenous DNA integrations have recently raised concerns for clinical safety. To eliminate these unwanted editing byproducts, we need to explore the underlying mechanisms for the formation of diverse editing outcomes from the perspective of DNA repair. Here, we describe the involved DNA repair pathways in sealing Cas enzyme-induced DNA double-stranded breaks and discuss the origins and effects of unwanted editing byproducts on genome stability. Furthermore, we propose the potential risk of inhibiting DNA repair pathways to enhance gene editing. The recent combined studies of DNA repair and CRISPR-Cas editing provide a framework for further optimizing genome editing to enhance editing safety. CEP290 for Leber Congenital Amaurosis type 10The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) nucleases have been engineered to achieve efficient gene editing in mammalian cells –4 . AmoBesides the great potential of CRISPR-Cas editing tools, unwanted editing byproducts accompanied with intended editing products have also attracted great attention recently, since they lend additional uncertainty to genome editingThe generation of both intended products and unwanted editing byproducts during genome editing are stimulated by endogenous DSB repair pathways, and understanding how these repair pathways work in depth could help to reduce the side effects of unwanted byproducts during gene editing.In this review, we begin with the editing mechanism for CRISPR/Cas editing system and then describe the involved DSB repair pathways in the editing process. We next discuss the generation of unwanted genome editing products and propose possible solutions to improve the safety of gene editing.Streptococcus pyogenes Cas9 (SpCas9) with an NGG  PAM is the first and most widely used Cas9 for genome editing [Staphylococcus aureus Cas9 (SaCas9) was also developed for target sites with NNGRRT (R=“A” or “G”) PAMStreptococcus thermophiles Cas9 (StCas9), Campylobacter jejuni Cas9 (CjCas9),Francisella novicida Cas9 (FnCas9),Geobacillus stearothermophilus Cas9 (GeoCas9),Neisseria Meningitides Cas9 (NmeCas9), andStreptococcus canis (ScCas9) were subsequently engineered for genome editing (Acidaminococcus sp. Cas12a (AsCas12a) andLachnospiraceae bacterium ND2006 Cas12a (LbCas12a) show great potential in gene editingThe CRISPR-Cas enzyme is an RNA-guided endonuclease that induces DSB at the phage genome. The CRISPR-Cas enzymes have two distinct groups: class I, which applies multi-Cas proteins to achieve DNA cleavage; and class II, which applies a single endonuclease for DNA cleavage ,47 . Clediting –4,49 .editing A [51–5trans-activating crRNA (tracrRNA) pairing with crRNA for Cas9 ribonucleoprotein (RNP) packageCRISPR-Cas9 system contains a CRISPR RNA (crRNA) for targeting DNA and aIn addition to CRISPR-Cas nucleases, CRISPR-based base editors and prime editors were mainly developed for mutation corrections. The base editor consists of a Cas9 nickase, a DNA deaminase enzyme, and a uracil-DNA glycosylase inhibitor or uracil-DNA glycosylase, which converts C to T, C to G, or A to G without causing DSBs –13 . InDSBs are the most deleterious type of DNA lesions, leading to genetic mutations or complex chromosomal rearrangements associated with oncogenesis ,82–87 Classical non-homologous end joining (C-NHEJ) directly re-joins two broken ends and is considered to be the default choice for DSB repair in mammalian cells through cell cyclesDuring C-NHEJ, KU70-KU80 heterodimer immediately binds to the broken ends and recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and/or Artemis endonuclease to mildly process broken ends if needed –104 . NAlternative end joining (A-EJ) dominates end joining repair when core factors of C-NHEJ are deficient ,123 . ALeishmania were repaired by SSA, and thereby SSA was enhanced to achieve large fragment deletion up to 29 kbMMEJ prevalently contributes to the formation of indels during genome editing by generating short deletions between two microhomologous sequences B. MMEJ-mHR requires a homologous template to finish DSB repair and therefore is a relatively precise DNA repair pathway. HR is mainly active in the S and G2 phases in dividing cells, exhibiting a lower utilization rate in comparison with NHEJ in most cells. The deactivation of C-NHEJ makes Cas-induced DSBs prone to be repaired by A-EJ or HR –146 . HTo introduce intended mutations at target sites during gene editing, a double-stranded (ds) or single-stranded (ss) donor DNA is transfected with CRISPR-Cas to activate the HR pathway to induce homology-directed repair (HDR) B 154, . The hoGiven that the repair of Cas9-induced DSBs is consistent with the repair of endogenous general DSBs, it is inevitable that the sealing of Cas9-induced DSBs results in many diverse outcomes. Besides the intended mutations at the target site, other unwanted byproducts are routinely identified. CRISPR-Cas activities at off-target sites are well explored by developed methods and a dozen of high-fidelity Cas9 variants have been engineered to reduce the off-target activities of CRISPR-Cas9 –179 . CSpCas9(1.1),SpCas9-HF1, and HypaCas9 were developed based on Cas9-DNA structures and Cas9 conformation change before cleavage [Off-target sites of CRISPR-Cas9 are highly homologous to the target sites with higher mutation tolerance at the PAM-distal regionleavage –179 . Sleavage ,186 . Tleavage ,173 . MThe juxtaposition of two DSBs can form translocation at a very low frequency. A single DSB generated by meganuclease I-SceI or CRISPR-Cas9 could join to any DSB induced by ion irradiation, implying that any two escaped DSBs can form translocation ,43,187During genome editing, DSBs at the target sites are the dominant DSBs and thereby the vast majority of editing outcomes are re-joinings of the two broken ends of the target DSBs. However, other broken ends that occur simultaneously within the edited cells may also have a chance to join with target DSBs to form translocations. These involved DSBs can be categorized into three types: other target DSBs, off-target DSBs, and general DSBs. Correspondingly, the translocations involving these DSBs are referred to as target translocations, off-target translocations, and general translocations, respectively. The target translocation mainly occurs in the multiplex gene editing system and multiple CRISPR-Cas-induced target DSBs join together to induce a high level of chromosomal translocations. The off-target translocations involving DSBs at off-target sites are also dependent on CRISPR-Cas enzymes. As for general translocations, general DSBs induced by various DNA metabolism activities arise randomly in the genome and can also be captured by CRISPR-Cas-induced target DSBs to form chromosomal translocations. These general DSBs may occur in certain physiological processes including V(D)J recombination or class switch recombination in lymphocytes –192 , oTRAC-TRBC-PDCD1 remained in the blood at even hundreds of days post-infusion into the patientsSeveral previous reports showed that target chromosomal translocations frequently arose during multiplex genome editing in CAR T manufacturing –200 . CUROS loci in HEK293T cellsChromosomal large deletions induced by CRISPR-Cas routinely occur at the target site and range from several hundred bases to megabases, resulting in the loss of a large chromosome fragment around the target site or even the entire chromosome ,44,193SWAPP,Mecp2, andDnmt3b), and moused muscle (Dmd)RAG1,DNMT1,EMX1,VEGFA, andC-MYC loci, for both Cas9 and its variants [Integration of exogenous DNA originating from vectors or viruses into the genome was another concern of genome editing ,SpCas9-HF1, FeCas9, and HypaCas9, are indeed able to effectively reduce off-target activitiesThe great improvement of CRISPR-Cas nucleases in clinics shows great potential in the treatment of intractable diseases. Yet DSB is a double-edged sword: off-target damages, chromosomal translocations, large deletions are other non-negligible unwanted editing byproducts that consist of up to 10% of total editing events. The high-fidelity Cas9 variants, especially eMany methods employ inhibitors for DNA repair proteins to change the choice of DNA repair pathways in editing cells ,211 . Htables


The coronavirus disease 2019 (COVID-19) pandemic caused by the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has cast a notorious damage to the public health and global economy. The Stimulator of Interferon Genes (STING) is a crucial element of the host antiviral pathway and plays a pivotal but complex role in the infection and development of COVID-19. Herein, we discussed the antagonistic mechanism of viral proteins to the STING pathway as well as its activation induced by host cells. Specifically, we highlighted that the persistent activation of STING by SARS-CoV-2 led to abnormal inflammation, and STING inhibitors could reduce the excessive inflammation. In addition, we also emphasized that STING agonists possessed antiviral potency against diverse coronavirus and showed adjuvant efficacy in SARS-CoV-2 vaccines by inducing IFN responses. The corresponding virus of COVID-19 was identified as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, which contains a positive single-stranded RNA genome (Coronavirus disease 2019 (COVID-19) has rapidly spread across the world since the end of 2019, resulting in over 500 million confirmed infected cases and over 6 million deaths so far -CoV and bat coronavirus HKU4 and so on –17. The The innate immune system is the first line of defense against evading pathogens . It recoSting-/- mice were more sensitive to vesicular stomatitis virus (VSV), a negative-stranded virus, and the production of type I IFNs was decreased in Sting-/- mice  exhibited antiviral activity against SARS-CoV-2 . MeanwhiThe generation of syncytia provides a possible mechanism for delayed IFN response in COVID-19 patients, indicating that the production of type I IFN is inhibited at early stage of SARS-CoV-2 infection, but then substantially activated at the late stage , 53.Although STING activation presents antiviral potential, the sustained STING signaling results in excessive amounts of type I IFNs. Indeed, patients with severe COVID-19 exhibited robust type I interferon response, which was associated with acute respiratory distress syndrome, lung injury and poor clinical outcome –57. TherBy profiling the transcriptome and secreted cytokines of SARS-CoV-2-infected lung epithelial cells, Neufeldt and co-workers found that the NF-κB and pro-inflammatory pathway was up-regulated in infected lung epithelial cells, while the antiviral IFN response was not enhanced . These oDifferent from other studies on lung epithelial cells or tumor cell models, Domizio and colleagues focus on skin manifestations in SARS-CoV-2 infection . They foSince viral proteins suppress the STING pathway in the early stage of infection, while the micronucleus and DNA damage caused by cell fusion in the host activate the STING signaling to suppress viral infection, treatment with STING agonists in the early stage of COVID-19 provides be a potential antiviral strategy.Recently, Li and co-workers found that SASR-CoV-2 infection induced delay of IFN response to evade innate immunity, which could be controlled by type I IFN treatment . SubsequSimilarly, the diABZI analogue, diABZI-4, was proved as well to prevent SARS-CoV-2 replication in ACE2-A549 cells and in 3D-cultured embryonic stem cell–derived induced alveolar type II (iAT2) cells . Intranain vivo and in vitro. The activation is transient, but can prevent lung tissue damage from abnormal inflammation. Therefore, STING agonists may provide alternative strategy for the treatment of COVID-19 in the early stage  was reported to induce more potential antibody response compared with S protein-loaded VLPs. The virus neutralizing capacity of resulting antibodies was improved as well . An intrIn addition to cGAMP, a few novel STING agonists were also used as adjuvants in SARS-CoV-2 vaccine. For instance, Wu and co-workers synthesized the analogue CDGSF by modifying cyclic di-GMP (CDG) with one phosphorothioate and one fluorine moieties . The flupan-sarbecovirus vaccine (CF501 is a new STING agonist and was found to show potent adjuvant efficacy in  vaccine . Compare vaccine .It is well established that the STING pathway elucidates a double-edged effect on COVID-19. At the early stage of infection, STING signaling is suppressed by vital proteins containing 3CL, ORF3a and ORF9b, resulting in the impaired innate immune response , 41. In XR collated the literature and wrote the review, and ZA established that the structure of the review and revised the article.


The results indicate that elevated preoperative VCAM-1 levels were not only associated with worse oncological outcomes but also predicted biologically and clinically aggressive disease. In DCA, the reference models for lymph node metastasis, non-organ-confined disease, recurrence-free survival, and cancer-specific survival prediction resulted in significant improvements after addition of VCAM-1.This consecutive cohort study enrolled 1036 patients with clinically nonmetastatic advanced UCB having undergone RC to investigate the relationship between preoperative plasma VCAM-1 levels and the established features of UCB invasion, metastasis, and survival outcomes.This study demonstrates that addition of VCAM-1 improved the discriminatory power of predictive/prognostic models significantly. Therefore, VCAM-1 might be a valuable biomarker in determining the need for perioperative chemotherapy and the extent of lymphadenectomy required. VCAM-1 is associated with ease of procurement, low cost, high sample homogeneity, and potential to improve early outcome prediction and prognosis. However, further study is warranted for VCAM-1 to be determined as a candidate biomarker worth integrating into prospective clinical trials to enhance currently available tools used for risk stratification in patients with UCB, and assess its value in the era of immunotherapy.


Drosophila with high bioactivation. Milk derived from five bean varieties, namely black bean (Phaseolus vulgaris), red kidney bean (Phaseolus vulgaris), mung bean (Phaseolus aureus), peanut (Arachis hypogaea) and soybean (Glycine max) did not induce DNA mutations in Drosophila with high bioactivation, indicating their genome-safe properties. All bean milks showed anti-mutagenicity against the food-derived mutagen, urethane, in vivo with different degrees of inhibition. In the co-administration study, larvae were treated with each bean milk together with urethane. Soybean milk showed the highest anti-mutagenicity at 27.75%; peanut milk exhibited the lowest at 7.51%. In the pre-feeding study, the larvae received each bean milk followed by urethane. Soybean milk exhibited the highest anti-mutagenic potential, followed by red kidney bean and black bean milks. Total phenolic and antioxidant data revealed that the anti-mutagenicity of both red kidney bean milk and black bean milk might be derived from their phenolic or antioxidant properties; other phytochemicals may contribute to the high anti-mutagenicity observed in soybean milk. Further investigations on the anti-mutagenicity of bean milks against other dietary mutagens are required to develop bean-based products with potent anti-mutagenic properties.The consumption of a nutritious diet including phytochemicals can minimize mutations as the primary cause of carcinogenesis. Bean consumption supplies calories, minerals and phytochemicals but their anti-mutagenic properties in vivo remain little understood. Hence, the present study aimed to study the mutagenicity and anti-mutagenic properties of five bean milks using the somatic mutation and recombination test (SMART) involving  A mutation is a change in DNA sequences or chromosomal architecture influenced by exposure to chemicals known as mutagens. There are two types of mutations—germline mutations and somatic mutations. Germline mutations occur in eggs and sperm and can be passed on to offspring; somatic mutations occur solely in body cells . MutatioO-galactoside and myricetin 3-O-glucoside pyrene induced mutations (over 70% inhibition) in Salmonella typhimurium TA98 and TA100 with S9 liver homogenate [Phaseolus vulgaris L.) belongs to the same family as red kidney bean and black bean, and the seed coat inhibited aflatoxin B1-induced DNA mutations in S. typhimurium TA98 and TA100 with bioactivation system [We further explored the anti-mutagenic properties of each bean milk against a model food mutagen, urethane, using three types of strategies, namely co-administration, type I pre-feeding and type II pre-feeding studies, as depicted in mogenate . Urethann system ,40.O-glucosides of delphinidin, petunidin and malvidin; hydroxycinnamic acids, diglycosides of kaempferol and quercetin were found in dark red kidney beans [N6-ethenoadenosine and 3,N4-ethenocytidine DNA adducts [Anti-mutagenic compounds inhibit mutagenic agents through several mechanisms, namely (i) inactivation of mutagens, (ii) binding to mutagens before they induce DNA damage, and (iii) antioxidant potency . Hence, ey beans . By contey beans ,45. Uret adducts ,47,48. T adducts ; geniste adducts . Hence, Although we stated that all tested bean milks lacked mutagenicity, our findings cannot be extrapolated to the bean seeds as a whole since our samples were prepared by imitating home bean milk. As a result, the seed cakes were not tested. Further research using whole bean seed may be able to overcome this limitation. The present study provides the prompt idea that some bean milks made from soybean, red and black kidney bean could inhibit urethane-induced mutations. Urethane can be found in alcoholic beverages and fermented food products. It has been reported that pickled vegetables and salted fish consumption are linked to an increased risk of gastric cancer occurrence . Thus, dDrosophila with high bioactivation and found to be genome-safe. High anti-mutagenic properties against a food-derived mutagen, urethane, were observed in milk produced from black bean, red kidney bean and soybean. Anti-mutagenicity shown by black bean and red kidney beans was associated with their TPCs and antioxidant activities; other compounds in soybean might also contribute to anti-mutagenicity against urethane. More thorough investigations on the anti-mutagenic effects of bean milks against other food mutagens are required to enhance the health benefits of beans and develop bean-based functional foods for mutation reduction.Milks made from five bean varieties commonly consumed in Thailand, namely black bean, red kidney bean, mung bean, peanut and soybean, were assessed using


To survive in adverse environmental conditions, plants have evolved sophisticated genetic and epigenetic regulatory mechanisms to balance their growth and abiotic stress tolerance. An increasing number of non-coding RNAs (ncRNAs), including small RNAs (sRNAs) and long non-coding RNAs (lncRNAs) have been identified as essential regulators which enable plants to coordinate multiple aspects of growth and responses to environmental stresses through modulating the expression of target genes at both the transcriptional and posttranscriptional levels. In this review, we summarize recent advances in understanding ncRNAs-mediated prioritization towards plant growth or tolerance to abiotic stresses, especially to cold, heat, drought and salt stresses. We highlight the diverse roles of evolutionally conserved microRNAs (miRNAs) and small interfering RNAs (siRNAs), and the underlying phytohormone-based signaling crosstalk in regulating the balance between plant growth and abiotic stress tolerance. We also review current discoveries regarding the potential roles of ncRNAs in stress memory in plants, which offer their descendants the potential for better fitness. Future ncRNAs-based breeding strategies are proposed to optimize the balance between growth and stress tolerance to maximize crop yield under the changing climate. Owing to the sessile nature, plants are exposed to diverse abiotic stresses due to climate change, such as drought, salinity, and extreme temperatures (cold and heat), high light, ozone, which pose a serious threat to crop productivity worldwide . To copeNon-coding RNAs (ncRNAs) are a class of RNAs which are transcribed in the genome but do not encode proteins. Recent genome-wide transcriptome analyses have revealed that in addition to the well-known rRNA, tRNA, small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs), a large number of other ncRNAs, including 18-30 nucleotide (nt) small RNAs (sRNAs), medium-sized ncRNAs (31-200 nt), and long non-coding RNAs (lncRNAs) (> 200 nt), are expressed in plants. These ncRNAs differ in their biosynthesis and modes of action on target genes. Despite the differences, the regulatory pathways of ncRNAs are interconnected and either synergistically or antagonistically regulate the expression of target genes or protect the genome from both external and internal threats . ncRNAs In this review, we summarize recent findings and the current progress on ncRNAs-mediated prioritization towards plant growth or tolerance to abiotic stresses, with a focus on hormone-based signaling crosstalk. We further highlight the potential roles of ncRNAs in the interplay between growth and stress responses in plants during transgenerational memory. We then propose perspectives in ncRNAs-based breeding strategies to make growth and stress tolerance compatible to maximize crop yield.MIR genes which are usually located in intergenic regions with some found in the intronic sequences of protein coding genes  (sRNAs in plants can be divided into microRNAs (miRNAs) and small interfering RNAs (siRNAs) according to their synthesis pathways and modes of function. miRNAs are non-coding small RNAs with a length of 20-24 nt and are encoded by ng genes . The plavergence  and mosthardtii) . miRNAs hardtii) . siRNAs hardtii) .PHAS) in a phased manner upon miRNA-mediated cleavage , can function in trans to modulate the expression of target mRNAs or non-coding transcripts , maintenance of genome stability and regulation of gene expression . PhasiRNcleavage  and drivcleavage . Some phnscripts . The prenscripts .Glycine max L.) nodulation  and SPL11 are critical for the somatic heat stress memory , celery (Apium graveolens), and cotton (Gossypium hirsutum) (Arabidopsis by repressing the expression of its targets AUXIN RESPONSE TRANSCRIPTION FACTOR 10/16/17 (ARF10/16/17) under heat stress, which may contribute to thermotolerance through the activation of HEAT SHOCK PROTEIN (HSP) genes (ARF10/17 expression under high temperature stress (Helianthus annuus) miR396-resistant HaWRKY6 in Arabidopsis decreases plant thermotolerance (COPPER/ZINC SUPEROXIDE DISMUTASE1 (CSD1), CSD2 and COPPER CHAPERONE FOR SOD1 (CCS) to promote the accumulation of ROS, which contributes to the induction of heat stress transcription factors (HSFs) and HSPs that are required for Arabidopsis thermotolerance  . OverexpP) genes . Howevere stress . These rolerance . Heat stolerance . Interessitivity .C-REPEAT BINDING FACTORS (CBFs) and INDUCER OF CBF EXPRESSION1 (ICE1), the core regulators in cold acclimation  also increases plant chilling and heat tolerance, which may be caused by inhibiting the expression of the target gene GAMYB-like 1 (Triticum aestivum) in Arabidopsis represses the expression of AtUBC24, thereby inhibiting the AtUBC24-mediated degradation of AtICE1 protein. The increased expression of AtICE1 induces the CBFs signaling pathway, resulting in the activation of antioxidant enzymes to enhance plant chilling and freezing tolerance  increases, which positively regulates plant drought tolerance by changing root architecture in Arabidopsis  through silencing SPL13 and WD40-2. Observed effects include reduced water loss, enhanced root growth, stomatal conductance, chlorophyll content and photosynthetic carbon assimilation , miR160 cleaves MdARF17 and relieves MdARF17-mediated inhibition of MdHYL1 expression. MdARF17-MdHYL1 form a positive feedback loop to regulate the stability of Mdm-miR160, which improves plant drought resistance by promoting adventitious root development and increasing the root-shoot ratio  miR156c expression in tobacco improves salt and drought tolerance without changes in plant architecture  module contributes to plant salt tolerance by maintaining ROS homeostasis during salt stress , and ethylene (ET)  .The miR1bidopsis . Overexpstresses . Transgestresses . Transgend veins . TogethePopulus spp.), the miR390-TAS3 module modulates lateral root growth under salt stress (Panicum virgatum L.) enhances plant cold tolerance, tillers and biomass yield in both greenhouse and field tests , cold stress induces a substantial and global impact on transposable element (TE)-related hc-siRNA-directed RdDM, which results in the demethylation of mCHH. Cold-induced differentially-methylated regions (DMRs) may be correlated with the transcription changes in ethylene biosynthesis-related CsACO3 and an Arabidopsis RAP2.4-like ethylene-responsive (AP2/ERF) transcription factor, which may contribute to the temperature-dependent sex determination in cucumber (Rosa hybrida), cold stress up-regulates mCHH levels in the promoter of RhAG, an AGAMOUS homolog, which may result in the repressed expression of RhAG and the cold-induced increase in petal number (ALLANTOINASE (ALN), a negative regulator of dormancy, is associated with seed dormancy in Arabidopsis -PYRROLINE-5-CARBOXYLATE DEHYDROGENASE (P5CDH)-SRO5 generates nat-siRNAs that are involved in salt resistance (KOKOPELLI (KPL) and ARIADNE14 (ARI14) generate a sperm-specific NAT that produces cis-nat-siRNAs to regulate double fertilization  upon cold treatment and modulate the vernalization process required for flowering in some species. COOLAIR is induced by cold signal and target the 3’ end of FLC mRNA to degrade it during vernalization. COLDAIR is located in the first intron of FLC and can directly interact with CURLY LEAF (CLF) in the POLYCOMB REPRESSIVE COMPLEX 2 (PRC2) protein complex, recruiting the PRC2 complex to the FLC gene locus. PRC2, which catalyzes the trimethylation of lysine 27 of histone H3 (H3K27me3), inhibits FLC expression by enriching H3K27me3 and thus promotes flowering in Arabidopsis  in  pathway . Through pathway . In uplaignaling . OverallPhytohormones are endogenous signal molecules, which play important roles in plant development, metabolism and stress responses. By regulating the expression of target genes, ncRNAs affect the metabolism, distribution and perception of hormones, as well as participating in the regulation of plant hormone signaling pathways in development and stress responses . The phyMYB101 and MYB33, thereby affecting seed germination and stress response through the ABSCISIC ACID INSENSITIVE 3 (ABI3)-dependent pathway (PHBULOSA (PHB), which promotes the expression of BG1 (hydrolysis of abscisic acid-glucose ester to ABA) and ABI4, thus leading to the accumulation of ABA and activation of ABA signal transduction, drought and cold resistance  increases salt sensitivity and drought tolerance in an ABA-dependent manner in Arabidopsis (ABA-RESPONSIVE ELEMENT-BINDING TRANSCRIPTION FACTOR3 (ABF3) and CSP41b, a chloroplast RNA binding protein  proteins, and ARFs . miR393 pressure . miR160,elopment . miR160 e stress . miR390 ugh ARF4 . In poplugh ARF4 . In riceugh ARF4 , implyinugh ARF4 .OsCYP51G3, an obtusifoliol 14α-demethylase, is more sensitive to salt stress and displays typical phenotypic changes relating to phytosterol and BR deficiency, including shorter cells, erect leaves, dwarfism and semi-sterile pollen grains  Raf.], ptr-miR396b is induced by cold stress and guides the cleavage of 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID OXIDASE (ACO) mRNA. ptr-miR396b positively regulates cold tolerance through repressing ethylene synthesis while simultaneously promoting polyamine synthesis, which ultimately results in enhanced ROS scavenging  to drive early flowering and compromised immunity. Thus, heat stress induces transgenerational inheritance of phenotypical changes through a cooperative epigenetic network involving transcription factors, histone demethylation and tasiRNAs, thereby ensuring successful reproduction and stress adaptation   are used for targeted miRNA modification, including knock-out of  systems . Secondl systems . SIGS cafera L.) . Thirdlyolerance . Using a in rice . As well in rice . Moreove in rice . RNA int in rice , and canPseudomonas putida affects the growth, development, and stress response of Arabidopsis thaliana by affecting the biogenesis and action of miRNAs in the root system , the National Natural Science Foundation of China (32070564 to JL), and Natural Science Foundation of Yunnan Province .Due to space limitations, we apologize to our colleagues whose important work are not cited in this review.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In oncology, miRNAs have raised great attention due to their aberrant expression and pivotal roles in the pathogenesis of multiple malignancies including osteosarcoma. MiRNAs can be transported by exosome, the nano-extracellular vesicle with a diameter of 30–150 nm. Recently, a growing number of studies have demonstrated that exosomal miRNAs play a critical role in tumor initiation and progression, by exerting multiple biological functions including metastasis, angiogenesis, drug resistance and immunosuppression. In this review, we aim to depict the biogenesis of exosomal miRNAs and summarize the potential diagnostic and therapeutic functions of exosomal miRNAs in osteosarcoma.MiRNAs are a group of non-coding RNA molecules that function in mRNA translational inhibition  Osteosarcoma is one of the primary bone malignancies that always occur in children, adolescents and young adults . It is aExosomes are nano-extracellular vesicles (EVs) with diameters ranging from 30 to 150 nm. They are widely and stably distributed in the majority of biological fluids including serum, plasma, urine, saliva, synovial fluid and cerebrospinal fluid . It is nMiRNAs are a group of ncRNA molecules that function as either oncogene or tumor suppressor via base-pairing with complementary sequences in target mRNA at post-transcriptional level . MiRNAs Exosomes originate from two main phases. Firstly, the intramembranous budding of plasma membrane forms early endosomes, then the endosomal membrane invaginates into surrounding lumina with biomolecules to form intraluminal vesicle (ILVs). Multivesicular bodies (MVBs) are later endosomes with dozens of ILVs. Some of these MVBs are fused with autophagosomes to generate amphisomes, and finally combine with lysosomes to degrade the cargos wrapped inside, which can inhibit the transport of cargos like miRNAs and proteins through exosomes . While t+ multivesicular endosomes (MVEs) and induce the formation of ILVs and exosomes. Meanwhile, RAB31 can also recruit TBC1 domain family member 2B (TBC1D2B) to de-activate RAB7 to inhibit the fusion of MVEs with lysosomes and assist the exosomes secretion  can direct EGFR localization to CD63ecretion .MiRNAs are small endogenous single-stranded RNAs that contain about 19–25 nucleotides in length without protein-coding capacity. MiRNA genes are transcribed by two RNA polymerases, namely, the pol II and pol III. The results of the transcription are some precursors of miRNAs called primary miRNAs (pri-miRNAs) that contain a hairpin structure where miRNA sequences are inserted . FollowiCells have special mechanisms to selectively sort miRNAs into exosomes, and subsequently transmit specific information between each other. Basically, proteins that participating in the sorting of miRNAs can be classified into two categories: RNA-binding proteins (RBPs) and membrane proteins , Figure.Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a group of RBPs that assist with the sorting process of miRNAs into exosomes. The hnRNPA2B1 protein was found to bind miR-198, miR-17, miR-93 via recognizing AGG/UAG motifs . AnotherFragile X mental retardation 1 (FMR1) was found to help control exosomal miRNAs’ loading during inflammation. Ann L. Wozniak and colleagues showed that overexpression of FMR1 resulted in the enrichment of miR-155 in EVs. Furthermore, the immunoprecipitation of FLAG-tagged rab-interacting lysosomal protein (RILP) constructs showed that cleaved RILP fragment (cRILP) interacted with Hrs, and when RILP was cleaved into cRILP by using LPS/ATP, the interaction of FMR1 and Hrs increased. These experiments suggested that cRILP recruit FMR1 to the MVBs to regulate the loading of miR-155 .Besides, Y-Box binding protein 1 (YBX-1) was reported to be involved in sorting miR-223 into exosomes through liquid chromatography and mass spectrometry . AnotherMajor vault protein (MVP) is a kind of ribonucleoprotein that mainly locates in the cytoplasm and associates with the cytoskeleton, and a small amount is around the nuclear membrane and pore complex . Recent Neural sphingomyelinase 2 (nSMase2) is a hydrolase enzyme that involved in sphingolipid metabolism reactions . NobuyosCaveolin-1 (Cav-1) is the major resident scaffolding protein that acts on forming caveolae. It was recently found that deletion of Cav-1 reduced the content of hnRNPA2B1 both in cells and microvesicles (MVs). Furthermore, the levels of hnRNPA2B1-binding miR-17, miR-93 were also decreased . MeanwhiVacuolar protein sorting 4 (Vps4) is important in the sorting of MVBs by triggering constriction and cleavage of ESCRT-III helical filaments . After iTumor cells may secrete much more exosomes than normal cells, and tumor-derived exosomes can enroll in the cancer development by facilitate local and distant communication between cells via transporting multiple biomolecules . Among tTumor growth depends on angiogenesis to a large extent. Exosomal miRNAs selected from several types of cells, such as MSCs, tumor cells, stromal cells, and endothelial cells can promote angiogenesis . For insCertain conditions of cellular stress like hypoxia are reported to alter the miRNA composition of exosomes, modulate tumor microenvironment and promote angiogenesis . In lungOn the contrary, some exosomal miRNAs can exhibit inhibitory effect on angiogenesis as well. Expression of miR-9 was obviously reduced in exosomes derived from nasopharyngeal carcinoma (NPC) cells and plasma samples. Moreover, exosomal miR-9 expression was negatively correlated with microvessel density in NPC, indicating a potential inhibitory role of exosomal miR-9 in angiogenesis. Further studies suggested that this process was mediated by directly targeting MDK and regulating phosphoinositide-dependent kinase-1 (PDK1)/Akt pathway .via exosomal miRNAs has been well documented in various cancers. In gastric cancer, exosomal miR-21-5p promoted peritoneal metastasis via directly targeting SMAD7-3′-UTR and inducing mesothelial-to-mesenchymal transition (MMT)  driven EMT (The induction of metastasis on (MMT) . In HCC,on (MMT) . Epithelon (MMT) . Accumulon (MMT) . In lungiven EMT .via exosomal miRNAs to form pre-metastatic niche. For instance, breast tumor cell-derived exosomal miR-21 could regulate osteoclastogenesis to form pre-metastatic microenvironment for bone metastasis  and dual specificity protein phosphatase 14 (DUSP14), respectively . Second,tastasis . In colotastasis . Fibrobltastasis . Exosomatastasis , while etastasis .Drug resistance, especially chemoresistance, is one of the most challenging issues in the treatment of cancer patients . It can Tumor-derived exosomes are reported to participate in both reversal and induction of drug resistance. In breast cancer, overexpression of miR-567 could promote trastuzumab-induced cell death, while silencing miR-567 decreased cell death, suggesting that miR-567 promotes chemosensitivity of trastuzumab treatment. Mechanistically, miR-567 was incorporated into tumor-derived exosomes and reversed drug resistance via inhibiting autophagy-related 5 (ATG5) expression . BesidesExosomal miRNAs derived by tumor-associated macrophages (TAMs) mostly accompany in inducing drug resistance. Cytidine deaminase (CDA) is the major enzyme of gemcitabine inactivation since overexpressed CDA converses into dFdUridine to inactivate gemcitabine. In pancreatic adenocarcinoma (PDAC), transfer of miR-365 in MDEs induced gemcitabine resistance via modulating pyrimidine metabolism and CDA expression . In gast+ cancer-associated fibroblast-derived miR-22 promoted tamoxifen resistance via targeting PTEN and estrogen receptor 1 (ESR1)  .via exosomal miR-1246 to induce an anti-inflammatory microenvironment, recruit immunosuppressive Treg cells and promote tumor progression  .Exosomes miR-221-3p has been identified to participate in the metastasis and drug resistance in various cancers. For instance, in cervical squamous cell carcinoma, miR-221-3p in exosomes released by tumor cells can downregulate vasohibin-1 (VASH1) and activate Akt and ERK pathway to induce lymphangiogenesis and lymphatic metastasis . In breaAs to osteosarcoma, pulmonary metastasis is the leading cause of death . MiR-221MiR-675 was upregulated in exosomes secreted by metastatic osteosarcoma cells. These exosomes mediated the transfer of miR-675 from osteosarcoma cells to stromal cells, and miR-675 downregulated the expression of calneuron 1 (CALN1), resulting in the promotion of migration and invasion of tumor cells .Exosomal miR-1307 was highly expressed in serum of ovarian cancer patient compared to benign or healthy counterparts. Its expression was obviously correlated with tumor staging. More importantly, it could be used as independent diagnostic tool with enhanced accuracy when compared with conventional biomarkers, such as CA-125 and HE4 .More recently, miR-1307 was found to be abundantly expressed in osteosarcoma exosomes and cells, as well as human osteosarcoma tissues. Among them, tumor cell-derived exosomal miR-1307 induced the cell proliferation, migration and invasion. Mechanistic assays showed that miR-1307 can directly bind to the 3′-UTR of ArfGAP with GTPase domain, ankyrin repeat and PH domain 1 (AGAP1) and inhibit its expression to promote tumorigenesis .MiR-208a has a close relationship with tumor metastasis and radio-resistance. In lung cancer, miR-208a was significantly upregulated in serum after radiotherapy, and serum miR-208a was translocated into lung cancer cells via exosomes. Further studies showed that miR-208a may directly bind to the 3′UTR of p21 to reduce its expression and activate the Akt/mTOR pathway. Consequently, exosomal miR-208a enhanced the proliferation and radio-resistance of lung cancer .While in osteosarcoma, exosomal miR-208a secreted by bone marrow MSCs was found to promote the viability, clonogenicity and migration of osteosarcoma cells. Furthermore, exosomal miR-208a inhibited programmed cell death protein 4 (PDCD4) and then activated ERK1/2 signaling pathway to enhance the aggressiveness of osteosarcoma .Till now, only two exosomal miRNAs were found to inhibit the progression of osteosarcoma, and they may show the potential in the treatment of osteosarcoma in the future.MiR-206 has been found to be closely related to the occurrence and progression of several kinds of cancers. In NSCLC, miR-206 can inhibit the expression of coronin 1C (CORO1C) to negatively regulate the tumor metastasis . In intrIn osteosarcoma cell, miR-206 was poorly expressed. Exosomal miR-206 derived by bone marrow MSCs can be internalized by osteosarcoma cells. Dual-luciferase reporter gene assay further showed that miR-206 can directly bind to 3′-UTR of transformer two beta homolog (TRA2B), and thereby suppress its expression. Since TRA2B is associated with EMT-related proteins , miR-206 encapsulated in exosomes could also impede the osteosarcoma metastasis by downregulating TRA2B-induced EMT. Moreover, functional assays revealed that exosomal miR-206 could facilitate osteosarcoma cell apoptosis, and inhibit proliferation, migration, and invasion . AccordiMiR-15a is located at 13q14.3 of the human genome, and acts as a tumor suppressor in different types of cancers . In HCC,In osteosarcoma cell, miR-15a was also found to be downregulated. Moreover, low expression of miR-15a indicated an unfavorable survival rate in osteosarcoma patients. Further assays showed that serum-derived exosomes with miR-15a can be internalized by osteosarcoma cells and then miR-15a directly binds to GATA binding protein 2 (GATA2) to inhibit its expression. Since GATA2 can induce the transcription of murine double minute 2 (MDM2) to suppress the p53 signaling pathway, serum-derived exosomal miR-15a can promote apoptosis and cell cycle arrests of osteosarcoma cells via GATA2/MDM2/p53 axis, as demonstrated in It is well known that PI3K/Akt, p53, Wnt, Notch, NF-κB and MAPK are important pathways in osteosarcoma pathogenesis . A plethHowever, the crosstalk of exosomal miRNAs with Notch, NF-kB and MAPK pathways have not been identified in osteosarcoma yet. Of note, only studies concerning the interaction between miRNAs and these pathways were reported. MiR-338-3p targeted RNX2/CDK4 and inhibited MAPK pathway to suppress tumor growth . MiR-129Circulating exosome miRNAs may be promising cancer biomarkers. First, it requires only noninvasive or minimally invasive procedure and could be easily accessed through body fluid, most frequently peripheral blood. Second, it is repeatable and inexpensive. Third, it could offer real-time information and thus reflect the tumor cell evolution . Fourth,Recently, some studies have explored the potential diagnostic and therapeutic values of dysregulated exosomal miRNAs in osteosarcoma . YoshidaExosomes could be released by a variety of types of cells and convey bioactive cargoes to modulate crosstalk between donor and recipient cells. MiRNAs are selectively sorted into exosomes as one of the essential components . As aforin vitro and in vivo. MiR-15b reconstitution can reverse chemotherapy resistance in osteosarcoma (First, some aspects regarding the mechanism of exosomal miRNAs in drug resistance and immune disturbance in osteosarcoma remain unclear. Up to now, only studies on the association between miRNAs or other bioactive substances in exosome and drug resistance are noted in osteosarcoma. For example, miR-15b can modulate MDR in human osteosarcoma both osarcoma . Similarosarcoma . Besidesosarcoma . Currentin vitro assay. While animal studies or multi-center, large clinical cohort are still lacking. Therefore, more in vivo experiments or clinical studies with large sample size are still necessary for further validation.Second, it should be noted that most of previous studies on exosomal miRNAs in osteosarcoma are based on Last, the aberrant expression of exosomal miRNAs may be detected for efficient clinical diagnosis or prognosis prediction. Recently, tremendous abnormally expressed exosomal miRNAs have been identified in osteosarcoma due to the advancement of high-throughput NGS. However, only a few of these miRNAs have been annotated and extensively studied. While other miRNAs remain unexplored. Despite a set of breakthroughs have been made in this field, more rigid studies are still needed to further unveil the specific clinicopathologic significance and underlying mechanism of exosomal miRNAs in osteosarcoma.


HPV-associated head and neck squamous cell carcinoma (HNSCC) is a cancer entity with unique biological and clinical characteristics that requires more personalized treatment strategies. As the backbone of conventional therapeutics, radiation is now harnessed to synergize with immunotherapy in multiple malignancies. Accumulating preclinical and clinical data have suggested the potential of radioimmunotherapy in eliciting local and systemic anti-tumor response via direct killing of tumor cells and immunogenic cell death. However, this effect remains uncertain in HPV-associated HNSCC. Owing to its intrinsic radiosensitivity and distinct tumor microenvironment, HPV-associated HNSCC may represent a good candidate for radioimmunotherapy. In this review, we provide a detailed illustration of the biology, the genomic features, and immune landscapes of HPV-associated HNSCC that support the synergism between radiation and immune agents. The interaction between radiotherapy and immunotherapy is described. We also highlight the present evidence as well as ongoing trials using different combination strategies in the recurrent/metastatic or definitive settings. In addition, we have summarized the challenges and outlook for future trial design, with special emphasis on radiotherapy optimization and novel therapeutic options to incorporate. With the global prevalence of human papillomavirus (HPV) as a most common sexually transmitted infection, the incidence of HPV-associated head and neck squamous cell carcinoma (HNSCC), particularly oropharyngeal carcinoma (OPC), has dramatically increased over the past decades. Currently, HPV-associated OPC accounts for 33% of all cases globally, with a highest prevalence reported in Lebanon 85%) and Sweden (70%) ,2. Radio% and SweClinically, HPV-associated HNSCC is featured by a proclivity towards male, younger age of onset, less consumption of tobacco and alcohol, specific anatomic site in oropharynx, and a histology of non-keratinized squamous cell carcinoma . As a diHigh-risk HPV strains, predominantly HPV16, represent the major driving factors that mediate the oncogenesis of HPV-associated HNSCC. Despite lacking direct evidence of precancerous lesions induced by HPV infection, HPV-associated HNSCC possibly resembles HPV-driven ano-genital cancers in their development that involves progression from normal epithelium to dysplasia, carcinoma in situ and eventually invasive cancer ,12. In lDue to the common smoking history, HNSCC presents a relatively high tumor mutational burden (TMB) that ranks in the upper quartile among different cancer types . AccordiHPV-associated HNSCC, primarily OPC, also exhibits unique immune microenvironment that harbors more pro-immunological features. Compared to its HPV-negative counterparts, HPV-positive HNSCC displays a T-cell-inflamed phenotype with increased CD4+ T-cells, CD8+ T-cells and cytokine production, as well as higher diversity of T-cell receptor repertoire ,23,24. TFollowing T-cell activation, HPV-associated HNSCC develops a negative feedback loop to forge an immunosuppressive microenvironment with upregulated expression of CTLA4 , CD39 anAs discussed above, the genomic and immunologic features of HPV-associated HNSCC support the rationale of immunomodulatory agents, ICIs that block PD-1, PD-L1 or CTLA-4 in this population of patients. Given the pre-existing tumor-specific immune cells in HPV-positive tissues, the ICIs can effectively reverse the immunosuppressive microenvironment by releasing the “brake” and reactivating effector T-cells, thus reinvigorating the anti-tumor response. p = 0.01]  . When ap = 0.01] . These d = 0.01] ,35,36.p = 0.01) and chemoradiation  when compared to HPV-negative disease [HPV-associated HNSCC is generally believed to be more sensitive to conventional chemotherapy and radiotherapy (RT). Direct evidence came from a trial by Fakhry et al., in which patients with HPV-positive HNSCC had higher ORR to induction chemotherapy  . In biopConversely, radiation can exert strong immunomodulatory effects on tumor microenvironment, which is regarded as the 6th “R” in radiobiology . MultiplTaken together, the interplay of the immune response and radiotherapy warrants the application of radioimmunotherapy in malignant tumors, even those traditionally deemed with a “cold” tumor microenvironment. HPV-positive HNSCC, with its natural advantages in radiosensitivity and immune-sensitivity, might represent a good candidate for radioimmunotherapy to maximize the therapeutic ratio.Given its compelling immunomodulatory effects, radiotherapy has been applied in a metastatic setting in multiple malignancies to elicit systemic immune response against tumor, or putative “abscopal effect”, while attaining proper local control. As was described by R.H. Mole in 1953, an abscopal effect refers to a phenomenon whereby localized RT induced a tumor shrinkage in distant sites that were not exposed to irradiation . Howeverp = 0.86). When regarding the virus status of HPV or Epstein–Barr virus (EBV), subgroup analyses in the SBRT plus nivolumab arm found a numerically but not statistically superior response in virus-negative patients compared to that in virus-positive cases  [In HNSCC, however, a phase II trial (NCT02684253) from Memorial Sloan Kettering (MSK) Cancer Center failed to replicate the ORR advantage with the addition of single-lesion SBRT (9 Gy for 3 doses) to nivolumab  . These rAlthough generally recognized with favorable prognosis, non-metastatic HPV-associated HNSCC is in fact a heterogenous entity with wide diversity of therapeutic effect and survival risk ,68. As mThe JAVELIN Head and Neck 100 trial (NCT02952586) was the first randomized phase 3 study to combine an ICI with CRT in high-risk locoregionally-advanced HNSCC (LA-HNSCC). With the inclusion of HPV-positive OPC  and HPV-positive non-OPC, as well as HPV-negative HNSCC (stage III-IVb), this trial was designed to evaluate the PFS benefit of concurrent and adjuvant avelumab based on standard CRT. However, the trial was terminated early due to the failure to reach its primary endpoint. Although with fair tolerability, avelumab exerted no improvement in PFS or OS, either in the whole population, the HPV-positive or HPV-negative subgroups. A non-significant trend towards higher PFS was observed only in those with a high PD-L1 expression . Comparep = 0.0118) and satisfactory outcomes . KEYNOTE-412 expanded this design into a phase III randomized trial. With enrollment of LA-HNSCC including p16-positive OPC (T4 or N3), KEYNOTE-412 was intended to answer the question whether escalating treatment intensity by adding pembrolizumab to CRT will translate into survival benefits eventually. The preliminary results will be released later in 2022.A phase Ib trial (NCT02586207) reported the safety and efficacy of concurrent and adjuvant pembrolizumab in conjunction to CRT  in LA-HNThe ECOG ACRIN EA3161 (NCT03811015) is a phase II/III trial that aimed to investigate the survival benefit of maintenance nivolumab following definitive chemoradiation in PFS and OS amongst high-risk HPV-positive OPC, including those with more than 10 pack-years of smoking history and advanced stage of T1-2N2-N3 or T3-4N0-3. Another phase III trial, IMvoke010 (NCT03452137), randomized high-risk LA-HNSCC including stage III HPV-positive OPC to receive adjuvant atezolizumab after local radiotherapy or surgery. As the patient population in both trials features a high tendency of local progression and distant relapse, the addition of adjuvant ICI might hopefully help eradicate residual or micro-metastatic cancer clones.The IMMUNEBOOST (NCT03838263) trial introduced two cycles of neoadjuvant nivolumab to CRTs in high-risk HPV-positive OPC, also selected based on tumor stage  and smoking history (>10 pack-years). The primary endpoint is the completion rate of full treatment. Interim analysis presented at the 2021 ASCO meeting showed the feasibility of this protocol, and the trial is continuing , awaitinA series of clinical trials assessed the feasibility of replacing concurrent standard-of-care agents with ICIs during radiotherapy in HPV-associated HNSCC. Single-agent ICI was used in the PembroRad trial, KEYCHAIN trial (NCT03383094), NRG-HN004 (NCT03258554), NRG-HN005 (NCT03952585) and CITHARE trial (NCT03623646) with similar design. The PembroRad trial has completed enrollment and the preliminary results were reported at the ESMO 2020 annual meeting. After enrolling 60% of patients with OPC (46% p16+), this trial found RT-pembrolizumab with less grade ≥ 3 acute toxicities but disappointingly no outcome improvement over RT-cetuximab , which hThe REACH study (NCT02999087) compared dual-agents of avelumab and cetuximab with cisplatin  in cisplatin-fit cohort and with cetuximab in cisplatin-unfit cohort among LA-HNSCC patients receiving definitive RT. With the inclusion of 61% cases with OPC (35% p16+), the REACH study showed a significantly lower rate of distant failure with RT-avelumab-cetuximab in the cisplatin-unfit cohort, suggesting the effect of ICI in eliminating potential micro-metastasis . NCT0316The NCT03715946 trial was designed to de-escalate the standard post-operative CRT by introducing ICI in p16-positive OPC with intermediate-high risk. Using concurrent and adjuvant nivolumab, the investigators expected to safely omit concurrent cisplatin and meanwhile reduce the RT dose to 45–50 Gy in 25 daily fractions.Another important scenario for radioimmunotherapy-based de-escalation would be the combination of ICIs and induction chemotherapy to select patients for subsequent de-intensified RT. The OPTIMA-II NCT03107182) is a phase II trial proposed on the heels of OPTIMA, which reported favorable outcomes and reduced toxicities in HPV-positive OPC, based on induction chemotherapy with carboplatin/nab-paclitaxel and ensuing de-intensified RT  is a pha. The CheWhen applied prior to surgery, ICI alone is limited to induce satisfactory complete (pCR) or major (MPR) pathologic response in HNSCC including HPV-associated diseases ,86. ReceIn general, radiation synergize with immunotherapy largely in a dose-dependent manner. Ablative RT, typically given with fractional dose >10 Gy for 1 to 5 fractions, mainly provides local tumor control via ICD-mediated mechanisms . HoweverThe fundamentally differing effect of high-dose and low dose- RT on immune response has led to the development of a new technique of “RadScopal” that integrates high-dose RT to generate “in situ vaccination” and low-dose RT to reverse stroma immunosuppression  and thusAccumulating evidence has supported the feasibility of multi-site irradiation in a metastatic setting. As was illustrated in detail by Chang et al. , multi-s3 metastases followed by pembrolizumab resulted in a local control comparable to that targeting the entire metastatic volume [Partial irradiation of metastases has also been proposed, based on preclinical studies that showed non-inferiority of irradiation to 50% of the volume of the tumor to that of the full volume. In the phase I NRG-BR001 trial that enrolled 79 metastatic patients including four with HNSCC, Luke et al. reported that partial SBRT to > 65 cmc volume . This reElective nodal irradiation (ENI), usually known as the prophylactic delivery of a radiation dose to the clinically uninvolved but at-risk nodal areas, targets subclinical metastases in draining lymph nodes (DLN) and is routinely used in HNSCC in the definitive setting, including HPV-associated HNSCC. However, with the growing importance of DLN as a major location of T-cell priming, larger-field ENI might hinder this process by directly killing CD8+ T-cells. In fact, ENI has failed to improve outcomes in multiple malignancies including esophageal squamous cell carcinoma , early-sTiming of ICIs to irradiation is of essential importance to exert a synergistic efficacy. According to Dovedi et al., effective anti-tumor response and long-term tumor control can be acquired only when PD-L1 blockade was administered concurrently with RT . RetrospTheoretically, any agent that acts against the intrinsic resistance of HNSCC might synergize with radioimmunotherapy to expand the therapeutic ratio. Based on the potential mechanisms of resistance, the combinational strategy could involve several aspects: (1) chemotherapy  that indOf note, the unique biological features of HPV-associated HNSCC could provide insight into disease-specific strategies. For instance, DNA immunotherapy targeting E6/E7 stimulated potent anti-tumor immune responses in a phase Ib/II trial , whetherIn summary, current evidence indicates that HPV-associated HNSCC has higher sensitivity to RT and immunotherapy. Given the synergetic effect of RT and ICIs, HPV-associated HNSCC may represent a favorable candidate for radioimmunotherapy due to its special biological, genomic and immunologic landscape. Preliminary studies, although rare, have indicated the tolerance and efficacy of radioimmunotherapy in HPV-associated HNSCC. Ongoing trials will further provide valuable insight into the potential of radioimmunotherapy in triggering abscopal effect in the metastatic setting and facilitating personalized treatment in the definitive setting, as well as the safety profiles in HPV-associated HNSCC. There are open questions regarding the optimal RT scheduling, volume, and sites to irradiate and the combinational agents to elicit the synergetic effect of RT and immunotherapy, future investigations in these fields should be encouraged.


Neuroendocrine tumors (NETs) have become increasingly common. There are several effective treatment options for advanced NETs. However, there are limited clinical trial data and published practical information on how these different treatments should be sequenced. This review assesses randomized, controlled clinical trial data in advanced NETs to provide an expert perspective on treatment sequencing for important clinical scenarios, ranging from local disease to high-volume metastatic NETs. The best practices provided in this review may be useful for clinicians considering treatment options and sequencing for their patients with advanced NETs.Neuroendocrine tumor (NET) incidence has grown. The treatment landscape for advanced NETs is rapidly evolving, but there are limited head-to-head data to guide treatment sequencing decisions. We assessed the available clinical data to aid practicing clinicians in their routine clinical decision-making. Clinical trials have demonstrated efficacy benefits for new therapies in advanced NETs. Emerging long-term data from these trials have enabled clinicians to make more accurate risk-benefit assessments, particularly for patients receiving multiple lines of therapy. However, clinical data specifically regarding treatment sequencing are limited. In lieu of definitive data, treatment sequencing should be based on disease-related factors  and patient-related characteristics . Clinical decision-making in advanced NETs remains highly individualized and complex; important evidence gaps regarding treatment sequencing remain. Given this, advanced NET management should be a joint effort of multidisciplinary teams at referring and high-volume centers. Additional clinical trial and real-world evidence are needed to meet the challenge of understanding how to sequence available NET therapies. Until these trials are conducted, the best practices provided in this review may serve as a guide for clinicians making treatment sequencing decisions based on the available data.  Neuroendocrine tumors (NETs) are rare and heterogenous neoplasms with continued rising incidence ,2. The i177Lu]Lu-DOTA-TATE , the tyrosine kinase inhibitor sunitinib, the mammalian target of rapamycin (mTOR) inhibitor everolimus, and radioligand therapy  with 17LuLu-DOT177Lu-DOTATATE for somatostatin receptor-positive gastroenteropancreatic NETs (GEP-NETs) [Although surgery is possible for localized NETs and in selected metastatic NETs, many patients are diagnosed with inoperable advanced or metastatic disease requiring medical therapy . SSAs arEP-NETs) ,11,12,18EP-NETs) . HoweverEP-NETs) ,12,13,20We review the pertinent clinical trial data of patients with advanced NETs to guide clinical decision-making and treatment sequencing.177Lu-DOTATATE, and cytotoxic chemotherapy. Included data pertain only to eight randomized, double-blind, controlled Phase III trials  along w  [The designs, types of eligible patients, and endpoints assessed differed to varying extents across the nine randomized controlled studies  6,7,8,9,29,30,314 weeks)  were the4 weeks) ,19,23,24177Lu-DOTATATE also received IM octreotide [Direct comparison between the trials is not possible because of variations in patient selection and study methodology. For instance, regarding the interventions, SUNNET patients could receive SSAs at the investigator’s discretion , RADIANTtreotide .Seven of the nine trials showed a benefit in time to tumor progression (PROMID)  or PFS . p value narrowly missed the prespecified boundary denoting statistical significance [The remaining two trials, RADIANT-2 and SPINET, did not show statistically significant PFS benefits for the interventional arm. In the RADIANT-2 study of patients with advanced NETs associated with carcinoid syndrome, the combination of everolimus plus octreotide prolonged PFS versus octreotide alone, but the study primary endpoint was not met as the ificance . Neverthificance  and RADIificance . p = 0.769) in the entire population, 21.9 versus 13.9 months in the subgroup with typical carcinoid lung NETs, and 13.8 versus 11.0 months in the subgroup with atypical carcinoid lung NETs [The SPINET study (lanreotide vs. placebo) was unique by enrolling patients with somatostatin receptor-positive typical and atypical carcinoid lung NETs . Enrollmung NETs .Per protocol exploratory analysis of PROMID , CLARINEExtensive follow-up is required to demonstrate significant gains in overall survival (OS) owing to the often indolent nature of advanced NETs. In five  at the data cutoff [p = 0.094) [In SUNNET, 9 deaths were reported in the sunitinib group (10%) versus 21 deaths in the placebo group (25%), which translated into a hazard ratio for death in favor of sunitinib  .177Lu-DOTATATE arm, and 26 deaths had occurred in the high-dose octreotide LAR arm, which represented a 60% lower risk of death in the 177Lu-DOTATATE arm  [177Lu-DOTATATE arm and 99 of 114 patients (87%) in the high-dose octreotide LAR arm entered long-term follow-up [177Lu-DOTATATE arm and 36.3 months  in the high-dose octreotide LAR arm  [In NETTER-1, at the time of prespecified interim analysis of OS, 14 deaths had occurred in the = 0.004) . Of the ollow-up . Final Oollow-up . During ollow-up . The med177Lu-DOTATATE (18%) [Objective response rate was a secondary efficacy endpoint in the clinical trials. This measure is less useful than PFS for population-based treatment decision-making, given the low frequency and small range of responses observed, but measuring treatment response does have utility on an individual basis. Of the seven trials showing a benefit in time to tumor progression  or PFS ,10,11,19Both short- and long-term safety data are required for clinicians to make more accurate risk assessments when selecting initial and subsequent therapies for advanced NETs. Adverse events (AEs) associated with each therapy over long-term use were entirely consistent with those emanating from the primary clinical trials and no new safety signals were detected during follow-up . There wIn SUNNET, patients received sunitinib and placebo for a median duration of 4.6 months and 3.7 months, respectively . The meaIn RADIANT-3 and RADIANT-4, median duration on everolimus was approximately double that of placebo ,10. The 177Lu-DOTATATE was characterized in the NETTER-1 study, which encompassed 5 years of patient follow up [177Lu-DOTATATE arm received all four infusions of 177Lu-DOTATATE, with 7% requiring a dose reduction. A numerically smaller proportion of patients in the 177Lu-DOTATATE arm than control arm had AEs resulting in premature withdrawal (6% vs. 9%) [177Lu-DOTATATE arm versus no patients in the control group [177Lu-DOTATATE. Although two of 111 patients (1.8%) in the 177Lu-DOTATATE arm developed myelodysplastic syndrome, no patients developed myelodysplastic syndrome or acute myeloid leukemia during long-term follow-up [177Lu-DOTATATE pharmacovigilance during real-world use is required to provide more clarity on the potential for myelodysplastic syndrome and acute myeloid leukemia, especially with respect to 177Lu-DOTATATE dose level, dose timing, and retreatment. There was no evidence of long-term nephrotoxicity in NETTER-1 [The potential for acute, subacute, and long-term AEs with ollow up ,30. In t vs. 9%) . Transieol group . During Patient-reported outcome (PRO) data can reveal if the overall efficacy and safety profiles of therapy reflect patient experience and perceptions. The main PRO instrument used in PROMID , SUNNET 177Lu-DOTATATE (in NETTER-1) translated into demonstrable improvement in health-related quality of life (HRQoL), as evidenced by a substantially longer time to clinically meaningful deterioration in EORTC QLQ-C30 scores vs. high-dose octreotide LAR [Only the efficacy and safety of tide LAR . Octreottide LAR ,7,8,44.®) and North American Neuroendocrine Tumor Society guidelines for advanced NETs recommend use of SSAs as first-line systemic therapy followed by targeted therapy (everolimus or sunitinib), 177Lu-DOTATATE, or chemotherapy as later-line options depending on tumor type, grade, SSTR expression, distribution, and bulk of disease [The NCCN Clinical Practice Guidelines in Oncology  that determines the patients’ initial treatment plan and any required changes to this plan when disease control is in jeopardy. The treatment plan should be based on disease-related factors and patient-related characteristics, such as comorbidities and patient preferences. How these factors and clinical trial data may guide treatment sequencing decisions in commonly encountered disease scenarios are summarized in 177Lu-DOTATATE. Despite these gaps, the current clinical data and our clinical experience have allowed us to provide a framework for treatment sequencing decisions across many different clinical scenarios, but more data are needed to fully support well-informed clinical decision-making for patients with NETs.It should be acknowledged that there are several evidence gaps that hinder well-informed clinical decision-making in certain contexts. For example, there are currently limited head-to-head data for continuation of SSAs beyond disease progression, use of combination therapy, optimal treatment sequences, and potential for retreatment with NETs have seen a significant advance in therapeutic drug development in the last decade. This has ushered us into a new era in which “how to sequence therapies” is a foremost challenge. New and emerging clinical trial data of agents for advanced NETs have demonstrated improved PFS versus control, with efficacy benefits preserved across clinically relevant patient subgroups. Long-term clinical trial follow-up has also evaluated the safety profile of each agent comprehensively, enabling clinicians to make more accurate risk assessments when selecting therapy. Clinical trial data and real-world data are now needed to meet the challenge of understanding how to use these valuable medicines optimally, and several studies are ongoing that may fill important knowledge gaps . In the


Nonalcoholic steatohepatitis (NASH) is a common chronic liver disease with increasing prevalence rates over years and is associated with hepatic lipid accumulation, liver injury, oxidative stress, hepatic inflammation, and liver fibrosis and lack of approved pharmacological therapy. Alanyl-glutamine (Ala-Gln) is a recognized gut-trophic nutrient that has multiple pharmacological effects in the prevention of inflammation- and oxidative-stress-associated diseases. Nevertheless, whether Ala-Gln has a protective effect on NASH still lacks evidence. The aim of this study is to explore the influence of Ala-Gln on NASH and its underlying mechanisms. Here, C57BL/6 mice were fed a methionine- and choline-deficient (MCD) diet to establish the model of NASH, and Ala-Gln at doses of 500 and 1500 mg/kg were intraperitoneally administered to mice along with a MCD diet. The results showed that Ala-Gln treatment significantly attenuated MCD-induced hepatic pathological changes, lowered NAFLD activity score, and reduced plasma alanine transaminase (ALT), aspartate transaminase (AST) and lactate dehydrogenase (LDH) levels. Ala-Gln dramatically alleviated lipid accumulation in liver through modulating the expression levels of fatty acid translocase (FAT/CD36) and farnesoid X receptor (FXR). In addition, Ala-Gln exerted an anti-oxidant effect by elevating the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX). Moreover, Ala-Gln exhibited an anti-inflammatory effect via decreasing the accumulation of activated macrophages and suppressing the production of proinflammatory mediators. Notably, Ala-Gln suppressed the development of liver fibrosis in MCD-diet-fed mice, which may be due to the inhibition of hepatic stellate cells activation. In conclusion, these findings revealed that Ala-Gln prevents the progression of NASH through the modulation of oxidative stress and inflammation and provided the proof that Ala-Gln might be an effective pharmacological agent to treat NASH. Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases with a prevalence of up to 20–30% worldwide, which has become a serious public health problem . NAFLD cL-glutamine is a conditionally essential amino acid in the human body, which can be acquired from a variety of foods including grains, dairy products, fish, and poultry . In factIn this research, we aimed to explore how Ala-Gln affects the progression of NASH. Here, we revealed the protective role of Ala-Gln in NASH by regulating oxidative stress and inflammation, which suggests that Ala-Gln is a promising pharmacological agent for NASH.C57BL/6 male mice, 6–8 weeks old, were acquired from Vital River Laboratory Animal Tech Co., Ltd. . All the mice were housed under thermostatic condition (21–23 °C) with a 12 h light–dark cycle with free access to food and water. To establish a NASH model, the model group with or without Ala-Gln treatment was fed with a methionine- and choline-deficient (MCD) diet, while the normal control group with or without Ala-Gln treatment was fed with a standard diet. The Ala-Gln control group was intraperitoneally injected with Ala-Gln (1500 mg/kg) each day. The low-dose Ala-Gln group was treated with MCD diet and Ala-Gln  each day. The high-dose Ala-Gln group was treated with MCD diet and Ala-Gln  each day. All the animal studies were performed according to the approved guidelines of the animal ethics and welfare Committee of Ningbo University .Ala-Gln was synthesized and provided by Xiamen university as previously described . Oil RedLiver tissue samples were fixed with 4% paraformaldehyde (PFA) at 4 °C for 24 h, paraffinized, and then cut into sections (5 μm), which were stained with HE to assess liver morphological characteristics, and the NAFLD activity score was quantitated by a comprehensive evaluation of histopathological changes including steatosis, hepatocyte ballooning, inflammation, and fibrosis, as reported previously . Masson After they were deparaffinized and rehydrated, paraffin-embedded liver sections were heated in sodium citrate solution for antigen retrieval and then immersed in 3% hydrogen peroxide. The blocking was performed with goat serum, followed by incubation with anti-F4/80 antibody or anti-α-SMA antibody overnight at 4 °C. Thereafter, secondary antibodies were applied to the sections after washing at room temperature (RT) for 1 h incubation. After washing, the sections were visualized with 3,3-diaminobenzidine (DAB) and co-stained with hematoxylin. Quantification of F4/80- and α-SMA-positive pixels was performed using ImageJ software after imaging using a light microscope .Plasma alanine transaminase (ALT), aspartate transaminase (AST), and lactate dehydrogenase (LDH) levels were analyzed using commercially available kits from Nanjing Jiancheng Bioengineering Institute  to assess the degree of liver injury. Hepatic malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities were detected by biochemical assay kits  to evaluate the degree of oxidative stress. Total triglycerides (TG) levels in the liver were measured by TG assay kit  to estimate the accumulation of liver fat.The levels of TNF-α, IL-1β, RANTES, and MCP-1 in mouse peripheral plasma were detected using Elisa assay kits  following the manufacturer’s protocols.Proteins form liver tissues were extracted by lysis buffer from Beyotime Biotechnology  with protease inhibitors from Roche . After quantification by BCA assay kit , protein samples were separated on SDS-PAGE and transferred onto PVDF membranes . Membranes were blocked with 5% skimmed milk at RT for 1 h and then incubated over night at 4 °C with anti-α-SMA antibody (1:1000) and anti-GAPDH antibody . Next, all membranes were washed and then incubated with HRP-conjugated goat anti-rabbit or goat anti-mouse antibody for 1 h at RT. Finally, protein levels were visualized using ChemiDoc MP Image System .RNA extraction kit from Tiangen Biotech Co., Ltd.  was used to isolate RNA from liver tissues in accordance with the instruction. The reverse transcription kit  was used to reverse transcribe mRNA to cDNA. A qPCR PreMix kit  was used to analyze the relative amounts of mRNA with reference to GAPDH. Primer sequences were described in p-values less than 0.05 were considered statistically significant.All the statistical differences were calculated by the software GraphPad Prism version 8.3.0. All data are presented as mean ± SEM. Statistical significance among multiple groups was analyzed using one-way analysis of variance (ANOVA) or Kruskal–Wallis test. To investigate the therapeutic effect of Ala-Gln in NASH, different doses of Ala-Gln (500 or 1500 mg/kg) were administered to MCD-diet-induced NASH mice. First, we examined the effect of Ala-Gln on liver histopathological changes in MCD-diet-fed mice by HE staining. Compared to those of the normal-diet-fed mice, HE staining showed that the hepatic lobules in MCD-diet-fed mice were structurally disturbed, together with marked vacuolar degeneration and inflammatory cells infiltration. In contrast, both doses of Ala-Gln treatment showed significant improvement in liver pathological morphology A. ALT, ATo confirm the regulatory effect of Ala-Gln on hepatic steatosis in MCD-fed mice, we assessed the size and distribution of lipid droplets in the liver using ORO staining. As illustrated, abundant lipid accumulation was observed in the livers of MCD-fed mice. However, Ala-Gln dramatically decreased hepatic fat deposition, while higher doses showed a more pronounced improvement than lower doses, which tended to be more normal A. MeanwhOxidative stress is the main pathological mechanism of NASH and plays an essential role in its onset and progression . To deteInflammatory response has been reported to be a major contributor to NASH progression . To valiNASH is typically characterized by progressive destruction and regeneration of hepatocytes, with the development of fibrosis being the most common phenomenon . To explAmong above profibrogenic genes, α-SMA is a marker of hepatic stellate cells activation, which contributes to liver fibrosis progression. From the results of IHC staining and Western blotting, we could observe that α-SMA protein expression was significantly elevated in MCD-diet-fed mice in comparison with the mice fed a normal diet, while Ala-Gln treatment significantly attenuated these elevations . These rHerein, we demonstrated a protective effect of different doses of Ala-Gln treatment on NASH. The MCD-diet-feeding model is a model that frequently used to investigate the mechanisms of NASH and to screen pharmacological therapies against NASH, as it can mimic various clinical features of human NASH . By usinThe pathogenesis of NASH is closely related to lipid accumulation in liver . From HEDuring the development of NASH, the excessive lipid accumulation could cause the dysfunction of mitochondrial function and then exacerbate hepatic oxidative stress, which in turn promotes the aggravation of NASH . RecentlInflammation is recognized as an important mediator contributing to the progression of NASH . AccumulAnother intriguing finding of this study was that Ala-Gln could markedly ameliorate fibrogenesis in liver. Growing evidence has demonstrated that persistent hepatic inflammation could lead to the occurrence of hepatic fibrosis, which is a wound-healing response characterized by collagen deposition in liver . In lineIn summary, our present study provided evidence that Ala-Gln is effective for treating NASH in a dose-dependent pattern; the proposed mechanism might be that it could effectively regulate oxidative stress and inflammation. The results support the possibility that Ala-Gln may diminish oxidative stress by enhancing SOD and GPX activities, reducing lipid accumulation via regulating the expression of CD36 and FXR, alleviating inflammation by decreasing the number of activated macrophages and proinflammatory mediators, and suppressing the progression of liver fibrosis through inhibiting hepatic stellate cells activation. Together, these findings demonstrated that Ala-Gln treatment can be considered as a potential strategy in the management of NASH.