However, TF-induced reprogramming is often restricted due to cell fate safeguarding mechanisms, which require a better understanding to increase reprogramming efficiency and achieve higher fidelity. The germline of the nematode Caenorhabditis elegans has been a powerful model to investigate the impediments of generating neurons from germ cells by reprogramming. A number of conserved factors have been identified that act as a barrier for TF-induced direct reprogramming of germ cells to neurons. In this review, we will first summarize our current knowledge regarding cell fate safeguarding mechanisms in the germline. Then, we will focus on the molecular mechanisms underlying neuronal induction from germ cells upon TF-mediated reprogramming. We will shortly discuss the specific characteristics that might make germ cells especially fit to change cellular fate and become neurons. For future perspectives, we will look at the potential of C. elegans research in advancing our knowledge of the mechanisms that regulate cellular identity, and what implications this has for therapeutic approaches such as regenerative medicine.Cell fate conversion by the forced overexpression of transcription factors (TFs) is a process known as reprogramming. It leads to de-differentiation or  Thereby, either induced pluripotent stem cells, or other specific types by direct conversion can be generated, respectively  can induce reprogramming to dedifferentiate or ectively . The proectively . As a coectively . The limectively . The gerC. elegans development, the germline is set apart from the soma by the 16–24 cell stage of embryogenesis  and happens to the oocyte closest to the spermatheca , unspecific neurons and intestinal cells. The induction of the mixed somatic fates is accompanied by tissue type-specific characteristics. These include filaments and adhesive structures resembling those found in normal muscles, pan-neuronal unc-119:GFP reporter expression typical for neurons and auto-fluorescent granules similar to those of wild-type intestinal cells  in spr-5 let-418 double mutants  and Mi2 respectively) causes eristics . Germ ce mutants . The dem mutants . Moreove mutants . Again, lineages .gcy-5:GFP in most embryonic cells, broad CHE-1 mis-expression in adult worms allowed marker expression only in a small number of head sensory neurons but nowhere else in the animal.Cellular transdifferentiation by the forced overexpression of cell-fate inducing TFs is limited due to cell fate safeguard mechanisms. As described above, these protective mechanisms often rely on epigenetic regulation. As a result, TFs that can induce ectopic fates in highly plastic cells such as developing embryos, usually fail to induce conversion of germ cells to somatic identities . One faclin-53 in combination with CHE-1 overexpression in adult animals allowed induction of gcy-5:GFP in mitotic germ cells. The converted germ cells expressed markers for the pan-neuronal fate  as well as for the specific neuron sub-type , while expression of markers for other neuron sub-types were not observed. Moreover, the converted cells underwent drastic morphological changes adopting neuron-like nuclear morphology and growing axonal projections  but none of the ASE neuronal fate markers. These observations indicate that this conversion is different from undirected differentiation of germ cells into mixed somatic cell types as observed during teratoma formation. Instead, the TF-induced conversion upon depletion of LIN-53 is specific and directed toward distinct neuron sub-types depending on the overexpressed TF. Notably, although the mis-expression of the cell fate inducing TFs  and the RNAi mediated depletion of lin-53 were both in the entire adult body, neuronal induction occurs in the germline only. Hence, removal of LIN-53 allows direct conversion into distinct neuronal subtypes in what seems to be a germline-specific manner  was later identified as a strong cellular safeguard of somatic cell identity during reprogramming to neurons and induced pluripotent stem cells (iPSCs) , indicatlin-53 depletion in germ-cell-to-neuron reprogramming is phenocopied by the removal of other components of the Polycomb repressive complex 2 (PRC2)   with var2 (PRC2) . PRC2 is2 (PRC2) . This ob2 (PRC2) .The methyltransferase complex member RBBP-5 was recently identified as a novel germ cell reprogramming barrier in a screen to identify factors that increase LIN-53 depletion mediated reprogramming efficiency into GABAergic neurons . AlthougThe mechanisms by which RBBP-5 operates as a barrier to reprogramming remains to be determined. However, these results illustrate the high specificity of the molecular programs that define cellular sates and antagonize the induction of neuronal cell fates.C. elegans, gcy-5:GFP induction in the germline upon depletion . These factors are implicated in a number of biological processes such as proteostasis, cell shape, mitochondrial function, aging and nuclear factors  namely HMG-3, HMG-4 and SPT-16 . The stuhmg-4 and spt-16 allowed partial intestine-to neuron reprogramming, whereas depletion of hmg-3 allows germ-cell-to-neuron conversion. Interestingly, single-molecule fluorescence in situ hybridization (smFISH) revealed that intestinal cells switch to a stable neuron-like gene expression profile upon hmg-4 and spt-16 depletion- mediated reprogramming. However, the converted cells do not obtain a neuron-like morphology. Yet, depletion of hmg-3 allows extended conversion into neurons as illustrated by changes in nuclear morphology and the expression of multiple pan-neuronal and neuron-specific reporter genes . Notably, depletion of germline-specific FACT without CHE-1 induction led to an impairment of cell fate maintenance of the germline. Depletion of hmg-3 decreased the expression of germ-cell specific markers such as PIE-1, and P-Granule levels indicating that the permissiveness for germ cell to neuron reprogramming upon hmg-3 RNAi is created by weakening the starting cell fate.As a result, RNAi-mediated depletion of The same study also demonstrated that FACT’s function as a reprogramming barrier is conserved, as siRNA mediated depletion of the human FACT homologs SSRP1 and SUPTH16 enhanced reprogramming efficiency of human fibroblasts into iPSCs and induced neurons . ChromatC. elegans germ cells during development  identified SIN-3, SET-26 and OGT-1 as novel interacting partners. This finding indicates that MRG-1 might also be involved in repressive chromatin regulating complexes. Since these interaction partners all mediate chromatin regulation, they might contribute to MRG-1’s function as cellular safeguard of the germline. Indeed, germline . OverallA more recent study performing in-depth CoIP-MS additionally detected a strong interaction of MRG-1 with the Small Ubiquitin-like Modifier (SUMO) . It was in vitro  as a potential source of neuronal tissues for clinical therapy . For exain vitro .More recently, C. elegans, as mechanisms are conserved and analogous across species and findings can provide new avenues for future regenerative medicine applications.Generally, clinical application of GSCs is beneficial when compared to embryonic stem cells as it bypasses ethical concerns, risk of teratoma formation and immune rejection . StudiesC. elegans germ cells into neurons. Detailed molecular and morphological analyses have shown an ability to reprogramming germ cells into multiple specific neuronal subtypes upon depletion of reprogramming barriers. The studies described here, mainly focus on factors involved in chromatin regulation. At the level of chromatin regulation, numerous factors have been identified as reprogramming barriers that seem to act in separate pathways. This indicates the multiple independent levels of protection of cells to safeguard their identities. Moreover, the whole-genome RNAi screen by which FACT was identified as a cellular safeguard revealed other candidates implicated in multiple biological processes such as proteostasis, cell shape, mitochondrial function, and aging  will allow us to study direct reprogramming more dynamically. Applying single cell technologies such as scRNA-seq and scATAC-seq for IM conceptualized and wrote the manuscript together with BT. BT helped to conceptualize the manuscript, advised and supported the manuscript writing. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


Globally, chronic hepatitis B (CHB) infection is one of the leading causes of liver failure, decompensated cirrhosis, and hepatocellular carcinoma. Existing antiviral therapy can suppress viral replication but not fully eradicate the virus nor the risk of liver-related complications. Novel treatments targeting alternative steps of the viral cycle or to intensify/restore the host’s immunity are being developed. We discuss novel drugs that have already entered clinical phases of development. Agents that interfere with specific steps of HBV replication include RNA interference, core protein allosteric modulation, and inhibition of viral entry or viral protein excretion (NAPs and STOPS). Agents that target the host’s immunity include toll-like receptor agonists, therapeutic vaccines, immune checkpoint modulators, soluble T-cell receptors, and monoclonal antibodies. Most have demonstrated favorable results in suppression of viral proteins and genomic materials , and/or evidence on host-immunity restoration including cytokine responses and T-cell activation. Given the abundant clinical experience and real-world safety data with the currently existing therapy, any novel agent for CHB should be accompanied by convincing safety data. Combination therapy of nucleos(t)ide analogue, a novel virus-directing agent, and/or an immunomodulatory agent will be the likely approach to optimize the chance of a functional cure in CHB. Chronic hepatitis B infection (CHB) affects 292 million people globally , and is Partial cure refers to sustained virological suppression after stopping NA, which is a better treatment endpoint compared to virological suppression with long-term NA, as patients can remain drug-free if it is achieved. Functional cure is a superior treatment endpoint to partial cure, which refers to sustained seroclearance of hepatitis B surface antigen (HBsAg) with or without seroconversion of antibody to HBsAg (anti-HBs). NA has been shown to reduce intrahepatic covalently closed circular DNA (cccDNA) from 7.28 to 0.57 copies/cell upon 1 year of treatment, and even below detection limit upon ≥6 years of NA treatment, although expression of HBsAg was not completely suppressed  and myeloid-derived suppressor cells (MDSC) activity [Novel therapeutic approaches aim to achieve sustained viral suppression and restoration of anti-HBV immunity through a finite duration of therapy ,21. Theynd CD244 ,29. In Ctibodies . HBsAg aactivity ,32. Thesactivity . For earactivity ,34, and The replication of HBV involves many different steps, including viral entry, transcription, translation, encapsidation, DNA synthesis, and virion export. These are the target sites for anti-HBV drugs to act on — competing for the sodium taurocholate co-transporting polypeptide (NTCP), interference of RNA transcription, inhibition of functional capsid formation/encapsidation, DNA synthesis, and viral protein export. 50 47 nM) would affect bile acid transportation and there was once a concern that it would lead to increased risks of adverse reactions related to bile acid accumulation. Fortunately, effective viral entry inhibition can be achieved at much lower doses  [Sodium taurocholate co-transporting polypeptide (NTCP) is a hepatic bile acid cotransporter and is the receptor for HBV entry, which uses its surface lipopeptide pre-S1 for docking to the hepatocyte via NTCP . Myrcludabolism) , thus thabolism) . CRV431 abolism) . When giabolism) . The exaRNA interference (RNAi) is a naturally occurring and biologically conserved mechanism for specific post-transcriptional gene silencing ,42. RNAiMost siRNAs are given at monthly doses and achieve potent and robust on-treatment HBsAg responses. JNJ-3989 , VIR-221ISIS505358/GSK3228836 (GSK836) is a type of non-GaiNAc-conjugated ASO. GSK836 given subcutaneously on days 1, 4, 8, 11, 15, and 22 led to significant reductions in HBsAg, with bigger magnitudes of decline in NA-treated patients (2.51 logs) compared to NA-naïve patients (1.56 logs). Moreover, 4 out of 16 patients had transient HBsAg loss during dosing, and prolonged HBsAg loss was observed in one NA-naïve patient and one NA-treated patient after dosing of GSK836  [The core protein is essential for capsid formation, encapsidation, and reverse transcription of pre-genomic RNA, virion formation, and cccDNA amplification. Core protein allosteric modulators (CpAMs) target the step of capsid formation by either forming aberrant unstable capsids (class 1) or empty capsids (class 2). Apart from reduction in the formation of mature virions, these dysfunctional capsids would also impair the nuclear recycling of rcDNA-containing nucleocapsids for cccDNA pool replenishment and epigenetic regulatory mechanisms in cccDNA transcription .RO7049389 , a class 1 CpAM, was given to treatment-naïve CHB patients at daily or twice-daily oral dosing for 4 weeks, which led to median serum DNA reduction of 2.66–3.2 logs and rebound back to the baseline level after the dosing period. No significant changes were observed in the HBsAg levels .GLS4, a class 1 CpAM, was coupled with ritonavir and compared with ETV monotherapy in the phase 2 trial. No patients in the ETV group achieved ≥1.5 log HBsAg decline at week 24 compared to 12.5% patients in the GLS4/ritonavir group. In addition, GLS4/ritonavir was more effective than ETV in suppression of HBV RNA  and HBcrAg  in previously treatment-naïve patients at week 24 .Most CpAMs in development can be administered orally and are used in combination with NAs . JNJ-637ALG-000184 (prodrug of ALG-001075) is another class 2 CpAM that has demonstrated 10- to 80- folds stronger antiviral activity at HBV DNA suppression compared to other CpAMs currently in clinical development in cell line models . ALG-001Nucleic acid polymers (NAPs) prevent release of HBsAg subviral particles from infected hepatocytes. The mechanism of action was investigated in a cell-based assay using human HepG2.2.15 cells. Following endosomal release, NAPs destabilize subviral particle assembly and secretion (but not Dane particles) through interaction with some uncharacterized host protein. Intracellular HBsAg accumulation is prevented by proteosomal and lysosomal degradation, which is enhanced by NAPs. Dane particles and other viral markers such as HBeAg secretion are not affected because their assembly and secretion pathways are via the multivesicular body pathway but not the endoplasmic-reticulum-Golgi intermediate compartment (ERGIC) pathway for subviral particles ,62.REP 2139 or REP 2165 are NAPs and were investigated in combination with PEG-IFN and NA. In the phase 2 study, combination therapy with REP 2139/REP 2165 + TDF + PEG-IFN for 24 weeks was effective in causing HBsAg < 0.05 IU/mL (60% patients), with 35% patients achieving HBsAg seroclearance .S-antigen Transport-inhibiting Oligonucleotide Polymers (STOPS) share structural similarities with NAPs with stronger in vitro potency. In the preclinical studies using cell line models, ALG-010133 (STOPS) was 30- to 180-folds more potent than REP 2139 (NAP) in inhibition of HBsAg release . The intEYP001 is an orally bioavailable second-generation non-bile acid farnesoid X receptor (FXR) agonist. The FXR nuclear receptor is highly expressed in the liver and intestine. There are two FXR response elements located in the HBV Enhancer II and core promoter region, which stimulate HBV pregenomic RNA synthesis and viral replication . ModulatAs mentioned above, chronic viral antigen exposure weakens HBV-specific immunity and leads to virus-specific T-cell anergy, leading to immune exhaustion in the host. Novel therapies aiming to restore or enhance the host immunity are being actively developed .Toll-like receptors (TLRs) are pattern-recognition receptors (PRRs) that upon activation would stimulate various leukocytes in both the innate and adaptive system. GS-9620 (Vesatolimod) is a TLR7 agonist. In the phase 2 double-blind placebo-controlled trial, GS-9620 induced ≥2-fold expression of the interferon-stimulated gene (ISG15) especially in female subjects. However, no significant serum interferon (IFN) alpha expression or HBsAg decline was demonstrated . GS-9688HBV-specific T-cell function can be restored by autoantibodies that block the inhibitory molecules. Nivolumab is a monoclonal antibody against PD-1 that is approved for treatment of various malignancies. When given at a reduced dose in combination with GS-4774 , nivolumab led to a modest reduction in HBsAg level (0.16 to 0.3 log reduction) at 12 weeks, and one patient achieved HBsAg seroclearance that was preceded by ALT flare and an increase in peripheral HBsAg-specific T cells .IMC-I109V is a novel immunotherapy. It is a soluble bi-specific T-cell-engaging fusion protein comprised of a soluble affinity-enhanced T-cell receptor fused to a humanized anti-CD3 single chain variable fragment. As a type of immune-mobilizing monoclonal T-cell receptor against virus (ImmTAV), IMC-I109V TCR recognizes HBsAg presented by specific human leucocyte antigen (HLA)-A*02:01 on the surface of infected hepatocytes. Upon engagement of TCR with HBsAg, the effector domain will bind to CD3 on any surrounding T cell of a different family, which is redirected to the complex, thereby engaging the global T-cell population and compensating for the defective HBV-specific CD8 cells in CHB. In vitro study has confirmed that ImmTAV-Env  can redirect T cells from healthy and HBV-infected donors toward HCC cells containing integrated DNA. The induced cytokine release was suppressible by corticosteroid, and the redirected T-cells induced cytolysis of HBV-infected cells and antigen-positive HCC cells, leading to reduction of HBeAg and loss of cells expressing viral RNA . IMC-I10Therapeutic vaccines for CHB make use of different vaccination doses and frequencies to stimulate an anti-HBV immune response. Different viral components have been used, including DNA or peptide vaccines, vector or cell-based vaccines, and combinations of core, X, and polymerase antigens in addition to HBsAg. GS-4774, a yeast-based vaccine that consists of highly immunogenic recombinant HBcAg, HBsAg, and HBx epitopes, led to HBsAg decline ≥0.5 logs at week 24 in 3 out of 50 CHB patients who received it every 4 weeks, and no patient experienced a functional cure . ABX-203GC1102 is a recombinant monoclonal hepatitis B immunoglobulin (HBIg) with enhanced affinity to HBsAg. HBsAg seroclearance was achieved in 22.2% CHB patients whose baseline HBsAg were ≤1000 IU/mL after 7 weeks of treatment with GC1102 . VIR-343SB9200 (Inarigrivir) is a dual agonist of retinoic acid inducible gene-1 and nucleotide-binding oligomerization domain that consists of host PRRs that activate the innate immune pathway. SB9200-induced HBsAg decline ≥ 0.5 logs from baseline in 22% patients . HoweverWith the efficacy and safety data of the individual novel agents mentioned above, the approach of combining two or more novel agents to reduce viral antigen load, inhibit viral replication, and stimulate immunity is being actively explored.The triple combination of RNAi (monthly injections for three doses of JNJ-3989) + CpAM  + NA  led to mean 1.7 logs reduction in HBsAg on day 113 in 12 CHB patients. In addition, other viral products including HBV DNA, HBV RNA, and HBcrAg were profoundly suppressed. This combination therapy was in general well-tolerated with no serious or severe adverse events reported. Mild ALT flares were observed in five patients and were attributed to therapeutic flares [ClinicalTrials.gov identifier: NCT04225715). One of them is a similar example as the above with triple combination with RNAi (RG6346) + CpAM (RO7049389) + NA. Other examples include: triple combination of RNAi (RG6346) + TLR7 agonist (RO7020531) + NA, triple combination with RNAi (RG6346) + PEG-IFN + NUC, and triple combination with CpAM (RO7049389) + TLR agonist (RO7020531) + NA. The approach of sequential use of therapeutic vaccines after antigen knockdown by RNAi has been explored in mice models that showed encouraging results in suppression of viral burden and stimulation in the number of functional HBV-specific T cells and production of HBV-neutralizing antibodies [A few more combination therapies are currently being evaluated in a phase 2 study that evaluates the safety and efficacy of multiple combination therapies  which is beneficial for subsequent immune stimulation therapy . In tran vaccine . AlthougThe currently preferred treatment endpoint for CHB is functional cure. Novel agents work on different steps of viral replication or the host’s immune system, in order to reduce viral burden, inhibit viral replication, and restore host immunity. The therapeutic effects of both virus-directed and host-immunity-directed agents are intercalated and are equally crucial for achieving a functional cure. Most agents currently in clinical phases of development demonstrated favorable results in suppression of viral proteins and genomic materials, with initially promising results of enhancing the functional cure of CHB. With an increasing number of novel agents entering clinical phases of development, it is expected that each agent should demonstrate favorable safety data, and long-term follow-up data in the subjects that participated in these trials will be an important consideration. Most trials aimed to achieve broad eligibility by recruiting CHB patients with variable duration of infection and viral activity. However, it is likely that specific subgroups of patients will benefit most from selected agents so that a finite duration of therapy  instead of lifelong NA treatment can become feasible. For all trials mentioned, no cirrhotic patients were included and this will need to be addressed when more safety data are available. Combination therapy with two or more novel agents theoretically leads to synergistic therapeutic effects. NA and/or PEG-IFN are still the backbone of these combination regimens. The best cocktail of therapies is being sought, and different regimens are likely to be needed for different patient populations with various viral factors , and host factors .


Transcriptomic analysis revealed a gene expression signature representing progenitor/hepatoblast features in the post-progression tumor tissue, with an increased expression of imprinted genes and decreased expression of cytochrome P450 family genes. Finally, tumor mutational burden and MHC class I expression in tumor cells were both increased in the post-progression tissue, suggesting that neoantigen depletion or loss-of-antigen presentation were unlikely causes of acquired resistance in this patient. Atezolizumab plus bevacizumab combination therapy enabled our patient to receive hepatectomy and achieve long-term remission. A comparison of paired tumor tissues suggested that immune-excluded TME and tumor dedifferentiation may have contributed to acquired resistance to the combination.We reported a patient with unresectable hepatocellular carcinoma (HCC) who initially received 15 cycles of atezolizumab plus bevacizumab combination and had best tumor response of partial response, but later experienced disease progression. After subsequent surgical resection, the patient enjoyed long-term disease-free status at the last follow-up 19 months after surgery. By investigating paired tumor tissues (pretreatment and post-progression samples) with immunohistochemistry, multiplex immunofluorescence, RNA sequencing, and DNA sequencing, we explored the dynamic changes in the tumor microenvironment (TME) and potential mechanisms underlying acquired resistance to the combination. In the post-progression HCC tissue compared with the baseline tissue, the expression of PD-L1 in tumor-infiltrating immune cells and the abundance of CD8The online version contains supplementary material available at 10.1186/s40164-021-00237-y. Atezolizumab, an PD-L1 monoclonal antibody, plus bevacizumab, an anti-VEGF monoclonal antibody, (atezo/bev) has become a new standard-of-care as the first-line therapy for patients with advanced hepatocellular carcinoma (HCC) [A 63-year-old man was diagnosed with intermediate-stage HCC and initially received transarterial chemoembolization. His disease progressed with an enlarged right hepatic tumor and the occurrence of multiple new hepatic tumors. He was enrolled in a clinical trial and received atezo/bev . Small h+ T cells in the tumor area had decreased. The multiplex immunofluorescence assay also revealed reduced density of CD8+ T cells in the tumor center but increased density in the surrounding stroma in the tissue obtained at the time of disease progression, indicating an immune-excluded phenotype. In addition, quantification based on a digital pathology algorithm indicated that expression of the activated fibroblast marker, FAP, was significantly increased in the disease progression sample, whereas the blood vasculature marker, CD31, was decreased. MHC class I expression on tumor cells was increased in the post-progression tissue  and potential mechanisms underlying acquired resistance to atezo/bev. Immunohistochemistry (IHC) analysis revealed that the expression of PD-L1 in tumor-infiltrating immune cells and the abundance of CD8sue Fig. A.A gene expression signature representing progenitor/hepatoblast features was found to be increased at the time of disease progression, indicating that tumor cells might have undergone dedifferentiation process to acquire resistance to atezo/bev. Supporting this hypothesis, a significant re-expression of several imprinted genes was also found in tissue collected upon disease progression, and genes of the cytochrome P450 family, which are typically highly expressed in well-differentiated hepatocytes, were found to be downregulated in the post-progression tumor tissue Fig. B. ConsisEffective systemic therapy may serve as a “conversion” therapy and enable patients with unresectable HCC to become amenable to curative locoregional therapies , 4. Anti+ T cells in the tumor area had decreased, and an immune-excluded TME had emerged in the post-progression HCC tissue [We analyzed HCC tissues before and after atezo/bev treatment. The expression of PD-L1 in tumor-infiltrating immune cells and the abundance of CD8C tissue . The obsC tissue . Our tra+ T cells infiltration was correlated with PD-L1 expression, but not with TMB, further echoing our findings in this case [Although PD-L1 expression and TMB were reported to be associated with responses to immune checkpoint blockade in multiple cancer types, their roles as predictive biomarkers of immune checkpoint blockade have not been defined in many other cancer types. A study showed CD8his case . Future his case .In summary, atezo/bev therapy enabled our patient to receive hepatectomy and achieve long-term remission. The potential resistance mechanisms of the combination therapy identified in our study warrant further investigations.Additional file 1: Materials and Methods; Table S1. Summary of mutations identified in tumor tissues collected at baseline and at disease progression.


The value of mechanical circulatory support (MCS) in cardiogenic shock, especially the combination of the ECMELLA approach (Impella combined with ECMO), remains controversial.2: 22 kPa) with severe respiratory acidosis (pH: 6.9) and a rising norepinephrine rate occurred within a few hours. Due to a Horovitz-Index < 100, out-of-centre veno-venous extracorporeal membrane oxygenation (vv-ECMO)-implantation was performed. A CT-scan done because of anisocoria revealed an extended dissection of the right vertebral artery. While the initial left ventricular function was normal, echocardiography revealed severe global hypokinesia. After angiographic exclusion of coronary artery stenoses, we geared up LV unloading by additional implantation of an Impella CP and expanded the vv-ECMO to a veno-venous-arterial ECMO (vva-ECMO). Clinically relevant bleeding from the punctured femoral arteries resulted in massive transfusion and was treated by vascular surgery later on. Under continued MCS, LVEF increased to approximately 40% 2 days after the initiation of ECMELLA. After weaning, the Impella CP was explanted at day 5 and the vva-ECMO was removed on day 9, respectively. The patient was discharged in an unaffected neurological condition to rehabilitation 25 days after the initial admission.A previously healthy 33-year-old female patient was submitted to a local emergency department with a flu-like infection and febrile temperatures up to 39 °C. The patient was tested positive for type-A influenza, however negative for SARS-CoV-2. Despite escalated invasive ventilation, refractory hypercapnia (paCOThis exceptional case exemplifies the importance of aggressive MCS in severe cardiogenic shock, which may be especially promising in younger patients with non-ischaemic cardiomyopathy and potentially reversible causes of cardiogenic shock. This case impressively demonstrates that especially young patients may achieve complete neurological restoration, even though the initial prognosis may appear unfavourable. Mechanical circulatory support (MCS) may contribute to improve the grave prognosis of cardiogenic shock. The avenue of combination of several systems such as extracorporeal membrane oxygenation (ECMO) with the Impella (ECMELLA) gave rise to complex MCS approaches which might contribute the reduced mortality despite higher complication rates . However2: 22 kPa) with severe respiratory acidosis (pH: 6.9) occurred within a few hours. A CT scan of the lungs showed swollen bronchioles, however, acute respiratory distress syndrome (ARDS) like infiltrates were absent. Reversible causes like tension pneumothorax, cardiac tamponade or pulmonary embolism were ruled out. Because of a transitory anisocoria the radiological diagnostics were supplemented with a native CT-scan of the brain, showing a mild cerebral oedema. In correspondence with a highly reduced pulmonary compliance, persisting hypercapnia and a Horovitz-Index < 100, out-of-centre veno-venous-ECMO (vv-ECMO)-implantation was performed due to a presumed refractory status asthmaticus. Immediately after ECMO-implantation, multiple episodes of tachycardia due to atrial fibrillation (Afib) had to be terminated by electrical cardioversion and amiodarone. However, the Afib episodes contributed to progressive haemodynamic instability. The transoesophageal echocardiography (TOE) initially showed hyperdynamic ventricular contractility with preserved left ventricular ejection fraction (LVEF), compatible with a sepsis-like constellation. Though hypercapnia was rapidly declining during ECMO-therapy, anisocoria and hypotension continued. This prompted a further whole-body CT-scan, which ruled out aggravation of cerebral oedema or any abdominal or thoracic bleeding after ECMO-cannulation as possible causes. The patient was transferred to our university hospital with a high norepinephrine rate (1.6 mcg/kg/min) for further complex intensive care treatment. We initiated an empiric treatment with meropenem and clarithromycin in accordance with a presumed septic shock following concomitant community-acquired pneumonia. Aiming at diagnosing the origin of persisting anisocoria, a third CT-scan of the brain with contrast medium was carried out, which now revealed an extended dissection of the right vertebral artery. Somatosensory evoked potentials (SEP) were conducted, showing an extremely reduced cortical action potential (N20) in agreement with a distinct brainstem damage. Due to rising doses of norepinephrine , who may develop maximal plasticity of LV recovery , 4. The Periprocedural bleeding after percutaneous interventions using large-bore catheters such as implantation of peripheral percutaneous left ventricular assist device  is associated with increased mortality, blood transfusions, length of Stay and health care costs . The incDespite the possibility of substantial periinterventional complications, which may also require massive blood transfusion and surgical treatment, this case adds to our knowledge on the potentially decisive role of aggressive MCS, including ECMELLA, in rare patients with cardiogenic shock due to clinically suspected myocarditis. Myocarditis and pericarditis have been associated in ca. 14% and 19% of COVID-19 disease, respectively , 12.Concerning the initial neurological status, it was uncertain, how to classify the described clinical findings in the context of the extended dissection of the vertebral artery and a presumably hypoxic brain damage. Based on recommendations related to prognostic biomarkers, the neurological prognosis in our patient was expected to be rather poor, especially due to distinctly elevated and increasing NSE, a generalized cerebral oedema and latency-delayed, reduced cortical action potentials during conduction of evoked potentials. Therefore, it may be critical to withhold or withdraw maximum life-supporting therapies in the early stage of assumed hypoxemic brain damage especially in young adults because of data primarily derived from older patients . At 6-moAggressive MCS, including vva-ECMELLA, may have a decisive role in survival after cardiogenic shock, and may successfully bridge to improvement of LVEF and to complete neurological restoration especially in young patients, despite potential periinterventional substantial complications and even though the initial prognosis may appear unfavourable. A broader, standardized implementation of MCS programs including ECMO and Impella following newly developed recommendations is warranted to meet the need of early MCS treatment in cardiogenic shock and the rapidly growing numbers of refractory respiratory failure especially during the current COVID-19 pandemic , 21, 22.


The COVID-19 global pandemic is entering its second year. In this short report we present additional results as a supplement to our previous paper on COVID-19 and common respiratory virus screening for healthcare workers (HCWs) in a tertiary infectious disease referral hospital in Manila, Philippines. We sought to understand what etiologic agents could explain the upper/lower respiratory tract infection-like (URTI/LRTI-like) symptoms exhibited by 88% of the 324 HCWs tested. Among the patients who had URTI/LRTI-like symptoms, only seven (2%) were positive for COVID-19, while 38 (13%) of the symptomatic participants were identified positive for another viral etiologic agent. Rhinovirus was the most common infection, with 21 (9%) of the symptomatic participants positive for rhinovirus. Based on these results, testing symptomatic HCWs for common respiratory illnesses in addition to COVID-19 should be considered during this time of global pandemic. The COVID-19 global pandemic is caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and continues into a second year , there hWe have reported that at SLH in Manila only 2% of HCWs were positive for SARs-CoV-2 as screened by RT-PCR (8 out of 324 HCWs screened) . At thatSARS-CoV-2 is projected to circulate in the population indefinitely , and thuHCW participants were tested for the presence of the following respiratory viruses: COVID-19, influenza A and B, human metapneumovirus (HMPV), respiratory syncytial virus (RSV), parainfluenza types 1 to 4, coronavirus 229E, coronavirus OC43, rhinovirus, adenovirus, and bocavirus.Viral RNA from nasopharyngeal and oropharyngeal swab specimens were extracted using a QIAamp Viral RNA Mini Kit , following the manufacturer’s instructions . Real-tiWe limited our analysis to those who exhibited URTI/LRTI-like symptoms. We categorized participants as “having URTI/LRTI-like symptoms” when they exhibited at least one of the following: fever, cough, sore throat, runny nose, shortness of breath, or loss of smell and taste. The epidemiological and clinical characteristics of these HCWs were compared between those who tested positive for the respiratory viral panels other than COVID-19 and those who tested negative for any of the panel of tests. The values were expressed as absolute numbers and percentages for categorical variables, and mean with standard deviation (SD) and median for continuous variables. Fisher’s exact test and chi-square test were used to test for associations between categorical variables, and the Mann−Whitney test was used to compare discrete variables between classifications of categorical variables. A p-value of ≤0.05 was considered statistically significant. Stata SE ver. 16.1  was used for all analyses.Our previous publication detailed the epidemiological and clinical characteristics of the 324 HCW staff tested for SARS-CoV-2 at SLH in Manila . Here wep = 0.04).Excluding SARS-CoV-2 from the analysis , most paIn the present study more health care workers exhibiting URTI/LRTI-like symptoms tested positive for common respiratory viruses, particularly rhinoviruses , than paAmong those who had coinfections, only one HCW yielded positive results for three etiologic agents ; a 48-year-old, male who was tested on the fifth day of illness. He was categorized as low-risk exposure and presented with fever, sore throat, cough, fatigue and loss of appetite.The signs and symptoms exhibited by those who tested positive for respiratory viruses other than COVID-19 were indistinguishable from those observed among persons who tested positive for SARS-CoV-2 . Based uAlthough COVID-19 is a serious concern, our study showed that it was not the main pathogen responsible for respiratory tract infections among HCWs. Symptoms associated with SARS-CoV-2 and other respiratory viral etiologies are frequently shared. While it is important to continue regular screening and testing for COVID-19 among this high-risk population, efforts should also focus on ensuring that HCWs have access to rapid molecular tests for other common respiratory illnesses.


Among them, remodeling TME to promote the endogenous immune response is one of the most attractive way to achieve a lasting CAR-T cells response.3 Nowadays, accumulating gene therapies emerge to remodel TME because of the relatively stable gene expression of TME.4 However, due to the lack of delivery specificity in most existing carriers , gene therapy stimulating the immune cells may also promote cancer progression. For example, PRR and IFN signaling in immune cells are immunostimulatory, while the same signaling in cancer cells drives cancer progression and immunotherapy resistance.1 Hence, a delivery system specific for immune cells is urgently needed for gene therapy targeting TME.Chimeric antigen receptor T (CAR-T) immunotherapy is a novel anti-tumor therapy through transferring genetically engineered T cells expressing a chimeric antigen receptor (CAR) specific for tumor antigens. It has shown remarkable efficacy in treating hematologic malignancies such as leukemia, lymphoma, and myeloma. However, CAR-T cells cannot exert persistent therapeutic effect on some solid tumors due to the cancer heterogeneity and suppressive tumor microenvironment (TME), which cause relapse from cancer cells with CAR antigen loss and rapid CAR-T cells exhaustion, especially for glioblastoma (GBM).Cell, Lexus et al. put forward that delivering the pattern recognition receptor agonists was an effective way to stimulate anti-tumor immunity.1 However, it’s hard to deliver them to immune cells directly and it often initiates cancer cell-intrinsic detrimental pathways on the contrary. Hence, Lexus et al. engineered CAR-T cells to deliver immunostimulatory non-coding RNA RN7SL1 with EVs, which functions as a damage-associated molecular pattern (DAMP) to activate RNA PRRs in immune cells.1 Lexus et al. considered that CAR-T cells could kill target cancer cells within the scope of the EVs and thus realize selective tumor-infiltrated immune cells delivery, which avoids the initiation of cancer cell-intrinsic detrimental pathways. In immune cells, RN7SL1 limits the accumulation of myeloid-derived suppressor cells (MDSCs) and inhibits TGF-β1 signaling pathway. Meanwhile, it also promotes the development of inflammatory dendritic cells (DCs) and contributes to an increase in effector-like T (Teff) cells and effector-memory-like T (Tem) cells. Due to the comprehensive endogenous immune activation, CAR-T cells efficacy can be improved and generate a positive immune cycle. Further, Lexus et al. designed to deliver peptide antigens to cancer cells by CAR-T cells and make CAR-T cell therapy persist regardless of CAR antigen loss. This strategy simultaneously employs CAR-T cells, enhances endogenous T cell function, and counteracts common suppressive mechanisms, offering effective combinatorial approaches to improve solid tumor response.1In a recent study in 5 This research from Lexus et al. just provides a new perspective for clinical transformation, taking immunotherapy for GBM to the next level. In the future, more substances can be loaded by CAR-T cells for different purposes. For example, CAR-T cells may delivery SN-38, an active metabolite of irinotecan killing tumor cells but not immune cells, to achieve widely GBM killing effect; and CAR-T cells may carry PD-1 monoclonal antibody to specifically work on immune cells, increasing the sensitivity to PD-1 monoclonal antibody.T cells have good deformability and can penetrate the blood-brain barrier under certain conditions, and T cells with specific CAR antigens  can kill target GBM cells to a large extent despite the effect is not lasting. These all equip CAR-T cells delivery system with the potential to shift the treatment landscape and outlook for patients with GBM. First, CAR-T cells enter in tumor area and cause the first killing Fig. . MeanwhiIn summary, Lexus et al. have demonstrated that the gene delivery by CAR-T cells possesses multiple advantages over other therapeutic delivery methods in terms of timing and space distribution. As CAR-T cells did not show optional effects on some solid tumors especially for GBM, we thought the core purpose of CAR-T cell therapy should be transferred from single cancer killing to being a specific gene carrier targeting the TME. We believe that these findings will expand our understanding of CAR-T cells engineering, and point to future combinations of gene therapy and CAR-T cell therapy in GBM research and clinical management.


N-methyl-D-aspartate receptor (NMDAR) antagonist, has been employed clinically as an intravenous anesthetic since the 1970s. More recently, ketamine has received attention for its rapid antidepressant effects and is actively being explored as a treatment for a wide range of neuropsychiatric syndromes. In model systems, ketamine appears to display a combination of neurotoxic and neuroprotective properties that are context dependent. At anesthetic doses applied during neurodevelopmental windows, ketamine contributes to inflammation, autophagy, apoptosis, and enhances levels of reactive oxygen species. At the same time, subanesthetic dose ketamine is a powerful activator of multiple parallel neurotrophic signaling cascades with neuroprotective actions that are not always NMDAR-dependent. Here, we summarize results from an array of preclinical studies that highlight a complex landscape of intracellular signaling pathways modulated by ketamine and juxtapose the somewhat contrasting neuroprotective and neurotoxic features of this drug.Ketamine, a non-competitive  N-methyl-D-aspartate receptor (NMDAR) antagonism. A growing body of research seeks to identify one or more biologically active metabolites of ketamine , as well as clarify ketamine’s cellular actions that may cause it to function as a neurotoxic agent .Over the past several years, ketamine has garnered significant interest as a novel therapeutic within the research and lay/media communities for its efficacy as a rapidly acting antidepressant. In March 2019, the United States Food and Drug Administration approved esketamine nasal spray as an adjunct therapeutic for treatment-resistant depression . As ketaketamine  or agentketamine  that mayketamine . As clinsubanesthetic ketamine (0.5 mg/kg over ∼40 min) to serve as a fast-acting antidepressant in treatment-resistant depression was demonstrated first in a clinical trial published in 2000  block to allow cation entry. Ketamine inhibits NMDARs at sub-micromolar concentrations, and its inhibitory actions are enhanced by Mg2+ in a subunit specific manner , resulting in activity-dependent synaptic plasticity (that requires protein synthesis) and antidepressant-like or neuroprotective effects  . As such effects . In supp effects . Similar effects . Widman  effects .direct inhibition of PNs through NMDAR antagonism induces a rebound homeostatic synaptic plasticity (as opposed to excitation-induced activity-dependent synaptic plasticity), which in turn causes an enhanced excitatory synaptic drive onto these neurons -hydroxynorketamine possesses similar antidepressant effects but with substantially lower NMDAR affinity, and instead involves and early and sustained activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPARs)  . While t(AMPARs) , clinica(AMPARs) : one stuthoughts .N-methyl-D-aspartate receptors are positioned both at synaptic compartments and extrasynaptic compartments. While the precise quantitative contribution of extrasynaptic NMDAR signaling (activated by excess ambient glutamate) has been classically difficult to measure  and pro-survival  signaling pathways, More recent explorations of ketamine’s upstream membrane targets indicate that the full-fledged antidepressant actions of ketamine require AMPAR signaling. s from lipid raft to non-raft domains, resulting in an increase in intracellular cAMP and a subsequent phosphorylation and activation of transcriptional regulator CREB, ultimately upregulating production of brain-derived neurotrophic factor (BDNF). This effect was present after subunit GluN1 knockdown, demonstrating the possibility of yet another NMDAR-independent mechanism of ketamine  receptors, have been implicated as both a mediator and modulator of neuronal degeneration, and as an important cellular biomarker of the depressed state . Two maj21A/21A/β9A/9A knockin mice, which express constitutively active GSK-3 isoforms that are resistant to ketamine inhibition, demonstrated that GSK-3 inactivation is necessary for ketamine-induced upregulation of AMPAR GluA1 subunits associated with ketamine’s antidepressant effects . In a rat model of combined amyloid-beta and isoflurane-induced cognitive impairment, ketamine  rescued spatial memory deficits on the Morris Water Maze paradigm and promoted BDNF production through upregulation of the PI3K/AKT/GSK-3β pathway. Ketamine-treated rats also displayed a dose-dependent reduction in Aβ peptide accumulation . Another effects . In cont effects .The second intracellular pathway implicated in inducing BDNF upregulation in response to subanesthetic ketamine treatment involves the eukaryotic elongation factor 2 kinase (eEF2K) as responsible for BDNF upregulation . Autry eBdnf-knockout failed to produce antidepressant-like effects. These findings suggest that the mTOR/BDNF pathway, possibly through an NMDAR-independent mechanism, is critical for the antidepressant-like effects of ketamine  prevented widespread apoptosis caused by more anesthetic doses (20 mg/kg) via the upregulation of Activity-Dependent Neuroprotective Protein (ADNP) . This fiin vivo in a concentration-dependent manner  rat pups to ketamine (20 mg/kg per dose) decreased BDNF expression and the phosphorylation of AKT and ERK and increased apoptosis in the developing rat hippocampus, which corresponded with later cognitive impairments . Currently, the basic science literature on ketamine paints a mixed picture as ketamine is studied in the context of a variety of disease models, cell types, brain regions, and employing a range of administration protocols . It is therefore necessary to undertake more systematic studies of ketamine’s long-term safety that incorporate a multidisciplinary approach to incorporate measures of wellbeing, including cell death and protein (such as BDNF) levels for DC wrote the first draft of the manuscript. All authors contributed to manuscript revision and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


Seven autoantigens  were upregulated in whole blood samples. MPO and TSHR were overexpressed in both lung autopsies and whole blood tissue and were associated with more severe COVID-19. Neutrophil activation derived autoantigens  were prominently increased in blood of both SARS-CoV-1 and SARS-CoV-2 viral infections, while TSHR and PTPRN2 autoantigens were specifically increased in SARS-CoV-2. Using single-cell dataset from peripheral blood mononuclear cells (PBMCs), we observed an upregulation of MPO, PRTN3, and PADI4 autoantigens within the low-density neutrophil subset. To validate our in-silico analysis, we measured plasma protein levels of two autoantigens, MPO and PRTN3, in severe and asymptomatic COVID-19. The protein levels of these two autoantigens were significantly upregulated in more severe COVID-19 infections. In conclusion, the immunopathology and severity of COVID-19 could result in transient autoimmune activation. Longitudinal follow-up studies of confirmed cases of COVID-19 could determine the enduring effects of viral infection including development of autoimmune disease.Immune homeostasis is disturbed during severe viral infections, which can lead to loss of tolerance to self-peptides and result in short- or long-term autoimmunity. Using publicly available transcriptomic datasets, we conducted an  Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing coronavirus disease 2019 (COVID-19), appeared first in Wuhan, China, in December 2019 and has since rapidly spread globally –6. SeverMultiple factors are involved in the development of autoimmunity, including genetics, age, and environment . Betweende novo induction of several autoimmune diseases including systemic lupus erythematosus  and the European Bioinformatics Institute . Moreover, single cell transcriptomic datasets of sorted neutrophils were used. In addition, the expression of autoantigens following COVID-19 infection was compared to that following infection with three respiratory viruses: SARS-CoV-1, IAV, and RSV.For the purpose of this study, we used a list of 52 autoantigens established by Burbelo et al.  for diagRNA-sequencing platforms were used for COVID-19 studies, while microarray platforms were used for older datasets of SARS-CoV-1, IAV, and RSV Table 2.For COVID-19 whole blood transcriptomic dataset, we used processed sequencing data deposited under project number EGAS00001004503 . In thisTranscriptomic datasets of whole blood isolated from RSV and IAV infected patients (GSE17156)  and PBMCvia standard Ficoll-Paque density gradient centrifugation. Authors performed the single-cell RNA-seq library preparation using a Nextera XT DNA library preparation kit (Illumina FC-131-1096) with 1 ng of pooled library and dual-index primers. Sequencing was performed on a NovaSeq S2 instrument   . DiffereGene ontology enrichments analyses was performed using DisGeNET  and Genevia standard Ficoll-Paque density gradient centrifugation . Assays were preformed strictly following the manufacturer’s instructions. Each sample was assayed in duplicate, and values were expressed as the mean of 2 measures per sample. One-way analysis of variance (ANOVA) and post hoc Tukey multiple comparison analyses were applied.The plasma level of MPO and PRTN3 (PR3) for seven non-infected controls, eight severe, and eight asymptomatic COVID-19 patients was determined using commercially available human ELISA kit . The plasma used in our study was obtained from COVID-19 patients recruited from Rashid Hospital. Plasma was isolated from blood versus mild COVID-19 , PRTN3 , and PADI4  levels were significantly higher in severe COVID-19 blood  Figure 2We next used gene ontology databases to determine general diseases associated with these seven genes  and PRTN3  in severe compared to asymptomatic COVID-19 infection , and respiratory syncytial virus (RSV) infected patients at the peak of disease. For each condition, differential expression and LogFC were obtained by comparing the normalized gene expression of the infected group Herein we report the common autoantigens during COVID-19 viral infection. Fifty-two autoantigens, which are linked with 24 different autoimmune diseases, were tested . Seven ade novo autoimmune disorders  did not increase in the mild COVID-19 and hence they were associated with more severe infection.Following the results obtained through gene ontology enrichment analyses, single cell transcriptomics of PBMCs revealed the significant increase in MPO, PRTN3, and PADI4 mRNA levels within low-density neutrophils. In addition, analyses of cell counts provided by Wilk et al. study showed significant increase in both low-density and canonical neutrophils during severe COVID-19 infection compared to controls .The top seven autoantigens upregulated in the blood of severe COVID-19 patients, were associated with a wide range of vascular and inflammatory autoimmune disorders , 48. WilConfirming previous findings , 50, incNETosis induces inflammation and DNA deployment that could trigger autoimmunity. While NET produced by neutrophils, their clearance is achieved by macrophages efferocytosis. During ARDS, neutrophil count and lifespan is significantly increased, and the ability of macrophages to engulf NETs and apoptotic cells is significantly decreased, prolonging the lung injury induced by neutrophil blast. Pharmacologic treatment could be used to enhance NETs clearance. Macrophage NETs efferocytosis could be restored by AMP-activated protein kinase (AMPK) activator such as Metformin or application of neutralizing antibody against HMGB1 . TherefoTSHR is expressed by thyroid epithelial cells, and various extra-thyroidal tissue including the adipose, peripheral blood cells, and fibrocytes . DerivedTSHR is targeted by autoantibodies during Graves’ disease . SARS-CoThe majority of COVID-19 patients are expected to show one or more residual symptoms months after recovering from the infection , 68–70. de novo reactive arthritis and cutaneous vasculitis 10 days after recovery of all COVID-19-related symptoms  can be found in the article/supplementary material.The studies involving human participants were reviewed and approved by Dubai Health Authority. The patients/participants provided their written informed consent to participate in this study.RaH, QH, NSS-A, FSS-A, conceived and designed the experiments. NSS-A, FSS-A, RaH, RiH, SH and SA analyzed the data. All authors contributed to the article and approved the submitted version.This research has been financially supported by Tissue Injury and Repair (TIR) group operational grant (Grant code: 150317); COVID-19 research grant (CoV19-0307) and (CoV19-0308), Seed grant (Grant code: 2001090275); and by collaborative research grant (Grant code: 2001090278) to RH, University of Sharjah, UAE; and by a Sandooq Al Watan Applied Research & Development grant to RH (SWARD-S20-007); and by Al Jalila Foundation Seed Grant (AJF202019); and by Prince Abdullah Ben Khalid Celiac Disease Research Chair, under the Vice Deanship of Research Chairs, King Saud University, Riyadh, Kingdom of Saudi Arabia.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.


Genetically encoded fluorescent indicators, combined with optical imaging, enable the detection of physiologically or behaviorally relevant neural activity with high spatiotemporal resolution. Recent developments in protein engineering and screening strategies have improved the dynamic range, kinetics, and spectral properties of genetically encoded fluorescence indicators of brain chemistry. Such indicators have detected neurotransmitter and calcium dynamics with high signal-to-noise ratio at multiple temporal and spatial scales in vitro and in vivo. This review summarizes the current trends in these genetically encoded fluorescent indicators of neurotransmitters and calcium, focusing on their key metrics and in vivo applications. The mammalian brain supports sophisticated mental processes such as cognition and memory within complex circuits of interconnected neurons. While the canonical transmission of information starts with the initiation and propagation of voltage waveforms throughout a neuron’s surface, a substantial amount of neural communication between neurons relies on chemical transmission over a range of scales. Local membrane depolarization opens voltage-gated calcium channels, permitting an influx of calcium ions. The calcium ions then activate various vesicle transport proteins to induce neurotransmitter exocytosis. Neurotransmitters then reach their postsynaptic targets by traversing the extracellular space in multiple ways. Neurotransmitters crossing the synapse can target ligand-gated ion channels and cause an immediate conformational change, allowing fast and spatially confined synaptic transmission . NeurotrThe interrogation of complex and diverse neural processes requires a detailed examination of how specific neuron types contribute to neural circuit functions. Such investigations have demanded tools that allow the noninvasive detection of neural activity with genetic specificity in vivo. This challenge is now partially met by recently engineered genetically encoded voltage indicators (GEVIs) and genetically encoded fluorescent indicators of brain chemistry. A recent review summarized the latest progress on the development of multiple categories of GEVIs in detail . Here, wThe combination of optical imaging and genetically encoded fluorescent indicators provides three distinct advantages in vivo: (1) neuron-type specificity, (2) cellular resolution, and (3) high temporal resolution. In conjunction with cell-type-specific promoters, targeting sequences, or animal driver lines expressing recombinase in specific cell types, genetically encoded indicators selectively express in targeted neural populations. First, lentivirus and adeno-associated viral (AAV) vectors are widely used as vehicles for gene delivery in neuroscience studies. Serotype and cell-type-specific promoters determine the transduction efficiency and targeting of transgene expression, respectively. Recent studies of viral vectors gave insight into serotype and promoter optimization, supporting the in vivo expression of genetically encoded indicators with high efficiency and neuron-type specificity ,4. SeconGenetically encoded fluorescent indicators report neural chemistry with high spatial resolution and reach in conjunction with optical imaging. Modern in vivo imaging methods, such as one-photon, multiphoton, and light-sheet microscopes, have enabled closed-loop neuroscience and the noninvasive recording of thousands of neurons at multiple spatial scales and depths ,13,14. SFinally, optical imaging has sufficient temporal resolution (~1800 Hz) to examine biochemical dynamics in neural circuits ,19. AlthAch (GACh3.0) [Recent protein engineering methodologies, including rational design, directed evolution, and computational approaches, have greatly advanced the development of genetically encoded fluorescent indicators. First, early protein engineering relied more heavily on rational design, which grafts known mutations onto homologous sequences or generated novel mutations based on established knowledge of protein structure. For example, the incorporation of known mutations in the cpGFP component of GCaMP resulted in GCaMP1.6 , and theGACh3.0)  shortly GACh3.0) . FinallyGACh3.0) . These cGACh3.0) , computaGenetically encoded indicators typically consist of a sensing domain and optical reporters. These indicators fall into classes based on their sensing domains. This work reviews bacterial periplasmic-binding protein (PBP)-based indicators a,d, G-prOn the other hand, single-wavelength indicators typically comprise a circularly permuted FP linked to the sensing domain d–f. CircE. coli-derived glutamate-binding PBP. Its function derives from its Venus Flytrap Domain (VFTD), which undergoes titular closing upon ligand binding. The first FRET architecture PBP indicators  is an s (FLIPE  and GluSs (FLIPE ) coupleds  into a loop of the interdomain hinge region of GltI  Figure d. Additi2AAR-cam, placed the donor of a CFP-YFP FRET pair at the third intracellular loop (ICL3) of the α2A-adrenergic receptor and the acceptor at the C-terminus of the receptor [GPCRs are endogenous proteins in many species that bind neurotransmitters with high specificity and change their conformation after binding. Protein engineers have employed GPCRs as the sensing domain in multiple genetically encoded indicators to detect various neurotransmitters. The first GPCR/FRET-based norepinephrine indicator, αreceptor    Table 1Table 1. ectively ,23,38,39Calcium flux is a proxy for neural activity. GECIs report such dynamics and are one of the most mature approaches in neuroscience. The architecture of GECIs runs in parallel to the architecture of neurotransmitter indicators. GECIs typically consist of a calcium-binding domain, a binding domain target peptide, and a reporter element based either on a single FP or a FRET pair.The first widely used FRET-based GECIs were the Cameleon family, which inserted were calmodulin (CaM) and M13  in between either a BFP-GFP or a CFP-YFP FRET pair  Figure c. Multip2+ indicators employed red-shifted FPs. Such indicators take advantage of decreased tissue scattering with a longer wavelength to enable high-resolution imaging at superior depths in scattering tissue. For example, one-photon light-sheet imaging of the near-infrared iGECI [2+ dynamics at 400 μm depth in acute brain slices.Modern engineering further expanded the spectral diversity of FRET-based calcium indicators. Green-red  and neared iGECI  can dete2+ affinity and measured small changes in Ca2+ concentration [2+-binding protein in cardiac and skeletal muscle that has less interference with the cellular regulatory protein network. TnC was the sensing domain in TN-L15 [2+ affinity in the neurons’ physiological range and had a larger dynamic range than the dynamic range of YC 3.6  covered a 100-fold range in Cantration   and cpEGFP/CaM linkers . Later engineering strategies improved Ca2+ affinity, dynamic range, and kinetics by optimizing the Ca2+-sensing domain, which employed high-throughput screening and additional protein structure information. These mutations near the calcium- and peptide-binding interface of CaM iteratively increased the dynamic range  enabled the visualization of compartment-specific acetylcholine signals in the Drosophila olfactory system, and bulk cholinergic dynamics during the sleep–wake cycle in mice.GPCR-cpFP-based neurotransmitter indicators have matched the capabilities of PBP-based indicators in vivo. For example, under two-photon illumination, dLight1.1 and dLight1.2 indicators enabled the robust and chronic detection of relevant dopamine transients in multiple mouse behaviors . In the Single-fluorophore GECIs have been prevalent in live animal experiments. The two-photon imaging of GCaMP1 visualized an odor-evoked activity map in the Drosophila brain . The oneThe cpFP class of GECIs also initiated investigations of information distribution, synaptic transmission, and plasticity within subcellular compartments. GCaMP6s expressed in the axonal boutons of neurons in the mouse primary visual cortex revealed a differentiated orientation and direction selectivity within projections targeting multiple higher-order visual areas . GCaMP5 2+ transients of ChR2-evoked muscle contraction in C. elegans [The spectral diversity of single-fluorophore GECIs have allowed integrated optogenetics/imaging and multichannel imaging in vivo. For example, simultaneous neural activation via channelrhodopsin-2 (ChR2) and imaging via RCaMP reported Ca elegans . The pro elegans . The class of FRET-based indicators have found fewer applications in vivo due to their smaller dynamic range and lower SNR compared to peer cpFP-based indicators. The most successful live animal application for these indicators has been the imaging of action potential-induced calcium flux. YC2.6, YC3.6, D3cpv, and Twitch can all report action potentials in mouse cortical neurons in vivo ,56,91. HIn this review, we have presented a broad overview of the existing genetically encoded neurotransmitter indicators and GECIs. Compared with other methods for neural recording, genetically encoded indicators possess several advantages. Their genetic specificity, enhanced brightness, dynamic range, and SNR enable the large-scale recording of neural chemistry at multiple temporal and spatial scales in vitro and in vivo. Advances in protein engineering and high-throughput screening have accelerated the indicator optimization pipeline in the past two decades. These technical advances have engendered genetically encoded biochemical indicators that not only support existing dissections of neural circuitry underlying behavior, but also serve future explorations of brain chemistry. Based on current trends in neuroscience, wherein neural activation and in vivo imaging are often conducted simultaneously, one possible future research direction in genetically encoded indicator development is pushing the fluorescent indicators toward red or NIR wavelengths. This red-shift would provide various benefits over green or yellow indicators, including deeper imaging due to the red light’s decreased scattering, decreased phototoxicity, and increased compatibility with blue-light-activated optogenetic actuators.


Nevertheless MSCs derived from aged donors or patients with age-related diseases are senescent . In addinescence . Althougnescence . SenesceLiu et al. and Zhou et al. summarize the current knowledge of phenotypic and biological properties of senescent MSCs and strategies for monitoring and rejuvenation, including gene modification and pretreatment strategies . They also systematically review the molecular mechanisms underlying MSC senescence including epigenetic changes, autophagy, mitochondrial dysfunction and telomere shortening. There is accumulating evidence that autophagy acts as a positive and negative regulator of MSC senescence . Sirtuin 3 (SIRT3), an NAD + -dependent deacetylase, regulates a variety of physiological and pathological processes including aging and aging-related diseases. Ma et al. have shown that reduced Sirt3 expression contributes to age-related natural senescence and H2O2-induced premature senescence of rat bone marrow (BM)-MSCs by stimulating cellular reactive oxygen species (ROS) production and DNA injury . Overexpressing Sirt3 partly reversed the senescence-associated phenotypic features of natural and premature senescent MSCs by alleviating ROS generation and upregulating SOD2 expression. Qin et al. reveal that knockout of NO synthase 2 significantly promoted the adipogenic, but not osteogenic, differentiation capacity of rat MSCs . Notably, they also showed that knockout of NOS2 in MSCs resulted in significant obesity in rat fed a high-fat diet.There are twelve manuscripts in this Research Topic, highlighting the current understanding of the potential mechanisms underlying MSC senescence and novel strategies to rejuvenate the senescent MSC. nescence . RastaldYin et al. summarize recent progress in understanding the impact of hyperglycemia on senescence of MSCs and strategies to suppress this senescence in a hyperglycemic environment . Chen et al. discuss the current knowledge of the role of MSC senescence in the development of myelodysplastic syndromes and show that targeting senescent MSC is a potential strategy for MDS treatment . Conley et al. report that MSCs isolated from obese subjects display a lower proliferative capacity than those derived from age-matched non-obese subjects. In addition, these obese-MSCs exhibited a senescent phenotype as evidenced by increased p16, p53, IL-6, and MCP-1 gene expression . Furthermore, co-culture of injured HUVECs with MSCs from non-obese subjects resulted in the formation of tube-like networks but not with MSCs from obese subjects, indicating that pro-angiogenic properties were impaired. Cell source also affects the function of MSCs. Yigitbilek et al. compared the function of liver-derived MSCs (L-MSCs) and adipose tissue-derived MSCs (A-MSCs) from donors matched for gender, age, and body mass index . Although both L-MSCs and A-MSCs exhibited a similar senescent phenotype manifested by similar cell cycle arrest and senescence-associated secretory phenotype genes, L-MSCs displayed an enhanced immunomodulatory capacity, while A-MSCs possessed better pro-angiogenic and vascular reparative potency.It has been reported that the physiological and pathological condition of the donor plays a critical role in regulating MSC senescence . Yin et Dong et al. report that elevated lncRNA lnc-CYP7A1-1 induced human BM-MSC senescence as evidenced by decreased cell proliferative ability, cell survival and migratory ability . They also found that inhibition of lnc-CYP7A1-1 rejuvenated aged BM-MSCs and improved their therapeutic efficacy in a mouse model of myocardial infarction. There is emerging evidence that MSC-derived exosomes display protective effects against a variety of human diseases . Compared with young-MSC-exosomes, aged-MSC-exosomes exhibited an impaired ability to promote endothelial tube formation and inhibit cardiomyocyte apoptosis in vitro. Transplantation of aged-MSC-exosomes also exhibited decreased cardioprotective effects in a mouse model of myocardial infarction. MicroRNA array and PCR analysis revealed dysregulation of miR-221-3p in aged-MSC-exosomes but restoration of miR-221-3p expression rescued aged-MSC-exosome reparative function.Recently, non-coding RNA including lncRNAs and miRNA have been reported to be involved in mediating MSC senescence . Dong etdiseases  althoughSharma et al. examined the effects of short-term navitoclax treatment, a chemotherapeutic drug reported to effectively clear senescent cells, on bone mass and osteoprogenitor function in aged mice . Interestingly, they found that despite clearance of senescent cells, navitoclax treatment significantly reduced the trabecular bone volume fraction in aged mice and impaired the calcified matrix production by aged BMSC-derived osteoblasts, indicating that the therapeutic effect of navitoclax on age-related bone loss was limited. Future studies including larger-scale studies in rodents and larger animal models are urgently needed to definitively assess the potential therapeutic efficacy of navitoclax in age-related diseases.It is well documented that clearance of senescent cells can improve tissue function and extend lifespan during aging . To thisIn summary, this Research Topic comprises twelve outstanding manuscripts of original research and comprehensive reviews. We summarize the most recent findings regarding the molecular mechanisms underlying MSC senescence and potential strategies for rejuvenation.


Vector-borne flaviviruses are responsible for nearly half a billion human infections worldwide each year, resulting in millions of cases of debilitating and severe diseases and approximately 115,000 deaths. While approved vaccines are available for some of these viruses, the ongoing efficacy, safety and supply of these vaccines are still a significant problem. New technologies that address these issues and ideally allow for the safe and economical manufacture of vaccines in resource-poor countries where flavivirus vaccines are in most demand are urgently required. Preferably a new vaccine platform would be broadly applicable to all flavivirus diseases and provide new candidate vaccines for those diseases not yet covered, as well as the flexibility to rapidly pivot to respond to newly emerged flavivirus diseases. Here, we review studies conducted on novel chimeric vaccines derived from insect-specific flaviviruses that provide a potentially safe and simple system to produce highly effective vaccines against a broad spectrum of flavivirus diseases. Flavivirus (family: Flaviviridae) comprises over 70 distinct members and is separated into four distinct groups: mosquito-borne (circulating between mosquitoes and vertebrates); tick-borne (circulating between ticks and vertebrates); no known vector  and insect-specific flaviviruses (which are only found in mosquitoes and cannot infect vertebrates) , dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV) and tick-borne encephalitis virus (TBEV). Despite their geographical limitations and the relatively small reported case numbers, several other species also cause severe or lethal diseases in humans. As such, they still pose a threat as emerging or re-emerging pathogens. Examples include St Louis encephalitis virus (SLEV), Murray Valley encephalitis virus (MVEV), Rocio virus (ROCV), Kyasanur Forest disease virus (KFDV), Omsk hemorrhagic fever virus (OHFV) and Powassan virus (POWV).The genus ebrates) . The FlaAedes aegypti mosquitoes, is endemic in tropical and subtropical regions in Africa and South America and causes an estimated 130,000 cases with 78,000 deaths annually ..Aedes aeAedes vigilax mosquitoes captured in Sydney, Newcastle and Brisbane . Mo. MoAedesdetected , and Ausectively  79,80].,80.Aedes in 2014 . Hidden  in 1975  81]..Aedes viWhile we and others have shown that ISFs cannot replicate in a range of vertebrate cells lines ,106,107,KUN) concluded that a blockage of ISF replication in vertebrate cells occurred pre-entry when infectious particles were not recovered following vertebrate cell infection ) failed to result in detectable levels of viral genome amplification [Wild-type BinJV and BinJ/VIF-prME chimeras failed to initiate replication in a wide range of vertebrate cell lines at 37 °C, including cells lacking components of the innate immune response. This observation indicates that BinJV replication in vertebrate cells is inhibited at temperatures above 36 °C, as observed for other flaviviruses ,113,114,fication .KUN-prME were cultured in wild-type human A549 cells and ZAP-knockout A549 cells at 34 °C, replication was only detected in the ZAP-KO cells [The lack of efficient ISF replication in vertebrate cell lines, even at lower temperatures, high MOI and immune-deficient cell lines, suggests that additional barriers inhibit steps in the virus replication cycle and are potentially mediated by cellular antiviral responses. However, this remains to be tested in primary cell cultures. Our recent studies incriminate the zinc-finger antiviral protein (ZAP) as a crucial player in this context . It appeKO cells .KUN-prME, BinJ/ZIKV-prME, BinJ/DENV2-prME were compared to their wild-type VIF (WT VIF) counterparts  using a range of antibody-binding assays including ELISA and IFA [d) curves. In each instance, the Kd correlation between chimeric virus and parental virus was high, with lines of best fit developing R2 values of 0.998 (BinJ/WNVKUN-prME), 0.930 (BinJ/ZIKV-prME) and 0.886 (BinJ/DENV2-prME), indicating high levels of antigenic authenticity in the structural proteins of BinJ/VIF-prME chimeras [KUN-prME particles (3.9Å) and BinJV/ZIKV-prME particles (7.1Å) were indistinguishable from the virions of their WT VIF counterparts [To demonstrate that chimeric ISF particles authentically display the structural proteins of VIFs on their surface, BinJ/WNV and IFA . An extechimeras . Furtherterparts ,116.The demonstration that chimeric viruses, based on the insect-specific alphavirus Eilat virus, could be used as safe and effective experimental vaccines against alphavirus pathogens such as chikungunya virus (CHIKV), Eastern equine encephalitis virus and Venezuelan equine encephalitis virus ,118, als−/−), vaccination with one or two small unadjuvanted doses of purified BinJ/ZIKV-prME chimera (2 or 20 µg) induced robust neutralizing antibody responses against wild-type ZIKV (ZIKVPVRABC59 strain) and protected against infection (viraemia) and pathology (weight loss or tissue pathology) after challenge with wild-type ZIKV . This pure 6 in .KUN-prME. When 6–8-week-old CD1 mice were immunised with low doses (1 or 5 µg) of unadjuvanted, purified BinJ/WNVKUN-prME, a single dose was sufficient to induce a robust neutralizing antibody response against both WNVKUN (NSW2011 strain) and the more virulent WNVNY99 strain [NY99, with no clinical pathology observed in vaccinated mice [Efficacious protection was also demonstrated against a lethal WNV challenge when mice were vaccinated with BinJ/WNV9 strain . Furtherted mice .AG129 mice immunized with a BinJ/DENV2-prME chimera were similarly completely protected against lethal challenge with a mouse adapted strain of DENV-2  . In thes−/− mice with two 5 µg doses of BinJ/YFV17D-prME induced neutralizing antibodies and protected mice against infection, weight loss and liver pathology after YFV 17D challenge [O-desacyl-4′-monophosphoryl lipid A, a non-toxic derivative of the lipopolysaccharide from Salmonella minnesota; and QS-21, a saponin fraction extracted from Quillaja saponaria Molina [17D-prME compared to the other BinJ/VIF-prME vaccines, which did not require an adjuvant, is not clear.Vaccination of female IFNARhallenge . In conta Molina . The rea17D chimeric vaccine, the BinJ/VIF-prME vaccines described above did not require multiple doses or even an adjuvant to induce protective immunity in the mouse model. A significant contributor to this potent immunogenicity is likely the structural and antigenic authenticity of the chimeric particles, compared to their WT VIF counterparts, as demonstrated by the high-resolution cryo-EM analysis and mAb Kd binding studies discussed earlier. By comparison, many chemically inactivated or recombinant preparations of VLP vaccines have significantly reduced immunogenicity due to the loss of structural and antigenic integrity of the particles [As discussed above, except for the BinJ/YFVarticles ,125. ThuNevertheless, this potent immunogenicity is unusual for vaccines that do not replicate in vertebrate cells and indicates the chimeric virus particles may have self-adjuvanting properties. One possibility is that the chimeric particles enter susceptible cells after vaccination by binding to cellular receptors via the VIF structural proteins on the chimeric particle surface. Subsequently, early stages of viral replication may be initiated, and RIG-I recognition of viral dsRNA intermediates then mediate the production of inflammatory cytokines  that enhance the humoral response ,127. AltKUN-prME by UV treatment and compared its protective efficacy with untreated particles by immunizing and challenging with a lethal dose of WNV. Although neutralizing titres were slightly lower in mice receiving the inactivated particles compared to mice receiving the live virus, this treatment did not significantly affect vaccine efficacy with all mice protected from disease after a single unadjuvanted dose [To test this theory, we completely inactivated BinJ/WNVted dose . This reted dose ,129. SinDuring licensing of live-attenuated vaccines, consideration should be given to the risks involved in the uncontrolled release of the vaccine virus into the environment. This risk is particularly pertinent to genetically modified (GM) vaccine viruses. Because mosquitoes can transmit wild-type flaviviruses, the possibility of vaccine virus transmission by these arthropod-borne vectors should be investigated. For example, the recombinant, live-attenuated Chimeri-Vax vaccines were shown to be highly attenuated in mosquitoes. Although low infection levels were observed in the midgut following a blood meal, these vaccine viruses were not transmitted by mosquitoes as no dissemination through the mosquito occurred ,132,133.KUN-prME with UV-C did not reduce its efficacy as a vaccine in the mouse model. No difference was observed in the binding of a panel of mAbs to the UV-inactivated particles compared to the non-treated vaccine preparation [Some jurisdictions may have strict regulatory requirements for the intentional release of GMOs and may not accept vaccination of humans and animals with a “live”, genetically modified vaccine. Thus, inactivation methods to eliminate the replication of the chimeric virus in mosquito cells may be required. As discussed above, the inactivation of BinJ/WNVparation , suggestThe BinJV chimeric viruses described here are potentially an ideal vaccine platform. These viral particles are antigenically indistinguishable from wild-type vertebrate-infecting viruses, replicate to high titres in mosquito cells and fail to replicate in vertebrate cells under standard conditions. However, live-attenuated and killed vaccines must be grown in GMP-approved cell lines or embryonated chicken eggs that meet strict guidelines. To date, the BinJ/VIF-prME chimeras have only been shown to grow efficiently in the C6/36 mosquito cell line, which is not GMP-approved. Proteins in mosquito saliva are known to aggravate the reaction of some individuals to mosquito bites and enhance the immune response . Indeed,Aedes albopictus cells (C7/10 line), mass spec analysis of purified preparations detected only low levels of three mosquito proteins, which were not linked to known mosquito allergens and are likely to be integral components of alphavirus particles [Aedes albopictus bites every two weeks before being injected with purified EILV/CHIKV showed only minor inflammation at the inoculation sites [To address these issues with the chimeric alphavirus vaccine candidate (EILV/CHIKV), which is also grown in articles . Additioon sites . These dThe antigenic authenticity of the BinJ/VIF-prME chimeric viruses compared to wild-type VIF particles described above also demonstrates this platform is ideal for producing safe, accurate diagnostic antigens against virulent pathogens, even those that generally require a high level of biocontainment (BSL3 or higher). Indeed, the apparent utility of the chimeric viruses has been demonstrated using immune sera in various ELISA formats, microsphere immunoassays and virus neutralization assays showing similar results between chimeric and wild-type derived VIF antigens . FurtherThe ability to safely grow BinJ/VIF-prME chimeras to high titres in facilities with minimal biocontainment conditions provides a simple platform for preparing antigenically authentic particles as vaccines or diagnostics for globally important flavivirus pathogens such as YFV, ZIKV, DENV and WNV. This platform is highly amenable for its adoption in resource-poor countries that bear the heavy burdens of these diseases and urgently need a continued supply of inexpensive vaccines and diagnostic reagents, but have limited capacity for vaccine manufacturing.


Despite this scientific ,Host factors associated with reduced vaccine efficacy and durability are well established and include older age in adults, chronic inflammation, CMV latent infection and immunosuppression.Information on COVID-19 vaccine immunogenicity and immune response durability in people living with HIV (PLWH) is not yet established, as these persons were mostly excluded from large prospective vaccine studies.PLWH represent a distinctive group among immunosuppressed patients. HIV infection is characterized by a reduction of the number of CD4 T-cells, predominantly the mucosal protective CD4 T-helper 17 (Th17) cells that are rapidly infected and depleted from the digestive track and to a lessen extend from the respiratory track. Such immune damage alters epithelial barrier, resulting in microbial translocation and systemic inflammation that further induce T-cell activation. Although long-term antiretroviral therapy (ART) prompts CD4 T-cell recovery, some immune defects persist such as immune activation, low CD4/CD8 ratio and risk of developing inflammatory non-AIDS comorbidity. As cell-mediated immunity, including CD4 helper T-cells, has emerged as a critical contributor to the COVID-19 vaccine response, the influence of combined immunosuppression and immune activation in PLWH remains a poorly addressed question.A handful number of studies have investigated responses to COVID-19 vaccines in PLWH. An observational study involving 14 PLWH on ART assessed the anti-Spike antibody titres following two doses of mRNA vaccines. Encouragingly, immunogenicity was not different from previous results in HIV-uninfected participants, suggesting that mRNA vaccination should succeed in PLWH with viral suppression, regardless of their CD4 T-cell count.The Lancet Regional Health – Europe, Lombardi et al. assessed in a single clinical site in Milan, Italy, the immunogenicity of the mRNA-1273 vaccine among PLWH.In this issue of ,However, the study did not address vaccine effectiveness and durability of humoral, nor cellular immune response, key factors for real life vaccine success. Recent data in French health care workers indicate that post-vaccination antibody titer decreases faster in vaccinated people than in COVID-19-infected individuals who then received vaccination.Vaccine response might, in any population, wanes due to developments of variants escaping T-cells and antibody selective immune pression (i.e.: antigenic drift)To address long-term benefit of COVID-19 vaccines in PLWH, we need to establish large prospective cohorts to assess level and durability of humoral and cellular immunogenicity to current and future variants, combined with their safety and tolerability.The authors declare no conflict of interest.


In the U.S., non-Hispanic Black individuals are disproportionately represented amongst COVID-19 mortalities. The COVID-19 vaccines are poised to change this outcome; however, inequitable access and decades of medical mistreatment have resulted in healthcare mistrust and an associated low uptake within this group. Loma Linda University (LLU) houses the largest mass vaccination site in San Bernardino County (SBC) California; nevertheless, there has been a perpetual low representation of Black vaccinees. To increase the number of Black persons vaccinated, a selected team at LLU leveraged a community-academic partnership model to address vaccine hesitancy and increase access to the COVID-19 vaccines. The objective of this study was to evaluate the number of Black persons vaccinated in community settings compared to the mass clinic. LLU developed a tiered approach to increase COVID-19 vaccinations within Black SBC communities. The first tier engaged faith leaders with the academic community in disseminating COVID-19 health information, the second included culturally representative LLU healthcare professionals in the delivery of COVID-19 educational webinars, and the third was to conduct low barrier, remote-site vaccination clinics, within targeted Black communities. Following these efforts, we compared the number of Black individuals vaccinated in the LLU mass clinic to those vaccinated in the community remote-site clinics. The remote-site COVID-19 vaccination clinics commenced in February 2021. From February 1 until April 30, 2021, 24,808 individuals were vaccinated in the LLU mass clinic with a first dose (Pfizer or Moderna) or single dose (Janssen) of a COVID-19 vaccine, however, only 908 (3.7%) were Black vaccinees. Contrastingly, the LLU remote site clinics vaccinated 1,542 individuals with a first or single dose of a COVID-19 vaccine. Of those vaccinees, 675 (44%) were Black. The multi-tiered community approach (remote-site vaccination clinics) resulted in a necessary overrepresentation of Black vaccinees, previously underrepresented in the LLU traditional mass vaccination clinic effort . Further research is warranted to examine the key elements to increase vaccinations amongst minoritized groups. COVID-19 Vaccination Comparisons Between ModelsThis table includes data from the Loma Linda University Mass Vaccination Clinic and the Remote-Site Vaccination Efforts compared to the San Bernardino County DemographicsAll Authors: No reported disclosures


Although coronavirus-disease-2019 (COVID-19) impacted everyone in some ways, it disproportionally impacted the older population. Given their increased vulnerability to severe illness and mortality, the ongoing pandemic has created greater distress, anxiety, and fear among the older population. In Nepal, a South Asian country nestled in the Himalayas between India and China, most stories of older adults are untold – both in the pre-COVID-19 and the COVID-19 era. This study aimed to explore the perceived fear of COVID-19 among Nepali older adults. A cross-sectional study was conducted between July-September 2020 among 847 older adults (≥60years) residing in three districts of eastern Nepal. The seven-item Fear of COVID-19 Scale assessed the perceived fear of COVID-19; higher scores on the scale (ranges 7 to 35 indicated greater fear. A sizeable proportion of the participants' reported being afraid (35%), anxious (32%), uncomfortable (24%), clammy (14%), and sleepless (12%), while 28% were fearful of losing their life due to COVID-19. In adjusted regression analysis, older age group, Dalit (minority) ethnicity, and remoteness to the health facility were associated with greater fear of COVID-19. Surprisingly, pre-existing health conditions were inversely associated with fear of COVID-19. Greater fear of COVID-19 amidst the pandemic, although anticipated, urges us to reflect on the most vulnerable groups' psychological needs not just during COVID-19 but in the future events of pandemics and public health emergencies. Fear during emergencies could be battled with accurate and effective information as well as better preparedness and psychosocial interventions.