A family of structurally homologous PTS-regulatory-domain-containing virulence regulators (PCVRs) has been recognized in divergent bacterial pathogens, including Streptococcus pyogenes Mga and Bacillus anthracis AtxA. These paradigm PCVRs undergo phosphorylation, potentially via the PTS, which impacts their dimerization and their activity. Recent work with predicted PCVRs from Streptococcus pneumoniae (MgaSpn) and Enterococcus faecalis (MafR) suggest they interact with DNA like nucleoid-associating proteins. Yet, Mga binds to promoter sequences as a homo-dimeric transcription factor, suggesting a bi-modal interaction with DNA. High-resolution crystal structures of 3 PCVRs have validated the domain structure, but also raised additional questions such as how ubiquitous are PCVRs, is PTS-mediated histidine phosphorylation via potential PCVRs widespread, do specific sugars signal through PCVRs, and do PCVRs interact with DNA both as transcription factors and nucleoid-associating proteins? Here, we will review known and putative PCVRs based on key domain and functional characteristics and consider their roles as both transcription factors and possibly chromatin-structuring proteins.Bacterial pathogens rely on a complex network of regulatory proteins to adapt to hostile and nutrient-limiting host environments. The  In Firmicutes, the channels Figure 1channels . PEP, a channels .carbon catabolite repression (CCR) (via the following steps: enzyme I (EI) receives phosphates from PEP on the histidine-191 residue and relays it to histidine-15 on HPr. From phospho-His15-HPr, the phosphate can be transferred to a sugar-specific enzyme IIA (EIIA) protein (PTS-regulatory domain (PRD)-containing transcription factors and antiterminators. See Görke and Stülke for a comprehensive review of CCR and the PTS  . CCR shuon (CCR) . This acon (CCR) . Unlike on (CCR) , which o protein . An inev the PTS . This recis-regulatory elements  that will be discussed later. The internal PRD domains  contain histidine residues (230 and 289 for PRD-1 and 342 and 399 for PRD-2) which in MtlR, are spatially conserved and subject to PTS-phosphorylation (Gat) containing a conserved cysteine at position 419, which is immediately followed by a distal EIIA-like binding domain which contains a conserved histidine at position 599  and Enterococcus faecalis MafR/EF3013 (PDB 3SQN), show similarities in tertiary structure to established PRD-containing regulators  crystal structures of AtxA (PDB 4R6I) as well as two homologs, gulators . This raanthrax toxin activator (AtxA) is a 53-kDa, DNA-binding protein that is encoded by atxA located between the cya and pagA genes on the Bacillus anthracis pXO1 plasmid (capBCADE) as well as the tripartite toxin genes encoding protective antigen (pagA), lethal factor (lef), and edema factor (cya) located on the pXO1 plasmid . Loss of both AtxA orthologues (AtxA1 and AtxA2) attenuated virulence in a capsule-dependent manner, thus highlighting AtxA as a master regulator which enables the virulence of pathogenic Bacilli (The  plasmid . AtxA wa plasmid . AtxA ha plasmid . Virulennfection . Bacillu Bacilli .multiple gene activator of the Group A streptococcus (Mga) is a 62-kDa DNA-binding protein and a major stand-alone virulence regulator conserved in all strains of S. pyogenes  and the collagen-like protein A (sclA) (scpA (encoding the C5a peptidase), sic (encoding the secreted inhibitor of complement), and fba (encoding the fibronectin-binding surface adhesin)   and a cladhesin) . The actadhesin) , while madhesin) . Numerouc target .One potential problem with the hypothesis that PCVRs are descended from a common ancestral PRD-containing protein lies in that they generally lack sequence similarity. Nevertheless, the more obvious structural similarities of PCVRs, specifically at the secondary and tertiary levels of order, suggest that PCVRs are indeed orthologues and a product of either divergent or convergent evolution. Despite having a crystal structure available for AtxA (PDB 4R6I), Mga has proven exceedingly difficult to express to levels that would allow it to be purified for crystallography. To determine the homology of PCVR functional domains, a Phyre2 analysis was used comparing user input amino-acid sequences to those of known three-dimensional structures . AtxA waMga (Mga) was absolutely essential for binding (conserved Mga domain (CMD-1) that is found in Mga as well as some S. dysgalactiae Mga orthologues  residue and in the case of MgaSpn, a conserved leucine (L) residue as well.As previously mentioned, both AtxA and Mga share predicted domain organization with MtlR, including two N-terminal DNA-binding domains sharing HTH-motif structural homology Figure 2Mga . McIver  binding . Mga alshologues . In S. pgulation . HoweverGat) in E. coli  is poorly understood , AtxA could no longer be immunoprecipitated in a phosphorylated form -inducible atxA expression (PTS-independent) had no altered ability to express the B. anthracis toxin lef, regardless of the presence or absence of an intact PTS. In contrast, atxA under the control of its native promotor was unable to induce lef expression in a PTS-null  genetic background. Additionally, atxA expression was significantly decreased in a ptsHI double mutant. EIIB was not investigated for in vitro phosphorylation of AtxA, thus its role in this process still needs to be explored.There is little direct evidence that PTS proteins  interact with AtxA. However, a series of experiments using site-directed mutagenesis to induce phospho-mimetic (H to D) and phospho-ablative (H to A) amino acid substitutions in AtxA revealed that H199D (in the PRD-1) activated AtxA while H379D (in the PRD-2) had an inhibitory effect . Specifition 199 . In addited form . Taken tted form . They foin vivo, Valdes and colleagues demonstrated that Mga does undergo variable phosphorylation in vivo (mga-2) and M1 (mga-1) could be phosphorylated by the PTS in vitro using purified PEP, EI, and HPr  in the PRD-1 of both isotypes of Mga prevented multimerization , both of which are encoded on the 96-kb pXO2 plasmid and have been implicated in regulating virulence   . Their p (RALPs) . Each ofin vitro . Additionfection . InteresGat-like domain (S. pyogenes contains a histidine residue (H392) in its PRD-2 that aligns with the H379 in AtxA, thus opening up the possibility that PTS-mediated phosphorylation occurs in RALPs  completely ablated the ability of RivR to form dimers, and inhibited RivR from regulating its target genes. This suggests the EIIBGat-like domain is primarily responsible for multimerization, which is consistent with AtxA and Mga , shares a high degree of amino acid sequence similarity to S. pyogenes Mga (Mga of Streptococcus pneumoniae (MgaSpn). Like Mga, MgaSpn plays an important role in early infection by modulating the production of bacterial adhesins encoded within the rlrA pathogenicity islet that provide S. pneumoniae the capacity to colonize the nasopharynx and progress to pneumonia (rlrA positively regulates the other genes of the rlrA islet and is homologous to RofA and Nra of S. pyogenes (rlrA islet has significant homology to the type-1 fibronectin-collagen-T-antigen (FCT) encoding region of S. pyogenes  and PDB prediction software shows that it forms a dimer with the C-terminal regions in close proximity to each other with the N-terminal regions facing out. Phyre2 alignments with AtxA show that MgaSpn contains secondary structures that are consistent with two N-terminal HTH-domains, two central PRDs, and a C-terminal EIIbGat-like domain  that was initially characterized by X-ray crystallization (PDB 3SQN) based solely on homology and biochemical analysis  and Group C streptococci  exhibit the most homologs (both having one Mga and three RALP homologs) whereas more distantly related viridans streptococci, such as the α-hemolytic S. pneumoniae or the γ-hemolytic S. mutans having few or no Mga/RALP homologs. These findings support the hypothesis that PCVRs originated from a common ancestral protein that either evolved from a classical PRD-containing regulator or a PRD-containing regulator-like protein that arose convergently. These similarities suggest that the Mga/RALP PCVRs provided a greater fitness advantage to ancestral streptococci that evolved into human pathogens, whereas streptococci that succeeded as commensals did not experience the same selective pressure to retain them.As more AtxA/Mga family proteins are identified, it has become more apparent that PCVRs are a diverse class of regulatory proteins that are widespread in Gram-positive pathogens. In the streptococci, numerous structural homologs of Mga and the RALPs have been previously discovered . The S. ng BLAST Figure 4S. pyogenes grown in either a glucose rich medium (THY) or glucose-limited medium (C media) showed a distinct difference in the Mga regulon under these two different growth conditions , which transported 63% of the other carbon sources tested  and 32P-labeled (radioactive) PCVR immunoprecipitation assays have provided some insight as to whether or not PCVRs are phosphorylated inside the cell  have shown promise for studying pHis phosphorylation in a quantitative manner. One issue with previous approaches was that any proteins containing pHis would often contain other phosphorylated residues that could result in false positives. Using a pHis-mimicking hapten , Jung-min and colleagues were able to develop and optimize a stable pan-pHis antibody that could enrich for proteins from a cell lysate using immunoprecipitation, and then determine the phosphorylation state with high-resolution nano-flow ultra-performance liquid chromatography-mass spectrometry . Indeed,E. coli .3+ immobilized metal affinity chromatography (IMAC) columns coupled with a more streamlined workflow to overcome the limitation of high acidity inherent in traditional IMAC chromatography  and novel pHis-containing proteins.Potel and colleagues have now developed a new methodology using Fetography . This altography . Using tin vivo would be a powerful tool to help direct subsequent phospho-mimetic/-ablative mutagenesis experiments to better understand how novel PCVRs function with respect to the PTS. pHis-LC-MS/MS would also be useful in determining which sugars and cognate EII complexes are essential in PTS-mediated PCVR signaling. Importantly, this same experimental approach could be used to simultaneously assess how specific PTS-sugars impact the regulation of PCVRs or other proteins in any bacterium of interest.These advancements provide a means by which to rapidly uncover the phosphorylation state of the entire proteome, including PCVRs. Obtaining direct evidence of which histidine residues undergo phosphorylation histone-like nucleoid structuring proteins (H-NS), structural maintenance of chromosomes proteins (SMC), factor for inversion stimulation (Fis), integration host factor (IHF), and heat-unstable protein (HU) . In eukaryotes, this organization is sustained through histones, which provide structural order to nucleic acid while undergoing PTMs that modulate gene expression . Prokaryein (HU) . A recenein (HU) .cis-regulatory elements. For instance, transitioning of H-NS from a dimeric to a long oligomeric state has been shown to enhance repression of its regulated genes  promoter in vitro in a phosphorylation-dependent manner and, although the binding region contained stem-loop structures, there was no specific binding site identified  on Host-Pathogen Interactions.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.


The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) shows a promising therapeutic potential in cancer treatment as it exclusively causes apoptosis in a broad spectrum of cancer cells through triggering the extrinsic apoptosis pathway via binding to cognate death receptors, with negligible toxicity in normal cells. However, most cancers, including glioblastoma multiforme (GBM), display TRAIL resistance, hindering its application in clinical practice. Recent studies have unraveled novel mechanisms in regulating TRAIL-induced apoptosis in GBM and sought effective combinatorial modalities to sensitize GBM to TRAIL treatment, establishing pre-clinical foundations and the reasonable expectation that the TRAIL/TRAIL death receptor axis could be harnessed to treat GBM. In this review, we will revisit the status quo of the mechanisms of TRAIL resistance and emerging strategies for sensitizing GBM to TRAIL-induced apoptosis and also discuss opportunities of TRAIL-based combinatorial therapies in future clinical use for GBM treatment. Programed cell death via apoptosis is an integral part of normal physiological mechanisms to eradicate unwanted or damaged cells and also functions as a natural barrier against cancer development and progression . HoweverGBM is a highly malignant primary brain tumor and the most aggressive type of gliomas arising from the central nervous system (CNS) . DespiteApart from tumor-treating fields, no other therapeutic interventions used in the new diagnosis or at recurrence exhibit possibilities to prolong overall survival in randomized trials . SeveralTumor cells including GBM have evolved multiple strategies to escape programmed cell death, underscoring that the reactivation of cell death programs, which are frequently inactivated in GBM, is a reasonable avenue to overcome this Achilles′ heel of GBM . Among tTRAIL was identified independently by two groups in 1995 and 1996 as a 281-amino acid type II transmembrane protein in the human form or 291-amino acid protein in the murine form and found to rapidly induce extensive apoptosis in a variety of transformed cell lines ,27. One The signal transduction of TRAIL-induced apoptosis is becoming increasingly clear with extensive investigations. It is now known that upon stimulation by TRAIL, trimeric TRAIL-crosslinked TRAIL-R1/R2 undergo homotrimerization and employ their intracellular death domains (DDs) to recruit the FAS-associated death domain protein (FADD), which subsequently recruits pro-caspase-8 through the death effector domains (DEDs), collectively forming the death-inducing signaling complex (DISC) to activate the pro-caspase-8. The activated dimeric caspase-8 is released to cleave and activate the effector caspase-3 that is sufficient to trigger extrinsic apoptosis in some cells (type I). Whereas in other cells (type II), the activation of the mitochondrial pathway is also required for apoptosis induction. Under this circumstance, caspase-8 cleaves the Bid (BH3 interacting-domain death agonist), and then BAK (BCL2 antagonist/killer) and BAX (BCL2 associated X) oligomerize in the outer mitochondrial membrane to form lipid-containing pores. Consequently, cytochrome c is released, together with Apaf-1 (apoptotic peptidase activating factor 1) and pro-caspase-9, assembling an apoptosome, which in turn enhances caspase-3 cleavage and the activation of other effector caspases, resulting in cleavage of a plethora of cellular proteins and ultimately the execution of apoptosis ,30,31. HIn addition to apoptotic signaling, it is well characterized that TRAIL can trigger various non-apoptotic signaling pathways to induce a plethora of biological responses. A typical example is that TRAIL activates the receptor-interacting serine/threonine protein kinase 1 (RIPK1) and RIPK3 signaling to induce necroptosis , anotherAmong the death receptor-ligand systems, the TNFR1-TNFα and CD95-FasL axes are excluded from further therapeutic exploitations, owing largely to the lethal toxicity of TNF or CD95 agonists . On the For other solid tumors, however, dissatisfactory results from clinical trials of recombinant TRAIL and agonist antibodies against TRAIL-R1/2 have been obtained, and the drug’s short half-life, poor pharmacokinetics, and resistance are thought to be the major contributors . RegardlStudies using different analyses have shown that TRAIL receptors, including TRAIL-R1 and TRAIL-R2, as well as “decoy” receptors TRAIL-R3 and TRAIL-R4 that lack the cytoplasmic “death domain” to induce the apoptotic pathway, are all expressed with varying degrees in several GBM cell lines ,46,47. AYet, some in vitro observations in GBM cell lines have shown that the levels of TRAIL receptors are not necessarily correlated with TRAIL sensitivity ,54, suggCancer cells, including GBM, have evolved multiple strategies to rewire TRAIL-induced apoptotic signaling in order to evade apoptosis. For example, GBM cells display very low level of caspase-8, which is correlated with resistance to TRAIL-induced apoptosis. Additionally, in tumors obtained from GBM patients, caspase-8 expression is also very low, suggesting that the TRAIL pathway may not be functional in GBM due to insufficient caspase-8 activation . MoreoveIn addition to well-established TRAIL-induced apoptotic signaling, increasing evidence has unraveled novel mechanisms that either promote or inhibit TRAIL-induced apoptosis in GBM , offerinSince resisting apoptosis is a hallmark of human cancers, it may not be so surprising that few mechanisms are dedicated to promoting TRAIL-induced apoptosis in GBM. One study has reported that nuclear factor-κB (NF-κB) activation via overexpression of constitutively active IκB kinase complex (IKK) β (IKK-EE) significantly increases TRAIL-mediated apoptosis. Conversely, the inhibition of NF-κB by overexpression of the dominant-negative IκBα superrepressor (IκBα-SR) decreases TRAIL-induced apoptosis in GBM cells . MechaniThe X-linked inhibitor of apoptosis (XIAP) exerts anti-apoptotic functions via inhibiting the activation of caspases. As expected, inhibition of XIAP with Embelin enhances TRAIL-mediated apoptosis in GBM cells . A genomSimilar to most tumors, GBM tumors display a range of TRAIL sensitivity, but the majority harbor innate resistance to TRAIL-induced apoptosis, which prevents the clinical application of TRAIL . The mecA recent study reveals that genetic or pharmacological inhibition of karyopherin β1 (KPNB1) potentiates TRAIL-induced apoptosis selectively in GBM cells partially through accelerating caspase-8 cleavage via downregulating c-FLIP , proposiAs described, caspase-8 is a master regulator in TRAIL-induced apoptosis. Understandably, genomic alterations in CASP8 contribute to TRAIL resistance in GBM cells . In GBM-Except canonical regulators of caspase-8 activation during TRAIL-induced apoptotic signaling, recent studies have unveiled some previously unprecedented regulators of caspase-8 activation in GBM and inspired new insights for designing TRAIL-based therapy. For example, it has been proven that the knockdown of PIM kinases decreases phosphorylation of p62 and sensitizes TRAIL-induced apoptosis via enhanced caspase-8 recruitment to and activation at the DISC; in line with this, p62 ablation facilitates TRAIL-induced caspase-8 activation, revealing an inhibitory role of p62 in TRAIL-mediated apoptosis in GBM . Thus, PAnother example is A20 ubiquitin ligase, which was illustrated to mediate ubiquitination of RIP1, through which it inhibits caspase-8 dimerization and cleavage and TRAIL-induced apoptosis in tumor-initiating cells isolated from GBM patients . A20 is DISC formation is a key upstream event in TRAIL-induced apoptotic signaling. A recent insight into this mechanism is provided by a study manifesting that in TRAIL-sensitive GBM cells, TRAIL-R2 is the only consistently expressed functional receptor that, upon TRAIL binding, homotrimerizes and recruits FADD and caspase-8 for assembling the DISC in the lipid rafts of plasma membrane, wherein caspase-8 is cleaved and initiates apoptosis. However, in non-raft fractions of plasma membrane of TRAIL-resistant GBM cells, TRAIL-R2-mediated DISC is modified by RIP, c-FLIP, and PED/PEA-15, resulting in caspase-8 cleavage inhibition. On the contrary, silencing RIP, c-FLIP, or PED/PEA-15 redistributes the DISC from non-rafts to lipid rafts, thereby eliminating caspase-8 cleavage inhibition and TRAIL resistance . Given tmiRNAs also attract attention as candidates to lower TRAIL resistance. miR-21 is elevated in GBM and its knockdown increases apoptotic activity. It has been found that the combined suppression of miR-21 with a secretable TRAIL causes an increase in synergistic apoptosis in human GBM cells in vitro and in vivo . In spitThe alteration of tumor suppressor p53 is the most common molecular abnormality in GBM, which has been demonstrated in 60–70% cases of GBM patients . The relAn interaction was reported between Beclin 1, a key regulator of autophagy, and survivin that belongs to a member of the anti-apoptotic protein family. Further, Beclin 1 knockdown sensitized GBM cells to TRAIL-induced apoptosis, which was antagonized in the presence of survivin introduction, suggesting that Beclin 1 enhances TRAIL resistance in GBM cells through maintaining the level of survivin . These rMost chemotherapies are designed to destroy cancer cells by inducing DNA damage, which may be repaired by the intrinsic DNA damage response machinery, and as a result, cancer cells will survive . RemarkaTRAIL pro-apoptotic signaling is subjected to epigenetic regulation . In GBM During cellular stress, the eukaryotic initiation factor 5B (eIF5B) promotes the translation of mRNA encoding the anti-apoptotic factor XIAP . A recenTo maximize the potential of TRAIL in treating GBM, most research has focused on developing methods to sensitize GBM to TRAIL treatment through two major directions: increasing TRAIL bioavailability via constructing efficient TRAIL delivery system and enhancing TRAIL tumoricidal activity through combining sensitizing drugs .The clinical application of TRAIL is largely hindered by its short serum half-life and lack of efficient delivery approaches. In recent years, developing nanoparticles as carriers in gene therapy has been considered as an effective approach to increase TRAIL delivery to tumors as transfected cells will specifically secrete TRAIL into the tumor microenvironment. However, another huge obstacle for gene delivery to GBM in the brain is to cross the blood–brain barrier, and most delivery vehicles fail to generate high gene transfection efficiency in vivo . By deveMost chemotherapeutic drugs kill cancer cells predominantly by triggering the apoptotic program. Increasing evidence has shown that several chemotherapeutic drugs treated in combination with TRAIL can result in the reversal of GBM resistance to TRAIL-mediated apoptosis. For example, combined TRAIL plus paclitaxel have cooperative anti-GBM efficacy in vivo, particularly with no discernable toxicity to normal tissue . AnalogiCompared with chemotherapeutic drugs, efficacious synergistic effects of non-chemotherapeutic agents and TRAIL may be uncommon. However, evidence has indicated that lovastatin, a lipid-reducing drug, enhances TRAIL-induced GBM cell apoptosis synergistically . AnotherAside from the abovementioned inhibitors of the Akt-mTOR-S6K1 pathway, KPNB1, and PIM kinases, a large growing body of studies have also shown that a variety of inhibitors that do not belong to therapeutic drugs but sensitize GBM to TRAIL-induced apoptosis. For instance, histone deacetylase inhibitors (HDACIs), such as MS275, suberoylanilide hydroxamic acid and valproic acid, sensitize GBM cells to TRAIL-induced apoptosis in vitro and in vivo through c-myc-downregulated c-FLIP , suggestOwing to excusive the tumoricidal property revealed by a large amount of pre-clinical and clinical studies, it is believed that targeting the TRAIL/TRAIL-R1/R2 axis holds great promise to be harnessed in combinatorial therapies for treating cancers, including GBM, a deadly cancer without efficacious therapeutic options. However, employing either recombinant human TRAIL or agonist antibodies against TRAIL-R1/2 to reactive the extrinsic apoptosis pathway in cancer cells for cancer therapy has yielded undesirable outcomes in previous clinical trials, casting a shadow over the future clinical applications of this strategy. Currently, seeking methods to overcome TRAIL resistance for enhancing TRAIL efficacy is a research focus. As with many cancers, the majority of GBM tumors are generally resistant to TRAIL-induced apoptosis largely due to several aberrations in genetics that result in low or loss of expression of apoptotic genes and simultaneous overexpression of anti-apoptotic genes, which comprise the TRAIL-induced apoptotic signaling pathway. Attempts to understand the mechanisms of TRAIL-induced apoptotic signaling in GBM have unraveled novel regulators in promoting or inhibiting TRAIL resistance, mainly through modulating the levels or activation of TRAIL-R1/R2, c-FLIP, caspase-8, and DISC. These studies provide novel therapeutic targets that can potentially interfere the resistance mechanisms to overcome GBM resistance to TRAIL-based therapies. Another strategy to improve the therapeutic efficacy of TRAIL and sensitize TRAIL resistance in GBM is through developing more effective approaches of delivering a sufficient amount of TRAIL to tumor lesions in the brain. Recent studies have shown the advantages of nanoparticles in increasing the delivery efficiency of TRAIL to sensitize GBM to TRAIL treatment. In addition, sensitizing GBM to TRAIL-induced apoptosis has proven effective by multiple preclinical studies through the combinatorial treatment of TRAIL with other agents, such as some commonly used chemotherapeutic and non-chemotherapeutic drugs and synthetic inhibitors. According to these progresses in overcoming TRAIL resistance in GBM, we expect more clinical trials will participate to test the therapeutic potency and safety of TRAIL-based combination modalities in GBM treatment. Finally, however, despite the abovementioned advances, how GBM tumors acquire TRAIL resistance is still not fully understood, and the mechanisms underlying synergistic effect of TRAIL and chemotherapeutic or non-chemotherapeutic drugs remain largely unexploited. Addressing these challenges is needed to overcome TRAIL resistance for maximizing the therapeutic potential of TRAIL in treating GBM.


RNF34 was localized in the cytoplasm and endosomes, where RFFL was equally colocalized. RNF34 ablation increased the PM density as well as the mature form of ∆F508-CFTR rescued at low temperatures. RFFL ablation, with the exception of RNF34 ablation, increased the functional PM expression of ∆F508-CFTR upon a triple combination of CFTR modulators (Trikafta) treatment by inhibiting the K63-linked polyubiquitination. Interestingly, simultaneous ablation of RNF34 and RFFL dramatically increased the functional PM ∆F508-CFTR by inhibiting the ubiquitination in the post-Golgi compartments. The CFTR-NLuc assay demonstrates that simultaneous ablation of RNF34 and RFFL dramatically inhibits the degradation of mature ∆F508-CFTR after Trikafta treatment. Therefore, these results suggest that RNF34 plays a crucial role in the CFTR periQC, especially when there is insufficient RFFL. We propose that simultaneous inhibition of RFFL and RNF34 may improve the efficacy of CFTR modulators.The peripheral protein quality control (periQC) system eliminates the conformationally defective cystic fibrosis transmembrane conductance regulator (CFTR), including ∆F508-CFTR, from the plasma membrane (PM) and limits the efficacy of pharmacological therapy for cystic fibrosis (CF). The ubiquitin (Ub) ligase RFFL is responsible for the chaperone-independent ubiquitination and lysosomal degradation of CFTR in the periQC. Here, we report that the Ub ligase RNF34 participates in the CFTR periQC in parallel to RFFL. An  The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel expressed on the plasma membrane (PM) of epithelial cells , where iRNF34 (CARP1) is a caspase 8/10-associated Ub ligase with high homology to RFFL . Both RNHere, we report that RNF34 functions for the periQC of CFTR. This is shown by RNF34 selectively interacting with unfolded proteins and directly facilitating their ubiquitination. Like RFFL, RNF34 is localized in endosomal compartments. Discussed in further detail, RNF34 ablation minimally affected the ∆F508-CFTR upon Trikafta treatment. However, simultaneous ablation of RNF34 and RFFL dramatically increased the functional expression of ∆F508-CFTR after Trikafta treatment by inhibiting the K63-linked polyubiquitination and degradation in the post-Golgi compartments. Thus, RNF34 determines the CFTR periQC, and simultaneous inhibition of RFFL and RNF34 may further improve the efficacy of CFTR modulators.The Materials and Methods are provided in the Supplementary material for this paper.E. coli to characterize the function  on the ∆F508-CFTR rescued by low-temperature incubation. Similar to RFFL, RNF34 KD significantly increased the PM levels of low-temperature rescued ∆F508-CFTR-HiBiT in BEAS2-B cells . In suppUnderstanding that the periQC system limits the efficacy of CFTR modulators ; next, wTo quantitatively measure the effect of RNF34 and RFFL on CFTR degradation, we have developed an assay which accurately quantifies the kinetic CFTR degradation in live cells. NanoLuc (NLuc) was fused with CFTR in the C-terminus (CFTR-NLuc) and stably expressed in BEAS-2B tet-on cells. As expected, WT CFTR-NLuc was expressed in mature and immature forms while ∆F508-CFTR-NLuc was only expressed in its immature form . The NLuIn vitro experiments revealed that, like RFFL, RNF34 directly interacts with unfolded NBD1 and selectively ubiquitinates the unfolded NBD1 and Luc. Cellular localization analysis demonstrated that RNF34 was colocalized with RFFL in the endosomal compartments consistent with their proximal localization (in vitro, RNF34 may ubiquitinate not only the unfolded CFTR but also other unfolded proteins located in the endosomal compartments. Although the involvement of the periQC mechanism remains unclear, it has been reported that endosomal RNF34 facilitates the GABAA receptor degradation by ubiquitinating the γ2 subunit (This study demonstrates that RNF34 is involved in the CFTR periQC. lization . Moreovelization . Given t subunit .In this study, we found that the RNF34 KD increased the PM expression and mature form of low-temperature rescued ∆F508-CFTRs, a similar phenotype observed in the RFFL KD. In comparison, unlike RFFL, RNF34 ablation failed to increase the functional PM expression of ∆F508-CFTR upon Trikafta treatment. The Trikafta treatment seems to completely correct the ∆F508-CFTR misfolding, including NBD1 and the NBD1-MSD interface . Low-temWe found that the mature ∆F508-CFTR induced by Trikafta treatment was still ubiquitinated and less stable than the WT CFTR. These results are consistent with a recent study that reported the CFTR phenotypes upon treatment of VX-661/VX-445 . AdditioRNF34 ablation failed but synergistically increased the PM levels of ∆F508-CFTR rescued by Trikafta under the RFFL KO. This result suggests that RNF34 and RFFL act through different mechanisms. Consistent with these results, the mature ∆F508-CFTR poly-ubiquitination was equally synergistically inhibited by the RFFL/RNF34 DKO. The CFTR-NLuc assay, which quantifies the kinetic CFTR degradation in live cells, clearly demonstrated that RNF34 was not essential for the unfolded CFTR degradation in the post-Golgi compartments when RFFL is active. However, when RFFL is insufficient, RNF34 plays a significant role in the periQC machinery that is crucial for CFTR degradation. We cannot exclude the possibility that RNF34 participates in the ERQC of CFTR as RNF34 ablation increases the immature ∆F508-CFTR. Future studies will be required to clarify this possibility.In summary, we identify RNF34 as a new component of the CFTR periQC machinery. RNF34 determines the ubiquitination of unfolded CFTR in the endosomal compartments in parallel to RFFL. We propose that simultaneous inhibition of RFFL and RNF34 may facilitate the functional PM expression of ∆F508-CFTR even upon Trikafta treatment. However, future studies will be required to validate the impact on ∆F508-CFTR in CF-HBE cells where both RFFL  and RNF3


All methods were able to detect prior infection with COVID-19, albeit with different relative sensitivities. The VNT and SARS-CoV-2-N ELISA methods showed a strong correlation yet provided increased detection rates when used in combination. A pVNT correlated strongly with SARS-CoV-2 VNT and was able to effectively discriminate SARS-CoV-2 antibody positive and negative serum with the same efficiency as the VNT. Moreover, the pVNT was performed with the same level of discrimination across multiple separate institutions. Therefore, the pVNT is a sensitive, specific, and reproducible lower biosafety level alternative to VNT for detecting SARS-CoV-2 antibodies for diagnostic and research applications. Our data illustrate the potential utility of applying VNT or pVNT and ELISA antibody tests in parallel to enhance the sensitivity of exposure to infection.SARS-CoV-2 virus was first detected in late 2019 and circulated globally, causing COVID-19, which is characterised by sub-clinical to severe disease in humans. Here, we investigate the serological antibody responses to SARS-CoV-2 infection during acute and convalescent infection using a cohort of (i) COVID-19 patients admitted to hospital, (ii) healthy individuals who had experienced ‘COVID-19 like-illness’, and (iii) a cohort of healthy individuals prior to the emergence of SARS-CoV-2. We compare SARS-CoV-2 specific antibody detection rates from four different serological methods, virus neutralisation test (VNT), ID Screen To investigate the reproducibility of pVNT for diagnostic activities, the pVNT was analysed using a SARS-CoV-2 pseudotype virus generated at three different institutions (APHA (institution 1), The University of Kent (institution 2) and The University of Sussex (institution 3)), with all analysis carried out at APHA.To further analyse the use of pVNT as an alternative to traditional VNT, a panel of serum from the UHS+ve COVID-19 confirmed patients was selected based on the titres from VNT, NP-ELISA, and Whole Antigen ELISA. The panel consisted of 23 serum samples, including those with (i) high VNT titres . All (100%) of the high, medium, and low VNT titre sera  were identified as containing neutralising antibody by pVNT using the pseudotype virus generated at all three institutions C–E. A toAntibody detection and quantification methods are critical to assessing asymptomatic infection, post clinical convalescence, and post-vaccine responses to COVID-19. Population-based studies have given an intriguing insight into potential levels of herd immunity across asymptomatic and symptomatic individuals. However, what the outputs mean regarding population level protection is hard to define. Several population-level studies have attempted to use serological outputs to indicate the level of virus spread, both in the presence and absence of clinical disease, as well as serological titres following severe infection and convalescence. A report detailing serological responses in 61,000 individuals from Spain demonstrated that 5% of sampled individuals had antibodies specific for the spike and nucleoproteins of SARS-CoV-2 . InteresHere, we have assessed three cohorts utilising three different assays to evaluate the relevance of biological testing for antibody responses and, for a small cohort, the longevity of the antibody response. Previous studies have demonstrated that ELISA outputs can correlate quite well with neutralising antibody responses ,36 and dOur data illustrate the potential usefulness of applying VNT and ELISA antibody tests in parallel to enhance the sensitivity of infection detection. This enhanced sensitivity being largely due to ELISA tests identifying infected individuals before neutralising antibodies can be detected and also recognising that VNT can identify infected persons that test negative by ELISA. The level of enhanced sensitivity also depends upon the ELISA test used. We generated a relatively crude Whole Antigen ELISA using BPL-inactivated virus-infected whole Vero E6 culture extract and compared it with a commercial ELISA test, the SARS-COV-2-N ELISA kit (IDvet). Both ELISAs had an equally high specificity of 97.8% and moderate sensitivity that was broadly equivalent to that of VNT testing. The parallel VNT and ELISA testing of 103 COVID patients showed that proportionally more patients were detected using the Whole Antigen ELISA in parallel with VNT (75%), compared to the SARS-COV-2-N ELISA in parallel with VNT (65%), both ELISA tests enhancing the sensitivity of VNT alone (53%).Interestingly, SARS-COV-2-N ELISA readouts correlated much better with VNT readouts than did the Whole Antigen ELISA. Whether this was due to a lower sensitivity of the SARS-COV-2-N ELISA that meant that SARS-COV-2-N-positives were more likely to be VNT-positive is possible; the ROC analysis suggested a lower SARS-COV-2-N sensitivity compared to the Whole Antigen ELISA, and sequential samples from COVID-19 symptomatic individuals assessed by SARS-COV-2-N ELISA also suggested a loss of sensitivity over time that was not observed using the Whole Antigen ELISA. The slightly higher sensitivity of the Whole Antigen ELISA compared to the SARS-COV-2-N ELISA may have been due to the nature of the crude antigen used, providing a broader target for polyclonal antibodies in serum samples.The Whole Antigen ELISA approach allows for flexibility in that new virus strains can be grown and used as antigen without exactly knowing what the antigenic alterations are. This is especially important considering the emergence of new SARS-CoV-2 viral variants that may exhibit different antigenic properties. In addition, this method lends itself to comparative testing alongside seasonal and other coronavirus crude antigens. However, for a wider diagnostic use, virus cultures would need to be grown to a much higher titre than we achieved in this study, to allow for a higher antigen dilution for ELISA—and each antigen batch to go further. Recombinant antigens avoid this issue, but in our experience, both S1 and Np proteins tested, despite showing positive responses in our sequential serum samples, had overall poor sensitivity in detecting antibody-positives among the larger PCR+ cohort.ELISA tests are relatively rapid tests compared to VNT, taking hours as opposed to days to set up and retrieve results, but ELISA results, while useful for diagnosis, may not define whether the antibody response detected is protective , whereas VNT is recognised as a gold standard test for the positive identification of biologically active neutralising antibody. Therefore, both ELISA and VNT tests have positive credentials to bring to diagnostics.The use of pseudotype viruses to probe the antibody responses of containment level 3 viruses has been used previously on a number of occasions ,14,15. H


The global pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is an unprecedented threat to the human health. A close association of the digestive tract is implied by the high frequency of gastrointestinal syndromes among COVID-19 patients. A better understanding of the role of intestinal microenvironment in COVID-19 immunopathology will be helpful to improve the control of COVID-19 associated morbidity and mortality. This review summarizes the immune responses associated with the severity of COVID-19, the current evidence of SARS-CoV-2 intestinal tropism, and the potential involvement of gut microenvironment in COVID-19 severity. Additionally, we discuss the therapeutic potential of probiotics as an alternative medicine to prevent or alleviate severe COVID-19 outcome. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel zoonotic coronavirus with human-to-human transmission route, and its recent emergence and rapid spread has resulted in a global pandemic of coronavirus disease 2019 (COVID-19) . The comMultiple clinical analyses have shown that there are high risks of severe COVID-19 in individuals with pre-existing comorbidities, such as obesity, diabetes and cardiovascular diseases . NotablyAccording to the studies investigating the gut microbiota dysbiosis among many COVID-19 patients, as well as the “gut-lung axis” phenomena demonstrated in animal models infected by various respiratory virus, probiotics have been proposed as an alternative strategy to prevent SARS-CoV-2 infection and ameliorate COVID-19 symptoms . In thisThe majority of COVID-19 patients showed mild to moderate symptoms, e.g., fever, cough, fatigue and shortness of breath. Some of the infected people with pneumonia suddenly deteriorated into severe and even fatal respiratory conditions . Reducedin vitro cell culture assays and ex vivo infection of primary cells indicate that SARS-CoV-2 replication is sensitive to IFN-I, whereas there may be a virus-mediated IFN antagonism to attenuate antiviral response by host cells. Despite the lack of IFN expression, those respiratory cell lines and NHBE cells showed strong chemotactic and inflammatory responses to SARS-coV-2 infection. Consistently, COVID-19 patients exhibited low levels of IFN-I and -III and high chemokine signatures (IFIT and IFITM genes), proinflammatory ISGs were uniquely and predominantly elevated in SARS-CoV-2 infected patients, as compared to CAP patients. Although a robust induction of ISGs at mRNA was observed, the protein abundance of IFNs in BALF cells and sera of COVID-19 patients were quite subtle, below the levels of detection sensitivities of conventional ELISA (Interferon (IFN) response to defend SARS-CoV-2 invasion was reported to be impaired with few production of IFNs while elevated expression of IFN-stimulated genes (ISGs), leading to the deficient control of early viral replication but promotion of the inflammatory responses . Upon ingnatures . Comparegnatures . Among tal ELISA . Howeveral ELISA . In CITEal ELISA . Given tal ELISA .+ T cells in the BALF were highly expanded and functionally competent with a tissue-resident memory T cell gene signature, while minimal infiltration of inflammatory monocytes into the BALF was observed  in PBMCs of COVID-19 patients than those of healthy individuals, and the peripheral pDCs of COVID-19 individuals showed impaired IFN-producing capacity. The level of expression of phosphorylated-ribosomal protein S6 (pS6), a classic target of mTOR activation, was decreased in pDCs, which may in part explain their impaired capacity of producing IFN-α, since mTOR signaling is critical for IFN-α production in pDCs downstream of Toll-like receptor (TLR) stimulation  subsets in the blood . Furthermulation . In ex vpatients .IL1RN and IL1B, were also significantly upregulated in COVID-19 patients   . In an iry cells . The cytry cells . These oCompared with moderate COVID-19 patients, severe COVID-19 patients frequently had hypoalbuminemia and relatively high levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), high-sensitivity C-reactive protein (hsCRP), ferritin, D-dimer and procalcitonin in the blood, suggestive of a significantly increased systemic inflammation . The hyp+ T cells and higher proportion of proliferating T cells within six major clusters of T and NK lymphocytes, than those from moderate COVID-19. Moreover, CD8+ T cells in BALFs from severely infected patients were less expanded than those in BALFs from moderate COVID-19 cases, indicating less cytotoxic T cell responses towards SARS-CoV-2 in severe COVID-19 than that with moderate infection  as well as higher abundance of opportunistic pathogens , indicating an intimate connection between uncontrolled viral infection in the intestine with gut microflora dysbiosis  had a significantly reduced risk of developing respiratory failure by around 87.5%, in comparison to the group without additional bacteriotherapy   . It was ne (DAT) . At the patients . Besidesfunction . To elimfunction .


The purpose of this study was to determine whether fear of re-injury is manifested in joint kinematics and muscle activation patterns during landings of a standardized rebound side-hop (SRSH), or in patient-reported outcome measures (PROMs), among individuals with anterior cruciate ligament reconstruction (ACLR).In this cross-sectional observational study, 38 individuals within 2 years post-ACLR were grouped into HIGH-FEAR  or LOW-FEAR  based on a discriminating question . These individuals and 39 asymptomatic controls performed the SRSH. Three-dimensional motion recordings were used to calculate trunk, hip, and knee joint angles at initial contact and range of respective joint motion during landing. Surface electromyography registered mean amplitudes and co-contraction indexes for thigh muscles during pre-activation (50 ms) and landing phases. PROMs of knee function, knee health, and physical activity were also analyzed.The HIGH-FEAR and LOW-FEAR classification was corroborated by distinct Tampa Scale of Kinesiophobia-17 total and subscale scores and revealed distinguishable muscle activation patterns. HIGH-FEAR demonstrated higher biceps femoris electromyography amplitude and higher anterior-posterior co-contraction index during landing than both LOW-FEAR and controls. However, there were no fear-related differences for kinematics or PROMs. Instead, both ACLR subgroups showed different kinematics at initial contact to controls; HIGH-FEAR with more trunk, hip, and knee flexion, and LOW-FEAR with more hip and knee flexion.Individuals with ACLR who had high fear of re-injury seem to have adopted a protective strategy with higher muscular activation patterns, presumably to stabilize the knee joint, compared with individuals with low fear of re-injury and controls. SRSH landing kinematics or knee-related PROMs may not be as sensitive to fear of re-injury.Fear of reinjury following anterior cruciate ligament injury should be evaluated as an independent psychological outcome throughout rehabilitation after ACLR for improved return to sport transition.If you have an anterior cruciate ligament injury treated with reconstructive surgery, you might have a high fear of reinjury, and that can change how you activate the muscles around your knee. Your physical therapist can do a simple screening test in addition to functional tests to help reduce your fear and improve your treatment outcomes. Therefore, individuals with a high fear of re-injury might adopt specific movement strategies as an attempt to increase knee control. Such strategies are likely characterized by stiff movements with a restricted range of motion,However, the existing research regarding associations between kinesiophobia and movement patterns among individuals with ACLR is currently limited to 2 studies, investigating gaitWe aimed to determine whether fear of re-injury was manifested in joint kinematics and muscle activation patterns during single-leg standardized rebound side-hop (SRSH) landings in individuals with ACLR with high fear compared with low fear and compared with asymptomatic non-injured controls. We also aimed to evaluate the subjective impact of fear of re-injury by comparing patient-reported outcome measures (PROMs) of knee function, knee health, and physical activity between the groups. We hypothesized that individuals with ACLR with higher fear would display a more defensive movement strategy with less joint motion and greater muscle activity in preparation for and during landing and accompanied by poorer PROMs than lower fear individuals with ACLR and asymptomatic controls. This study was a cross-sectional observational study approved by the regional ethical review board in Umeå, Sweden (Dnr. 2015/67-31). Before participating, all participants provided written informed consent and agreed with the declaration of Helsinki. Thirty-eight physically active individuals with ACLR within the last 2 years, stratified into either HIGH-FEAR or LOW-FEAR subgroups (see below), and 39 asymptomatic controls (CTRL) were included . ParticiAll participants filled out the PROMs before the hop testing and in the following order: 2000 International Knee Documentation Committee subjective knee form, Knee injury and Osteoarthritis Outcome Score, Lysholm Scale, International Physical Activity Questionnaire, Tampa Scale for Kinesiophobia-17 , and Tegner Activity Scale . ,,The TSK-17 is a 17-item scale commonly used to estimate movement-related fear.The SRSH is a recently published single-leg hop test developed for biomechanical evaluation.Kinematics were registered using motion capture  and time-synchronized force plates . A 6 degree-of-freedom model was constructed from 56 passive spherical markers attached with double-coated adhesive tape on the skin at anatomical landmarks, as previously described.Qualisys Track Manager  and Visual3D  were used for data processing and calculation of kinematic outcome measures. Marker data were filtered with a critically damped digital filter with a cut-off frequency of 15 Hz before calculating the dependent variables. The Cardan rotation sequence XYZ was used .Surface EMG was sampled at 1680 Hz  using silver-silver chloride, pre-gelled bipolar surface electrodes  from biceps femoris, semitendinosus, vastus medialis, and vastus lateralis muscles. After shaving, scrubbing, and cleaning the skin surface with isopropyl alcohol, electrodes were placed over the muscle belly at an inter-electrode distance of 20 mm following SENIAM guidelines.,,, average measures form) of 0.90 to 0.99 for trial-to-trial peak values for all muscles in all groups. EMG outcomes investigated were mean amplitudes for each muscle and co-contraction indexes (CCIs) for anterior-posterior and medial-lateral muscles, both during a 50-millisecond pre-activation phase before initial contact (force plates registered loading >20N) and during the landing phase ,EMG raw data were band-pass filtered (20 Hz–500 Hz) and applied a root mean square moving window of 20 millisecondslower/EMGhigher)*(EMGlower+EMGhigher),CCI = (EMGlower is the level of activity in the less active of flexor vs extensor (anterior-posterior CCI) or medial vs lateral muscles . where EMGFor 2 individuals in HIGH-FEAR, 1 individual in LOW-FEAR, and 3 individuals in CTRL, there were technical errors for EMG data for 1, 2, or all 4 muscles. These data were excluded from the EMG analyses. Data for kinematics and PROMs were analyzed for all individuals. P < .05 determining statistical significance. The ACL-reconstructed legs in HIGH-FEAR and LOW-FEAR were compared with the non-dominant legs among CTRL  for all between-group analyses in accordance with previous biomechanical studies from our research group.The funders played no role in the design, conduct, or reporting of this study. F = 3.7, P = .030, ES = 0.09 [small]), hip flexion , and knee flexion , after controlling for sex. Specifically, HIGH-FEAR had more trunk flexion at initial contact than CTRL (P = .031), and both HIGH-FEAR and LOW-FEAR had more hip flexion  and knee flexion (P < .001 for both) at initial contact than CTRL. No group differences were found for the other trunk, hip, or knee kinematic outcomes at initial contact or during landing   . There wF = 14.6, P < .001, ES = 0.29 [large]) and anterior-posterior CCI  during landing. These outcomes indicated fear-related differences between the 2 ACLR subgroups. HIGH-FEAR had higher mean activation for biceps femoris and higher anterior-posterior CCI compared with both LOW-FEAR  and with CTRL  during landing   .P = .003) and the TSK-17 subscale Activity Avoidance  both with (P = .001) and without (P = .038) Q9 that divided the groups . Both HIGH-FEAR and LOW-FEAR had lower ratings for all PROMs than CTRL , except for the Knee injury and Osteoarthritis Outcome Score Subscale Activities of Daily Living, where LOW-FEAR and CTRL had similar ratings (P = .240). Tegner Activity Scale ratings and International Physical Activity Questionnaire scores were similar between groups . Both HIGH-FEAR and LOW-FEAR groups reported lower current Tegner ratings than their pre-injury ratings . The classification into HIGH-FEAR and LOW-FEAR using Q9 was corroborated by higher median scores for HIGH-FEAR for the TSK-17 total score  may additionally have increased the tibiofemoral compression forces, considering the function of the knee joint to act as a damper during impact. Consequently, our results may suggest that individuals with high fear of re-injury might expose themselves to a greater risk of accelerated knee joint osteoarthritis in the long term.Contrary to our hypothesis and the significant fear-related group differences for muscle activation patterns during the landing phase, we found no fear-related differences for any kinematic outcome or muscle activations during the 50-millisecond pre-activation phase. However, the higher reactive biceps femoris muscle activation and anterior-posterior CCI during landing in HIGH-FEAR compared with LOW-FEAR and controls suggest a knee protective strategy to stabilize the joint.,The only comparable study with ours that we know of that analyzed a sport-mimicking maneuver in relation to fear of re-injury (or kinesiophobia) analyzed the 2-legged drop-vertical jump test.,,,,The lack of significant fear-related group differences for any of the PROMs (except those of the TSK-17) suggests that fear of re-injury is an independent outcome that does not necessarily relate to self-reported knee function and health among individuals with ACLR within the first 2 years post-surgery. However, this interpretation may only be valid for certain knee-demanding sport levels because both HIGH-FEAR and LOW-FEAR had lower current Tegner ratings  compared with their pre-injury Tegner ratings (both median 8). Studies with larger sample sizes than ours (n = 135–1362),,,Individuals with ACLR displayed different kinematics in preparation for SRSH landing compared with asymptomatic controls. The increased flexion angles observed at initial contact for both ACLR subgroups  were possibly a preparatory strategy aimed to maintain knee control during landing. A flexion strategy results in a lower center of mass, which in turn assists in completing the task. Another reason for greater knee flexion angles at initial contact may be to protect the graft because it results in smaller patellar tendon insertion angles, greater hamstrings insertion angles, lower anterior tibial shear force, and lower tibiofemoral peak compression forces, all of which are considered to decrease ACL strain.Our findings link subjectively reported fear of re-injury to muscle activation patterns as suggestive of a knee-protective strategy. Fear of re-injury, evaluated in this case by a single question, constitutes a simple clinical screening tool that should be considered in relation to functional assessment  and biomechanical evaluation throughout rehabilitation for individuals with ACLR to facilitate the return to sport transition and to decrease the risk of a secondary injury. Our results also strengthen a recent consensus statement on sport-related injuries that recommends a holistic approach to optimize recovery,There is no gold standard measure of fear of re-injury following ACL rupture. The single question from TSK-17 that we used is not ACL specific, but the question  was answered considering the ACL injury. Our choice of only using the Q9 from TSK-17 to classify fear of re-injury should be considered when comparing our results with similar studies that used total scores for different TSK versions. Although the TSK-17 has been adapted to fit knee-injured patients better,Our findings show that individuals with ACLR with higher self-reported fear of re-injury exhibit atypical muscle activation patterns when performing single-leg SRSH. Fear of re-injury was associated with higher biceps femoris muscle activation and higher anterior-posterior co-contraction during landing yet poorly associated with anticipatory muscle activation patterns, kinematics, and self-reported knee function, knee health, and physical activity. Further, individuals with ACLR adapted, irrespective of fear of re-injury, a whole-body flexion strategy to prepare for landing compared with asymptomatic controls. Our study highlights that those individuals with ACLR should be evaluated for fear of re-injury throughout rehabilitation as a complement to functional assessment and biomechanical evaluation.PTJ-2020-0785_R1_SupplementaryVideo_pzab218Click here for additional data file.Supplementary_video_SRSH_pzab218Click here for additional data file.


MicroRNA (miRNA) expression in the brain is altered in neurodegenerative diseases. Recent studies demonstrated that selected miRNAs conventionally regulating gene expression at the post-transcriptional level can act extracellularly as signaling molecules. The identity of miRNA species serving as membrane receptor ligands involved in neuronal apoptosis in the central nervous system (CNS), as well as the miRNAs’ sequence and structure required for this mode of action remained largely unresolved.Using a microarray-based screening approach we analyzed apoptotic cortical neurons of C56BL/6 mice and their supernatant with respect to alterations in miRNA expression/presence. HEK-Blue Toll-like receptor (TLR) 7/8 reporter cells, primary microglia and macrophages derived from human and mouse were employed to test the potential of the identified miRNAs released from apoptotic neurons to serve as signaling molecules for the RNA-sensing receptors. Biophysical and bioinformatical approaches, as well as immunoassays and sequential microscopy were used to analyze the interaction between candidate miRNA and TLR. Immunocytochemical and -histochemical analyses of murine CNS cultures and adult mice intrathecally injected with miRNAs, respectively, were performed to evaluate the impact of miRNA-induced TLR activation on neuronal survival and microglial activation.We identified a specific pattern of miRNAs released from apoptotic cortical neurons that activate TLR7 and/or TLR8, depending on sequence and species. Exposure of microglia and macrophages to certain miRNA classes released from apoptotic neurons resulted in the sequence-specific production of distinct cytokines/chemokines and increased phagocytic activity. Out of those miRNAs miR-100-5p and miR-298-5p, which have consistently been linked to neurodegenerative diseases, entered microglia, located to their endosomes, and directly bound to human TLR8. The miRNA-TLR interaction required novel sequence features, but no specific structure formation of mature miRNA. As a consequence of miR-100-5p- and miR-298-5p-induced TLR activation, cortical neurons underwent cell-autonomous apoptosis. Presence of miR-100-5p and miR-298-5p in cerebrospinal fluid led to neurodegeneration and microglial accumulation in the murine cerebral cortex through TLR7 signaling.Our data demonstrate that specific miRNAs are released from apoptotic cortical neurons, serve as endogenous TLR7/8 ligands, and thereby trigger further neuronal apoptosis in the CNS. Our findings underline the recently discovered role of miRNAs as extracellular signaling molecules, particularly in the context of neurodegeneration.The online version contains supplementary material available at 10.1186/s13024-021-00498-5. Neurodegenerative disorders are characterized by progressive neuronal injury and loss. Innate immune receptors such as Toll-like receptors (TLRs) can contribute to such central nervous system (CNS) injury by triggering an inflammatory response, but also by mediating cell-autonomous damage . To datelet-7b miRNA was detected extracellularly in the injured CNS .


The emergence of various novel therapies over the last decade has changed the therapeutic landscape for multiple myeloma. While the clinical outcomes have improved significantly, the disease remains incurable, typically in patients with relapsed and refractory disease. Chimeric antigen receptor (CAR) T-cell therapies have achieved remarkable clinical success in B-cell malignancies. This scope of research has more recently been extended to the field of myeloma. While B-cell maturation antigen (BCMA) is currently the most well-studied CAR T antigen target in this disease, many other antigens are also undergoing intensive investigations. Some studies have shown encouraging results, whereas some others have demonstrated unfavorable results due to reasons such as toxicity and lack of clinical efficacy. Herein, we provide an overview of CAR T-cell therapies in myeloma, highlighted what has been achieved over the past decade, including the latest updates from ASH 2020 and discussed some of the challenges faced. Considering the current hits and misses of CAR T therapies, we provide a comprehensive analysis on the current manufacturing technologies, and deliberate on the future of CAR T-cell domain in MM. While these new therapeutics have led to better disease control, myeloma remains largely incurable, and high-risk patients do not benefit much from this armamentarium of therapies7. Drug resistance is inevitable and disease relapse remains a great clinical challenge.Multiple myeloma (MM), which accounts for 10% of blood cancers, is a malignancy of plasma cells originating in the bone marrow. Increased understanding of disease biology over the years has led to parallel improvement in treatment modalities, evidenced by the emergence of the novel therapeutics such as proteasome inhibitors , immunomodulatory drugs  and U.S. Food and Drug Administration (FDA)-approved monoclonal antibodies (daratumumab and elotuzumab)9. Chimeric antigen receptor (CAR) T-cell therapy is one of the rapidly emerging and highly promising immunotherapeutic options that has shown unprecedented results in B-cell malignancies13. It prolongs patients’ survival and remission, even for some whom standard treatments have failed14.Immunotherapy, which was previously deemed a favorable concept, has now evolved into a practical cancer treatment and its progress in the past decade has revolutionized the cancer therapy landscape16  for the treatment of acute lymphoblastic leukemia (ALL) and axicabtagene ciloleucel/Yescarta, (Gilead/Kite) for diffuse large B-cell lymphoma (DLBCL). Most recently (February 2021), CAR T therapy scene hits another milestone when liso-cel/Breyanzi (Juno/BMS) has also received the FDA-approval for DLBCL treatment, owing to its remarkable efficacy and low incidence of high-grade toxicityThese unprecedented results in B-cell malignancies have spurred a tidal wave of CAR T research in other cancers, including MM. This review details the general development of CAR T-cell therapy and its role in paradigm shift for myeloma treatment. Our topics of discussion range from the current state of its clinical development to the latest technologies adopted in CAR manufacturing for myeloma. Here, we also deliberated on the future perspectives and proposed ways to potentially improve the CAR T-cell research, as we maneuver towards revolutionizing the CAR T scene in myeloma.17. Subsequent research remained lacklustre until another decade later, the second-generation CAR T cells directed against CD19 emerged with proven efficacy in preclinical B-cell models18. This sets the precedence for the promising venture of CD19-CAR T therapy in ALL and chronic lymphocytic leukemia (CLL)19.The history of CAR T-cell therapy dates back to more than two decades ago, when the first ever CAR T cells were created against 2,4,6-trinitrophenyl Fig. . Althoug20. This unique CAR T-cell property can help mitigate the common limitations of T-cell receptor (TCR)-induced immunity, including the Major Histocompatibility Complex (MHC) loss on tumor cells and low antigen-binding affinity of T cells23.CAR T-cell therapy is able to reprogram host’s immune system to attack tumor cells without the physiological need for HLA-presentation. T lymphocytes are biologically engineered to express monoclonal antibodies (moAb) recognizing tumor-associated antigens Fig. . Engagem17. The current and conventionally produced CAR T products incorporate either one (second-generation) or two (third-generation) co-stimulatory domains  to promote efficient CAR T-cell signaling, persistence, and efficacy27. The more sophisticated fourth-generation CAR, T-cell redirected for universal cytokine-mediated killing (TRUCK), consists of an added transgene encoding for a pro-inflammatory cytokine, which when induced by signaling molecules, is released to mediate cytotoxicity29.Figure 30. This is followed by immunophenotyping analyses to ensure successful endowment of T cells with CARs and cytolytic activities. These CAR T cells will then undergo ex vivo expansion within a bioreactor, containing growth factor-enriched media, before finally being frozen for storage or immediately transported to the clinic for infusion.CAR T-cell manufacturing is a laborious process which takes 2–4 weeks for completion in a Good Manufacturing Practice (GMP) setting  and A Proliferation-Inducing Ligand (APRIL), to BCMA promotes growth and proliferation of plasma cells in the bone marrow. Although the expression of BCMA is heterogenous33, it is universally present in all MM cells34 and its overexpression carries important prognostic value36.CAR T research in myeloma is still at its infancy, comparatively to lymphoma and ALL Fig. . The mos31. This was followed by the first-in-human Phase I clinical trial to test the efficacy of the BCMA-targeted CAR T cells in relapsed/refractory multiple myeloma (RRMM) (NCT02215967). The overall response rate (ORR) was a good 81%, with 63% demonstrating very good partial (VGPR) or complete response (CR)38. Consequentially, there was a widespread effort to develop novel anti-BCMA CARs and to fine-tune the old CARs.The first BCMA-directed CAR was developed less than a decade ago, showing preclinical evidence of functional targetability40. The first significant breakthrough in CAR T-cell development for myeloma was reached when bb2121 (Bluebird Bio) was granted a breakthrough-therapy designation award by the FDA and Priority Medicine (PRIME) eligibility award by the European Medicines Agency (EMA) in 2017. This was precipitated by its excellent efficacy and durable responses. Latest reported ORR was 73% with significantly improved PFS (median PFS 8.8 months) in the RRMM patients42. Bb2121 has been heralded as the latest front-runner for FDA’s approval for MM treatment. Its successor bb21217, designed with the same CAR construct but is being cultured in the presence of PI3K inhibitor bb007 ex vivo, has recorded a decent ORR of 55%, associated with enrichment of memory T-cell phenotype and high-peak expansion43. Additionally, JNJ-4528, the bi-epitope CAR T-cell product containing two BCMA-targeting domains, aimed to enhance the antigen binding affinity, has also shown an impressive 94.8% ORR in the CARTITUDE-1 trial45. This study provides compelling evidence that a single low-dose infusion of the CAR T product was sufficient to induce early, deep and durable response. JNJ-4528 takes after the exact construct of LCAR-B38M that is being evaluated in another geographical cohort (China) in the LEGEND-2 trial. The study reported a promising ORR of 82.4% with a good safety profile in the high-risk patient subgroup46.Table 47. ORRs to these treatments were a remarkable 64, 100, and 82%, respectively. The efficacy of JCARH125, in particular, was reported to be associated with T-cell product that is of high CD3+ purity and enrichment of early-memory phenotype and polyfunctionality48.Some other noteworthy BCMA-CAR T products are the human-derived and fully humanized CARs , designed to reduce the graft-versus-host disease (GVHD) and to prolong the persistence of the T cells49.Described in Table Alas, the promising results from anti-BCMA CAR T-cell therapies do not come without its own set of challenges.50.The underlying mechanisms for therapeutic resistance have remain largely unclear, but tumor heterogeneity, and antigen escape have been implicated47. Indeed, BCMA often loses its expression upon disease relapse after the first CAR T infusion, suggesting the selection for BCMA−negative MM clones by the CAR T cells53.Heterogeneously expressed BMCA at the intra-tumor level can lead to preferential targeting of cells with high BCMA while sparing those with low/zero BCMA expression, resulting in the outgrowth of the latter clones50 or it can be shed into the blood circulation ), mediated by γ-secretase47. Both can cause dampening of tumor cell recognition54. Although lower sBCMA level is indeed associated with good ORR56, it does not always correlate with CAR T dose–response and its expression remains low at the time of relapse with increase disease burden52, indicating that mechanism of resistance can extend beyond tumor intrinsic factors.For BCMA antigen escape, one of the most well-described ways is the erroneous physiology of the BCMA antigen. BCMA can be inadvertently transferred from tumors to T cells in a process called trogocytosis, causing T-cell fatricide52. This was corroborated by another study which revealed an association of genomic instability with biallelic deletion of BCMA loci upon CAR T-cell infusion51. Both alleles were intact before CAR T treatment, therefore, suggesting the occurrence of branching evolution over treatment course and acquisition of genomic aberrancy by MM cells in selecting for BMCA-negative cells.In relation to this, molecular BCMA aberration was recently highlighted by a study using longitudinal single-cell transcriptomic and bulk genomic analysis. They identified that resistance to bb2121 in a patient was associated with biallelic loss of BCMA. Majority of the MM cells demonstrated heterozygous deletion of chr16p, containing the BCMA locus, concomitantly with nonsense mutation of the other allele, rendering a biallelic inactivation of BCMA20. Despite being a plasma cell marker, BCMA is also co-expressed on normal B-lymphocytes, therefore, BCMA CAR T-cell therapy could also introduce on-target/off-tumor effects, where the common manifestations include B-cell aplasia, neutropenia, and immunosuppression that leads to increased infection risks. In the same aforementioned meta-analysis study, an average CRS and neurotoxicity prevalence of 80.3% and 10.5%, respectively, were reported49. While toxicities are frequent events, toxicity-related lethality in the patients are controllable. High-grade toxicities are more often observed in patients with heavy tumor burden or on high CAR T-cell dosage38. Toxicities are typically managed with readily available drugs such as IL6R-antagonist (tocilizumab) for CRS, and corticosteroids for neurologic symptoms20.Toxicity such as cytokine release syndrome (CRS) and neurotoxicity, mediated by pro-inflammatory cytokines, upon CAR T-cell activation, is another persistent problem. CRS manifests as fever, nausea, and flu in mild cases, but could escalate to hypotension, cardiac arrest, and liver failure in severe cases. On the other hand, neurotoxicity could range from mild confusion and delirium, to severe obtundation, seizure, and white matter degradationThe emergence of next breakthrough molecule is perpetually anticipated. Various surface antigens have been explored in this pursuit. This section documents the progress made by some of the more well-studied myeloma antigens Table .Table 3N58. Anti-SLAMF7 CAR T cells deriving from elotuzumab showed preclinical evidence of rapid cytolysis in primary myeloma samples and elimination of extramedullary cells in xenografts59. SLAMF7-CAR T cells incorporating on/off suicide gene for enhanced safety and tolerability was later developed for clinical investigations (NCT03958656 and NCT03710421). A European group has also engineered a novel SLAMF7 CAR T-cell model utilizing non-viral gene transfer approach of the Sleeping Beauty transposon system. Their proprietary product is currently being tested in Phase I/II CARAMBA trial (https://www.caramba-cart.eu/patients/)60.The attractiveness of employing signaling lymphocytic activation molecule F7 (SLAMF7) for CAR targeting stems from elotuzumab, the first SLAMF7 humanized moAb that was FDA-approved for RRMM61.Signifying a milestone, the allogenic anti-SLAMF7-CAR T cell (UCARTCS1) became the first ‘off-the-shelf’ CAR T-cell product in MM gaining FDA-approval for clinical trial (MELANI-01). UCARTCS1 was manufactured using healthy allogenic T cells with the TALEN gene editing technology to eliminate endogenous TCR and SLAMF7 expressions, intended to reduce the risk of GVHD and T-cell fratricide62.Importantly, a novel anti-SLAMF7/BCMA bispecific CAR T-cell product is under preclinical development with aim of increasing tumor coverage and overcoming antigen loss. These CAR T cells with single chain antibody containing two ligand-binding domains (one for SLAMF7 and another for BCMA) connected in tandem, showed enhanced activity, compared to T cells expressing individual CARs. In combination with PD1 checkpoint inhibitor, it confers accelerated tumor clearance in vivo65, underlies the development of anti-CD38 CAR T cells. Preclinical evidence of effective anti-myeloma effects of anti-CD38 CAR T-cell therapy67 has led to the initiation of various clinical trials, one of which investigates anti-CD38 CAR T cells as monotherapy in RRMM (NCT03464916), while others explore its combination with different CAR T cells, including anti-BCMA, anti-CD19, anti-CD138, anti-CD56, and anti-NY-ESO-13. A dual-specificity anti-CD38/BCMA CAR T-cell product is being evaluated (NCT03767751).The clinical success of anti-CD38 moAb (daratumumab and the second-generation isatuximab) in MM68 thus, resistance to the anti-CD38-CAR-T may be expected. There is also a likelihood for on-target/off-tumor toxicity as CD38 is also expressed on activated T cells , NK cells and normal prostate, neuronal, and muscle cells.It is noteworthy, however, that CD38 is generally not highly expressed on myeloma cells and its expression can be downregulated in advanced disease;54, making it a plausible therapeutic target. A proof-of-concept pilot study was conducted with anti-CD19 CAR T cells, Kymriah. Out of 12 patients treated, six achieved VGPR, two achieved PR, and two other experienced progressive disease (NCT02135406)13. Importantly, a cocktail CAR T product of anti-CD19/anti-BCMA produced an impressive 100% ORR (NCT03455972). Despite more than 1000-fold cell expansion at the peak data-point, patients experienced only mild (Grade 1–2) CRS and no neurotoxicity.Although CD19 is typically absent in matured plasma cells, minor subsets of myeloma cells with unique propagating properties express low CD19, associated with drug resistance and relapse-promoting properties34. Both TACI and BCMA share the same activating ligands, APRIL and BAFF, which when bound to their receptors, confer MM growth and survival. Based on this biological nature, ligand-based CAR T cells directed against APRIL have been developed to concurrently target both the TACI and BCMA signaling pathways70. In an in vivo model, APRIL-directed CAR T cells eradicated BCMA+TACI− and BCMA−TACI+ tumors, but monospecific anti-BCMA CAR T cells failed to contain the proliferation of the BCMA− cells, suggesting the potential application of APRIL-directed CAR T cells in cases associated with BCMA loss69. More recently, another APRIL-targeting CAR T-cell product with a refined molecular design has been reported70. This trimeric APRIL-based CAR T cells have enhanced binding to BCMA and TACI receptors with increased cytolytic activities, compared to its monomeric counterpart. Despite these promising preclinical data, the only clinical trial on APRIL-targeting CAR T cells conducted till date was terminated due to insufficient efficacy (NCT03287804).Transmembrane activator and CAML interactor (TACI), like BCMA, is a member of the TNF superfamily71.There is only one reported CAR T-cell product in development that specifically targets TACI surface antigen. It is the bispecific TACI/BCMA CAR T cells, that possess efficient in vitro and in vivo cytotoxicity towards MM cells72, rendering a good two-pronged approach.Besides myeloma cells, TACI is also found on the immunosuppressive regulatory T cells (Tregs). Targeting TACI, thus, may not only cause cytotoxicity through direct cytolysis, but also by indirectly manipulating the hostile microenvironment imposed by Tregsn = 5), thus results may not be sufficiently powered for meaningful interpretation. Anti-CD138 CAR T cells have also been incorporated into other CAR T-cell cocktail  trials but no substantial data can be obtained (NCT03778346 and NCT03196414).CD138 is a highly expressed surface molecule on MM cells, used clinically as a selection marker for plasma cells. The Chinese PLA Hospital tested a second-generation 4-1BB/CD3 CD138-targeted CAR T-cell product as a monotherapy in MM; however, only modest therapeutic response was reported (NCT01886976). This data was obtained from a small sample size  involved in plasma cell biology are being investigated preclinically73.Amidst the intensive research on CAR T-cell therapy, various other target antigens have also been examined, some of the worth mentioning ones are G-protein-coupled Receptor Class C Group 5 Member D (GPRC5D), CD56, Lewis Y, CD44v6, and Kappa-light chain. These molecules were studied because of their surface expression and prognostic implications74 in contrast, the FCARH143 trial (NCT03338972) found differential BCMA expression on tumor cells of long-term responders versus relapse patients prior to the treatment. In the many BCMA clinical trials conducted till date, the baseline BCMA is or is not an inclusion criterion and if it is, the range encompasses a mere observable expression (~1%) to a high >50%  revealed no correlation between baseline BCMA expression and the response rate;51. However, it is crucial to consider the accompanying challenges. For example, in (b), controlling the ratio of positivity of each CAR within the T cells may be technically challenging. The difficulty to define the expression level of either of these CARs on a single T cell would create a highly heterogenous pool of CAR T cells that may impinge on its tumor selectivity and ultimate potency. Furthermore, as with single-targeting CARs, the dual/multi-targeting CARs can also elicit selective pressure that could be stronger due to enhanced immune response, resulting in simultaneous escape of both antigens. Beyond technical complexities, another apparent challenge is the risk of high-grade CRS that comes with augmented T-cell activation from concurrent antigen targeting75.In overcoming clonal selection for single antigen loss, dual/multi-antigen targeting has been approached by either (a) infusing patients with multiple CAR T-cell products in a cocktail regimen or (b) having double ScFVs for different antigens within one T cell. Some of the known antigens that are being co-expressed with BCMA CAR are SLAMF7, CD19, and CD38 Table . This st20.To ameliorate the toxicities arising from hyperactivated CAR T cells and on-target/off-tumor specificity, some CARs have been modified to contain an inhibitory/suicide gene, which when required, can be triggered to switch off T-cell signaling to avert ensuing toxicity. Suicide genes such as truncated EGFR (NCT03070327 and NCT03093168), iCasp9 (NCT03958656 and NCT03125577), and HSV-TK (NCT04097301) are currently being investigated in myeloma. Other safety strategies such as bispecific T-cell engager and tandem CAR have yet to be explored in myeloma and are therefore worth looking into76. Although the former is associated with swift and robust cytolytic activities, its brisk signaling kinetics was associated with faster T-cell exhaustion, thus, resulting in less durable responses. They lack ongoing persistence compared to their 4-1BB counterpart, making them a likely inferior choice for a CAR construct66. The lesser preference for CD28-CAR in MM is evident  were tested by infusing the product cocktail into RRMM patients, post-ASCT. The ORR was 100%, with low grade CRS54. This then begs the question of whether the response was truly attributable to the activity of third-generation CARs or was it an effect of dual-antigen targeting with successful ASCT. A study making a parallel comparison between the second- and third-generation CARs in a single experimental setup may be imperative. Likewise, the concept of fourth-generation CARs is well-received for its role in immunomodulation and reprogramming of tumor microenvironment; however, the knowledge on its efficacy compared to second- and third-generation CARs is limited by the lack of clinical pursue28. A search on the ClinicalTrial.gov registry’s portal revealed 16 trials on fourth-generation CARs, two of which are for myeloma. These trials explore the efficacy of single or multi-antigen CAR T-cell compounds  (NCT03778346 and NCT03271632). While novel designs intended to increase CAR T-cell efficacy in myeloma are crucial, practical elements should also be considered, as higher innovations are often more labor-intensive and less cost-effective, not to mention the accompanying toxicity profile28.Second-generation CARs are leading the pack in CAR T-cell research and investigation of the third- and fourth-generation CARs in myeloma is scarce. Although the third-generation CARs should logically possess superior performance than its predecessor, most clinical data, at least in B-cell malignancies, demonstrated minimally augmented efficacy78. Bbb21217 displayed higher levels of CCR7 and CD27 compared to bb2121, suggesting higher levels of memory-like T cells, and lower levels of CD57, a marker of T-cell exhaustion. The latest update on bb21217 indicated 55% ORR with the CAR T cells being detectable up to 18 months43. Additionally, the contribution of immunosuppressive factors in tumor microenvironment to the lack of durability of CAR-T cells is also notable. A new fully human BCMA-CAR harboring a dominant negative domain against the immunosuppressive TGF-β was recently reported to exhibit robust proliferation and persistence, and remains functionally impervious to hostile TGF-β-rich microenvironment79.Short clinical remissions in MM have been attributed to low persistence of the BCMA CAR-T cells. To circumvent this, bb21217 that was derived from its precursor bb2121, was cultured ex vivo in the presence of PI3K inhibitor bb007, to enrich T cells with memory-like phenotype. The benefit of this approach was proven, when bb21217, but not bb2121, induced tumor clearance during a second tumor challenge within the same mice, without the need for re-administration of the product74, corroborating the reports in the CD19-targeted CAR T-cell therapy in lymphoma and ALL81. JCARH125 was infused into the RRMM patients (EVOLVE) with a designated CD4:CD8 ratio of 1:1. Latest clinical data reported robust expansion of these products at all dose levels, with patients’ ORR (82%) closely associated with the composite T cells82.Another manufacturing optimization that has also been attempted to amplify the potency of CAR T cells in myeloma is the T-cell composition. A higher CD4:CD8 T-cell ratio utilized at the pre-transduction and expansion stage was associated with greater in vivo BCMA-targeted CAR T-cell expansion and response84. Additionally, another non-viral system, the Sleeping Beauty, was used to introduce SLAMF7-CAR into the effector cells and has demonstrated good integration60.Gene delivery method is another variable in MM CAR T manufacturing, of which virus transduction is most conventionally opted. Given that this system comes with the risk of random insertional mutagenesis and is often associated with laborious and costly manufacturing procedures, other non-viral ways of introducing CARs into T cells have been introduced. The first non-viral CAR T-cell product for myeloma was developed by Poseida Therapeutics utilizing the piggyback-transposon technology (P-BCMA-101). This mRNA electroporation method demonstrated >95% efficiency and a more stable and higher CAR expression, coupled with a prolonged activity from the expansion of T cells with memory stem cell phenotype, when compared to those delivered by virus80. Integrating the less immunogenic humanized ScFVs into T cells is therefore a pragmatic approach. Indeed, the fully humanized BCMA-targeted CAR T cells (FCARH143 and JCARH125) developed by MSKCC exhibited rapid expansion, eradication of large tumor burden, and more durable patient response78. A novel, fully human, heavy-chain-only anti-BCMA binding domain (FHVH33) instead of a full ScFV, that was engineered to avert host immune cells’ recognition, has been shown to induce deep and durable responses with manageable toxicities53.Another CAR T manufacturing consideration is the origin of the moAb. One of the described therapeutic limitations of the otherwise successful CD19-targeted CAR T cells in lymphoma was T-cell fratricide due to host immune anti-murine CAR responsesThe quest to identify novel targetable myeloma antigens is crucial; however, those that have been deliberated thus far did not seem able to uphold the potential of BCMA’s. As a single agent, non-BCMA CAR T cells lacked meaningful outcomes. Refinements to these CARS such as rendering them bispecific often requires BCMA being the accompanying targeted antigen. With the impending FDA-approval of bb2121, studies on elucidating the underlying resistance mechanisms and ways of circumventing it, is indispensable.86. Oral administration of GSI with CAR T cells is currently being investigated in a Phase I trial (NCT03502577). Out of 10 evaluable patients, 30 and 50% achieved sCR/CR and VGPR, respectively87. Interestingly, all-trans retinoic acid (ATRA), that was reported to rescue daratumumab sensitivity by increasing CD38 expression88, was also found to be able to augment BCMA expression through an epigenetic modulation and enhances its recognition by BCMA-CAR T cells86. Combination treatment with ATRA and GSI led to higher BCMA density on MM cells, resulting in maximized reactivity. It is, again, imperative that we first clarify the inconclusive notion on the importance of baseline BCMA in conferring responsiveness. Not only is it necessary to ascertain the percentage of plasma cells having BCMA positivity, knowledge on the antigen density in each of the BCMA+ cells is also critical for discerning the BCMA threshold responsible for efficient CAR T-cell recognition.Synthetic biology is crucial for engineering more potent BCMA-targeting CAR T cells, nevertheless, maintaining tumor-associated-BCMA expression and preventing antigen loss, is required for their execution of potency. In this instance, pharmacological means have been explored. γ-secretase inhibitor (GSI) treatment was able to increase BCMA density on tumor surface alongside decreased sBCMA concentration and improved CAR T-cell anti-tumor activity89 and HER2-positive solid tumors90. Effective CD19-specific CAR T-cell-mediated cytotoxicity was observed in cases where artificial CD19 expression was successfully created on the surface of these CD19-low/negative tumor cells. Such innovative strategy may also be considered for MM  could synergize with bortezomib in drug-resistant MM cells93. Various signaling inhibitors such as BRD4i and MEKi were shown to boost CAR T cell’s functionality by hampering their immunosuppressive effects but these have never been studied in myeloma95. Plausibly in a drug combination study, one could investigate whether or not to incorporate an agent concomitantly with the CAR T-cell infusion or for maintenance post-CAR T therapy, which may be useful for patients with huge MM burden.Current standard-of-care has proven that combination of multiple agents targeting different mechanisms is the more effective way for disease management. However, investigations into BCMA CAR T therapy in combination with the diverse portfolio of anti-myeloma agents are rather limited. A preclinical work combining CAR T cells with lenalidomide found that the latter could enhance the persistence and activity of the former5. Attempt into investigating bb2121 in high-risk newly diagnosed patients has been initiated in KarMMa-4 trial (NCT04196491)96.The impressive results obtained thus far from CAR T-cell therapy trials in advanced and heavily pretreated MM patients have prompted the question of whether CAR T cells should be used as an earlier-line therapy. The lower disease burden in newly diagnosed or early-stage diseases may represent higher chance of achieving tumor clearance. In these cases, CARs with CD28, instead of 4-1BB co-stimulatory domain, may be a better choice as they elicit faster immune response. Swift activity of CD28-equipped-CAR T cells would be useful for prompt therapeutic effects, thereby, better controlling the disease at an early stage, preventing progression. This would appear to be even more critical for high-risk patients such as double-hit or functional high-risk myeloma whose disease often advances quickly even with modern treatment strategy98. These products were conceived using different technologies including the non-gene editing method to eliminate TCR (CYAD-211) and single-step platform to concurrently knock-out TCR while knocking-in CAR (PBCAR269A)99. Another BCMA allogeneic compound (ALLO-605) that is being evaluated pre-clinically incorporates additional chimeric features to enhance cytokine signaling100. Besides addressing the T-cell quality, off-the-shelf accessibility of these products also means that we can by-pass the long and complex autologous manufacturing process, thus, allowing for more rapid frontline therapies. The sustained availability of these allogenic CAR T cells will be useful for eradication of residual malignant cells, without the need for multiple rounds of painstaking autologous T-cell harvesting from the patients. In short, a cell bank with universal CAR T cells for allogeneic cell transfer would provide more flexibility in the application, faster delivery to the patients with less cost-intensive manufacturing.Additionally, myeloma has long been associated with quantitative and functional deficient T cells, particularly in the progressive, refractory disease. Acquisition of T cells from healthy donors is an attractive solution to the problem of insufficient and inferior CAR T cells. There are a few known allogenic compounds in MM that have gained IND clearance from the FDA, namely, UCARTCS1 (SLAMF7), CTX120 (BCMA), ALLO-715 (BCMA), CYAD-211 (BCMA), and PBCAR269A (BCMA)101. Several preclinical studies have demonstrated cytotoxic activity and MM growth inhibition using CAR NK cells against various targets, including CS1, CD138, BCMA, and NKG2D ligands102. There is currently one ongoing BCMA-CAR NK Phase 1/2 study in RRMM (NCT03940833). Furthermore, leveraging on the stem cells reprogramming system, an innovative CAR NK model, harboring a recombinant IL-15 signaling complex, was generated from induced pluripotent stem cells (iPSC). It has shown therapeutic efficacy alongside good synergism with moAb drugs 103. With the reported safety properties of CAR NK cells over CAR T cells, we envision that the CAR NK therapy research field in MM will gain an incremental interest that will allow for the diversification of MM cell therapy product portfolio, although its potential drawbacks are also to be cautiously weighed.Of late, CAR NK in myeloma is slowly gaining traction, owing to its better safety profile, versatility in its mechanisms of killing, and lower risk of GVHD, thereby higher feasibility for allogenic manufacturing that translates into cost effectiveness104. Considering this and MM heterogeneity, it would therefore be interesting to observe how different genomics and transcriptomics, such as different TC classes and RNA editing signatures, could dictate expression of surface targets that can be of use for immune targeting.On top of bio-engineering and diverse clinical testings, basic mechanistic research is also of importance as it provides insights into the fundamentals of immune response. Understanding the basic biology of T cells would enable us to produce ‘fitter’ CAR T cells that possess enhanced proliferation and longer endurance. Edging on the advancement of CRISPR-Cas9 technology, a genome-wide screening process can be performed to identify novel genes such when manipulated, could confer enhanced resilience and capabilities to the CAR T cells. Epigenetic mechanisms such as chromatin accessibility, DNA methylation states, RNA landscape in T cells, immune profiling, and identification of immune checkpoint signature are some of the key areas for further investigation. Recoding the T-cell behavior via gene knock in/out approach would be helpful in addressing the issue of innate T-cell defects and inefficient pool of effector cells. To understand disease recurrence in a highly heterogeneous pool of MM cells, leveraging on the advent of single-cell sequencing technology may be able to help us obtain a high-level understanding of the temporal evolution on genetic and epigenetic aspects of the immune cells. A recent single-cell study of a plasma cell leukemia patient revealed distinct CAR T-cell subsets having different expression, proliferation, and cytotoxicity, indicating stage-specific changes along the developmental trajectory37. Logistic factor involving time elapses between apheresis and delivery of manufactured CAR T cells back to patients is critical as the current turnaround time of 2–4 weeks renders CAR T-cell therapy unsuitable for patients with rapidly progressing disease. One emerging technology addressing this lengthy manufacturing issue is GraCell Bio’s propriety FasT CAR platform, in which product release can be achieved within 22–36 h using an expedited protocol to activate and transduce T cells without the need for further ex vivo expansion. A dual BCMA-CD19 CAR T therapy was developed using this platform and the first-in-human study has demonstrated an effective 93.8% ORR105. Such finding should be a precursor to further identification of key bottlenecks along the whole CAR T-cell manufacturing pipeline (Fig. Beyond the basic biology, ex vivo and scaling processes also play a big part in CAR T-cell productionThe CAR T-cell field in MM has indeed come a long way since the first BCMA-targeted CAR was developed in 2013. In our constant pursuit to develop personalized medicine, CAR T-cell therapy is perhaps the ultimate, as nothing can be more personalized than a treatment that harnesses the patient’s own immune system for tumor destruction. BCMA has gained the ‘holy grail’ recognition for the CAR T-cell field in myeloma and as long as continuous effort is invested into circumventing the barriers that have limited the success of current BCMA-directed CAR T-cell products, we may soon be able to produce an optimized product for MM that could be within the same realm of success as CD19-targeting CARs in lymphomas and ALL. Although the emergence of novel drugs has brought about significant improvement in patients’ outcome, these synthetic agents are physiologically eliminated from the body over time, making it difficult to produce a durable response without repetitive administration. CAR T cells, in constrast, is deemed a ‘living drug’ as they would be induced as long as targeted tumor antigen exists, thus making its therapeutic effect sustainable. If its efficacy is long-standing enough, we may well be on our way to having patients with lasting remission. With this and the anticipation for FDA’s approval of bb2121 this year, we have every single reason to remain hopeful and optimistic that CAR T-cell therapies may one day render MM a chronic but highly manageable and curable disease.