Human umbilical cord blood (UCB) represents a valuable source of hematopoietic stem cells, particularly for patients lacking a matching donor. UCB provides practical advantages, including a lower risk of graft-versus-host-disease and permissive human leukocyte antigen mismatching. These advantageous properties have so far been applied for stem cell, mesenchymal stromal cell, and chimeric antigen receptor T cell therapies. However, UCB-derived professional antigen-presenting cells are increasingly being utilized in the context of immune tolerance and regenerative therapy. Here, we review the cell-specific characteristics as well as recent advancements in UCB-based cell therapies focusing on dendritic cells, monocytes, B lymphocytes, innate lymphoid cells, and macrophages. Ever since the 1980s, umbilical cord blood (UCB) has been used as a rich source of allogeneic hematopoietic stem cells (HSC); these are primarily used for autologous pediatric transplantation or allogeneic transplantation in the absence of a fully human leucocyte antigen (HLA)-matched donor ,3. This The more recent surge in novel therapeutic technologies primarily focuses on the optimization of stem cell expansion and utilization of T lymphocytes and natural killer cells (NK) for chimeric antigen receptor (CAR)-based treatments. However, UCB cells have a rich and complex cell composition beyond the aforementioned cell subsets, each with their unique properties. Here, we review the immune cell composition of professional antigen-presenting cells (APC) in UCB and their clinical relevance to date.Numerous groups have demonstrated that UCB constitutes a rich source of HSCs. Likewise, early progenitors giving rise to hematopoietic colonies of different cell lineages, possessing long-term self-renewal properties, are more frequent in UCB samples in comparison with adult bone marrow (BM) or mobilized peripheral blood. Mouse studies also revealed that cord-blood-derived HSCs boast a higher hematopoietic reconstitution capacity in vivo in comparison with their adult BM counterpart ,5. In ad+ CD16−, intermediate CD14+ CD16+, and non-classical CD14− CD16+ monocytes . Un. Un14]. eriences . The underiences ,24. Thiseriences . Epigeneeriences . In parteriences .+ and CD16− monocyte behaviors. Among the known constitutively expressed extravasation markers [+ CD16− and CD14+/− CD16− monocytes [Due to their diminished production of pro-inflammatory cytokines, neonatal monocytes have been suggested to act as a double-edged sword in the context of viral infections. One of the more recent examples can be seen in neonatal Zika virus (ZIKV) infections, whereby monocytes provide a potentially favorable environment for viruses. C-type lectin domain family 5 member A, a classical flavivirus immune receptor, is highly expressed in neonatal monocytes and acts as a viral entry receptor for ZIKV . This, t markers , CD31 an+ and CD8+ T cell stimulation via antigen presentation. CD141+ DC possess a high intrinsic capacity to cross-present antigens via MHC class I to activate CD8+ T cells and NK cells whilst producing comparably low levels of IL-12p70 [+ DC, on the other hand, express a wide range of retinoic acid inducible gene I receptors, toll-like receptors (TLRs), lectins, and nucleotide-binding oligomerization domain (NOD)-like receptors similar to monocytes, producing high levels of IL-12p70, and can act as potent activators of CD8+ T cells and TH1, TH2, and TH17 populations [DC comprise a heterogeneous group of BM-derived cells arising from lympho-myeloid hematopoiesis that mediates the interaction between sensing and activation of both the innate and adaptive immune system . DC consIL-12p70 ,41,42. Culations ,44,45,46ulations ,48. Thes+ and CD1c+ DC in the initiation and resolution of inflammation [+ C-X3-C motif chemokine receptor 1low monocyte excavation into inflamed or infected tissue, TH1-associated cytokines favor inflammatory MoDC generation [+, MHCII+, CCR7+, CD11b+, CD205+, CD206+, and CD209+ and their lack of cDC-associated  markers [+ and CD8+ T cell activation [2 [As mentioned above, DC can derive from monocytes (MoDC), which play a key role in inflammation. Although distinct from DC, MoDC play a complementary role to CD141ammation , acting neration . These M markers . Followitivation . In vitration [2 . The rel+ DC [+ DC can be split into DC2 and DC3 [−CD1c− double-negative subset associated with a monocyte-like cell type; DC5 corresponds to a newly defined AXL receptor tyrosine kinase+ DC subset that can act as a precursor for CD1c+ DC [Further advances in gene expression studies have allowed for DC subset classification not only in terms of these functional properties, but also based on lineage, which correlates with the differential expression of transcription factors ,51,52. T+ DC ; CD1c+ D and DC3 ; DC4 repCD1c+ DC ,55; and CD1c+ DC .+ T cell proliferation similar to adult DC [+ T cells [+ T cell activation [+ T cells can be enhanced by blocking IL-10 using an α-IL-10R antibody [H1 and TH2 responses but predominantly induces a TH2 recall response later in life [The previously discussed DC subsets can be found in UCB; however, they differ in their frequency and function to adult peripheral blood ,34,56. Iadult DC . Further T cells . Still, tivation . Accordiantibody . Antigen in life ,64.+ CD4+ T cells with TH1 displaying a higher sensitivity to FasL-mediated apoptosis than TH2 cells [+ cDC in UCB and, therefore, the killing of activated CD4+ TH1 cells results in the polarization of T cells towards a TH2 phenotype associated with lower incidences of GvHD [Studies addressing the lower incidence and severity of GvHD following allogenic UCB transplantation uncovered that neonatal DC express significantly higher amounts of the Fas ligand (FasL) in comparison with adult DC, leading to higher rates of apoptosis in co-cultured T cells . UCB cDCH2 cells . It has  of GvHD . Taken tH1 T cell maturation and polarization, they are still able to induce T cell subsets. For instance, UCB-derived MoDC are able to induce melanoma antigen-specific CD8+ T cells, promoting IFNγ secretion and cytolytic activity towards tumor cells in vitro [H17 T cells [As in adult peripheral blood, UCB monocytes can differentiate into DC (UCB-MoDC). Standard differentiation with GM-CSF and IL-4 produces a similar phenotype to adult MoDC, yet UCB-derived MoDC express lower levels of CD80, DC-specific intercellular adhesion molecule-3-grabbing non-integrin, and MHC molecules . While Iin vitro . Various T cells ,70,71. A T cells . ConsideMΦ describe a heterogeneous group that is essentially omnipresent in tissues, taking on a range of functions, including both induction and resolution of inflammation, regeneration, and homeostasis. This dual nature is maintained in a delicate balance with phagocytosis contributing to inflammatory processes as well as innate and adaptive responses while the same processes restore homeostasis and promote tissue repair . AccordiH nomenclature: type 1 (M1) activated MΦ and, alternatively, activated type 2 (M2) MΦ [H1 cells. M1 MΦ are associated with the release of nitric oxide and citrulline, enabling avid cytotoxic activity as well as other downstream toxic metabolites associated with the M1 “killing machinery” [H2 responses, promote the killing of encapsulated parasites, possess high levels of scavenger, mannose, and galactose-type receptors, and are crucial to tissue remodeling and angiogenesis [2A receptor agonists, promotes IL-10 and vascular endothelial growth factor secretion, and provides pro-angiogenic properties [As discussed above, monocytes represent a heterogenous population, raising the question of whether specific subsets give rise to particular MΦ . In shor (M2) MΦ ,77. In vchinery” ,78. In cchinery” . In geneogenesis . M2 MΦ rogenesis ,82. M2b ogenesis ,82. IL-1ogenesis ,84. Receoperties ,85,86. Ioperties , with mooperties .H1, TH2, and TH17 lymphocytes [Reports addressing the function of MΦ from neonatal monocytes are rare. However, in a recent study, Drescher et al. note that neonates suffer more frequently from sepsis-related morbidity than adults, hinting at a differential immunological response towards invading pathogens . MΦ polaphocytes . Additiophocytes . Interesphocytes ,94. S100phocytes . It has phocytes . Exposurphocytes . Human Hphocytes ,96,97. Hphocytes . CumulatH (TFH) cells and DC, antigen-activated B cells undergo somatic hypermutation, a mutation rate ~106 greater than the normal rate of mutation in the genome, of the variable V-(D)-J antibody gene segment to improve its antigen specificity and affinity [B lymphocytes describe the center of the adaptive humoral immune system responsible for the production of antigen-specific Ig or antibodies against invasive pathogens. Each B cell expresses a unique antigen receptor, the B cell receptor (BCR), which constitutes a membrane-bound form of its antibody . Recogniaffinity ,101.H cells have a profound influence on B cells through the expression of CD40L and the secretion of IL-4 and IL-21, supporting B cell proliferation and differentiation into plasma cells or germinal center B cells [H cells. However, soluble proteins, haptens, bacterial polysaccharides, and LPS can stimulate B cells in the absence of TH. T independent antigens (TI) can be subdivided into TI-1 and TI-2 antigens, with TI-1 encompassing intrinsic B-cell-activating activity independent of the BCR specificity, whereas TI-2 initiate cross-linking, accumulation, and cross activation of BCRs in mature B cells [B cells develop in specialized microenvironments of the fetal liver and BM from HSCs through a complex interplay of genetic programs and cytokine milieus. The major stages associated with B cell development include the HSC, the multipotent progenitor (MPP), and the common lymphoid progenitor, followed by the progenitor B cell (pro-B cell), the precursor B cell (pre-B cell), and the immature B cell . For an  B cells ,105. For B cells ,106. Bot B cells ,106,107. B cells . After t B cells . Memorie B cells ,109,110. B cells ,111.H2 responses, either limiting or enhancing antibody responses, depending on the antigen-specific requirements of the specific B cell [+ memory B cells, due to a lack of antigen exposure, are almost non-existent while CD27− naïve B cells represent a predominantly transitional cell subset that has only recently emigrated from the BM and only contains a few mature naïve B cells [+CD27+CD43+CD38lo/int phenotype [− B1 cells tend to be more focused on the expression of IgM while CD11b+ B1 cells appear to be more reactive to T cells [+CD24hiCD38hi regulatory B cells [The abrupt transition from a sterile intrauterine environment to a new one confronts the immune system of neonates with a challenging situation. Though different to their adult counterparts, neonatal innate immune cells are able to respond appropriately after birth, but are short lived and ineffective for prolonged immune protection . Early ic B cell ,123. Neo B cells . The lon B cells ,125. The B cells . While t B cells ,126. A f B cells . Natural B cells . Similar B cells  and havehenotype ,130. B1 henotype ,132. It henotype . The exihenotype ,134. The T cells . However B cells . While r B cells . Upon st B cells . These p B cells .Innate lymphoid cells (ILCs) describe a recently defined family of immune cells that mirror the phenotypes and functions of T cells . In contH2-cell- and TH17-cell-associated cytokines [+, CD56+, and CD94+ in humans, and partially overlap with other ILC subsets [H2 cytokines, such as IL-5, IL-6, IL-9, and IL-13, in response to IL-25 and IL-33 [ILCs derive from the BM-situated common lymphoid progenitor and can be split into three groups based on their phenotype and secreted molecules: group 1 ILCs (NK and ILC1 cells), group 2 ILCs (ILC2 cells), and group 3 ILCs (ILC3 cells and lymphoid tissue inducer cells) ,140. Grond IL-33 ,142. In nd IL-33 . ILC2s and IL-33 ,145. Humnd IL-33 .Though scarce, the discussed ILC subsets can also be identified in healthy peripheral pediatric and adult blood through strenuous gating strategies involving the exclusion of cell-surface markers identifying other immune cells ,147. Sti3CSK4 [Toxoplasma gondii, which can be recognized by TLR2:1 [The search for ILC subsets and their progenitors has only recently expanded to UCB using RNA sequencing and flow cytometric approaches. So far, only three studies have addressed the phenotypical characterization and functional properties of ILCs in human UCB, unveiling distinct differences between adult and neonatal blood. Frequencies of ILCs are significantly higher in UCB compared with adult and pediatric peripheral blood . Interes3CSK4 . So far,y TLR2:1 ,150. In y TLR2:1 . This iny TLR2:1 . CurrentCell therapies based on UCB have gained increasing momentum in patient care, primarily in the treatment of pediatric hematological malignancies but also in oncology and immune reconstitution applications. In addition to current clinical applications, much effort is put into the investigation of potential approaches in pathophysiology, including muscle regeneration, myocardic ischemia, and myeloablation . Though + monocytes in mice in the context of oxygen and glucose deprivation following hypoxic–ischemic injury [+ monocytes in contrast to adult monocytes [+ monocytes, 48 h post-stroke led to a significant reduction in infarct size and promoted functional recovery in contrast to CD14+-monocyte-depleted controls [+-monocyte-mediated neuroprotection remains to be elucidated. However, it has been implied that UCB monocytes tend to take on M2 MΦ-associated alternative activated phenotypes that in turn induce alternative activation of local myeloid cells [H1:TH2 and TH17:TH2 ratios in vivo following UCB-derived CD14+ monocyte injection [+ monocyte administration lead to an increase in neuro-inflammatory responses and an increase in TH subsets in treated mice [+ compartment contains both monocyte-derived suppressor cells as well as other, potentially activated, monocyte subsets, which were both removed in the previous model [Cellular therapies harnessing the potential benefits of UCB-derived monocytes have so far been focused on tissue recovery and repair, particularly involving disorders of the central nervous system. Saha and colleagues investigated the neuroprotective properties of UCB-derived CD14c injury . The obsonocytes . A similonocytes . Intravecontrols . While econtrols . The exaid cells ,172. Intnjection . Howeverted mice . The autus model . The queOther beneficial effects of UCB-derived monocytes can be seen in novel cell therapeutic approaches to the treatment of Alzheimer’s disease. Though pharmacological targets have been discovered in recent years, disease-modifying treatments are still in the making. Initial experimental approaches using low-dose infusions of UCB cells were able to ameliorate cognitive impairments and Amyloid-β plaque neuropathology in amyloid precursor protein/presenilin 1 transgenic mice . In a fo+ and CD8+ T cell numbers declining after 7 days of co-culture, similarly to conditioned media from IFNα-induced UCB-MoDC. In addition, IFNα-induced UCB-MoDC enhanced the cytotoxic capabilities of allogenic Vγ9γδ T cells in terms of A549 tumor cell lysis [In an in vitro model, Anh and colleagues studied the effects of IFNα-induced UCB-MoDC on allogenic adult T cells . Here, Ill lysis . Previoull lysis . With Vγ+ monocytes, possessing properties from both MΦ and microglia [+ monocytes and DUOC-01 cells were evaluated in terms of remyelination of the brains of NOD/severe combined immunodeficient (SCID)/IL-2Rγnull mice following cuprizone-feeding-mediated demyelination [+ monocytes accelerate remyelination, they do so to a significantly lower degree than DUOC-01 cells due to transcriptional changes initiated by the manufacturing process of the latter [MΦ represent crucial components of the innate immune system due to their phagocytic activity, antigen presentation, and flexible phenotypes in mice and humans . Similaricroglia . As a prlination . Followilination . These fe latter ,167,176.e latter . HoweverRecent developments in MΦ cellular therapies will potentially soon harness the benefits of UCB and potentially affect the outcome of neonatal sepsis as well as novel strategies for immunotherapies. One approach can be seen in the “education” of MΦ to either enhance or mitigate inflammatory immune responses . In a renull-3/GM/SF xenograft mouse models, CAR-MΦ were able to maintain the M1 phenotype, while control-donor-matched MΦ were converted to M2. Furthermore, CAR-MΦ presented antigen-specific phagocytosis, were able to stimulate local M2 MΦ to switch to a M1 phenotype, were able to recruit and stimulate CD3+ T cells to tumor sites, and significantly increased survival rates [Novel advances in MΦ-based cell therapeutics can also be seen in the development of human chimeric antigen receptor (CAR) MΦ for cancer immunotherapy . CAR T cal rates . The que+ cells in order to enhance the rapid generation of tumor-specific cytotoxic T cell responses and enhanced Graft-versus-leukemia in vivo [+ cells were selected, expanded, and differentiated into Wilms Tumor 1 (WT1) antigen-carrying UCB-derived DC under good manufacturing practice (GMP) conditions [+ and CD8+ expansion in an allogeneic MLR setting [+ cells, which are differentiated with Flt3-L, SCF, GM-CSF, and IL-4 followed by exposure to a gold-standard maturation cocktail, a WT1 peptivator, as well as WT1 mRNA electroporation [+ cells are used to generate a Langerhans cell-like dendritic cell subset-based vaccine for the treatment of adult AML [+ and subsequent exposure to either a combination of GM-CSF and IL-4 or TGFβ1 were used to generate clinically relevant numbers of mature CD1a+ myeloid DC and CD207+ Langerhans cells, respectively [−/− mouse model [H1-associated IL-12β secretion over TH2 and pro-inflammatory cytokine responses [+ myeloid DC-mediated cell therapies in adults.DC are key elements in modern cellular therapies with applications harnessing their multifaceted prowess as cellular vaccines in the treatment of, e.g., malignant cancers . For ins in vivo . Becausenditions . Within  setting . In thei setting . The so-poration . The CBDporation . In a sidult AML . Expansiectively . Both suse model . Both UCesponses . These pIn a different approach, UCB-derived DC are being employed as effector cells for the development of cytokine-induced killer cells (CIK) . CIKs de+ T cell stimulation in an in vitro lung cancer model [+Vγ9 and CD3+CD8+ but not CD3+CD4+ T cells was induced by pulsed UCB-derived MoDC as well as a combination of UCB-derived MoDC and exosomes [In a recent study, UCB-derived DC were exploited for their production of exosomes and application in anti-tumor immunity . Exosomeer model . Exosomeer model . It is nexosomes . While iexosomes . Pre-cliWidespread use of UCB as a source of HSCs and mesenchymal stromal cells for the treatment of leukemia or inherent metabolic diseases has been complemented by the exploitation of UCB-derived immune cells in regenerative therapy. Due to pre-clinical studies, UCB has been recognized as a feasible and standardized cryopreserved source of regenerative cells. UCB offers several advantages over other sources of HSC and immune cell subsets, including a lower rate of chronic GvHD in the case of a higher HLA disparity and the benefit of being an “off-the-shelf” resource that can be collected painlessly. However, depending on the desired cell population, limitations in cell dosage and delayed engraftment remain the main challenges in UCB-based cellular therapies. This bottleneck requires efficient ex vivo expansion strategies to enable HSC multiplication or the expansion of clonogenic cell subsets.In summary, UCB provides an invaluable source of APC for the field of regenerative medicine. The exploration and development of Phase I–III clinical trials to evaluate the potential of UCB-derived APC populations remains an essential goal in translational research and medicine.


The vasoconstriction to PE and the vasodilatation to ACh were not different in WT and D3R−/−; in contrast, the contractile responses to 7-OH-DPAT were significantly weaker in D3R−/−, though not abolished. L741,626 did not change the contractile response induced by 7-OH-DPAT in WT or in D3R−/−, whereas SB-277011-A significantly reduced it in WT but did not in D3R−/−. D3R mRNA (assessed by qPCR) was about 5-fold more abundant than D2R mRNA in aorta from WT and undetectable in aorta from D3R−/−. Following transduction with lentivirus (72-h incubation) delivering synthetic microRNAs to specifically inactivate D2R (LV-miR-D2) or D3R (LV-miR-D3), the contractile response to 7-OH-DPAT was unaffected by LV-miR-D2, while it was significantly reduced by LV-miR-D3. These data indicate that, at least in mouse aorta, D3R stimulation induces vasoconstriction, while D2R stimulation does not. This is consistent with the higher expression level of D3R. The residual vasoconstriction elicited by high concentration D3R agonist in D3R−/− and/or in the presence of D3R antagonist is likely to be unrelated to DRs.Dopamine receptors (DRs) are generally considered as mediators of vasomotor functions. However, when used in pharmacological studies, dopamine and/or DR agonists may not discriminate among different DR subtypes and may even stimulate alpha1 and beta-adrenoceptors. Here, we tested the hypothesis that D2R and/or D3R may specifically induce vasoconstriction in isolated mouse aorta. Aorta, isolated from wild-type (WT) and D3R Dopamine (DA) is a catecholamine neurotransmitter involved in a variety of functions, including locomotor activity, cognition, motivation and reward, food intake, and endocrine regulation. The DA transmission in central nervous system (CNS) has been highly studied, mainly because its dysfunction is implicated in severe pathological conditions, such as schizophrenia, Parkinson’s disease, and Tourette’s syndrome.1- and D2-like, on the basis of their biochemical and pharmacological properties [1-like receptors, comprising D1 and D5 receptors , mainly activate adenylate cyclase activity; D2-like receptors, comprising D2, D3, and D4 receptors , mainly inhibit adenylate cyclase activity and regulate some ionic channels. D1R is the most abundant subtype in the CNS; D5R is found at a much lower level than D1R, mainly in the hippocampus and thalamus. D2R and D3R are expressed in the striatum, olfactory tubercle, islands of Calleja, nucleus accumbens, substantia nigra pars compacta, ventral tegmental area, and the pituitary gland. D2R and D3R are pre- and post-synaptic, unlike D1R and D5R, which are mainly post-synaptic receptors. D4R is found in the basal ganglia and in the frontal cortex, medulla, amygdala, hypothalamus, and mesencephalon, at levels below the other DRs [−/− appear more resistant to stressful procedures than WT littermates [DA exerts its action by binding to five distinct seven transmembrane domain/G-protein coupled receptors, grouped in two subfamilies, Dther DRs . Currentther DRs . Anotherther DRs ,9. Furthther DRs ,11 or etther DRs ,13. Actither DRs ,15. D3R−termates ,17, showtermates ,19.Though abundant in the CNS, DA receptors (DRs) are also found outside the brain. The discovery of the renal vasodilating function of DA led in fact to an extensive collection of experimental and clinical data concerning its potential role in cardiovascular system. Currently, DA itself and fenoldopam are approved in the US and EU as drugs supposedly acting on cardiovascular DRs. Detection of DRs out of CNS, however, has not been paralleled by demonstration of peripheral DA concentrations high enough to activate them. Furthermore, when exogenously administered to sustain blood pressure and heart activity, DA is likely to activate alpha and beta adrenoceptors ,21, whil−/−) to assess the role of D3R in arterial vasoconstriction. In particular, we used isolated mouse aorta, from wild-type (WT) and D3R−/− as an intact, native system, to assess the vasomotor responses to the D3R agonist 7-hydroxy-2-dipropylaminotetralin (7-OH-DPAT), in the presence and in the absence of the selective D3R antagonist SB-277011-A and in the presence and in the absence of the preferential D2R antagonist L741,626. The results show that 7-OH-DPAT induces a vasomotor response, which seems specifically related to the stimulation of D3R because it is reduced in D3R−/− and it is reduced by D3R antagonism in WT.In this study, we took advantage of D3R null mice (D3R−/−) and wild-type (WT) littermates  were fed on standard laboratory chow and were allowed free access to water in an air-conditioned room, at 22 ± 2 °C, with a 12-h light/12-h dark cycle. Mice D3R−/− were tenth to twelfth generation of congenic C57BL/6J mice, generated by a back-crossing strategy [D3R null mice , cut in segments (2 mm length), and mounted on the 40-μm diameter stainless steel wire of a wire myograph  for isometric record of contractile force. After mounting, each preparation was equilibrated unstretched for 30 min, in PSS, at 37 °C and aerated with 95% O2|5% CO2. The normalized passive resting force and the corresponding diameter were then determined for each preparation from its own length–pressure curve, as previously described [3, 25; MgSO4, 1.2; KH2PO4, 1.2; CaCl2, 1.2; glucose, 10; EDTA, 0.025, pH 7.4 at 37 °C) [Mice were killed by COescribed . Contract 37 °C) . After w8 TU/mL, ICM—Plateforme de Vectorologie, Paris, France), coexpressing under the drive of the ubiquitous PGK promoter the eGFP and a miRNA, specifically directed against the mRNA of D2R or D3R, was used to downregulate D2R or D3R in mouse aorta [6 TU in 200 μL DMEM), carrying synthetic miRNA specific for D2R, D3R, or the control, scrambled sequence, for 72 h, in a cell culture incubator, at 37 °C and 5% CO2. At the end of the incubation period the arterial segments were mounted in a wire myograph to examine the vasomotor responses. The expression level of DR mRNA following lentivirus-miRNA-induced knockdown was assessed by real-time quantitative polymerase chain reaction (see below).A lentiviral vector . Amplifications were carried out in a Light Cycler 1.5 instrument (Roche Diagnostics). Quantification was obtained by the ΔCt comparative method.Total RNA extraction of mouse aortic segments was carried out by TRIzol . Single-stranded cDNA was synthesized with SuperScript III (Invitrogen) by random priming. Aliquots of cDNA were amplified in parallel reactions with external standards at known amounts, using specific primer pairs for D3R, the short and the long splice variant of D2R , and GAPDH (reference gene). Each PCR reaction  contained 0.5 μM primers, 1.6 mM MgPhenylephrine, acetylcholine, 7-OH-DPAT, and L741,626 were from Sigma–Aldrich . These drugs were dissolved at 10 mM in aqueous stock solutions, except L741,266, which was dissolved in ethanol. The stock solutions were further diluted in water or directly in physiological salt solution, as required to reach the final concentration. SB-277011-A was from Tocris ; it was dissolved at 10 mM in aqueous stock solution and then further diluted as appropriate. p < 0.05. The concentration–response curves to 7-OH-DPAT with or without the antagonist were carried out in parallel, i.e., they represent comparisons between the very same run/challenge. PCR data were expressed as mean ± SEM. Data in concentration–contraction curves were expressed as a percentage of KCl-induced vasoconstriction against a log molar concentration of drug. Each set of data points was curve-fitted by a non-linear regression, best-fit, sigmoidal dose–response curve, with no constraints using GraphPad Prism . Each curve represents the mean of at least six individual preparations from a minimum of three or more mice. For each set of experiments, n is expressed as number of preparations. Arterial segments from the same animal were represented in the different experimental conditions. For example, we typically cut one aorta of mouse in eight segments, one was incubated with vehicle (control), the other three with the antagonist , each experiment run in duplicate. Whole curves were compared by two-way analysis of variance (ANOVA). Statistical significance was set at −/−, were mounted in a wire myograph and first constricted by exposing to a 100 mM KCl-depolarizing solution. After wash out and recovery, the preparations were challenged with cumulative concentrations of the alpha-adrenergic agonist phenylephrine (PE), to induce vasoconstriction, followed by cumulative concentrations of acetylcholine (ACh) to assess endothelium-dependent vasodilatation. The first contraction induced by KCl was not different among the two groups and was further used to normalize the subsequent phenylephrine-induced vasoconstriction, as in previous studies [−/−. In both groups, the maximum contraction evoked by PE slightly overcame that induced by KCl. Furthermore, ACh relaxed the pre-existing PE-induced tone by about 80%, indicating that functional integrity of endothelium was preserved.Aortic segments, from either WT or D3R studies . As show−/−; this contraction was, however, weaker than that induced by PE, barely reaching 40–50% of KCl-induced contraction. The maximum effect was clearly not yet reached with 10 μM 7-OH-DPAT; however, we did not test higher concentrations because, based on the nanomolar affinity of 7-OH-DPAT for dopamine receptors reported in literature [−/− were smaller than in preparations from WT. This difference was statistically significant at 3 and 10 μM (p < 0.01).Preparations were subsequently exposed to cumulative concentrations of the D3R agonist 7-hydroxy-2-dipropylaminotetralin (7-OH-DPAT). As shown in terature , the spe−/−, neither SB-277011-A nor L741,626 significantly changed the contractile response to 7-OH-DPAT , or with the preferential dopamine D2R receptor antagonist L741,626  reduced the contractile responses to 7-OH-DPAT A,C; SB-2-OH-DPAT B,D. This−/−, confirming that the effect of 7-OH-DPAT on contractile response was due to the presence of D3R and not to D2R. Finally, as expected, D3R mRNA was not detectable in D3R−/−.To evaluate the expression levels of D3R and D2R receptors in aortic segments, we measured the mRNA levels of two different splicing variants of D2R dopamine receptors  and of D3R by RT-PCR. As shown in Aortic segments were exposed, in vitro, to lentiviruses carrying synthetic miRNA specific for D2R and D3R, to assess their contribution to contraction following their knockdown (see below). The expression level of DR mRNA was determined by RT-PCR, following lentivirus-miRNA-induced knockdown, and confirmed a specific knockdown, below 50% of basal levels, as shown in The contribution of specific DR receptor subtypes to 7-OH-DPAT-induced vasoconstriction was further investigated in aortic segments where DR were knocked down by lentiviral delivery of synthetic miRNA. As shown in −/−), we tested the hypothesis that D3R subtype may induce vasomotor effects in isolated mouse aorta. We first analyzed the vasomotor responses unrelated to DA, to characterize the functional baseline of our model. The vasoconstriction induced by KCl and PE as well as the vasodilatation to ACh were not different in WT and D3R−/−, indicating that our system was suitable for assessing vasomotor effects specifically related to D3R. We then tested the vasomotor effect of the selective D3R agonist 7-OH-DPAT [−/−. This compound evoked concentration-dependent contractile responses not only in preparations from WT, but also in preparations from D3R−/−, albeit significantly weaker. This observation indicated that the contractile effect of 7-OH-DPAT was attributable in part to D3R stimulation, in part to other, undefined, receptors. Generally speaking, agonists show less selectivity than antagonists. In particular, molecular modeling as well as radioligand binding studies report 7-OH-DPAT as having a 20-100-fold selectivity for D3R over D2R [Despite the large amount of data available on DR in CNS, both in terms of expression and function in the context of DA transmission, less is known on DR in periphery . In arte-OH-DPAT , in WT aover D2R ; thus, cover D2R . Table 1−/−. This latter observation further supports the view that part of contractile response induced by 7-OH-DPAT in mouse aorta is due to D3R stimulation, while another part, persistent in the presence of the D3R antagonist and in mice not expressing functional D3R, is unrelated to D3R. Notice that L741,626, considered a D2R antagonist, was also able to inhibit 7-OH-DPAT-induced contraction, but in WT only; this suggests that the inhibition by L741,626 occurred as a result of D3R antagonism, which is not surprising, based on the reported affinity of L741,626 for D2R and D3R.We then further analyzed the DRs involved in 7-OH-DPAT-induced contraction by testing two antagonists, L741,626 and SB-277011-A, respectively, selective for D2R , delivering synthetic microRNAs to specifically inactivate D2R (LV-miR-D2) or D3R (LV-miR-D3), the contractile response to 7-OH-DPAT was unaffected by LV-miR-D2, while it was significantly reduced by LV-miR-D3. These data, showing that variation in D3R mRNA expression is linked to the functional response induced by 7-OH-DPAT, are entirely consistent with data comparing functional responses in aorta from WT and D3R−/−. Again, the residual vasoconstriction elicited by high-concentration D3R agonist in D3R−/− and/or in the presence of D3R antagonist is likely to be unrelated to DRs. We did not try to analyze the DR antagonism according to Schild [We then tried to estimate the abundance of DR subtypes in mouse aorta. However, because the available antibodies do not clearly discriminate between D2R and D3R, which also have close molecular size, making it difficult to resolve them in SDS-PAGE, we used RT-qPCR to estimate DR mRNA abundance. D3R appeared about 5-fold more abundant than D2R in samples from WT mRNA and, as expected, was undetectable in aorta from D3Ro Schild , becauseDRs have been identified so far in several arteries, such as aorta, renal, coronary, pulmonary, mesenteric, and cerebral arteries of different species, including humans . FurtherIdentification of DR in vessels was initially carried out through functional and/or radioligand binding studies, using agonists and antagonists not specific and selective enough to provide univocal and unambiguous conclusions. More recently, DR expression in arteries has been assessed at the mRNA and/or at the protein level. While mRNA expression does not directly correspond to the receptor protein in the plasma membrane, at least it provides an estimate of the relative abundance of different subtypes, with the advantage of absolute specificity, not afforded by pharmacological studies. In contrast, antibodies do not resolve the different DR proteins on the cell surface, nor do they in immunoblots, because the molecular size of DR subtypes is relatively close . Data onPsychiatric patients seem to be at increased risk for cardiovascular events, such as 2- to 5-fold greater risk of coronary heart disease and a 2- to 3-fold greater risk of cardiac mortality ,57,58. AIn this context, understanding the direct effect of DR stimulation in critical arterial districts, such as the cerebral and the coronary ones, may not only help in improving the safety of chronic drug treatments acting on DRs, but also define novel pharmacological targets to treat ischemic conditions.−/− and/or in the presence of D3R antagonist is likely to be unrelated to DR stimulation. Additional studies are needed to determine whether or not D3R receptors contribute to vascular tone in other vascular beds/species. Furthermore, the endogenous agonist responsible for the stimulation of these D3R remains elusive, given the negligible amount of DA available in periphery, mostly spilling from sympathetic nerve endings and/or adrenal medulla, though some may also come from endothelium. These data may be useful to understand the safety of drug treatments acting on DRs and/or to find novel drug targets for cardiovascular diseases.In conclusion, our data indicate that, at least in mouse aorta, D3R stimulation induces vasoconstriction, while D2R stimulation does not exert significant vasomotor effects. These functional data are consistent with mRNA expression assessment and knockdown, showing higher D3R expression in WT aortic tissues and reduction of contractile responses following D3R knockdown. The residual vasoconstriction elicited by high concentration D3R agonist in D3R


The influenza A virus (IAV) PA-X protein is a virulence factor that selectively degrades host mRNAs leading to protein shutoff. This function modulates host inflammation, antiviral responses, cell apoptosis, and pathogenesis. In this work we describe a novel approach based on the use of bacteria and plasmid encoding of the PA-X gene under the control of the bacteriophage T7 promoter to identify amino acid residues important for A/Brevig Mission/1/1918 H1N1 PA-X’s shutoff activity. Using this system, we have identified PA-X mutants encoding single or double amino acid changes, which diminish its host shutoff activity, as well as its ability to counteract interferon responses upon viral infection. This novel bacteria-based approach could be used for the identification of viral proteins that inhibit host gene expression as well as the amino acid residues responsible for inhibition of host gene expression. Orthomyxoviridae family, and cause influenza disease [Influenza A viruses (IAV) are single-stranded, negative-sense, segmented viruses belonging to the  disease ,2. IAVs  disease ,2,3.In the last centuries, different IAV subtypes have been responsible of influenza pandemics, including 1889 (H2N2), 1918 (H1N1), 1957 (H2N2), 1968 (H3N2), 1977 (H1N1), and 2009 (H1N1) ,5,6,7. TIAV segment 3 encodes a single, unspliced mRNA which encodes both the polymerase acidic (PA) and the PA-X proteins . The N-tNotably, PA-X modulates host inflammation, antiviral responses, cell apoptosis, T-lymphocyte-signaling pathways, and virulence  in a strMany residues have been shown to be relevant for PA-X’s endonuclease activity, including the catalytic residue K134 and the bivalent cation-binding residue D108 . MoreoveIn this work, we used an innovative bacterial-based approach and took advantage of previous observations indicating that genes cloned under a T7 promoter in plasmids were expressed in bacteria, to identify amino acid residues important for 1918 PA-X’s ability to induce cellular shutoff. This approach represents a very convenient, feasible, and easy method, and is based on the ability of PA-X protein to inhibit bacteria host gene expression and therefore growth. Notably, we have identified novel amino acid residues important for the shutoff activity of 1918 PA-X. This approach could be used to identify other viral proteins inhibiting host gene expression as well as the amino acid residues responsible for their shutoff activity, including other PA-X proteins encoded by different IAV strains.2.Human embryonic kidney (HEK) 293T cells  were cultured and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% PSG  at 37 °C with 5% CONdeI/F: 5′-AATTCATATGgaagactttgtgcgacaa-3′  and 1918/DelCFS/R: 5′-ctctctcggactgacaaaggaatcccagag-3′ (including the 1-nucleotide deletion introduced during the frameshift leading to the PA-X expression) for PCR1, and primers 1918/DelCFS/F: 5′-ctctgggattcctttgtcagtccgagagag-3′ (including the 1-nucleotide deletion introduced during the frameshift leading to the PA-X expression) and 1918/PAX/SalI/R: 5′-AATTGTCGACttacttctttggacattt-3′  for PCR2. Furthermore, an overlapping PCR using as templates PCR1 and PCR2 products and primers 1918/PA-X/NdeI/F and 1918/PAX/SalI/R was performed. The final PCR product was digested with NdeI and SalI restriction enzymes and cloned in the pGBKT7 plasmid digested with the same restriction enzymes. The pGBKT7-PA-X plasmid was transformed into chemically competent DH5 α Escherichia coli , and the transformants were plated on Luria broth (LB) agar plates containing 50 µg/mL kanamycin A and incubated for 24 h at 30 °C. An empty pGBKT7 plasmid and a pGBKT7 plasmid encoding the NS1 gene from A/Puerto Rico/8/34 H1N1 strain were included in separate transformations as internal controls. Bacterial colonies were selected and grown in LB containing 50 µg/mL kanamycin A at 30 °C for 24 h and plasmid DNA were purified using the E.Z.N.A Plasmid Mini Extraction kit  based on the manufacturer’s instructions. Mutant PA-X were cloned into the mammalian expression plasmid pCAGGS containing an HA epitope tag at the N-terminus. To this end, the pGBKT7 plasmids encoding the PA-X variants were used as templates for PCR amplification using the 1918 /PAX/SmaI/F: 5′-AATTCCCGGGATGgaagactttgtgcgacaa-3′  and 1918/PAX/XhoI/R: 5′-AATTCTCGAGttacttctttggacattt-3′  primers. The final PCR product was digested with SmaI and XhoI restriction enzymes and cloned in a pCAGGs plasmid encoding an HA epitope tag digested with the same restriction enzymes. Sequences for all plasmids containing PA-X mutants were confirmed (ACGT Inc.). Primers used for the generation of plasmid constructs and sequence analysis are available upon request.The pGBKT7 plasmid encoding the wild-type (WT) PA-X gene from A/Brevig Mission/1/1918 H1N1 (GenBank accession number DQ208311.1) was generated using specific primers and standard molecular biology methods. To this end, a pPolI plasmid encoding the 1918 PA gene , was used as template for performing two PCRs with the primers 1918/PA-X/4 cells/well, 24-well plate format, triplicates) using Lipofectamine 2000  with increasing concentrations  of pCAGGS expression plasmids encoding WT 1918 PA-X together with 250 ng of pCAGGS plasmids expressing the green fluorescent protein (GFP) or Gaussia luciferase (Gluc). Empty pCAGGS plasmid was included as an internal control and to normalize the total amount of transfected plasmid DNA. At 24 h post-transfection (h p.t.), cellular GFP expression and Gluc activity from tissue culture supernatants were evaluated using a fluorescence microscope, or a Biolux Gaussia luciferase assay kit  and a GloMax microplate reader , respectively. A similar protocol was used to evaluate the ability of 1918 PA-X mutants to inhibit host gene expression.Host shutoff activity of 1918 H1N1 WT PA-X on host protein synthesis was determined by transiently transfecting HEK293T cells  were transiently co-transfected using a calcium phosphate mammalian transfection kit , with 10 ng/well of pCAGGS plasmids encoding the WT or mutant 1918 PA-X proteins, or an empty plasmid as a control, together with 20 ng/well of a simian virus 40 (SV40)-driven Renilla luciferase (Rluc) expression plasmid and 50 ng/well of a plasmid expressing Firefly luciferase (Fluc) under the control of the ISRE promoter (pISRE-Fluc) [To evaluate inhibition of interferon-stimulated response element (ISRE) promoter activation, HEK293T cells (5 × 10RE-Fluc) . At 24 hRE-Fluc) . At 21 hHEK293T cells from the host shutoff assays above were harvested and lysed in passive lysis buffer  for 20 min, and proteins from whole cell lysates were separated by denaturing electrophoresis using 12% SDS-polyacrylamide gels. Next, proteins were transferred onto nitrocellulose membranes  using a Bio-Rad Mini Protean II electroblotting apparatus at 100 V for 2 h. After transfer, membranes were blocked for 1 h with 5% dried skim milk in 1X PBS containing 0.1% Tween 20 (T-PBS) and incubated with primary anti-HA epitope tag  or anti-GFP  rabbit polyclonal antibodies (P)Ab overnight at 4 °C. Mouse monoclonal (M)Ab against β-actin  was included as a loading control. Secondary horseradish peroxidase (HRP)-conjugated Abs  specific against mouse MAbs or rabbit PAbs were used to detect the membrane-bound primary Abs. Proteins were detected by chemiluminescence using SuperSignal West Femto substrate  following the manufacturer’s specifications and imaged using a ChemiDoc Imaging System .5 cells/well, triplicates) were transiently transfected, using LPF2000, with 1 μg of the indicated pCAGGS expression plasmids encoding 1918 PA-X WT or mutants. At 24 h p.t., cells were fixed and permeabilized with 4% (vol/vol) formaldehyde and 0.5% (vol/vol) Triton X-100 , respectively, before blocking with 1X PBS containing 2.5% FBS for 1 h. Thereafter, cells were incubated with an anti-HA epitope tag PAb  and then with a FITC-conjugated donkey anti-rabbit secondary Ab . Cell nuclei were stained with DAPI . Representative images were obtained using an EVOS M5000 Imaging System .To assess cellular PA-X expression by indirect immunofluorescence, HEK293T  using the PyMOL molecular graphics system.Microsoft Excel  and GraphPad Prism software were used to analyze the data. Microsoft Excel was necessary to perform some of the calculations, and to visualize the raw data. One-way analysis of variance (ANOVA) was performed using GraphPad Prism software.To confirm whether the PA-X protein from 1918 possesses the ability to inhibit host gene expression in our assay, HEK293T cells were co-transfected with pCAGGS expression plasmids encoding GFP and Gluc, together with increasing concentrations of a pCAGGS plasmid expressing an N-terminally HA epitope tagged WT PA-X from the 1918 strain . At 24 hIn order to identify 1918 PA-X amino acid residues responsible for its host shutoff activity, we cloned the 1918 PA-X open reading frame into the pGBKT7 yeast expression plasmid using DH5α bacteria competent cells. Remarkably, an unnaturally low number of bacterial colonies were acquired compared to an experimental approach involving the same pGBKT7 empty plasmid or a pGBKT7 plasmid encoding influenza A/Puerto Rico/8/34 H1N1 NS1 gene (data not shown). We and others have previously found that genes cloned under a T7 promoter in plasmids were expressed in bacteria (data not shown) . TherefoOut of 15 bacterial colonies, all isolated pGBKT7 plasmids encoded 1918 PA-X genes with mutations, 5 of them containing unique single  mutations and 3 of them containing double  amino acid mutations . All of Upon sequencing of pBGKT7 plasmids containing the PA-X gene from the recovered bacteria, several mutations were identified, suggesting their importance for host shutoff activity. To investigate this, we assessed the ability of the identified 1918 PA-X mutants to inhibit host gene expression and compared them to that of the WT 1918 PA-X . Each ofNext, we evaluated the expression of 1918 PA-X mutants by immunofluorescence in HEK293T cells transfected with pCAGGS plasmids expressing each of the identified 1918 PA-X mutants . Empty aIn the case of the double I79V/Y161C, T98I/P103S, and H146Y/L187P mutants, two amino acid changes in 1918 PA-X protein could be involved in their impaired inhibition of host gene expression . To deteLastly, 1918 PA-X double and single mutants were evaluated by immunofluorescence, which displayed no signal for WT, I79C/Y161C, or its single mutants I79V or Y161C . HoweverTo analyze the effect of the amino acid changes identified in modulating innate immune responses, we used a well-established virus-based assay. To this end, HEK293T cells were co-transfected with pCAGGS plasmids expressing 1918 PA-X WT or mutants, together with a plasmid expressing Fluc under the control of an ISRE promoter A and a phttps://www.fludb.org/ (accessed on 30 December 2020)) until 30 December 2020, were aligned to study the conservation of the amino acid residues identified in our bacteria-based screening in the PA-X proteins of H1N1 IAV. Interestingly, the residues found in the PA-X WT proteins from H1N1 IAV are highly conserved and more than 99.7% of the H1N1 IAV isolates encode the WT sequence  in human H1N1 viruses isolated since 1918, including 2009 pH1N1 viruses . These dThe relevance for PA-X shutoff activity of three of the amino acid residues identified in our screening using the 1918 strain  were recently reported in another manuscript in which the authors used a yeast-based assay to identify amino acid changes involved in WSN H1N1 PA-X inhibition of host gene expression , furtherIn this study we validate a convenient and novel approach, based in the simply use of bacteria, for the identification of residues relevant for IAV PA-X’s inhibition of host gene expression. This system has allowed us to identify 1918 PA-X amino acid residues important for its shutoff activity and for the ability of the protein to impair IFN responses after viral infection, which likely has implications in virus pathogenesis. Interestingly, the importance of some of the identified 1918 PA-X residues for host shutoff activity were previously shown with other IAV strains, further validating the use of this bacteria-based approach to identify mutations affecting IAV PA-X’s inhibition of cellular host gene expression. These results demonstrate the feasibility of using this bacteria-based system to identify other viral proteins inhibiting host gene expression as well as amino acid residues involved in host shutoff of these viral proteins. Because of functional relevance and high conservation of these amino acids, it could be feasible to design novel compounds targeting these protein regions for the treatment of influenza infections.


To contain the spread of SARS-CoV-2, the mRNA vaccine of viral spike protein has proven to be highly effective, although the efficacy against the emerging variants is decreasing. Here, we show that the mRNA of spike protein with deletion of glycosites in the RBD or especially the S2 domain to expose more conserved epitopes can be used as a broadly protective vaccine against the wild type and variants of concern. The spike protein translated from such mRNA was not properly folded, and thus induced cell apoptosis and a strong T cell response. Our findings demonstrate the importance of the glycosylation effect on the development of broadly protective mRNA vaccines. + T cell responses with broader protection against the alpha, beta, gamma, delta, and omicron variants, compared to the unmodified mRNA. Immunization of such mRNA resulted in accumulation of misfolded spike protein in the endoplasmic reticulum, causing the up-regulation of BiP/GRP78, XBP1, and p-eIF2α to induce cell apoptosis and strong CD8+ T cell response. In addition, dendritic cells (DCs) incubated with S2-glysosite deleted mRNA vaccine increased class I major histocompatibility complex (MHC I) expression. This study provides a direction for the development of broad-spectrum mRNA vaccines which may not be achieved with the use of expressed proteins as antigens.Development of the messenger RNA (mRNA) vaccine has emerged as an effective and speedy strategy to control the spread of new pathogens. After vaccination, the mRNA is translated into the real protein vaccine, and there is no need to manufacture the protein in vitro. However, the fate of mRNA and its posttranslational modification inside the cell may affect immune response. Here, we showed that the mRNA vaccine of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein with deletion of glycosites in the receptor-binding domain (RBD) or especially the subunit 2 (S2) domain to expose more conserved epitopes elicited stronger antibody and CD8 In 1796, Edward Jenner created the first vaccine (cowpox) in the world to protect against smallpox and successfully rescued millions of people, and, since then, vaccination has been recognized as the best way to protect against pathogens –4. Of th5N-glycosites and 2 O-glycosites per monomer. As part of our efforts to develop broadly protective vaccines and antibodies against SARS-CoV-2 and the emerging variants, we have shown that immunization of wild-type (WT) S protein with all 22 N-glycosites trimmed down to N-acetylglucosamine (GlcNAc) as the mono-GlcNAc decorated S protein (Smg) elicited broadly protective immune responses, including antibody and CD4+ as well as CD8+ T cell responses against the alpha, beta, gamma, and delta variants. Further study showed that most of the conserved epitopes on S protein are located in the RBD and the HR2 domain of the S2 subunit, but these conserved epitopes are largely shielded by glycans to escape from the immune response , and the subunit 2 domain (S2) . Previou12response . So, remvariants . To tranN- and 2 O-glycosites in RBD, 11 N-glycosites in S1, and 9 N-glycosites in S2) to evade host immune response  (N-glycosites (from N to Q in the N-X-S/T sequon) and O-glycosites (from S/T to A) where the conserved epitopes in the RBD or the S2 region were shielded by the glycans (N-glycosite at N-801 or N-1194 in S2 were important for the integrity of S protein and its binding to the receptor. After confirming the expression of the variant prefusion S protein in the HEK293 cell line (N-glycosites deleted (S-(deg-S2)) or with all S2 N-glycosites except N-1194 deleted (S-(S2-1194)), showed lower IgG titer against the fully glycosylated WT S protein . This finding is consistent with our previous study of sequence alignment analysis revealing that deletion of the glycosites in RBD or S2 will expose more conserved epitopes in immune responses  . To stud glycans A. We preell line B, the mR protein C, S2  and WT S peptide pool to measure the IFNγ-secreting T cells by flow cytometry. The result showed that there was no significant difference in the number of IFNγ-secreting CD4+ T cells among all vaccinated mice -secreting T cells by enzyme-linked immune absorbent spot (ELISpot) analysis. It was found that S-(deg-S2) and S-(S2-1194) induced more GrzB-secreting cells than WT and S-(deg-RBD) did after incubation with full-length WT S A, RBD FB, and S2ted mice D, but thresponse E.+ and CD8+ T cell responses and related cytokines, with a stronger IFNγ-producing CD8+ T cell response in mice immunized with S-(deg-S2) and S-(S2-1194). Taken together, these results suggest that glycosylation on S2 significantly regulated the T cell response and expression of cytokines.To analyze cytokine expression, the medium from splenocytes incubated with full-length WT S peptide pool was measured by ELISA. It was shown that the splenocytes from S-(deg-S2) and S-(S2-1194) immunized mice secreted higher levels of T-helper-1 (TH1) cytokines , whereas the splenocytes from WT and S-(deg-RBD) immunized mice secreted higher levels of T-helper-2 (TH2) cytokines   16–20).–20.16–20SI Appendix, Fig. S2), suggesting that deglycosylation in S2 affected the folding of expressed S protein.To investigate how glycosylation on S2 affected immune response, HEK293 cells were transfected with the prefusion stabilized S protein expression plasmid of variants. It was shown that S-(deg-S2) and S-(S2-1194) did not express well, but the expressed levels of S-(deg-S2) and S-(S2-1194) proteins were restored, to some extent, after treatment with MG132, a proteasome inhibitor A. In addSince the increased unfolded S protein in the ER would trigger the unfolded protein response (UPR), the UPR marker proteins BiP/GRP78, XBP1, and p-eIF2α were examined in RNA transfected HEK293 cells 48 h after transfection –25. The SI Appendix, Fig. S3), suggesting that UPR regulated the expression of MHC I on DCs. To further study how other glycosylation sites affected the host immune response, we used S-(deg-RBD) vaccine as a model, as it induced a better antibody neutralization activity against the four variants of concern than the WT. The glycosites in RBD and N-801 (S-(deg-RBD-801)) or N-1194 (S-(deg-RBD-1194)) were deleted, as these glycosites were important for S protein expression, especially the glycosite N-1194 which was essential for the integrity of S protein and its receptor binding . After confirming the expression of the prefusion S protein from mRNA-LNP in HEK293 cell line , mice were immunized with varied mRNA vaccines. The result showed that S-(deg-RBD-801), S-(deg-RBD-1194), and S-(deg-RBD-122-165-234) induced lower IgG titer against fully glycosylated WT S .To study whether UPR leads to the biased immune response, major histocompatibility complex class I (MHC I) and class II (MHC II) on DCs, which are essential for presentation of the internalized molecules after processing, were measured by flow cytometry . After D binding . Since t binding , 27. Aftted WT S A and RBDted WT S B, but S-ted WT S C in ELISted WT S D, but wiSI Appendix, Fig. S5 A and B). After the isolated CD4+ and CD8+ T cells were incubated with bone marrow–derived DCs and WT S peptide pool to measure the IFNγ-secreting T cells by flow cytometry, it was found that there was no significant difference in the number of IFNγ-secreting CD4+ T cells , but the S-(deg-RBD-801), S-(deg-RBD-1194), and S-(deg-RBD-122-165-234) mRNA vaccines induced more IFNγ-secreting CD8+ T cells -secreting T cells were measured by ELISpot analysis. It was shown that S-(deg-RBD-801), S-(deg-RBD-122-165-234), and especially S-(deg-RBD-1194) induced more GrzB-secreting T cells than WT did in all peptide pools ) also showed a stronger CD8+ T cell response and a higher anti-S IgG titer against the five variants of concern than the WT mRNA vaccine, although the anti-S IgG titer against the WT strain is lower. The expression of S protein with deglycosylated S2 was shown to have a decreased level in the plasma membrane and an increased level of S protein expression in the ER, but the antibodies generated are more effective against the five variants of concern, although the level of antibody production was lower than that of WT ) is the most protective against the five variants of concern with regard to antibody neutralization activity and CD8at of WT –30. In aresponse . The UPRnfection –34. It wnfection –38. In aresponse . TherefoGlycosylation on the target antigens or pathogens affects antibody response , but howIn summary, we have developed a method, as demonstrated in this study, to address the impact of glycosylation on S protein folding and immune response in vitro and in vivo using the mRNA of S protein as an immunogen with deletion of specific glycosites. This strategy is especially useful for the study of the glycosylation sites which are essential for protein folding, because the target protein cannot be easily obtained and purified in vitro for such study. We have also demonstrated the development of broad-spectrum vaccines using the mRNA of S protein with deletion of specific glycosites to expose the conserved epitopes.Human embryonic kidney cells (HEK293) were maintained in Dulbecco's modified Eagle's medium (DMEM) (lnvitrogen) with 10% heat-inactivated fetal bovine serum (FBS) (Thermo Scientific) and antibiotics (100 U/mL penicillin G and 100 gm/mL streptomycin).The rabbit anti–SARS-CoV-2 S polyclonal antibody, and SARS-CoV-2 full-length S, S2, RBD, and variant proteins (293T cell expressed), were purchased from Sino Biologicals. Mouse monoclonal anti–β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and rabbit monoclonal anti-MHC II antibodies were purchased from Millipore. The rabbit monoclonal anti-Na/K ATPase was obtained from ABcam. The mouse monoclonal anti-SERCA2 and rabbit monoclonal anti-MHC I antibodies were obtained from Invitrogen. The rabbit monoclonal anti-BiP/GRP78, anti-XBP1, and anti–p-eIF2α antibodies were purchased from ABclonal. All commercial antibodies were validated for specificity by companies and us via Western blot. To obtain the deglycosylated protein, S, RBD, or S2 protein was deglycosylated in a buffer solution with PNGase F (Sigma) at 37 °C for 24 h in the dark. After deglycosylation, samples were purified and checked by Western blot  tail signal sequence was amplified by using TOOLS Ultra High Fidelity DNA Polymerase  with 1 μL of the DNA template in an mMESSAGE mMACHINE Kit (Thermo Scientific) at 37 °C for 1 h according to the manufacturer’s protocol. The mRNA was purified by RNA cleanup kit (BioLabs) according to the manufacturer’s protocol and stored at −80 °C until further use. For the formulation of mRNA-LNP, the mRNA was encapsulated in LNP using a self-assembly process in which an aqueous solution of mRNA at pH 4.0 was rapidly mixed with an ethanolic lipid mixture containing ionizable cationic lipid, phosphatidylcholine, cholesterol, and polyethylene glycol lipid (sn-glycero-3-phosphocholine (DSPC) (Sigma), cholesterol (Sigma), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) (Sigma), and 1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000) (Sigma). The mRNA-LNP was characterized and subsequently stored at −80 °C at a concentration of 1 mg/mL. After HEK293 cells were transfected with 10 μg of mRNA-LNP in six wells of a plate for 48 h, the total cell lysate was collected to monitor the expression of S by Western blot.The prefusion state of the S, the codon-optimized S gene of SARS-CoV-2, was synthesized by GenScript and cloned into pcDNA3.1 or pVax, and was stabilized by proline substitutions at positions K968 and V969 (S-2P) . The solol lipid . The comn = 5) were immunized intramuscularly with 50 μg of mRNA-LNP in phosphate-buffered saline (PBS) with 300 mM sucrose. Animals were immunized at week 0 and boosted with a second vaccination at week 2, and serum samples and spleens were collected from each mouse 1 wk after the booster immunization. The animal experiments were evaluated and approved by the Institutional Animal Care and Use Committee of Academia Sinica.BALB/c mice aged 6 to 8 wk old  was read by a microplate reader.Anti-S protein ELISA was used to determine IgG titer. Plates were coated with 50 ng per well of variant S protein as shown in g in a 96-well plate for 30 min at 37 °C. After incubation for 16 h, the culture medium was removed and replaced with fresh complete DMEM containing 2.5 μg/mL puromycin (Sigma). After treating the mixture with puromycin for 48 h, the culture medium was removed, and the cell viability was detected by using AlarmaBlue reagents according to the manufacturer’s instructions. The survival rate of uninfected cells was set as 100%, and the virus titer was determined by plotting the survival cells versus diluted viral dose.Pseudovirus was constructed by the RNAi Core Facility at Academia Sinica using a procedure similar to that described previously . Brieflycontrol − RLU Serum)/RLU control  at 16 h postinfection and continuously cultured for another 48 h. The expression level of luciferase gene was determined by using Bright-Glo Luciferase Assay System (Promega). The relative light unit (RLU) was detected by Tecan i-control (Infinite 500). The percentage of inhibition was calculated as the ratio of RLU reduction in the presence of diluted serum to the RLU value of no serum control using the formula . The S-protein three-dimensional (3D) structure model with representative glycan profile was constructed by CHARMM-GUI and OpenMM programs. The transmembrane region of the spike protein defined by UniProt was used in this study. The input of CHARMM-GUI includes the Protein Data Bank file 6VSB_1_1_1, the representative glycan profile, and parameter settings. Relative solvent accessibility (RSA) of the spike protein with and without representative glycans is calculated by the FreeSASA program. The probe radius 7.2Å was used in the FreeSASA program to mimic the average size of the hypervariable loops in the complementarity determining region of the antibody. The RSA value of each residue used in this study was the average RSA value from three protein chains. The definition of exposed/buried residues was the same as in the study by Kajander et al. .5 splenocytes from immunized mice were ex vivo restimulated with full-length S, RBD, and S2 peptide mix   in the GrzB ELISpot assays (R&D Systems) according to the manufacturer’s instructions, and spots were counted. For T cell subtyping, CD8+ T cells and CD4+ T cells were isolated from splenocyte suspensions using Dynabeads Untouched Mouse CD4 and CD8 Cells kit (Invitrogen) according to the manufacturer’s instructions. CD4+ or CD8+T cells (1 × 105) were subsequently restimulated with 5 × 104 syngeneic bone marrow–derived DCs loaded with full-length WT S peptide mix  . The purity of isolated T cell subsets was determined by flow cytometry to calculate the spot counts per 1 × 105 CD4+ or CD8+ T cells. For flow cytometry, cells were suspended in FACS buffer (2% (vol/vol) FBS in PBS) at a density of 106 cells/mL, and the antibody used in this study was anti-IFNγ (Abcam). Cellular fluorescence intensity was analyzed by FACS Canto (BD Biosciences) and FCS Express 3.0 software.A total of 5 ×10IFNγ, IL-2, IL-4, IL-6, IL-12, and IL-13 were measured by using the ELISA kit according to the manufacturer’s protocol .After HEK293 cells were seeded in a six-well plate, cells were transfected with 3 μg of plasmid by TransIT-LT1 Transfection Reagent (Mirus) and then incubated with 1 μM MG-132 (MedChemExpress) or DMSO at 37 °C for 24 h. The total lysate was collected, and the variant S expression was analyzed by Western blot.The in vitro translation was performed with the plasmid that encoded S-2P using Glycoprotein Expression in a Human IVT System (Thermo) according to the manufacturer’s instructions. The expression of S protein in different incubation time periods was monitored by SARS-COV-2 spike protein ELISA kit  according to the manufacturer’s protocol.After HEK293 cells were transfected with 10 μg of S mRNA with TransIT-mRNA Transfection Kit (Mirus) for 48 h, the plasma membrane and ER were isolated by Minute ER Enrichment Kit (Invent Biotech) according to the manufacturer’s protocol. The S protein in the plasma membrane, cytosol, and ER was analyzed by Western blot. Total lysate was collected, and the UPR markers XBP1, BiP/GRP78, and p-eIF2α were monitored by Western blot. The apoptosis cells were measured by APO-BrdU TUNEL Assay Kit (Thermo) according to the manufacturer’s instructions.HEK293 cells were transfected with 10 μg of mRNA that encoded the soluble version of variant S-2P with TransIT-mRNA Transfection Kit (Mirus) for 72 h; the S protein was purified from the supernatant using Ni-nickel-nitrilotriacetic acid (NTA) affinity column . The purified protein and total lysate were analyzed by Western blot to determine the protein level of S.DCs were isolated from mice by using M-pluriBead Cell Separation kit (PluriSelect) following the procedure from the company and incubated with 10 μg of mRNA-LNP in DC culture medium  at 37 °C for 48 h, then analyzed for MHC I and MHC II expression by flow cytometry.t test. Differences were considered significant at *P < 0.001, **P < 0.05. All data were analyzed using GraphPad Prism 6 software.All data were presented as means ± SEM. The numbers of sample and replicates of experiments were shown as mentioned in the figure legends. Comparisons between groups were determined using Student’s Supplementary File


Neither deletion of Pom152 nor its carboxy-terminal glycosylation sites altered the nuclear protein export rate of an Msn5/Kap142 protein cargo. These data narrow the Pom152 region sufficient for NPC localization and provide evidence that alterations in other domains may impact Pom152 targeting or affinity for the NPC.Pom152 is a transmembrane protein within the nuclear pore complex (NPC) of fungi that is important for NPC assembly and structure. Pom152 is comprised of a short amino-terminal region that remains on the cytosolic side of the nuclear envelope (NE) and interacts with NPC proteins, a transmembrane domain, and a large, glycosylated carboxy-terminal domain within the NE lumen. Here we show that the N-terminal 200 amino acids of Pom152 that include only the amino-terminal and transmembrane regions are sufficient for localization to the NPC. Full-length, glycosylation-deficient, and truncated Pom152-GFP chimeras expressed in cells containing endogenous Pom152 localize to both NPCs and cortical endoplasmic reticulum (ER). Expression of Pom152-GFP fusions in  Summary: Pom152 is a transmembrane nucleoporin important for nuclear pore complex structure and synthesis. Here we further characterize Pom152, showing that the cytosolic and transmembrane domains are sufficient for NPC localization. Saccharomyces cerevisiae, each NPC is composed of about 30 different proteins termed nucleoporins (Nups). Each Nup is present in multiples of eight copies within each NPC, resulting in the eightfold symmetry of the nuclear pores (Nuclear pore complexes (NPCs) are large, aqueous, proteinaceous channels that perforate the nuclear envelope of eukaryotic cells and regulate communication and transport between the nucleoplasm and cytoplasm . In the ar pores . Nups caar pores . Non-FG ar pores . The porPom152 is a type II transmembrane protein with an amino-terminal cytosolic domain, a single transmembrane (TM) domain spanning residues 176–195, and a carboxy-terminal lumenal domain that comprises most of the mass of the 1337 amino acid polypeptide . The carPOM152 or removal of its lumenal glycosylation sites does not significantly alter the kinetics of Msn5-mediated nuclear protein export from yeast nuclei, consistent with observations that pom152 mutants do not have nuclear protein import or mRNA export defects , the amino-terminal and TM sequences exclusively , a truncation of the carboxy-terminal 377 amino acids , or POM152 lacking glycosylation sites at residues 280, 398, 569, and 1099 in the C-terminal lumenal domain  and observed transformed cells by phase contrast and fluorescence microscopy. Localization of Pom152wt, pom152Δglyc and pom1521-200 to the cortical ER was reduced to the point of no longer being detectable, but pom1521-960 retained significant localization to the cortical ER  pom152Δ yeast.Some cells expressing each Pom152-GFP fusion exhibited what appear to be clusters of more intense fluorescence in regions of the nuclear envelope, suggesting localization of the tagged Pom152 protein to a subset of NPCs or an NPC clustering phenotype resulting from Pom152-GFP expression see B. In ordwt-GFP, Pom152ΔGlyc-GFP, Pom1521-200-GFP, and Pom1521-960-GFP. The data are expressed as the mean percentage of cells with fluorescent clustering at the nuclear envelope±the standard error of the mean, based on a minimum of three independent analyses of over 50 cells each for each strain. The mean percent clustering of Pom152wt-GFP was 7.1±3.3%, Pom152ΔGlyc-GFP was 11.3±4.7%, Pom1521-200-GFP was 7.3±1.8%, and Pom1521-960-GFP was 5.6±5.3%. A one-way ANOVA was performed and the differences between the log transformed average of cells exhibiting nuclear clustering were not statistically significant  between these Pom152-GFP constructs in wt yeast. In pom152Δ yeast, a similar ANOVA analysis was performed on the nuclear envelope localization of the same four Pom152-GFP constructs. This analysis revealed no statistically significant differences in fluorescence localization among the Pom152-GFP constructs . The mean percent clustering of Pom152wt-GFP was 22.4±9.4%, Pom152ΔGlyc-GFP was 11.1±6.8%, Pom1521-200-GFP was 9.0±3.2%, and Pom1521-960-GFP was 4.6±0.5%, based on a minimum of two independent analyses of greater than 35 cells each.In the wt yeast background, Pom152-GFP fusions examined for intracellular localization were Pom152pom152Δ yeast.While all strains exhibited some cells in which various Pom152-GFP fusions appear to cluster in small regions of the NE B, based pom1521-960 to the endoplasmic reticulum in both wt and pom152Δ yeast raised the possibility that the perceived NPC localization of the Pom152-GFP fusions might be due to localization to the nuclear envelope as a consequence of this membranous structure being contiguous with the cortical ER. To confirm NPC localization, we expressed the same four Pom152-GFP chimeras in nup133Δ cells that exhibit a temperature-sensitive, NPC clustering phenotype , a glutamine at residue 182 (Q182). We used site-directed mutagenesis to create two mutant pom1521-200-GFP fusions  and another in which Ser158-Phe159-Phe160 are changed to Ala residues . Yeast strains deleted for POM152 , pom152::KAN (P=2.49×10−2), rpom152::NAT (P=4.03×10−8), and pom152Δglyc (P=1.57×10−10) strains. There were no significant differences detected between wt and the pom152 mutant strains tested (P>0.01). These data suggest that neither a full deletion of POM152 nor removal of the lumenal glycosylation sites significantly alters Msn5-mediated nuclear protein export.Deletions of r export . To examyc cells . To bettyc cells . We theng method . The onlSaccharomyces cerevisiae. Carboxy-terminal residues 201-1337 of Pom152 localized within the NE lumen are not necessary for NPC localization, but removal of a portion of the carboxy-terminal domain may affect Pom152 targeting to and/or affinity for the NPC, as evidenced by continued cortical ER localization of pom1521-960-GFP, even after deletion of endogenous Pom152. Deletion of POM152 or removal of its carboxy-terminal glycosylation sites do not alter the rate of nuclear export of the shuttling transcription factor Crz1.In this study, we have presented evidence that amino-terminal amino acid residues 1-200 of the pore membrane nucleoporin Pom152, including only the cytosolic and transmembrane regions, are sufficient for targeting the protein to the nuclear pore complex in POM152. These data may suggest that co-expression of the recombinant Pom152-GFP fusions and endogenous Pom152 results in saturation of Pom152 sites within the NPC and that excess Pom152 not integrated into the nuclear pores is retained throughout the ER membrane. Alternatively, the observation of some Pom152-GFP in the ER membrane may indicate a lower affinity of the recombinant protein than endogenous Pom152 for the NPC, although the presence of some recombinant protein at the NE indicates that any reduction in affinity is not so great that Pom152-GFP chimeras are essentially excluded from NPCs. The observation that expression of Pom152-GFP, pom152Δglyc-GFP and pom1521-200-GFP in cells lacking endogenous Pom152 results in NE envelope localization but undetectable fluorescence in the ER suggests that the affinity of these recombinant proteins for the NPC is sufficiently high for efficient targeting and/or retention. It has been shown previously that the amino-terminal region of Pom152 binds directly to the pore membrane Nups Pom34 and Ndc1 and to the non-FG nucleoporin Nup170 to link the membrane ring complex at the pore membrane to the inner ring complex that provides the inner scaffolding of the NPC -like folds that interact within the NE lumen and self-assemble head-to-tail and antiparallel to form the lumenal membrane ring at the equator of each NPC  and flanking amino-terminal  residues in pom1521-200-GFP chimera did not reveal any changes in localization when expressed in wt and pom152Δ cells and appeared to be targeted identically to the pattern observed for pom1521-200-GFP. Pom152 and gp210/Nup210 both are large, single-pass pore membrane nucleoporins with a large lumenal domain and shorter cytoplasmic region , pom152Δglyc-GFP (1-960-GFP (pKBB521), and pom1521-200-GFP (pKBB520) fusions were generated by amplifying sGFP from pJK19-1 using PCR with PfuUltra™ HF  and primers that generated chimeric Pom152-sGFP PCR products for homologous recombination in yeast. For pKBB463 and pKBB464, primers KOL236 (5′- GAAATTACAGATGCTTATTGTTTTGCCAAAAATGATCTTTTTTTCAATAACGCTAGCAAAGGAGAAGAACTC-3′) and KOL307 (5′- ATTTCTGTGGATGTTCAAAAGTCTGCTTTTAACACACCTCTATAGACCGTCCTTTCGGGCTTTGTTAGCAGCC-3′) were used for amplification and the resulting PCR products were co-transformed into yeast strain BY4742 with either plasmid pPM1-HA (PshAI. Primers used to amplify sGFP from pJK19-1 were KOL407 (5′-AGAAGAAGTCTGTCAAGGGATGGAAGGTACGGTTGATTTGGCTCTATTTGGTTCTCCAGCTAGCAAAGGAGAAGAACTC-3′) and KOL307 for pKBB521 and KOL406 (5′-CAGATTTTAGCTATGCTACTATTGAACATTTTCATATCAAGCGATCACGAGTTCGTTGCTAGCAAAGGAGAAGAACTC-3′) and KOL307 for pKBB520, and PCR products were independently co-transformed into BY4742 with PshAI-linearized pPM1-HA. Cells containing recombinant plasmids were selected for on SD-Leu media and Pom152-sGFP fusions were confirmed by PCR and DNA sequencing. Site-directed mutagenesis with a Stratagene QuikChange Lightning Multi Site-Directed Mutagenesis Kit  was used to generate Y180L,Q182L-GFPpom152 (pKBB553) and S158A,F159A,F160A-GFPpom152 (pKBB556) from 1-200-GFPpom152 (pKBB520) using mutagenic oligonucleotides KOL434 (5′- GCCATGGGTTGTTTTACTCCTGATTTTAGCTATGC-3′) and KOL437 (5′- GTATTTCATTATAGATGCCGCCGCCCTGTATGTTTTACCATCC-3′), respectively, as per the manufacturer’s instructions. Successful mutagenesis was confirmed by DNA sequencing.Yeast strains  and plasglyc-GFP ; pKBB464 pPM1-HA  and pKBB pPM1-HA  linearizEcoRV (600=0.05–0.2) and visualized under direct fluorescence using a Nikon E400 microscope. KBY850 (nup133Δ) strains were shifted to 35°C for 1 h prior to microscopy. Images were captured using an RTKE Spot CCD camera and SPOT-RTKe Imaging software . A one-way ANOVA was conducted to determine if the frequency of Pom152 clustering at the nuclear envelope was different between the Pom152-GFP constructs tested in wt and in pom152Δ yeast. In the wt yeast background, there was one outlier, as determined by boxplot, and the data failed to meet normality as assessed by Shapiro–Wilk test and equality of variances as assessed by Bartlett's test. To satisfy ANOVA assumptions, the outlier was removed, and the data were log transformed. Following data processing, a Shapiro–Wilk test (P=0.23) and visual inspection of a histogram and a QQ plot revealed that the data were normally distributed. There was also equality of variances as assessed by Bartlett's test of homogeneity of variances (P=0.77). A one-way ANOVA was performed on this transformed dataset. In pom152Δ yeast, a similar one-way ANOVA analysis was performed. The visual inspection of a histogram and a QQ plot and Shapiro–Wilk test confirmed normal distribution (P=0.74). Bartlett's test confirmed homogeneity of variances (P=0.14), and no outliers were detected using boxplot analysis.Yeast strains  were co-EcoRV . For fluPOM152 mutant strains were performed as described previously  and at 2 min intervals after. Export assays were performed blind with each yeast strain assayed at least three times and greater than 100 cells observed at each timepoint. Robust regression analysis was performed using the Theil-Sen estimator (P-value adjustment for multiple testing is performed with Benjamini–Hochberg method (Kinetic analyses of Crz1-GFP nuclear export in wt and eviously . Brieflyeviously  was transtimator . The Crzstimator . A visuastimator  was usedg method .


E. coli cells typically consume D-glucose first and then D-xylose. Similarly, when E. coli BL21(DE3) is provided with both D-glucose and D-xylose under anaerobic conditions, glucose is consumed first, whereas D-xylose is consumed very slowly.Microorganisms can prioritize the uptake of different sugars depending on their metabolic needs and preferences. When both D-glucose and D-xylose are present in growth media, xylR gene , respectively, controlled the expression of the xyl operon. RT-qPCR analyses demonstrated that the XylR mutation caused a 10.9-fold and 3.5-fold increase in the expression of the xylA (xylose isomerase) and xylF (xylose transporter) genes, respectively, in the adaptively evolved JH001 and JH019 strains. A C91A adaptive mutation was introduced into a new BL21(DE3) background via single-base genome editing, resulting in immediate and efficient D-xylose consumption.When BL21(DE3) was adaptively evolved via subculture, the consumption rate of D-xylose increased gradually. Strains JH001 and JH019, whose D-xylose consumption rate was faster, were isolated after subculture. Genome analysis of the JH001 and JH019 strains revealed that C91A (Q31K) and C740T (A247V) missense mutations in the xylR mutations responsible for faster D-xylose consumption were identified, which may aid in the improvement of microbial fermentation technology.Anaerobically-adapted BL21(DE3) cells were obtained through short-term adaptive evolution and The online version contains supplementary material available at 10.1186/s12866-021-02395-9. Recent microbiome studies have increasingly focused on the correlation between added sugars in foods consumed by humans and changes in microbial communities . IntestiE. coli has a native xylose transport system that enables these bacteria to metabolize xylose [E. coli cells through the D-xylose/proton symporter XylE and the XylFGH ATP-dependent ABC transporter [e xylose , 10. D-xnsporter , 12. Thensporter .xylFGH and xylAB genes, which respectively encode xylose uptake and metabolism-related enzymes, are co-regulated by the XylR transcriptional factor, as well as the intracellular cyclic AMP (cAMP) concentration. XylR is a transcriptional activator that directly regulates the xylose operon by binding to the promoter of the regulatory region in the presence of D-xylose  [E. coli, a phenomenon known as carbon catabolite repression (CCR) [E. coli have also been performed by co-culturing a xylose transporter-deficient strain and a strain in which glucose transport-related genes  were deleted [The inducer) . Howeveron (CCR) , 16. In on (CCR) –20. In ton (CCR) . Inhibiton (CCR) . In the on (CCR) . Co-util deleted , 25. Add deleted , 27.xylFGH gene encodes the ABC transporter involved in xylose uptake. However, when this gene is deleted via adaptive evolution, xylose is absorbed through GatC, an alternative transporter [xylR mutation was identified in a mutant strain that readily consumed D-xylose [The nsporter . In anotD-xylose .E. coli wild-type strains when both glucose and xylose were present in the growth medium. E. coli strain BL21(DE) consumed D-xylose very slowly compared to other strains. Therefore, experimental evolution was performed to increase the D-xylose consumption rate of this strain. Moreover, we demonstrated the potential use of strain BL21(DE3) as a model for the development of microorganisms that can quickly consume D-xylose, which is an important milestone for the advancement of fermentation technology.Our study confirmed that the rate of xylose consumption after glucose depletion varied among E. coli BL21(DE3), BW25113, C strain, and MG1655 strains were anaerobically grown in a fermentation medium supplemented with D-glucose (12.5 mM) and D-xylose (12.5 mM). All four strains consumed D-glucose and appeared to fully consume the D-glucose within 4–6 h  cells with an increased D-xylose consumption rate through adaptive evolution by serially transferring cultures to fresh fermentation media. BL21(DE3) cells were anaerobically grown in a fermentation medium containing D-glucose (12.5 mM) and D-xylose (12.5 mM). The growth rate and D-glucose consumption rate of the bacteria did not change significantly after several transfers; however, the maximum D-xylose consumption rate increased gradually. The D-xylose consumption rate was 0.6 mM/h in the first passage, but gradually increased to 0.8, 1.3, and 1.4 mM/h in each subsequent passage  strain was 1.1 mM/h in D-glucose- and D-xylose-supplemented anaerobic media. In contrast, the D-xylose consumption rates of the JH001 and JH019 rates were 1.9 mM/h and 2.9 mM/h, which represented 1.7- and 2.7-fold increases compared to the BL21(DE3) strain  The difference between the wild-type and adaptively evolved strains was confirmed based on their organic acid and ethanol output during fermentation. When provided with both D-glucose and D-xylose, there was no significant difference in the amount of acetate, formate, and lactate produced by the bacterial strains. However, while the wild-type strain produced 3.2 mM of ethanol, the adaptively evolved strains produced 15.2 and 14.4 mM. Conversely, the JH001 and JH019 strains produced 5.3 and 4.8 mM of succinate, respectively, whereas the wild-type strain produced 12.2 mM.When provided with D-xylose only, neither the wild-type nor the adaptively evolved strains produced lactate, and acetate production was not significantly different. Moreover, similar to the D-glucose + D-xylose condition, ethanol production was further increased and succinate decreased in the adaptively evolved JH001 and JH019 strains  in the xylR gene and IS (insertion sequence) insertion in the open reading frame of the carB gene of the JH019 strain .Whole-genome sequencing analysis of the adaptively evolved strains with increased D-xylose consumption identified a C91A point mutation (Q31K missense in XylR protein) in the itution A47V in thund Fig. , we assuxylR point mutations. The expression levels of the xylA and xylF genes  were compared between the wild-type and adaptively-evolved BL21(DE3) strains grown in a fermentation medium containing both D-glucose (12.5 mM) and D-xylose (12.5 mM). Compared to BL21(DE3), the expression of the xylA and xylF genes in the JH001 strain were upregulated 11- and 3-fold, respectively. Similarly, in the case of the JH019 strain, the expression levels of the xylA and xylF genes increased 5- and 2-fold compared to the wild-type strain, respectively  strain  and MG1655 wild-type strains via the target-mismatched CRISPR/Cas9 method [Q31K cells were grown in fermentation media containing both D-glucose and D-xylose, D-xylose was completely consumed 2 h after D-glucose depletion  cells in the fermentation medium through enhanced D-xylose transport and metabolism.The single point mutation (C91A) of the 9 method . When BLcarB gene inactivation also identified in the JH019 strain affects the D-xylose consumption rate, a carB deletion mutation was introduced into the BL21(DE3) wild-type strain and the adaptively evolved strain JH001. D-xylose consumption rates were accelerated when the carB gene deletion was introduced into the BL21(DE3) cells , the physiology and metabolism of this model microorganism have been extensively studied under aerobic conditions, as these conditions are more easily implemented in laboratories. The priority at which different sugars such as D-glucose, lactose, L-arabinose, and D-xylose are consumed follows a hierarchical order, and carbon source preference is known to be regulated by intracellular cAMP-CRP and transcription factors [Although  factors , 31, 32.E. coli strains. Particularly, the BL21(DE3) strain exhibited the longest xylose consumption delay  recently reported that the expression of the D-xylose operon and subsequent D-xylose consumption were enhanced by R121C and P363S mutations in the xylR gene, which might induce compact folding of XylR and increase the DNA binding affinity of XylR, respectively [xylR gene of BL21(DE3) cells, and observed an accelerated consumption of D-xylose after D-glucose consumption. However, the D-xylose consumption rate of XylRR121C cells was not faster than that of XylRQ31K cells. These results indicate that the most suitable adaptive mutations can vary depending on the strain background  strain, gain-of-function mutations (Q31K and A247V) in the ectively . In our und Fig. .xylA and xylF genes was higher in the xyl operon of the JH001 and JH019 strains, indicating that the xylR mutations were responsible for the accelerated uptake and metabolism of D-xylose  in the evolved strains Table . AdditiocarB gene mutation, which was additionally identified in the adaptively evolved JH019 strain, affected the xylose consumption rate. Notably, the JH001 strain exhibited growth delays during D-xylose consumption . Strain BL21(DE3) is known to have defects in anaerobic metabolism due to its mutation in the fnr gene. Specifically, the DE3 episome is inserted within the genes encoding the molybdenum transport system in this strain [carB), is known to be required for hydrogenase maturation [We then tested whether the ion Fig. D. Howeveium Fig. . It is ss strain . Moreoveturation  and may E. coli strains, xylR adaptive mutations were obtained through experimental evolution. Our study demonstrated that E. coli strains can rapidly acquire genome mutations that enable them to consume D-xylose sugars even with a single base substitution, suggesting that a variety of variants may exist at the species or strain level. The gain-of-function point mutation in the xylR gene was introduced into a new background through target-mismatched CRISPR/Cas9 genome editing technology, resulting in E. coli strains capable of efficient D-xylose consumption under anaerobic conditions. Therefore, our findings provide an important basis for the development of fermentation biotechnology.In summary, to improve the anaerobic D-xylose consumption rate of the BL21(DE3) strain, which has a slower xylose consumption rate compared to other wild-type 3 = 10 g; NaH2PO4·H2O = 8.5 g; K2HPO4 = 15.5 g. Yeast extract (Cat. No. 212750) was purchased from Becton Dickinson . NaHCO3 (Cat. No. S6014), NaH2PO4·H2O (Cat. No. S9638), and K2HPO4 (Cat. No. P3786) were purchased from Sigma-Aldrich . The headspace of the fermentation bottles was filled with nitrogen gas, and sodium sulfide  was added to quench the dissolved oxygen, thus yielding strictly anaerobic conditions. Bacterial cells were grown anaerobically at 37 °C with constant mixing at 180 rpm. Additionally, 25 mM D-glucose , 25 mM D-xylose , or 12.5 mM D-glucose + 12.5 mM D-xylose were incorporated as carbon sources.The strains and plasmids used in this study are listed in Supplementary Table For serial transfer, 1 mL of seed cultures were added to a 100 mL fermentation medium containing 12.5 mM D-glucose and 12.5 mM D-xylose. Cells were grown anaerobically at 37 °C with constant mixing at 180 rpm. Cell growth and residual D-glucose and D-xylose were monitored throughout the experiment. Once D-glucose and D-xylose were fully depleted, serial passages were performed by diluting the culture to a 1:100 ratio in 100 mL of a fermentation medium containing 12.5 mM D-glucose and 12.5 mM D-xylose. To obtain adapted strains, the cultures were spread on LB plates to obtain pure isolates from fermentation broth.− 1 using 0.01 N H2SO4 . Cell growth was monitored by measuring the optical density of the culture media at 600 nm using an Ultraspec 8000 spectrophotometer . The cell cultures were diluted using phosphate buffered saline to measure the optical density. The maximum sugar consumption rate was calculated as the amount of sugar consumed divided by the fermentation time (mM/h) in the section where sugar was consumed most rapidly.Sugar and metabolite concentrations were measured using high-performance liquid chromatography  using an Aminex HPX-87H column  as described previously . The celEscherichia coli strains was purified with the Wizard Genomic DNA Purification Kit . The genome sequences of the adaptively evolved strains were obtained with an Illumina HiSeq 2500 sequencer. Pretreatment of the reads, reference mapping, and variant detection were carried out using the Genome Analysis Tool Kit (GATK). Reads shorter than 50 nt were filtered out after quality trimming using Trimmomatic Version 0.36 (Table E. coli BL21(DE3) (CP001509.3) were used for reference mapping. Genome sequencing data were deposited in the NCBI BioProject database under accession number PRJNA689415. Sanger sequencing was conducted to confirm the xylR sequence. xylR was amplified using the xylR_250F and xylR_100R primer pairs in Supplementary Table The genomic DNA of different xylR mutant strain, P1 vir phage lysates of kanamycin-resistant strain BW25113 ΔxylR (JW3541) from the KEIO collection were used to transduce the BL21(DE3) strain to generate JH003 strain.Mutations were transferred to other strains via standard P1 transduction . To obtaxylR C91A or C361T point mutation, oligo-directed mutagenesis was performed, and negative selection was carried out using the CRISPR-Cas9 system, as described in a previous study [E. coli cells was removed through P1 transduction, and temperature-sensitive sgRNA plasmids were removed by incubating the cells at 42 °C.To introduce us study . The gencarB mutation, P1 vir phage lysates of kanamycin-resistant strain BW25113 ΔcarB (JW0031) from the KEIO collection were used to transduce strains BL21(DE3) and JH001 as recipient cells to generate JH042 and JH044, respectively.To introduce a ΔxylA and xylF genes was confirmed using quantitative real-time PCR (qRT-PCR). The BL21(DE3) wild type strain and adapted strains were grown for 8 h under anaerobic conditions in fermentation media containing 12.5 mM D-glucose and 12.5 mM D-xylose or 25 mM D-xylose at 37 °C, and RNA was isolated using the RNeasy® Mini Kit . qRT-PCR primer sequences for target genes were designed using the IDT PrimerQuest®Tool  was added as a carbon source in the fermentation medium. (A) BL21(DE3); (B) BW25113; (C) C strain; (D) MG1655. Figure S2. Molecular structure of XylR dimer with D-xylose (PBD ID: 4FE7). Green, DNA binding domain; Pink, D-xylose; Red, Q31 (Q31K mutation site identified in JH001 strain); Blue, A247 (A247V mutation site identified in JH019 strain). Figure S3. Anaerobic cell growth and sugar consumption profiles of JH003 (BL21(DE3) ΔxylR) strain. (A) Cells grown in a fermentation medium containing both D-glucose (12.5 mM) and D-xylose (12.5 mM), (B) cells grown in a fermentation medium containing D-xylose only (25 mM). Figure S4. Anaerobic cell growth sugar consumption profiles of ΔcarB strains. (A) JH042 (= BL21(DE3) ΔcarB) grown in a fermentation medium containing both D-glucose (12.5 mM) and D-xylose (12.5 mM), (B) JH042 in D-xylose only (25 mM) medium, (C) JH044 (= JH001 ΔcarB) in D-glucose (12.5 mM) + D-xylose (12.5 mM) medium, (D) JH044 in D-xylose only (25 mM) medium. Figure S5. Anaerobic cell growth and sugar consumption profiles of JH036 (BL21(DE3) xylRC361T) strain in a fermentation medium containing D-glucose (12.5 mM) and D-xylose (12.5 mM). Table S1.E. coli strains and plasmids used in this study. Table S2. Primers used in this study.