T cell exhaustion is an alternative differentiation path of T cells, sometimes described as a dysfunction. During the last decade, insights of T cell exhaustion acting as a bottle neck in the field of cancer immunotherapy have undoubtedly provoked attention. One of the main drivers of T cell exhaustion is prolonged antigen presentation, a prerequisite in the cancer-immunity cycle. The umbrella term “T cell exhaustion” comprises various stages of T cell functionalities, describing the dynamic, one-way exhaustion process. Together these qualities of T cells at the exhaustion continuum can enable tumor clearance, but if the exhaustion acquired timeframe is exceeded, tumor cells have increased possibilities of escaping immune system surveillance. This could be considered a tipping point where exhausted T cells switch from an asset to a liability. In this review, the contrary role of exhausted T cells is discussed.  Exhau+TCF-1+ precursor exhausted T cells have stem cell-like properties as they have the capacity to proliferate, self-renew and further produce progeny populations of exhausted T cells, including terminally exhausted less proliferative PD-1+TCF-1–T cells , and these express αβ TCRs consisting of an α- and a β subunit, while γδ T cells express TCRs consisting of a γ- and a δ subunit  molecules. Each T cell expresses TCRs, typically consisting of a α and β chain. Stochastic V(D)J gene recombination during T cell maturation results in a broad variety of TCRs, estimated to contain 107-108 unique signatures , and these stem-like exhausted T cells are necessary for long-term T cell responses and efficacy of immunotherapy , and characterized to be TCF-1+, CXCR5+, PD-1int and TIM-3- -specific CD8+ T cell population was found in the spleens of mice chronically infected with the LCMV clone 13 strain. In contrast, in mice infected with the LCMV Armstrong strain having cleared the infection, GP33-specific memory CD8+ T cells did not express CXCR5. The study showed that during early phase chronic infection, both CXCR5+ and CXCR5- CD8+ T were found in the blood, but at later stages on infection (day 30 onwards), only the CXCR5−CD8+ T cells were found in the blood  models . In a LChe blood . In the eriphery .+ T cells. The study revealed that TDLN-localized tumor-specific PD-1+CD8+ T cells are capable of effectively initiate antitumor immune responses following TDLN-targeted PD-L1-blockade. Following PD-L1-blockade, TDLN-resident T cells induced TPEX cell accumulation at the tumor site, resulting in improved tumor control. These findings support the pivotal role of progenitor exhausted T cells in LNs as an extra-tumoral source of antitumor T cell activity.Dammeijer et al.  used LNs+CD8+ TPEX cells migrating from lymph nodes to tumor sites provide a therapeutic window for maintaining a proliferative T cell population and possibly preventing differentiation of a terminal exhausted state of T cells  cells and show that these cells are capable of directly suppressing T cell proliferation ex vivo.Ts cannot . As pre-s cannot , 62, 68,+ TIL perforin expression in colorectal cancer (CRC), even though these cells stored the highest levels of granzyme B. Furthermore, PD-1 expression correlated with impaired perforin production, and the intact perforin expression was restricted to tumor resident T cells  of antigens  and Fas/Fas ligand (FasL) pathway (death ligands) , 70. Out T cells . Yan et  T cells  have ides et al.  studied antigens , indicat+ T cells during tumor immune response have been studied in C57BL/6 mice inoculated with EG.7. The study showed that the number of activated antigen-specific CD8+ T cells decreased via apoptosis during prolonged tumor immune responses, but the number of T cells in FasL-dysfunctional gld mice were higher than in control mice , PD-1 expression has been observed to be associated with spontaneous and Fas-induced apoptosis  chronic infection, and these cells were dependent on glucose supplies. In contrast, non-chronic infection cytomegalovirus (CMV)-specific T cells that could utilize OXPHOS in the absence of glucose to optimize their energy supply -stress sensor XBP1. XBP1 can directly increase PD-1, TIM-3, and LAG-3 expression, thus immunosuppression of T cells  cells and CCR7+ PD-1+TIGIT+ T progenitor exhausted T (TPEX) cells. The TPEX population gave rise to a cell linage with reduced functionality compared to TSTEM cells. Interestingly, the TPEX cells had memory-like features, suggesting that exhaustion-like and memory-like characteristics could coexist in healthy individuals. This further indicates that T cell exhaustion could be a functional adaption to persistent antigen stimulation aimed at minimizing the risk of immunopathology, and simultaneously maintain a memory-like population. The role of exhaustion in limiting immunopathology is underlined in a study by McKinney et al.  in CAR T cells have been shown to prolong the survival of tumor-bearing mice, and NR4A inhibition has been pointed out as a promising cancer immunotherapy strategy (Nr4a TKO TILs (Expression of members of the transcription factor (TF) family NR4A is regulated downstream of TCR signaling . Beltra strategy . ExpressTKO TILs , indicatStudies suggest that varying tumor types might generate distinct exhaustion patterns in T cells. Woroniecka et al.  performeAs the cancer-immunity cycle points out, the balance between stimulating factors and inhibitors is critical . Tumor c4Immune checkpoint blockade (ICB) therapies such as antibodies targeting PD-1, PD-L1, CTLA-4 and LAG-3 aim to activate T cells by interrupting inhibitory signals Table 1.Some tumors have been shown particularly challenging to target due to T cell exhaustion. Chimeric antigen receptor (CAR) T cells have been proven to be a potent tool for targeting blood cancer, but the dense antigen environment in solid tumor often generates severe exhaustion of these cells, causing restricted efficacy. Studies have shown that combination therapies with ICBs and CAR T cells can improve T cell expansion and tumor reactivity .Tox gene in CAR T cells proved to improve persistence and tumor reactivity in tumor inoculated mice  expression in isolated LCMV DbGP33–41–specific CD8examined . MiR-29aexamined . Hence, Tox for augmented tumor control  have been introduced to the field of immunotherapy. BsAbs can bind two distinct targets on the same cell at the same time, enabling additive effects and novel therapeutic approaches . Dual Bsxpansion . In Septxpansion . Similarxpansion .5Throughout the cancer-immunity cycle, T cells are subjects of a variety of factors such as prolonged antigen stimulation and altered conditions of the TME, unavoidably shifting the T cells towards an exhausted state . Studies+ exhausted T cell state maintain a memory-like population with proliferation capacity (+ T cells giving rise to terminally exhausted cytotoxic T cells could serve as a type of safety mechanism. Excessive antigen density in the TME leading to short-term cytotoxic states restrain cytotoxicity to tumors sites and thus limits immunopathology in healthy tissues. Since terminally exhausted T cells have reduced proliferation capacity (T cell exhaustion has potential benefits, but also consequences. A stem-like TCF-1capacity , 11. Stecapacity , 11, theAB performed the literature review and wrote the manuscript. RL, AA, and AT provided critical discussion, revised, and approved the manuscript. AT conceived the review article.


In this study, non-classical HLA class I molecules HLA-E and HLA-G were introduced in an immortalized porcine liver endothelial cell line with disruption of five genes  encoding three major carbohydrate xenoantigens  and swine leukocyte antigen class I (SLA-I) molecules. Expression of HLA-E and/or HLA-G on pig cells were confirmed by flow cytometry. Endogenous HLA-G molecules as well as exogenous HLA-G VL9 peptide could dramatically enhance HLA-E expression on transfected pig cells. We found that co-expression of HLA-E and HLA-G on porcine cells led to a significant reduction in human NK cell activation compared to the cells expressing HLA-E or HLA-G alone and the parental cell line. NK cell activation was assessed by analysis of CD107a expression in CD3-CD56+ population gated from human peripheral blood mononuclear cells. CD107a is a sensitive marker of NK cell activation and correlates with NK cell degranulation and cytotoxicity. HLA-E and/or HLA-G on pig cells did not show reactivity to human sera IgG and IgM antibodies. This in vitro study demonstrated that co-expression of HLA-E and HLA-G on genetically modified porcine endothelial cells provided a superior inhibition in human xenoreactive NK cells, which may guide further genetic engineering of pigs to prevent human NK cell mediated rejection.Natural killer (NK) cells play an important role in immune rejection in solid organ transplantation. To mitigate human NK cell activation in xenotransplantation, introducing inhibitory ligands on xenografts  Pig-to-human xenotransplantation offers a promising solution to address the persistent organ shortage . Interspin vitro and different inhibition pathways have been revealed , is a functional marker for NK cell activation, which correlates with both cytokine secretion and NK cell-mediated cytotoxicity  with five-gene knockout (5GKO)  was usedtoxicity . The reaGGTA1/CMAH/β4galNT2/SLA-I α chain/β-2 microglobulin) was generated from ipLDEC, as previously described  and B2M (NCBI reference number: NM_004048.4) genes with self-cleaving peptide P2A DNA fragment, synthesized by Integrated DNA Technologies , and inserted downstream of the CMV promoter in an expression vector derived from pEGFP-N1, which had EGFP gene removed (HLA-G is a heterodimer protein consisting of a heavy chain and β-2 microglobulin (B2M) subunits encoded by two genes located on different chromosomes. A single chain gene was designed by linking the HLA-E heavy chain gene  was modified by replacing its original signal peptide DNA sequence with HLA-B*07:02 signal peptide  DNA sequence, then linked to B2M gene with P2A DNA fragment. This single-chain HLA-E gene was synthesized by IDT and subsequently cloned to the downstream of the CMV promoter in an expression vector derived from pEGFP-N1 , as described above. HLA-E and HLA-G expression were measured by the percentage of positive cells as well as the mean fluorescence of intensity (MFI). The percentage of HLA-E or HLA-G positive cells was compared between 5GKO.HLA-E and 5GKO.HLA-E.HLA-G cells or between 5GKO.HLA-G and 5GKO.HLA-E.HLA-G cells at each time point. HLA-E MFI was compared between 5GKO.HLA-E and 5GKO.HLA-E.HLA-G cells while HLA-G MFI was compared between 5GKO.HLA-G and 5GKO.HLA-E.HLA-G cells.2 incubator at 37°C overnight. 5GKO.HLA-E cells alone were used as a control. HLA-E expression on pig cells was measured by APC-conjugated mouse anti-HLA-E antibody staining and analyzed by an LSRFortessa flow cytometer (BD Biosciences). Experiments were repeated three times with similar results.HLA-G VL9 peptide (VMAPRTLFL) was synthesized at purity of 95.1% by GenScript Biotech . 5GKO.HLA-E cells were incubated with HLA-G peptides at final concentrations of 25 µM, 50 µM, and 100 µM in a CO2 incubator for 5 days. 5GKO, 5GKO.HLA-E, 5GKO.HLA-G, and 5GKO.HLA-E.HLA-G cells were plated at 5 ×104 per well in a Biocoat collagen I-coated 48-well plate  one day prior to co-culture. PBMCs were added to porcine cells at 5 ×105 per well and co-cultured for 2 hours at 37°C in a CO2 incubator. Cultured cells were then collected and stained with fixable viability dye eFluor 780 (Thermo Fisher Scientific) and fluorochrome-conjugated antibodies against human CD45, CD3, CD56, and CD107a (BioLegend). Cells were fixed with 2% PFA for 15 minutes at room temperature and subsequently analyzed using an LSRFortessa flow cytometer (BD Biosciences). 70,000 - 80,000 events were acquired in lymphocyte gate. After pre-gating on CD45+ live singlets, NK cell degranulation activity was assessed by the percentage of CD107a positive cells in a CD3-CD56+ cell population. Flow data were analyzed using FlowJo v10 software (BD Biosciences). The experiment was repeated three times to obtain technical replicates.NK cell degranulation assay was performed in a similar fashion as previously described , 38. CD15 porcine cells  were washed and incubated with 25% heat-inactivated human serum in EX-CELL 610-HSF serum-free medium  with 0.1% sodium azide for 1 hour at 4°C. Human sera were obtained from patients on the kidney transplant waitlist, 10 sera with high panel reactive antibody (PRA) and 10 sera with low PRA, for a total of 20 samples (n = 20). Each pig cell line was washed three times with EX-CELL 610-HSF serum-free medium and stained with goat anti-human IgG Alexa Fluor 488 or donkey anti-human IgM Alexa Fluor 647  for 30 minutes at 4°C, respectively. Cells were washed, fixed with 2% PFA for 15 minutes at room temperature, and subsequently analyzed using an LSRFortessa flow cytometer (BD Biosciences). Flow data were analyzed using FlowJo v10 software (BD Biosciences). Each pig cell line stained with goat anti-human IgG Alexa Fluor 488 or donkey anti-human IgM Alexa Fluor 647 was used as background and subtracted from each corresponding sera binding group. Human IgG and IgM bindings to pig cells were analyzed by stratification into low PRA and high PRA sera groups. The difference between low PRA and high PRA sera binding to each individual modified cell line was also compared.Human antibody binding to porcine endothelial cells was examined, as previously described . BrieflyStatistical analyses were performed using GraphPad Prism 9 software . A normality test was used to assess data distribution. An ordinary one-way ANOVA multiple comparisons test with Šídák’s correction was used to analyze the differences among multiple groups. Student’s t-test was used to analyze the differences between the two groups. A p-value less than 0.05 was considered statistically significant.Three porcine cell lines expressing HLA-E, HLA-G, and co-expressing HLA-E and HLA-G were successfully established  cells, could activate human NK cell. Despite the elimination of four xenoantigens , 5GKO cells maintained the capability to trigger human NK cell degranulation (-CD56+) was shown in -CD56+ population were shown in Human NK cell response to pig cell stimulation was examined by CD107a expression on NK cells. Our previous study demonstrated that 5GKO cells, like WT and TKO  binding to 5GKO, 5GKO.HLA-E, 5GKO.HLA-G, and 5GKO.HLA-E.HLA-G cells was examined. A total of twenty human sera samples including ten high PRA sera and ten low PRA sera from the patients on the kidney transplant waitlist were used in this experiment. No statistically significant differences in human IgG or IgM binding among groups were observed: IgG with low PRA sera (p = 0.66), IgG with high PRA sera (p = 0.88), IgM with low PRA sera (p = 0.88), and IgM with high PRA sera (p = 0.75)  and pathological  conditions . The rolIn this study, human antibodies (both IgG and IgM) did not react to HLA-E and HLA-G on pig cells. Even across the stratification of human sera with low PRA and high PRA, there was no appreciable increase in antibody binding to HLA-E and HLA-G molecules. These findings suggest that even in highly sensitized individuals, there would likely be no substantial increase in antibody-mediated rejection induced by porcine organs expressing HLA-E and HLA-G.In human allotransplantation, HLA-E and HLA-G expression can be used to predict transplant outcomes. For example, elevated HLA-G in allografts and in the circulation of recipients was associated with improved graft acceptance in solid organ transplantation . In contThe next step will be generating transgenic pigs co-expressing HLA-E and HLA-G. Targeting HLA-E and HLA-G genes to the safe harbor loci in the pig genome can control the copy number of transgene while avoiding undesirable position effects. Studies have shown that the porcine Rosa26 locus and elongation factor 1 alpha  locus are ideal for the integration of transgene for constitutive and ubiquitous expression , 50. CRIin vivo validation studies, will provide context for the clinical applicability of these GMs in pig-to-human xenotransplantation. The field of xenotransplantation is rapidly progressing, and systematically evaluating potential GMs to optimize pig-to-human compatibility will be crucial to addressing the organ shortage.In conclusion, our results provide valuable insight into potential mechanisms for overcoming human NK cell-mediated immune rejection in xenotransplantation. Further optimization of this approach, in addition to https://www.ncbi.nlm.nih.gov/genbank/, NM_001363567.2 https://www.ncbi.nlm.nih.gov/genbank/, NM_004048.4 https://www.ncbi.nlm.nih.gov/genbank/, NM_005516.6.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The studies involving human participants were reviewed and approved by the Institutional Review Board (IRB) of Indiana University (IRB#11013).AC-N, KF, AI, JW, and PL performed experiments, AC-N, WL, WZ, KL, YP, NH, and PL analyzed data. AC-N, WL, ZL, and PL interpreted data. AC-N and PL wrote the paper. DC provided guidance and critical review of the manuscript. PL and BE conceived of the study and provided funding. PL designed experiments and supervised the project. All authors contributed to the article and approved the submitted version.


We show that the proton pumping activity of AHA2 is stimulated by anionic phospholipids, especially by phosphatidylserine. This activation was independent of the cytoplasmic C-terminal regulatory domain of the pump. Molecular dynamics simulations revealed several preferential contact sites for anionic phospholipids in the transmembrane domain of AHA2. These contact sites are partially conserved in functionally different P-type ATPases from different organisms, suggesting a general regulation mechanism by the membrane lipid environment. Our findings highlight the fact that anionic lipids play an important role in the control of H+-ATPase activity.The activity of membrane proteins depends strongly on the surrounding lipid environment. Here, we characterize the lipid stimulation of the plant plasma membrane H Plasma membrane H+-ATPases belong to the P3 subfamily and are only found in plant, fungi, and protozoa . Theyl growth .+-ATPases found in the model plant A. thaliana, the isoform 2 (AHA2) is the most extensively studied. Phosphorylation of residues in the C-terminal R-domain of AHA2  can inhibit or induce binding of regulatory proteins that are believed to displace the autoinhibitory R-domains from the catalytic domain, resulting in activation of the pump . ,24. 4,24and 3W5C ,26,27,28at M8–10 ,30. Bothative MS . For theS. cerevisiae Pma1 with lipid raft domains and its interaction with sphingolipids, required for delivery to the plasma membrane . Partic ATPases ,34,35,36the pump ,37,38. W+-ATPases from maize, Zea mays, rice, Oryza sativa, and mung bean, Vigna radiata [+-pumps. Of the tested anionic lipids, PS showed the strongest stimulation of H+ pumping as compared to PA and PG, implying a specific lipid–protein interaction. Consequently, several regions with an increased anionic lipid density were identified using coarse-grain MD simulations of AHA2 embedded in different membrane compositions, each containing 10% of the anionic phospholipid PS in a background of neutral lipids. The identified sites were all located at the lipid–protein interface, and potential residues for anionic lipid–protein interaction sites were subsequently found by contact analysis. Of the five sites located at the cytoplasmic side, site B with M1K57 and M1K60 has previously been identified as possibly involved in the function and regulation of P-type ATPases [M1K60 also being conserved in the two yeast proton pumps S. cerevisiae Pma1 and N. crassa Pma1. The two residues have previously been identified in the report on the refined crystal structure of AHA2 by Focht et al. (2017) as possible interaction sites for anionic phospholipids. Furthermore, MD simulations showed this region to partition into the membrane interface, inducing a local depression speculated to facilitate solvent access to the H+ binding site (D684-N106 pair) [S. cerevisiae Pma1 [N. crassa Pma1, solved in the auto-inhibited E1 conformation [N. crassa Pma1, M1 is buried deeply within the membrane facilitating cytosolic access towards the H+ binding site, with K115, corresponding to M1K60 in AHA2, oriented to the lipid headgroup–aqueous interface as a so-called snorkeling residue [S. cerevisiae Pma1, where additionally the proton pumping cycle is found to involve movement of M1 towards the cytosolic leaflet of the bilayer in the E2P state [N. crassa Pma1 based on an SERCA homology model [Reconstitution of purified AHA2 into liposomes with defined lipid composition enabled the specific characterization of the potential role of lipids on the pump outside the complexity of the native environment. The proton pumping activity of the resulting AHA2-containing proteoliposomes was shown to vary depending on the presence of anionic lipids, with the strongest stimulation by PS. Within cells, PS is enriched along the secretory pathway, constituting 1.5 mol% of the glycerophospholipids in late secretory vesicles and 7 mol% in the plasma membrane, respectively . Additio radiata ,17,44, i ATPases . A segmet et al. 17 as posiae Pma1  and N. cormation . In N. c residue . A simil2P state . Such regy model . In thisA. thaliana Ca2+-ATPase ACA2, corresponding to M1K60 in AHA2, resulted in a deregulated pump with activity similar to the activated state [N. plumbaginifolia Pma2, corresponding to M1P68 in AHA2, which is conserved in P3 ATPases and is responsible for the 90° kink in the M1 speculated to facilitate solvent access to the H+-binding site [In addition to the structural data detailed above, mutational studies on residues located in the region of site B have also been reported. An E167K mutation in the related ed state . A similing site ,48. Thising site ,43. ConsM4D272A and M4D275A located adjacent to the lipid–protein contact residues at site C seems to be the only examples of mutagenesis studies of the putative anionic interaction sites identified in this work [M4D272 and M4D275 at the periphery of the contact site buried deeper into the membrane near a putative cation binding site identified by [+-binding site to the exoplasmic space. A similar role for the attraction of protons in the E1 conformation to the H+-binding site could also be envisioned for sites A2 and B.Along with the above-mentioned residues at site B, mutations of the his work . Howeverified by ,21,39. GS. cerevisiae Pma1 and N. crassa Pma1 revealed a hexamer arrangement, confirming previous reports on the oligomerization of P3 type ATPases [N. crassa Pma1 autoinhibition is enhanced or possibly even dependent on the hexamer arrangement [1, A2, and C could potentially be involved in mediating the formation of hexamers. In the structure of the S. cerevisiae Pma1, lipids in the exoplasmic leaflet were found to bind to M3/M7, at the interface between two monomer units, near the location of site C. Unfortunately, the lipid headgroups were not resolved, and therefore their exact composition remains unknown. MD simulations [N. crassa Pma1 identified two putative lipid binding sites near our site A1 (Site I) and site C (Site II) also located at the interface of two monomers. Whereas site I showed an accumulation of anionic PS, in accordance with the observations from the MD simulations on AHA2, site II showed preferred binding of PC. The identification of a putative PS binding site for N. crassa Pma1 fits well with reports showing the activity of the yeast proton pump to depend on the presence of anionic lipids [N. crassa Pma1 containing polymer nanodiscs [The recently reported structures of the yeast proton pumps  ATPases ,45,46,49 ATPases ,50. Even ATPases , while fc lipids  and the anodiscs . The effsn-glycero-3-phosphatidylcholine (PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (PG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (PE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (PS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid (PA), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- (N-NBD-PE) were purchased from Avanti Polar Lipids Inc. . N-dodecyl-β-maltoside (DDM) and n-octyl-β-d-glucoside (OG) were obtained from Glycon . The ionophores valinomycin and CCCP, the pH-sensitive dye ACMA, and all other chemicals and regents were from Sigma-Aldrich , if not stated otherwise. ACMA was dissolved in dimethylsulfoxide; valinomycin in ethanol.Phospholipids 1-palmitoyl-2-oleoyl-A. thaliana H+-ATPase isoform 2 (designated AHA2), containing a hexahistidine (6 × His) and a SNAP® tag at the N-terminal end of the protein, was overexpressed in the Saccharomyces cerevisiae strain RS-72  and purified according to previously published protocols [−1 pepstatin. The cells were lysed by mixing with glass beads, and the protein was solubilized and purified with DDM at a detergent:protein (w/w) ratio of 1:3 using batch-binding to a Ni2+-NTA resin. The purified protein was finally concentrated to 1–10 mg mL−1 using centrifugal concentrators with a cut-off at 100 kDa , frozen in liquid nitrogen and stored at −80 °C in storage buffer containing 50 mM Mes-KOH (pH 7), 50 mM KCl, 20% (v/v) glycerol, 1 mM EDTA and 1 mM DTT supplemented with 0.04% (w/v) DDM until further use.A 73 amino acid C-terminal truncated version of rotocols . All bufv/v) and trace amounts (0.5 mol%) of fluorescent marker lipid N-NBD-PE in a glass tube. In addition to binary lipid mixtures, a ternary lipid mixture containing 45:45:10 PC:PE:PS was included in the set-up. The solvent was removed using a rotary evaporator . The lipid film (10 mg) was re-hydrated in 667 µL reconstitution buffer  by vortexing in the presence of a glass bead (5 mm diameter) for 5 min above phase transition temperature of the lipids , yielding a final lipid concentration of 15–20 mM. The vesicle suspension was further processed by five freeze-thawing cycles (90 s in liquid nitrogen followed by 90 s in a 60 °C water bath) and extrusion (21 times) through a stack of polycarbonate membranes (pore size 200 nm) using a mini-extruder (Avanti Polar Lipids). The vesicles made from PC:PE were unstable (lipids felt out of solution after short term storage), possibly because of an increased amount of PE in the membrane and the increased phase transition temperature. Thus, in this case, all steps (hydration and extrusion) were performed above 30 °C without storage at 4 °C. Liposome solubilization by OG was monitored by measuring light scattering of the liposome-containing solution at 600 nm using a fluorometer , thereby determining the ‘onset’ and ‘total’ solubilization conditions [Top is the starting plateau of solubilization curve , Bottom is the final plateau of solubilization curve , V50 is the point of inflection, and slope refers to the slope at the point of inflection.Liposomes were prepared by re-hydration of a thin lipid film, followed by freeze-thawing and manual extrusion. Briefly, binary lipid mixtures were prepared by mixing appropriate volumes of the lipid stock solutions in chloroform/methanol . The protein/lipid/detergent mixture was subjected to gel filtration  by centrifugation . The eluate was incubated for 60 min at room temperature with 50 mg of prewashed SM-2 Bio-Beads  under end-over-end rotation to ensure detergent removal.To facilitate the insertion of AHA2 into liposomes, preformed liposomes (4–5 mM) were solubilized by an amount of OG that was just sufficient to result in a half-maximal scattering change at 600 nm (‘Turning Point’). Purified AHA2 (12.5 μg) was added at a protein-to-lipid ratio of 1:60 . H+ pumping was initiated by the addition of MgSO4 , and the H+-gradient dissipated by the addition of 5 μM CCCP. Fluorescence quenching was recorded over a period of 600–1200 s at 480 nm  at 23 °C using a PTI-Quantamaster 800. Fluorescence traces were normalized to the intensity measured directly after addition of MgSO4. Recorded traces were analyzed with a customized python script with the slope of a linear curve fitted to the first 30 s after magnesium addition used as a measure for proton pumping activity. Traces that upon magnesium addition did not show a drop in fluorescence  were considered inactive and excluded from analysis.Huenching . Proteol® 488, New England BioLabs Inc., Ipswich, MA, USA) followed by 20 pmol membrane permeable SNAP dye  in reconstitution buffer supplemented with 1 mM DTT or with each dye separately. Samples were analyzed by SDS-PAGE using 12% gels and visualized on a ChemiDoc XRS Imaging System  using the Image Lab™ software (https://www.bio-rad.com/zh-cn/product/image-lab-software?ID=KRE6P5E8Z&WT_mc_id=211202033049&WT_srch=1&WT_knsh_id=cr180107&gclid=EAIaIQobChMI0eKynPLxgAMVlZhmAh2HUArSEAAYASABEgJPdvD_BwE) and pre-programmed option for Coomassie stained gels or Alexa488/Alexa647 fluorophores, respectively [AHA2 orientation in liposomes was determined by site-specific labeling of the SNAP-tag. Proteoliposomes  were incubated for 2 h either sequentially, first with 20 pmol membrane impermeable SNAP dye (10 µM SNAP-Surfaceectively .v/v/v). Detergent and lipid standards were chromatographed on the same plate and applied without prior extraction by chloroform/methanol. For visualization, plates were stained with primuline  and photographed under long-wave UV light (Biorad ChemiDoc XRS Imaging System).Phospholipid phosphorus was assayed after heat destruction in presence of perchloric acid by the method of Bartlett (1959) . To checp values < 0.05 were interpreted as statistically significant. Based on the results, significance was indicated as following; with no statistical significance (ns) or statistically significant; p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.To analyze the kinetic data, customized algorithms were developed using python version 3.8.8 . Data armartinize 2.6 and an elastic network was applied to stabilize the backbone conformation [2. The protein was embedded separately in two lipid bilayers with insane [2. Three 3 µs production runs were started from the equilibrated systems using different random seeds for generating the initial velocities of the particles at 310 K.For all computational studies the coarse-grain MARTINI 2.2 ,62,63,64ormation  by conneh insane , containh insane ,68,69 at−1, separately for the membrane plane (xy-plane) and its perpendicular axis (z-axis).The “New-RF” MD parameters recommended by de Jong et al.  were useOverall translation and rotation of the protein were removed prior to analyses by structural alignment to the starting structure of the simulations. Analyses were conducted for each trajectory separately, and for the concatenated trajectories of the three analogous replicas. Contacts between protein residues and lipid headgroups were analyzed with GROMACS 2020.1 and custom Python scripts. A contact was counted when at least two beads, one from the protein residue and one from a lipid headgroup, were within 0.55 nm from one another and no other lipid was closer to the protein residue. The contacts were normalized by the number of timeframes to obtain the probability of finding a protein–lipid contact at any time. Two-dimensional lipid headgroup densities were computed with the GROMACS tool gmx densmap (grid spacing: 0.02 nm), separately for each leaflet and lipid species. Lastly, three-dimensional lipid densities were computed with MD Analysis ,72 consi+-ATPase isoforms 1 and 2, respectively, from Arabidopsis thaliana. Q42932, for the H+-ATPase isoform 2 from Nicotiana plumbaginifolia. Q7XPY2, for the H+-ATPase from Oryza sativa subsp. Japonica. P05030, for the H+-ATPase isoform 1 from Saccharomyces cerevisiae. P07038, for the H+-ATPase from Neurospora crassa. P04191, for the SERCA pump from Oryctolagus cuniculus and Q4H132, for the Na,K-ATPase from Squalus acanthias.Protein sequences were downloaded from the UniProt knowledgebase database (access January 2021) and aligned with the tool Clustal Omega ,74. Acce


Dopamine D2-likereceptors  control diversephysiological and behavioral functions and are important targets forthe treatment of a variety of neuropsychiatric disorders. Their complexdistribution and activation kinetics in the brain make it difficultto target specific receptor populations with sufficient precision.We describe a new toolkit of light-activatable, fast-relaxing, covalentlytaggable chemical photoswitches that fully activate, partially activate,or block D2-like receptors. This technology combines the spatiotemporalprecision of a photoswitchable ligand (P) with cell type and spatialspecificity of a genetically encoded membrane anchoring protein (M)to which the P tethers. These tools set the stage for targeting endogenousD2-like receptor signaling with molecular, cellular, and spatiotemporalprecision using only one wavelength of light. Motor impairmentsin Parkinson’s disease can be ameliorated with D2R agonists,7 antipsychotic medications are either D2R antagonistsor weak partial agonists,10 and D2R is a putative targetfor treating addiction.11 Nevertheless,D2R medications have several features that lower the clinical efficacy,quality of life, and medication compliance. First, D2R is widely expressedin the central and peripheral nervous system, having distinct functionsin different locations and cell types.12 Because D2Rmedications are freely diffusible, they cannot differentiate betweenthe D2Rs that confer clinical benefit versus the D2Rs linked to adverseside effects. Second, once administered, D2R medications are persistentlyactive until they are gradually metabolized or removed by the body,making it difficult to control the precise timing and level of D2Rdrug action at the target site. Finally, D2R medications bind off-targetproteins, such as other GPCRs and ion channels.The neuromodulator dopamine plays a centralrole in physiologyand behavior ,in vivo. Genetic manipulation ofD2R  occurs over long time scalesand can lead to compensatory and possibly deleterious effects in neuralcircuits. Chemogenetic DREADDs (mutant GPCRs exclusively activatedby designer ligands) and optogenetic opto-XRs  can be used to promoteG protein signaling with cell type and spatiotemporal precision, butthese tools are artificial proteins that cannot fully mimic the actionsof endogenous receptors and their natural ligands.14 Membrane-anchored chemical ligands (DARTs) have been used to targetAMPA and GABAA receptors with tethered agonists and antagoniststhat limit action to genetically targeted cells and this approachcould, in principle, be used to target endogenous D2Rs in specificlocations.16 However, DARTs are chronicallyactive, turning off gradually over hours to days as their proteincomponent is removed by the cell, and thus, do not allow for temporalcontrol to match the dynamics of dopamine signaling. LumiToxins (light-sensitivemembrane-anchored peptides) can be turned on and off with light, buttake minutes to turn off and are difficult to develop for dopaminereceptors, which bind small chemical ligands.17 Taken together, there remains a great need for improved therapeuticsthat target D2R.18The alternativesto conventional D2R medications are also limitedin their utility Figure S1A).25 However, photouncaging is irreversible, making it difficult toreliably and repeatedly turn on and off the target receptor, especiallyin living animals. In contrast, reversibly caged D2R modulators havebeen developed that contain photoswitches such as dithienylethenesor fulgimides.20 These are small moleculesthat undergo a reversible change from their thermodynamically stable trans state to their cis state within millisecondsin response to light, and then return to the trans state either in a light-driven manner or through thermal relaxationin the dark.26 Similarly, when appendedto or incorporated within existing receptor ligands, azobenzenes canswitch the ligands from the off-state to the on-state and back byreversibly distorting the ligand shape and ability to bind and activateor inhibit the target.Photopharmacologyoffers a means to toggle protein function onand off with precise spatiotemporal control, whereby a ligand’saffinity and/or efficacy for its endogenous biological target is modulatedthrough a synthetic photoswitch. Remote control of D2R has been achievedwith diffusible photocaged agonists based on dopamine itself and antagonistsbased on eticlopride or sulpiride  has atone end an azobenzene that switches the receptor ligand between anon- and off-state, at the other end a moiety for covalent attachmentto the protein tag ,and in the middle a polyethylene glycol (PEG) linker that allows thephotoswitchable ligand to reach for its attachment point to the ligandbinding site on the target protein. While this technology has enabledlight-control of dopamine receptors and a variety of other proteinsthat are fused to a protein tag,41 insertion of the tag into the receptor requires modification ofthe receptor primary amino acid sequence, which could alter its functionand requires either overexpression or genetic knock-in.Proximity photopharmacology enables selectiveprotein control withspatiotemporal and cell type specificity. In one version, the photoswitchableligand is attached to an engineered version of the receptor via aself-labeling protein tag  placedat a site in the receptor that is distal to the ligand binding site.40 Instead of attachmentto an engineered receptor, the PORTL (P) is covalently bound to anengineered membrane-anchor protein (M) that consists of an externallyfacing self-labeling protein tag fused to a single-pass transmembranesegment  D2 PORTLs: an agonist(agoMP-D2), a partial agonist p.ago(MP-D2), and an antagonist (blockMP-D2). Importantly, by incorporating a red-shifted fast-relaxingazobenzene into the pharmacophores used in each PORTL, we obtainedone-wavelength photocontrol of D2R, with a light-driven turn-on anda rapid turn-off in the dark. These novel MPs could be used to providecell type-specific and spatiotemporally precise control of endogenousD2R in vivo.We recentlysolved this problem with our Membrane-anchored PhotoswitchableRemotely Tethered Ligand (MP) approach, which combines the cell type-specificcontrol of endogenous receptors afforded by DARTs with the temporalprecision of photopharmacology.segment 1A.When segment 1A, the Msegment 1B. Recen42 Recentprogress has led to the development of bitopic compounds, which consistof a primary pharmacophore that binds the orthosteric binding site,a hydrophobic secondary pharmacophore that binds a secondary bindingsite, and an aliphatic linker that forms a bridge between the twopharmacophores.44 Importantly, the identity ofthe secondary pharmacophore and the length and substitution patternof the aliphatic linker can influence receptor affinity, subtype selectivity,and signaling bias.50 While most of the privileged scaffolds for the bitopic D2R ligandsshow polypharmacology across other non-dopaminergic GPCRs, our MPapproach will only render off-target activity to GPCRs of concernthat are expressed on cells targeted by the M.52 The hydrophobic secondary pharmacophore stood out as an ideal unitfor azologization, as previous work has shown that a fulgimide ordiarylethene photoswitch can be incorporated at this position .53 Furthermore,it has been shown that bitopic compounds can be elongated beyond thesecondary pharmacophore with a remote fluorophore or a second ligand,56 hinting that an analogous extension with a PEG linker and covalentbinding to the M anchor protein could be tolerated  or UV-A light (∼20% trans: ∼80% cis), limiting the dynamicrange of photoswitching.60 This limitationalso applies to variety of other commonly used photoswitches (fulgi(mi)de,diarylethene, stilbene, hemithioindigo, spiropyrane and the LOV2 peptide).67 In contrast, since many azobenzenes are purely trans when left to relax for long enough in the dark,57 azobenzene variants that undergo this thermal relaxationrapidly provide the advantage of being able to toggle between lightactivated majority cis and fully trans in the dark. Therefore, if the trans-configurationwas inert and the light-driven cis-configurationwas bioactive, the photoswitch would avoid a finite floor of activationin the light-driven off state. This can be particularly useful inthe MP approach, where the concentration of the genetically encodedM anchor protein may be difficult to control in vivo. Therefore, we based our design of the D2R photoswitches on a red-shifted,fast-relaxing azobenzene moiety that is flanked by an amine and amide(a push–pull azobenzene), analogous to that in a glutamatereceptor photoswitch that we developed previously.68An additionaladvantage of red-shifted, fast-relaxing azobenzenesis their photostationary state (PSS). Unsubstituted azobenzene isan isomeric mixture under visible light  with freshly prepared acidchloride from acid 1 furnished the final construct P-D2ago.71 Motivated by the recent successof branched PORTLs bearing multiple photoswitchable glutamates inincreasing the effective concentration and sensitivity to light, wealso synthesized a branched analog of agoP-D2 with two PEG[24]-azobenzene-2-aminoindanes . The trans isomer absorbance maximum shifted to 444 nm in 10% DMSO . P-D2ago can be reversibly switched without fatigue . The photostationarystate of P-D2ago was determinedby NMR . Under in situ irradiationwith a 415 nm high-power LED, 20% cis content wasachieved .UV/vis studies confirmed the desired switching properties of t440 nm S2. The tt440 nm S2. The t29 To provide orthogonality with our D1MP, we switchedthe SNAP-tag to the HaloTag, which reacts with P-D2ago  that enhances surface expression. P-D2ago 1.P-D2ago tethered to the M (MP-D2ago) on D2R in HEK293Tcells using a coexpressed G protein-activated inwardly rectifyingK+ (GIRK) channel as an effector . Cells were cotransfected with D2R, the M, and GIRK andthen labeled with P-D2ago. Followingwashout of unbound P-D2ago, thecells were patch-clamped in whole-cell configuration in high externalK+ (120 mM), held at a negative holding potential (−80 mV),and switched from the dark to 440 nm (blue) light and then back againto the dark. Consistent with receptor activation and consequent openingof GIRK channels, blue light (to drive isomerization to the cis configuration) elicited a large inward current thatwas rapidly reversed by turning off the light   maximally activated D2R (101% of dopamine current) andhad no effect on dopamine-induced current (ago-2X photoactivated D2R withgreater sensitivity to light (∼5-fold) and faster activationkinetics (∼2-fold) than the single photoagonist MP-D2ago  in a mannerthat was rapid, reversible, and repeatable over multiple light exposures. In addition, P-D2ago-2X had no effect on HEK293Tcells lacking either D2R  or the M . Taken together,these results indicate that ago-2XMP-D2 worksas intended.Consistentwith the photophysical properties of its parent compound, P-D2ago 3A, B, P-ago-2XMP-D2 be completelyinactive in its trans-configuration in the dark.To measure the activityof ago-2XMP-D2 in the dark, we applied theD2R inverse agonist spiperone, which is a competitive inhibitor thatalso suppresses constitutive receptor activity. As expected, in HEK293Tcells expressing D2R and GIRK, spiperone elicited a reduction in inward current, consistent with inhibition of constitutiveD2R activity . Importantly,there was no difference between the effect of spiperone in the darkon cells that contained or lacked ago-2XMP-D2 , indicating that the photoswitchis completely inactive unless photoconverted to the cis-configuration with blue light.To be useful as a tool for controlling D2R, itis critical that P-D2ago-2X toM by measuring the kineticsof complex formation. Using flow cytometry, we found that the M isfully labeled by 1 μM compound in just 10 min , ∼4 times faster than the binding of ourprevious D1R photoswitch to SNAP-tag.40 We also measured the concentration dependence of P-D2ago-2X labeling. Interestingly,although flow cytometry indicated that 10 nM of P-D2ago-2X was required to approximatelyhalf-label the M , only 1 nMof compound was needed to reach approximately half-maximal photoactivationof D2R in the GIRK assay ,suggesting that there was an excess of P-D2ago-2X, likely due to a combination ofthe inherent high affinity of the agonist moiety, 2-aminoindane, forD2R and an excess of M. This is in contrast to PORTLs developed previouslyfor D1R and metabotropic glutamate receptors that require micromolarlevels of compound to achieve full receptor activation under similarM expression and photoswitch labeling conditions.40P-D2ago-2X wasabout 10-fold more potent when labeling was extended from 1 h to overnightlabeling . The ability to workat low labeling concentrations could be particularly beneficial whenP-D2ago-2X is appliedto the brain in vivo.We next characterized the bindingof P-D2ago-2X on D2R when attached to two different positions:(i) to the HaloTag in the M anchor protein of ago-2XMP-D2, where it is not physically attached to D2R , placing it in permanent closeproximity to the receptor’s orthosteric binding site. Interestingly,the magnitude and kinetics of photoactivation of D2R by P-D2ago-2X in these two configurationswere similar . Thisindicates that the M density in the membrane is sufficient for theP to reach the target receptor.We next compared theeffect of d to D2R 1, and muago-2XMP-D2 on the other members of the D2-likereceptor subfamily. ago-2XMP-D2 was a strongpartial photoagonist of D3R . In the GIRK assay,in response to blue light (to photoswitch to the cis configuration), P-D2p.ago tetheredto the M (p.agoMP-D2) partially activatedD2R (57% of dopamine current), an effect that was reversed by switchingback to the dark . P-D2p.ago-2X attached to the M (p.ago-2XMP-D2) partially activated the receptorto a similar degree as the monovalent analogue (50% of dopamine current)but also partially inhibited dopamine-induced receptor activation, a four-carbon linker, P-D2block(C4), or a five-carbon linker, P-D2block(C5) . P-D2block(C3) tethered to the M, or block(C3)MP-D2, inhibited dopamine-induced D2R activation in the GIRKassay  tethered to the M,or block(C4)MP-D2, more robustly inhibitedthe receptor than block(C3)MP-D2 (41% reductionin dopamine current) and had no agonist activity  tethered to the M, or block(C5)MP-D2, was less effective than block(C4)MP-D2  S9. P-D2block(C5) S9. P-D2block(C5) S9. P-D2block(C5) S9. P-D2bP-D2block(C4) with two 2-methoxy-phenylpiperazine moieties (P-D2block(C4)-2X) (P-D2block(C4)-2X tethered to the M (block(C4)-2XMP-D2) enhanced photoblock ∼1.5-fold compared toits monovalent counterpart -2X) 6E. P-D2bago-2XMP-D2, the partial photoagonist p.ago-2XMP-D2, and the photoantagonist block-2XMP-D2.In summary,we developed a toolkit of photoswitchable ligands tocontrol D2R in a cell-specific manner. These include the full photoagonist 77 The toolsdescribed here complement our previously described tethered D1R photoagonistsince they can be tagged to orthogonal fusion proteins  and have distinct wavelength sensitivity, potentially allowingfor multiplexed experiments in complex biological systems.Dopaminergic signalingspans a variety of physiological processes.The five dopamine receptors are expressed in diverse patterns in theCNS and periphery, making it challenging to pinpoint the exact roleof each receptor in each cell type or neural circuit. While many approacheshave been developed over the years to control a receptor of interest,none have provided control over endogenous receptors with cellularand spatiotemporal precision. The combination of membrane-anchoredligands for cell and spatial specificity with photopharmacology forprecise temporal control provides a solution and offers a powerfulmeans to study GPCR signaling.in vivo use, the gene encoding the M componentmust be delivered to the desired brain region and cell type. In rodents,this can be accomplished with established genetic approaches, suchas the Cre-lox system, that enable ectopic expression of the M proteinin specific neurons.78 For example, werecently targeted striatal D1R-expressing direct-pathway medium spinyneurons (dMSNs) by injecting mice expressing Cre recombinase in thiscell type (D1-Cre mice) with a Cre-dependent adeno-associated virus(AAV) encoding the SNAP-tag version of M that goes with P-D1ago.40 Along these lines, the HaloTag versionof M developed here for the P-D2 switches can be targeted to D2R-expressingneurons in the striatum, including indirect pathway-medium spiny neurons(iMSNs) using A2a-Cre mice, or to cholinergic interneurons (ChIs)using ChAT-Cre mice, or to dopamine neurons that innervate the striatumusing DAT-Cre mice.80 Once the M is expressed, a canulais implanted into the brain and used to infuse the photochemical Pand guide the placement of the optical fiber to the target area, thusconfining the photocontrol in three ways: via local delivery of thevirus encoding M and the P, as well as by local illumination.For 81 It is also interesting to consider developmentof biased P variants that selectively activate specific signalingproteins downstream of D2R ,since cellularly and spatiotemporally precise control of one suchpathway at a time could greatly enhance our understanding of the relationshipbetween this receptor and behavior. Such an advance would requirethe development of new assay systems that can detect the activationof specific signaling proteins in response to MP stimulation withlight. Finally, our D2R MPs could be combined with neuronal activityreporters to enable the simultaneous control of dopamine receptorsignaling and detection of neural activity. This challenge has beenpartly solved by the advent of reporters for calcium, voltage, orneural signal release  that operatein red/infrared light and are thus orthogonal to our blue-light sensitiveD2R MPs.83Several technical advancements could further increase the functionalityof the MP system. The delivery of both the AAV encoding the M geneand the P would be considerably simplified by versions that crossthe blood-brain barrier . To optimize expression of M, onecan adjust the AAV titer or boost M cell surface targeting with ERexport signals. In the future, it will be interesting to concentrateM in presynaptic terminals or postsynaptically using specific subcellulartargeting motifs to boost M density and increase the specificity ofthe modulation. In addition, while tethering the P to the M proteinin specific cells avoids unwanted effects in off-target cells, itcould still bind off-target receptors in those target cells. Thiscould be solved through the substitution of photoligands that bindin the orthosteric site with more selective allosteric ligands.84 Dopaminereplacement with D2R agonists  is a standard-of-carefor treatment of Parkinson’s disease.85 However, as classical drugs, D2R agonists have two major problems:they reduce therapeutic efficacy and increase adverse side effects.First, they bind on- and off-target proteins beyond the striatum.Second, it is difficult, if not impossible, to apply the ideal therapeuticdose of D2R agonists once they are administered and metabolized bythe body. These problems could be solved by targeting ago-2XMP-D2 specifically to the striatum and fine-tuningits dose using light to mimic the actions of the dopamine that islost in Parkinson’s disease. In addition to Parkinson’sdisease, p.ago-2XMP-D2 and/or block-2XMP-D2 could potentially be used to treat schizophreniaby targeting therapeutically relevant D2Rs in the brain, thereby avoidingoff-target effects in both the central nervous system and peripheralorgans .The MPs developed here have the potentialto help uncover the roleof D2R in various dopamine-associated diseases and could potentiallybe used as precision therapeutics. For example, Parkinson’sdisease is a movement disorder that results from the degenerationof dopamine neurons that project from the substantia nigra compactato the striatum, leading to the under-activation of striatal dopaminereceptors that control movement.


Methods, 2.3. Amplification and Illumina MiSeq sequencing, the forward primer 708F-DEG has been incorrectly pasted as the same as the 18S-V9R primer. The paragraph previously stated:In the published article, there was an error. In section rbcL chloroplast gene. The primers used to amplify the 18S gene were the universal eukaryotic 18S-V9F (TTGTACACACCGCCCGTCGC) and 18S-V9R . Libraries for the rbcL barcode were built using the diatom-specific primers 708F-DEG (CCTTCYGCAGGTTCACCTAC) and R3-DEG (CCTTCTAATTTACCWACWACWG), both modified from Vasselon et al. (2017a).”“Two markers were chosen for metabarcoding, namely the 150 base pair (bp) 18S-V9 region and a ~ 312 bp barcode within the The corrected sentence appears below:rbcL chloroplast gene. The primers used to amplify the 18S gene were the universal eukaryotic 18S-V9F (TTGTACACACCGCCCGTCGC) and 18S-V9R . Libraries for the rbcL barcode were built using the diatom-specific primers 708F-DEG (AGGTGAAGYWAAAGGTTCWTAYTTAAA) and R3-DEG (CCTTCTAATTTACCWACWACWG), both modified from Vasselon et al. (2017a).”“Two markers were chosen for metabarcoding, namely the 150 base pair (bp) 18S-V9 region and a ~312 bp barcode within the The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


CLCN4 gene can lead to X-linked intellectual disability and Raynaud–Claes syndrome (MRXSRC), characterized by severe cognitive impairment and mental disorders. This study aimed to investigate the genetic defects and clinical features of Chinese children with CLCN4 variants and explore the effect of mutant ClC-4 on the protein expression level and subcellular localization through in vitro experiments.The dysfunction of the in silico prediction tools. Protein expression and localization of mutant ClC-4 were measured using Western blotting (WB) and immunofluorescence microscopy. The impact of a splice site variant was assessed with a minigene assay.A total of 401 children with intellectual disabilities were screened for genetic variability using whole-exome sequencing (WES). Clinical data, including age, sex, perinatal conditions, and environmental exposure, were collected. Cognitive, verbal, motor, and social behavioral abilities were evaluated. Candidate variants were verified using Sanger sequencing, and their pathogenicity and conservation were analyzed using CLCN4 variants in six unrelated patients with intellectual disabilities, including two recurrent heterozygous de novo missense variants (p.D89N and p.A555V) in three female patients, and two hemizygous missense variants (p.N141S and p.R694Q) and a splicing variant (c.1390-12T > G) that are maternally inherited in three male patients. The p.N141S variant and the splicing variant c.1390-12, delayed speech, autism spectrum disorders (ASD), microcephaly, hypertonia, and abnormal imaging findings. The minigene splicing result indicated that the c.1390-12T > G did not affect the splicing of CLCN4 mRNA. In vitro experiments showed that the mutant protein level and localization of mutant protein are similar to the wild type.Exome analysis identified five rare CLCN4 gene variants associated with X-linked ID. It expanded the gene and phenotype spectrum of CLCN4 variants. The bioinformatic analysis supported the pathogenicity of CLCN4 variants. However, these CLCN4 gene variants did not affect the ClC-4 expression levels and protein location, consistent with previous studies. Further investigations are necessary to investigate the pathogenetic mechanism.The study identified six probands with  Raynaud–Claes syndrome (MRXSRC) is a rare X-linked mental developmental disorder characterized by intellectual disability (ID) or global developmental delay (GDD), language delay, autism spectrum disorder (ASD), anxiety, hyperactivity, epilepsy, brain abnormalities, and facial dysmorphism –6. AlthoCLCN4 gene , leading to ClC-4 protein dysfunction. The CLCN4 gene was initially identified and cloned by van Slegtenhorst et al.  are present at the beginning of the third exon, while the last exon (exon 13) terminates with the poly(A) tail in all examined species. The coding sequence from exon 3 to 13, which exhibits nearly 90% sequence identity among various species, is highly conserved throughout evolution. The CLCN4 gene encodes ClC-4, an intracellular 2Cl−/H+ transporter  is caused by mutations in the t et al.  who discvolution . Di Nguynsporter , 10, 11.nsporter . It is a−/H+ exchangers found in endolysosomal membranes. The structures of ClCs are remarkably similar. Among the ClC family, ClC-1, ClC-2, and ClC-K proteins are located at the plasma membrane and distributed in different tissues. ClC-K has two highly homologous isoforms that require the β-subunit barttin. On the other hand, vesicular ClCs (ClC-3 through ClC-7) have been shown to facilitate the acidification of intracellular vesicles by shunting the H+-ATPase currents  and previous reports , 25, 27,t common . In thisWe screened 401 children with ID using genetic testing conducted from 1 December 2018 to 31 October 2022. We retrospectively included patients who met the following ID criteria: patients with an intellectual disability disorder with poor or absent speech, autistic features, delayed motor delay, and certain brain imaging abnormalities. The study was approved by the Ethics Committee of the Children's Hospital of Nanjing Medical University  and was conducted according to the Helsinki Declaration. Informed consent forms were obtained from all legal guardians, and we collected clinical information, demographic data, clinical progression details, neuroimaging findings, as well as blood samples from the probands and their family members.CLCN4 gene, Sanger sequencing was performed. This additional sequencing technique helped confirm the presence or absence of the identified variants, as predicted by in silico tools.A 2 ml peripheral venous blood sample was collected and placed into disposable vacuum tubes for genetic analysis. Genomic DNA was extracted from peripheral leukocytes using a DNA isolation kit  following standard protocols. The DNA samples were subjected to whole-exome sequencing (WES) and next-generation sequencing , sorting intolerant from tolerant (SIFT), mutation taster, PROVEAN, and CADD. Splice site variants were predicted using MaxEntScan and dbscSNV software. Alignments of CLCN4 and its orthologs were performed using Clustal Omega .The CLCN4 cDNA (GenBank NM_001830) was synthesized by Youbio Biotechnology Company. The recombinant plasmid was inserted into pcDNA3.1-3xFlag vectors by subcloning . Based on the construction of the expression plasmid of wild-type pcDNA3.1-3xFlag-C1C-4-WT, four mutants, namely pcDNA3.1-3xFlag-C1C-4-D89N, pcDNA3.1-3xFlag-C1C-4-R694Q, pcDNA3.1-3xFlag-C1C-4-A555V, and pcDNA3.1-3xFlag-C1C-4-N141S recombinant expression plasmid, were constructed using the Mut Express II Fast Mutagenesis kit . All constructs were confirmed by bidirectional sequencing.The wild-type full-length human 2. After the cells were 50–70% confluent, HEK-293T cells were transfected with purified plasmids (containing either wild-type or mutant CLCN4) using PolyJetTM (SignaGen) by the manufacturer's protocols. The final concentration of each plasmid after transfection was 0.01 μg/μL. Transfection was conducted for 4 h with 2 μg of pcDNA3.1-3xFlag-C1C-4-D89N, pcDNA3.1-3xFlag-C1C-4-R694Q, pcDNA3.1-3xFlag- C1C-4-A555V, pcDNA3.1-3xFlag-C1C-4-N141S, and pcDNA3.1-3x Flag-WT DNA, and the cells were harvested after 24 h.HEK-293T cells (human embryonic kidney cells) were transiently transfected with wild-type (WT) or mutant hClC-4. Briefly, 24 h before transfection, HEK-293T cells were seeded in six-well plates with 2 ml of DMEM in each well at 37°C in an atmosphere of 5% COt-test with GraphPad Prism software. All experiments were repeated at least three times, and data were presented as the mean ± SD.HEK-293T cells were rapidly washed with ice-cold PBS, and whole-cell lysates were generated using a lysis buffer containing protease inhibitors. The cell total proteins were extracted according to the manufacturer's instructions . Protein concentration was determined using a micro-BCA protein assay kit with BSA as a standard . Then, 20 μg of total protein was separated by 10% SDS-PAGE and transferred onto the PVDF membrane. The membranes were blocked by TBS-T (0.1% Tween 20 in TBS) containing 5% non-fat milk for 1 h at room temperature and then incubated with primary antibodies against Flag , GAPDH  overnight at 4°C, followed by the incubation of HRP-labeled secondary antibodies at room temperature for 1 h. Membranes were visualized by the chemiluminescence reaction. Band intensity was quantified using ImageJ software . Differences between groups were calculated using an unpaired 2 atmosphere at 37°C. The wild-type and four mutant ClC-4-3xFlag plasmid DNAs were transfected using PolyJetTM (SignaGen), according to the manufacturer's guidelines. Then, the cells were fixed with methanol for 20 min at 4°C followed by staining the cells' nuclei with DAPI . The cells were rinsed with PBS and incubated with the primary mouse anti-Flag  and rabbit anti-Na+/K+-ATPase antibodies  overnight at 4°C. The secondary antibodies were added and incubated for 2 h at 37°C. Imaging was performed on an inverted confocal laser scanning microscope using a 63/1.4 oil immersion objective. The excitation wavelength was 488 nm for the enhanced green fluorescent protein (EGFP), 543 nm for Cy3, and 340 nm for DAPI. Moreover, the wild-type and mutant ClC-4 proteins were stained with ClC-4-specific antibodies (green) to revalidate the effect of mutant protein on subcellular localization.To determine the localization of ClC-4, HEK-293T cells were grown in 24-well culture plates containing glass slides in DMEM with 10% FCS in a 5% COCLCN4 variants. MRXSRC was shown to be caused by variants in the CLCN4 gene according to previous research  who carried rare research  and the CLCN4 gene variants in these individuals.Brain magnetic resonance imaging (MRI) was performed in four patients , and the results revealed a diverse spectrum of abnormalities, as shown in CLCN4. We identified four missense variants  and a novel splice site variant (c.1390-12T > G) in the six patients  and p.A555V (P3 and P4). Three hemizygous variants that are maternally inherited were identified in three unrelated male patients: P2 carried a novel missense variant, p.N141S, P5 carried a novel splicing variant (c.1390-12T > G), and P6 carried the p.R694Q variant that had been reported in two healthy female carriers. According to the ACMG guidelines, the p.D89N, p.N141S, and p.R694Q variants were predicted to be of uncertain significance , while the p.A555V variant was predicted to be pathogenic (PS2 + PS4 + PM2_supporting + PP3)  .in silico predictions , all variants were most likely damaging and deleterious  was predicted to be of uncertain significance (PM2 + PP3). This variant is located at the 12th position of the splice acceptor site of exon 10 and has not been previously reported in any public genomic databases. CLCN4 variants do not alter the subcellular location of ClC-4 . The flag-specific monoclonal antibody revealed a protein band (84.9 kDa) with similar protein expression levels for all constructs . Subsequof ClC-4 .in vitro. We evaluated the effect of the variant on splicing by the pSPL3 minigene reporter in HEK-293T cells. The cDNA analysis prepared from HEK293 cells indicated that the c.1390-12T > G variant may disturb normal splicing in vitro. However, agarose gel electrophoresis performed by RT-PCR products showed that no skipping exons occurred , transmembrane (TM), and intramembrane (IM) domains. The TM domains affect the function of ion channels, whereas CBS domains affect the gating and localization of a transmembrane protein and regulate membrane components  and the splice site variant (c.1390-12T > G) in P6. Variants such as p.N141S and c.1390-12T > G are novel, and p.R694Q has been identified in two asymptomatic heterozygous females in a previous study. Importantly, p.D89N, p.N141S, and p.A555V variants were located in the TM domains of the ClC-4 protein. In a previous study, GOF variants were found, namely, p.D89N and p.A555V variants, which disrupted the gating process of ClC-4 that prevents inward currents , delayed speech, autism spectrum disorders (ASD), microcephaly, hypertonia, and abnormal imaging manifestations. The onset age of our patients was under 3 years old. The genetic analysis indicated that pathogenic variants of the CLCN4 gene, identified using multiple in silico prediction tools, may result in the loss of function of the ClC-4 protein. In P6, we identified a novel splice site variant c.1390-12T > G, which was predicted to lead to affect the normal splice of ClC-4 mRNA. However, the in vitro minigene splicing reporter assay revealed that the splice site variant had no significant effect on RNA splicing. It is possible that the in vitro minigene splicing experiments cannot fully simulate gene splicing in vivo. In in vitro experiments, we found that the four mutants  did not affect the ClC-4 protein level and localization. In the previous study, p.A555V did not affect the expression and location but reduced the ClC-4 currents , The studies involving humans were approved by Ethics Committee of the Children's Hospital of Nanjing Medical University . The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants' legal guardians/next of kin. Ethical approval was not required for the studies on animals in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used. Written informed consent was obtained from the individual(s), and minor(s)' legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.XZ and CW designed the study. SL drafted the article and performed the experiments. CW and SL performed the genetic analysis and bioinformatics evaluations. XZ and SL conducted the clinical evaluations. SL, PL, and WZha collected clinical data and follow-up. All authors analyzed the data and approved the final article.


A decreased proportion of CD56+ T cells was found in ICU patients with fatal outcome. Severe COVID-19 was accompanied by a decrease in the proportion of CD8+ T cells, mainly due to the CD56− cell death, and a redistribution of the NKT-like cell subset composition with a predominance of more differentiated cytotoxic CD8+ T cells. The differentiation process was accompanied by an increase in the proportions of KIR2DL2/3+ and NKp30+ cells in the CD56+ T cell subset of COVID-19 patients and convalescents. Decreased percentages of NKG2D+ and NKG2A+ cells and increased PD-1 and HLA-DR expression levels were found in both CD56− and CD56+ T cells, and can be considered as indicators of COVID-19 progression. In the CD56− T cell fraction, increased CD16 levels were observed in MS patients and in ICU patients with lethal outcome, suggesting a negative role for CD56−CD16+ T cells in COVID-19. Overall, our findings suggest an antiviral role of CD56+ T cells in COVID-19.The effectiveness of the antiviral immune response largely depends on the activation of cytotoxic T cells. The heterogeneous group of functionally active T cells expressing the CD56 molecule (NKT-like cells), that combines the properties of T lymphocytes and NK cells, is poorly studied in COVID-19. This work aimed to analyze the activation and differentiation of both circulating NKT-like cells and CD56 Coronavirus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) leads to complex immune dysregulation and affects different components of the antiviral cellular immunity. In particular, T cell immune response, which plays an essential role in infection control, may vary in patients with mild, moderate, and severe COVID-19 [+ T cells, the so-called NKT-like cells, constitute a subset of T cells that express both T cell receptor (TCR) and NK cell receptors, such as killer-cell immunoglobulin-like receptors (KIRs), Fc receptor FcγRIII (CD16), C-type lectin-like receptors , and others. Thus, NKT-like cells exhibit both adaptive and innate cell properties [+ cells at the late stages of differentiation, gamma delta (γδ) T cells, and mucosa-associated invariant T (MAIT) cells [+ T cells is closely correlated with their cytotoxic activity [+ T cell fraction and their immunostimulatory effector role were shown in patients infected with such viruses as HIV and hepatitis C [The specific adaptive response, induced by the recognition of a certain viral peptide presented by MHC molecules, is a hallmark of T cells. CD56operties . This suT) cells , all of activity . Changesatitis C .+ subset [+ cells constitute the majority of the NKT-like subset [+ T cells with more mature phenotype during SARS-CoV-2 infection [Over the past two years, most reports on COVID-19 have been highlighting lymphopenia as the main feature reflecting the severity of COVID-19 . T cell + subset ,13,14,15+ subset . It has e subset . Severalnfection ,19,20.+CD56+ cell fraction during COVID-19 in different cohorts of patients was observed in several studies [+ T cell frequency were shown to be independent of COVID-19 severity [+ T cell subset size as a biomarker of COVID-19 severity needs more detailed consideration.The reduction in the CD3 studies ,13,21, a studies ,23. The severity ,23. Thus+ T cells in patients with severe COVID-19, characterized by an increased expression of CD38 or/and HLA-DR molecules [There is some evidence of T cell hyperactivation during COVID-19, which is associated with a poor clinical outcome of this disease . Severalolecules ,24,25,26olecules . Increasolecules ,28. In solecules .+ T cells often express KIRs, which interact with HLA class I molecules to modulate NK cell functional response [+ T lymphocytes are mainly CD8+CD27−CD28− and express CD45RA and CD57 but not CCR7; thus, these are effector memory T cells re-expressing CD45RA (TEMRA) [+CD8+ T cells in SARS-CoV-2 or influenza-infected patients correlated with the disease severity and vasculitis, however the authors strongly supported the hypothesis that KIR+CD8+ T cells perform a regulatory role in infection diseases, including COVID-19, by controlling autoreactive T cell response [+CD56+ T cell subset can increase dramatically in cytomegalovirus (HCMV) infection [+ T cells expressing NKp30-activating receptor exhibits a high potential for natural killer cell activity against tumor formations, and that IL-15 is able to induce the expression of NKp30 in a population of CD8+ T cells de novo [+ T cells are often characterized by an effector memory phenotype enriched within virus-specific T cells [+CD8+ T cell population in the peripheral blood was shown to be CCR7+CD45RA+CD28+, indicating a naïve phenotype [+ are negative for NKp30 [+ T cells in COVID-19 has been poorly described.CD56response . KIR+ T  (TEMRA) . Elevateresponse . The frenfection , but it nfection . Unlike  de novo . NK-rece T cells . In conthenotype . Accordior NKp30 . The rol− and CD56+ T cell subsets in the context of their differentiation and activation, including both classical markers of T cell differentiation and expression of activating and inhibitory NK cell receptors in samples from severe and moderate COVID-19 patients . Additionally, we examined changes in the most cytotoxic CD8+CD56+ subset level and granzyme B expression in T cells against the background of COVID-19. Further, we compared these features between T cells from the survivors and non-survivors in the group of patients with severe COVID-19.The prediction of further challenges associated with the emergence of new strains of SARS-CoV-2 is problematic. The Wuhan variant , which dhighCD14− cells. The gating strategy for determination of the lymphocytes subsets and their surface markers are presented in Most reports on COVID-19 have highlighted lymphopenia ,37,38. A+) in the ICU and MS groups; moreover, the percentage of CD3+ cells was lower in the non-survivors from the ICU group , central memory , effector memory  T cells, and TEMRA (CCR7−CD45RA+) cells in this most cytotoxic subset  after 4 h co-culture with K562 cell line. NKT-like cells from patients with severe COVID-19 had higher CD107a surface level compared to recovered donors.− and more differentiated CD56+ T cells. The total T cells and CD56− T cells from the ICU, MS, and CCP groups had higher proportions of HLA-DR+ cells compared to HD. Within the CD56+ T cell subset, the HLA-DR expression level was also upregulated in COVID-19 patients compared to healthy individuals, while the CCP group had a similar level to the HD  [+ T cells are a substantial component of the cytotoxic T cell response to HCMV in healthy carriers [+CD56+ T cells are considered the most cytotoxic subset among all T lymphocytes [+ cells in CD56− but not in the CD56+ T cell subset in the ICU group compared to the HD, which suggests an increased survival ability of the CD56+CD8+ T cells during COVID-19 (+ T cells has been suggested previously [CD56oxicity) . For exacarriers . Currentphocytes . The NK-phocytes . We founCOVID-19 f,g. An ieviously .+CD62L−, among the CD8+ T cells has been shown in patients with COVID-19 compared with HD [+CD27−CCR7− was shown in the CD8+ T cell subset [+ TEMRA cells defined as CCR7−CD45RA+ were increased in patients with severe and critical COVID-19, but in mild patients, TEMRA CD8+ T cells were similar to controls [−CD45RA+ in all T cells and in CD8+ T cell subset, defined as CCR7−CD45RA+, did not differ between the patients with COVID-19 and the HD group  and are involved in mediate antibody-dependent cellular cytotoxicity (ADCC) ex vivo . MoreoveNK cells . The pertoxicity . Highly COVID-19 . In our COVID-19 , while iHD group f, which HD group , and in HD group . Given tCOVID-19 , the selCOVID-19 .+ T cells and NK cells was registered and considered as a signal of depletion and inhibition of the antiviral immune response [+ NK cell inhibition by the viral peptide SARS-CoV-2-Nsp13 in patients with COVID-19 leads to their activation through the HLA-E [+ and in the CD56− and CD56+ T cell subsets, we recorded a reduced expression of the NKG2A receptor compared to the HD group . Written informed consent of each participant was received.The groups of patients were formed in accordance with the inclusion criteria: the confirmed presence of SARS-CoV-2 infection by PCR examination of the upper respiratory tract secretions, by presence of a clinical and radiological symptoms . Saturation level SpO2 ≤ 93%, intermittent mandatory ventilation (IMV) supporting, treatment in the pulmonology department of FMBA, the absence or the presence of diabetes mellitus, obesity, cardiovascular disease CVD, and other comorbid conditions that worsen the prognosis for recovery did not prevent patients from being included in the study. The exclusion criteria: presence of a blood disease or an oncological disease.According to the severity of the COVID-19 two groups of patients were defined: (1) 34 COVID-19 patients of the intensive care unit (ICU patients) ; (2) 28 patients with moderate COVID-19 severity (MS) . The healthy donors were separated into two groups: (1) 33 COVID-19 convalescent plasma donors (CCP), recovered from COVID-19 less than a month ago, with a high SARS-CoV-2-specific antibody titer in plasma ; (2) 48 volunteers (HD), who had no signs of COVID-19 infection in the last 6 months .3. Separated peripheral blood mononuclear cells (PBMC) were immediately used in phenotype and viability analysis, functional test by flow cytometry; plasma was stored at −60 °C until used.The blood samples from EDTA-containing test tubes were diluted in PBS solution  in 1:1 and centrifuged in a ficoll gradient with a density of 1.077 g/cmPBMC samples were stained with the panels of fluorochrome-conjugated monoclonal antibodies, which are in the Viability staining with SytoxBlue Dead Cell stain was performed . The gating strategy is presented in Samples were analyzed using a MACSQuant 10 flow cytometer  equipped with lasers λ = 405 nm, λ = 488 nm, λ = 635 nm; threshold was set to cut off events with low CD45 staining.Data processing was conducted by FlowJo X 10.0.7r2 .Intracellular staining with Alexa Fluor 647—labeled antibodies to granzyme B  in preliminary fixed PBMC samples  was performed, after that samples were analyzed by flow cytometry.+CD56+ cells.Flow cytometry was employed to analyze the natural cytotoxic ability of NKT-like cells in PBMCs by determining their degranulation level. The PBMCs were stimulated with IL-2, 500 U/mL, for an overnight period . NKT-like cells degranulation was detected by the level of expression of the lysosomal marker LAMP-1 (CD107a) using CD107a-VioBlue monoclonal antibody , as described earlier . Cells wUnbiased analysis of serum cytokines was performed using Luminex xMAP multiplex technology and the MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel Kit, according to the manufacturer’s standard protocol . Data processing was carried out using Belysa software v1.1.0  at the Resource Center “Cell Technology and Immunology”, Sirius University of Science and Technology.t-tests for parametric data and the Mann–Whitney U test for nonparametric data were performed. Pearson’s correlation test for data with normal distribution and Spearman’s correlation test for abnormally distributed data was applied for correlation analysis.GraphPad Prism 8.00 software  and presented as mean ± standard deviation. The ANOVA test for parametric data was carried out using post hoc Tukey’s multiple comparisons test; Kruskal–Wallis test followed by Dunn’s multiple comparison test for nonparametric samples was used. For comparison analysis of two groups, − and CD56+ (NKT-like cells) fractions. The NKT-like cell subset was decreased in patients with lethal outcome of COVID-19, which implies an antiviral role of this cell subpopulation and emphasizes its prognostic value. Along with an increase in the expression levels of HLA-DR and PD-1, and elevated IL-6 serum concentration, an increase in the CD16+ cell proportion among CD56− T cells may be considered the factor associated with increased COVID-19-mediated mortality. Moreover, a decrease in the expression of the NKG2D and NKG2A NK cell receptors observed in T cells of COVID-19 patients can potentially serve as prognostic indicators of the disease severity. Along with the depletion and hyperactivation of T cells, an elevated granzyme B level and a tendency to an increased TCR-independent functional activity were observed in CD56+ cells of patients with COVID-19. We have shown that CD56+ T cells increase expression of the KIR2DL2/DL3 and NKp30 receptors during COVID-19. The elevated KIR2DL2/DL3 and NKp30 levels were maintained in newly generated CD56+ T cells after the disease recovery. KIR+CD8+ T cells were previously supposed to play a regulatory role in several infection and autoimmune diseases [+ T cell subset, may promote significant progress in the study of SARS-CoV-2 and other viral infections. Single cell analysis of T cells, focused on NK cell receptors and signaling molecules, such as KIRs, NKp30, NKG2C, CD16, NKG2D, and FcεRIγ, may provide valuable insights into the role of such “innate-like” T cell subpopulation in the development of COVID-19 and other viral infections.In this work we have studied the COVID-19-induced redistribution of T cell subsets with a special focus on CD56


Compared to their healthy counterparts, patients with type 2 diabetes (T2D) can exhibit an altered gut microbiota composition, correlated with detrimental outcomes, including reduced insulin sensitivity, dyslipidemia, and increased markers of inflammation. However, a typical T2D microbiota profile is not established. The aim of this pilot study was to explore the gut microbiota and bacteria associated with prediabetes (pre-T2D) patients, and treatment naïve T2D patients, compared to healthy subjects.Fecal samples were collected from patients and healthy subjects (from Norway). The bacterial genomic DNA was extracted, and the microbiota analyzed utilizing the bacterial 16S rRNA gene. To secure a broad coverage of potential T2D associated bacteria, two technologies were used: The GA-map® 131-plex, utilizing 131 DNA probes complementary to pre-selected bacterial targets (covering the 16S regions V3-V9), and the LUMI-Seq™ platform, a full-length 16S sequencing technology (V1-V9). Variations in the gut microbiota between groups were explored using multivariate methods, differential bacterial abundance was estimated, and microbiota signatures discriminating the groups were assessed using classification models.In total, 24 pre-T2D patients, 18 T2D patients, and 52 healthy subjects were recruited. From the LUMI-Seq™ analysis, 10 and 9 bacterial taxa were differentially abundant between pre-T2D and healthy, and T2D and healthy, respectively. From the GA-map® 131-plex analysis, 10 bacterial markers were differentially abundant when comparing pre-T2D and healthy. Several of the bacteria were short-chain fatty acid (SCFA) producers or typical opportunistic bacteria. Bacteria with similar function or associated properties also contributed to the separation of pre-T2D and T2D from healthy as found by classification models. However, limited overlap was found for specific bacterial genera and species.This pilot study revealed that differences in the abundance of SCFA producing bacteria, and an increase in typical opportunistic bacteria, may contribute to the variations in the microbiota separating the pre-T2D and T2D patients from healthy subjects. However, further efforts in investigating the relationship between gut microbiota, diabetes, and associated factors such as BMI, are needed for developing specific diabetes microbiota signatures. Type 2 diabetes (T2D) is a significant global health challenge, constituting over 90% of diabetes cases worldwide, and with more than 700 million adults estimated to be impacted by 2045 . Often tGut microbiota plays a pivotal role in metabolism, immunomodulation and overall human health, and disruptions to the balance of this community are associated with many diseases, including inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS), and metabolic diseases such as T2D . AbundanOver the last decade, the most common approach for microbiota profiling has been to target the nine hypervariable regions (V1–V9) of the bacterial 16S rRNA gene. This relatively short  gene region provides phylogenetic signatures on different taxonomic levels. The hypervariable regions are surrounded by highly conserved sequences, which are used for primer design.The aim of this pilot study was to explore the gut microbiota and bacteria associated with pre-T2D patients, and newly diagnosed, treatment naïve T2D patients, compared to healthy subjects. To secure a broad coverage of potential T2D associated bacteria, two technologies were used: The GA-map® technology platform 131-plex (GA-map® 131-plex), utilizing 131 DNA probes complementary to pre-selected bacterial targets (covering the 16S rRNA gene regions V3-V9), and the Long 16S using Unique Molecular Identifiers – Sequencing (LUMI-Seq™) platform, a full-length 16S sequencing technology (V1-V9).n = 24) or T2D (n = 18) were recruited, and native fecal samples collected, by three diabetes clinics in Norway . In addition, healthy adult subjects (n = 52) were recruited, and native fecal samples collected by Oslo Metropolitan University (OsloMet) in Oslo, Norway. The study was approved by the regional Norwegian ethical committee , and informed consent was obtained from all participants. All samples and information were de-identified before analysis. The samples collected by the clinics were sent non-frozen to Genetic Analysis (GA), Oslo, Norway by mail and frozen upon delivery. The samples collected by OsloMet were frozen upon delivery to the university, before transferal to GA . All fecal samples were frozen (between -40 °C and -80 °C) within 5 days after collection before further processing.Adult patients diagnosed with either pre-T2D . Criteria for the inclusion of T2D patients included analysis of blood glucose levels, specifically, hemoglobin A1c (HbA1c) ≥ 6.5%. Inclusion criteria for the pre-T2D patients included HbA1c of 6.0–6.4%. Criteria for the inclusion of healthy subjects included no history of diabetes or pre-diabetes, and HbA1c < 6.0%. Exclusion criteria for all groups included recent use of antibiotics (last 4 weeks), and a positive fecal calprotectin  test (> 200 mg/kg). See Table Total bacterial genomic DNA extraction was performed by GA, using a protocol previously described . Brieflyin-silico , and against bacterial DNA from a selection of culturable bacterial species in vitro.The GA-map® 131-plex utilizes a pre-targeted approach, based on DNA probe hybridization to bacterial 16S rRNA gene targets, to identify and characterize bacterial profiles from fecal samples. This research-only panel of DNA probes  was established to cover major bacterial observations made from the literature relating to the microbiota in healthy, IBS and IBD . DisruptThe same laboratory procedures as described for the standardized and CE-marked GA-map® technology platform Dysbiosis Test were followed , with a Synthetic-long read sequencing is now emerging in the microbiome space as a methodology for generation of reliable quality long fragments from Illumina short reads –14. In tp < 0.1) in abundance of the bacterial markers.To account for variable signal levels, the raw signal data (fluorescent intensities) was normalized using a hybridization control, as previously described , and bacMicrobiota signatures for separation of pre-T2D or T2D and healthy subjects were calculated using the caret (classification and regression training) R-package . To evalAfter obtaining full-length 16S sequences, QIIME scripts (version 2019.7.0) were used for collapse of 100% identical sequences . Each unp-value lower than 0.05 were considered as differentially abundant.Principal coordinates analysis (PCoA) and PerMANOVA, using the Bray–Curtis method, was used to explore variations in bacterial profiles between and within the groups . The above-mentioned methods were also used to explore any confounding effects on the data due to the clinical variables . Differential abundance analyses were conducted with the DESeq2 package from Bioconductor , 24. Basp-value < 1% or 10%) was performed to reduce the dimensionality. The cross-validation procedure was repeated 300 times.Microbiota signatures for the separation of pre-T2D or T2D and healthy subjects were calculated using one of the most commonly used classifiers, Random Forest , in a fip: 0.001, R2: 0.93) on the data, while the parameters clinical groups , age , sex  and F-cal , had little to no significant impact. For the LUMI-Seq™ data, PCoA revealed little discrimination of samples according to the clinical groups (data not shown). Further, PerMANOVA analysis showed no significant impact on the data of the parameters clinical groups , age , sex  and BMI , tested as one PerMANOVA, without interaction.For the GA-map® 131-plex data, the PCA score plot Fig. , indicatAs found by the combined results from the GA-map® 131-plex and LUMI-Seq™ data analysis, mainly bacteria from the phyla Bacillota (Firmicutes), were differentially abundant in pre-T2D and T2D compared to the healthy group Tables A&B. NoteFaecalibacterium prausnitzii and Roseburia intestinalis, major producers of the SCFA butyrate [Veilonella spp. and Dialister (D. invisus), that can produce SCFAs such as propionate and acetate [p < 0.1) in healthy than pre-T2D. From the classification modeling, the butyrate producing R. intestinalis and F. prausnitzii [Parabacteroides johnsonii, producer of the SCFAs succinate and acetate [F. prausnitzii and another major butyrate producer, Agathobacter rectalis  [Phascolarctobacterium and Dialister, that can produce e.g., propionate and acetate [Roseburia and Roseburia faecis [For instance, according to the GA-map® 131-plex data analysis Table , the Clobutyrate , and the acetate , 28, werusnitzii  were als acetate , contribrectale) , and the acetate , 28, wera faecis  also conDorea spp., gas- and SCFA-producing bacteria [Dorea spp. contributed to the separation of pre-T2D and healthy  in T2D , a lactate producing bacteria [Holdemanella biformis (Eubacterium biforme), contributed to the separation of T2D and healthy , numerous being opportunistic [Eisenbergiella massiliensis (Clostridia class), a potential SCFA producer that may be associated with obesity [Neglecta timonensis (Clostridia class), possibly associated with T2D [Lactobacillus [Enterococcus faecalis), a diverse bacterial class containing both commensal and opportunistic bacteria [E. faecalis , as well as Bacillota, a highly diverse and abundant group of gut bacteria [Further, based on the LUMI-Seq™ analysis Table , Bacillitunistic , as well obesity , 38, andwith T2D , were mowith T2D , was amobacillus  for the bacteria , contribbacteria , to the Shigella spp./Escherichia spp. (Gammaproteobacteria class) [Aeromonas sp., and the Bacillota Clostridium sp., a potential pathogen [Campylobacter sp. (Epsilonproteobacteria), typical pathogenic bacteria [Haemophilus sp./Mannheimia sp., contributed to the separation of T2D and healthy  and a class)  were morpathogen , were ambacteria , and thethy Fig. B. Pseudothy Fig. A.For the GA-map® 131-plex classification modeling, area under the curve (AUC) values of 0.88 or 0.77 were achieved for the separation of pre-T2D and healthy subjects, or T2D and healthy subjects, respectively. For the LUMI-Seq™ classification modeling, AUC values of 0.78 or 0.64 were achieved for the separation of pre-T2D and healthy subjects, or T2D and healthy subjects, respectively.The primary aim of this pilot study was to explore the gut microbiota and associated bacteria in pre-T2D and treatment naïve T2D patients, compared to healthy subjects. LUMI-Seq™ and the GA-map® 131-plex represent two methods which can be used for bacterial profiling and to identify bacterial biomarkers. The results represent a preliminary discovery of possible diabetes specific bacterial patterns.F. prausnitzii and Roseburia were found to be less abundant, and the gas- and SCFA-producing Dorea more abundant, in pre-T2D and/or T2D, and were also among the ten most discriminative bacteria separating the groups from healthy. Additionally, SCFA producing bacteria such A. rectalis  and H. biformis (Eubacterium biforme), were indicated by classification models to contribute to this separation.Differences in the abundance of SCFA producing bacteria in the phylum Bacillota (Firmicutes) between healthy subjects and pre-T2D and T2D patients were revealed. SCFA producing Bacillota were also among the top ten contributors for the separation of pre-T2D and T2D from healthy using classification models. For example, the SCFA producing bacteria Streptococcus, and typical opportunistic, pro-inflammatory bacteria from the phylum Pseudomonadota (Proteobacteria) were increased in pre-T2D, and as found by classification models, contributed to the separation of pre-T2D and T2D from healthy.Further, also typical opportunistic bacteria contributed to the differentiation between the groups. For instance, bacteria from the Bacilli class, such as the opportunistic Roseburia, F. prausnitzii and A. rectalis are major SCFA producing bacteria, particularly of butyrate, which have been negatively associated with T2D and/or pre-T2D [Dorea, and bacteria from the Bacilli class and phylum Pseudomonadota have been found increased in T2D and/or pre-T2D [Dorea may promote inflammation, and increased abundance of Dorea in T2D patients has also been negatively correlated with the abundance of butyrate-producing bacteria [ pre-T2D –47. Oppo pre-T2D , 45, 48.bacteria , 50.Bacteria and their microbial products can impact the development of T2D by various and connected mechanisms, for instance, by affecting gut permeability, inflammatory regulation, and glucose metabolism . ButA strength of this study is that DNA was extracted by the standardized GA-map® method, and DNA samples split, before analysis by the two analysis platforms, as the choice of extraction method may influence the end results , 56. ThuAnother strength is the inclusion of treatment naïve T2D patients, as it has been shown that the use of the common diabetes drug Metformin may affect the gut microbiota , 58. IncEven though the same criteria for age and BMI were used for the inclusion of patients and healthy subjects, the healthy subjects were younger and had lower BMI . Also, the pre-T2D group had a slightly higher median F-cal, and the LUMI-Seq™ analysis included 5 subjects that should have been excluded due to F-cal > 200. While PerMANOVA of the GA-map® 131-plex data, showed that BMI had a significant effect on the data, F-cal levels did not. This is perhaps not surprising, as diabetes is strongly associated with higher BMI/overweight [Turicibacter sanguinis, found elevated in T2D by LUMI-Seq™, cannot be detected directly by the GA-map® 131 plex – however, may be covered by the broad Bacillota marker. Another limitation may be the use of different statistical methods, chosen to fit the dataset in question. For instance, for the GA-map® 131-plex data, bacterial abundances were considered as significantly different if p.adj. < 0.1, as a limit of 0.05 gave limited results.The GA-map® 131-plex detects 131 DNA probes representing pre-selected 16S rRNA bacterial targets, while the LUMI-Seq™ platform entails full length 16S rRNA sequencing. Differences in the targeted 16S regions for the GA-map® 131-plex and LUMI-Seq™ , as well as the selected targets and the pre-determined taxonomic levels of the GA-map® method, may lead to differences in the phylogenetic resolution. This may be one reason for the two method’s limited overlap in genera and species of potential T2D associated bacteria. For instance, It is critical for researchers to take into consideration the strengths and limitations of different platforms and choose a system appropriate for their experimental design. The GA-map® platform offers the advantage of a standardized method, utilizing a pre-selected target approach, allowing for a reduced assay turn-around time and less resource-demanding data analysis. At the level of genera, the GA-map® technology exhibit strong correlation to MiSeq amplicon sequencing . Even thThis pilot study focused on a limited number of Scandinavian (Norwegian) participants only, and so the results and interpretation should be taken with caution. The recruitment and inclusion of treatment naïve T2D patients is especially challenging due to, following standard guidelines, the limited time between diagnosis and start of treatment. The lower number of pre-T2D and T2D patients may have affected the outcome and can be one explanation for the pre-T2D group seemingly having the most distinct microbiota composition. Differences in diet may be another factor affecting the results, as no detailed description of the diet was recorded. To strengthen the foundation for developing bacterial signatures for Type 2 diabetes, future studies should be larger, international, multi-site studies, to account for variation in inter-individual microbiota. Possible confounding factors that ought to be controlled closely include medication-use, diet, and lifestyle –63.Multiple studies have provided compelling evidence of an altered state in gut microbiota composition in pre-T2D and T2D individuals as compared to healthy subjects, with a strong correlation to insulin resistance and β-cell dysfunction, detected even prior to glucose abnormalities in these individuals , 64–66. This pilot study revealed that differences in the abundance of short chain fatty acid (SCFA) producing bacteria, and an increase in typical inflammation-associated or potentially pro-inflammatory or opportunistic bacteria, may contribute to the variations in the microbiota separating the pre-T2D and T2D patients from the healthy subjects. However, further efforts in investigating the relationship between gut microbiota, diabetes, and associated factors such as BMI, are needed for developing specific diabetes microbiota signatures.