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Chimeric antigen receptor (CAR) T cell therapy relies on T cells that are guided by synthetic receptors to target and lyse cancer cells. CARs bind to cell surface antigens through an scFv (binder), the affinity of which is central to determining CAR T cell function and therapeutic success. CAR T cells targeting CD19 were the first to achieve marked clinical responses in patients with relapsed/refractory B cell malignancies and to be approved by the U.S. Food and Drug Administration (FDA). We report cryo-EM structures of CD19 antigen with the binder FMC63, which is used in four FDA-approved CAR T cell therapies (Kymriah, Yescarta, Tecartus, and Breyanzi), and the binder SJ25C1, which has also been used extensively in multiple clinical trials. We used these structures for molecular dynamics simulations, which guided creation of lower- or higher-affinity binders, and ultimately produced CAR T cells endowed with distinct tumor recognition sensitivities. The CAR T cells exhibited different antigen density requirements to trigger cytolysis and differed in their propensity to prompt trogocytosis upon contacting tumor cells. Our work shows how structural information can be applied to tune CAR T cell performance to specific target antigen densities.

There is no ideal therapy for testicular damage induced by Cr(VI); however, bone marrow mesenchymal stem cells (BMSCs) transplantation may be a promising therapy. A Cr(VI) solution was administered to rats by intraperitoneal injection for 30 days, then BMSCs from donor rats were transplanted. Two weeks later, decreased activity and appetite, along with other pathological changes, were improved in the BMSCs group. The location of BMSCs in damaged testes was observed via laser confocal microscopy. Chromium content in the Cr(VI) and BMSCs groups significantly increased compared with that in the control group, but there was no significant difference between the two groups, as revealed by atomic absorption spectrometry. The ferrous iron and the total iron content of testes in the BMSCs group were significantly lower than those in the Cr(VI) group, as observed by Lillie staining and a tissue iron assay kit. Western blotting and immunohistochemical analyses revealed that the expression of Beclin 1, LC3B, 4-hydroxynonenal, and transferrin receptor 1 was decreased in the BMSCs group, compared with the Cr(VI) group. The expression of glutathione peroxidase 4 (GPX4), SLC7A11, p-AKT, mammalian target of rapamycin (mTOR), and p-mTOR in the BMSCs group was higher than that in the Cr(VI) group. Taken together, we propose that BMSCs repair Cr(VI)-damaged testes by alleviating ferroptosis and downregulating autophagy-associated proteins through the upregulation of AKT and mTOR phosphorylation.

Purpose To investigate the relationship between the susceptibility to type 2 diabetes and gut microbiota and explore the potential mechanisms. Methods Thirty-two SPF SD rats were raised as the donor rats, and divided into control, type 2 diabetes mellitus (T2DM) and Un-mod groups. Feces were collected and prepared as fecal bacteria supernatant A (T2DM group), B (Un-mod group) and Con (control group). Another seventy-nine SPF SD rats were separated into normal saline (NS) and ABX groups. ABX and NS groups were given antibiotics solution and normal saline, respectively. And then, the ABX group were randomly separated into the ABX1 (4-weeks ordinary diet), ABX2 (4-weeks HFD and STZ ip), FMT-A (fecal microbiota A transplantation, 4-weeks HFD and STZ ip), FMT-B (fecal microbiota B transplantation, 4-weeks HFD and STZ ip) and FMT-Con (normal fecal microbiota transplantation, 4-weeks HFD and STZ ip) groups. The NS group were randomly divided into the NS1 (4-weeks ordinary diet) and NS2 (4-weeks HFD and STZ ip) groups. The short-chain fatty acids (SCFAs) in feces were detected by gas chromatography using 16S rRNA gene sequencing. G protein-coupled receptor 41 (GPR41) and GPR43 were detected by Western blot and quantitative real-time polymerase chain reaction. Results g__Ruminococcus_gnavus_group were increased obviously in the FMT-A group (vs ABX2 or FMT-B group). Blood glucose levels, serum insulin, total cholesterol levels, triglyceride levels, and low-density lipoprotein cholesterol levels were evidently increased in the FMT-A group than ABX1 or ABX2 group. It was found that both the FMT-A and FMT-B groups had evidently higher contents of acetic acid and butyric acid (vs ABX2 group). In both FMT-A and FMT-B groups, GPR41 protein expression and GPR43 mRNA and protein expression increased. Conclusions G__Ruminococcus_gnavus_group may cause T2DM susceptibility, and T2DM susceptible flora transplantation can evidently increase the susceptibility to T2DM. It is possible that gut microbiota-SCFAs-GPR41/GPR43 play a role in the development of T2DM. A new treatment strategies and methods may be to develop pharmaceutical treatments that reduce blood glucose by controlling gut microbiota.

GITR is a TNF receptor, and its activation promotes immune responses and drives antitumor activity. The receptor is activated by the GITR ligand (GITRL), which is believed to cluster receptors into a high-order array. Immunotherapeutic agonist antibodies also activate the receptor, but their mechanisms are not well characterized. We solved the structure of full-length mouse GITR bound to Fabs from the antibody DTA-1. The receptor is a dimer, and each subunit binds one Fab in an orientation suggesting that the antibody clusters receptors. Binding experiments with purified proteins show that DTA-1 IgG and GITRL both drive extensive clustering of GITR. Functional data reveal that DTA-1 and the anti-human GITR antibody TRX518 activate GITR in their IgG forms but not as Fabs. Thus, the divalent character of the IgG agonists confers an ability to mimic GITRL and cluster and activate GITR. These findings will inform the clinical development of this class of antibodies for immuno-oncology.

The activation of thermogenic programs in brown adipose tissue (BAT) and white adipose tissue (WAT) provides a promising approach to increasing energy expenditure during obesity and diabetes treatment. Although evidence has been found that rutin activates BAT against obesity and type 2 diabetes mellitus (T2DM), its potential mechanism is not completely understood. In this study, we focused on the potential modulating effect of rutin on short-chain fatty acids (SCFAs) and the thermogenesis of BAT and WAT, aiming to elucidate the molecular mechanism of rutin in the treatment of obesity and T2DM. The results showed that rutin could significantly reduce the body weight and fasting blood glucose, inhibit fat accumulation, relieve hepatic steatosis and ameliorate the disorder of glycolipid metabolism in db/db mice. Moreover, rutin also increased the expression of uncoupling protein 1 (Ucp1) and other thermogenic genes and proteins in BAT and inguinal WAT (IWAT), indicating that rutin activated BAT and induced browning of IWAT. Importantly, rutin markedly enhanced the concentration of SCFAs (acetate, propionate and butyrate) and SCFA-producing enzymes (acetate kinase (ACK), methylmalonyl-CoA decarboxylase (MMD) and butyryl-CoA (BUT)) in feces of db/db mice. In addition, rutin significantly increased the mRNA expression of monocarboxylate transporter 1 (Mct1), catabolic enzyme acyl-CoA medium-chain synthetase 3 (Acsm3), carnitine palmitoyl transferase 1α (Cpt-1α) and Cpt-1β genes in BAT and IWAT of db/db mice, which is conducive to inducing adipocyte thermogenesis. In summary, our findings revealed that rutin played a variety of regulatory roles in improving glucose and lipid metabolism disorders, reducing hepatic steatosis, inducing browning of IWAT and activating BAT, which has potential therapeutic significance for the treatment of obesity and T2DM. Mechanistically, rutin activates the thermogenesis of BAT and IWAT, which may be associated with increasing the concentration of SCFAs.

The incidence of liver-related complications in type 2 diabetes mellitus (T2DM) is rapidly increasing, which affects the physical and mental health of T2DM patients. Mulberry leaf flavonoids (MLF) were confirmed to have certain effects on lowering blood glucose and anti-inflammation. In this study, the high-fat diet (HFD) + STZ method was used to establish T2DM rat model and the MLF was administered by gavage for eight weeks. During the experiment, body weight and blood glucose level were measured at different time points. The pathological changes of rat liver were observed by HE staining. The serum glucolipid metabolic indicators of serum, fasting insulin (FINS), and inflammatory factors levels were detected by ELISA. The expression levels of toll-like receptor 4 (TLR4), TNF receptor-associated factor 6 (TRAF6), myeloid differentiation factor 88 (MyD88), inhibitor of NF

The hydrophobic coupling between membrane proteins and their host lipid bilayer provides a mechanism by which bilayer-modifying drugs may alter protein function. Drug regulation of membrane protein function thus may be mediated by both direct interactions with the protein and drug-induced alterations of bilayer properties, in which the latter will alter the energetics of protein conformational changes. To tease apart these mechanisms, we examine how the prototypical, proton-gated bacterial potassium channel KcsA is regulated by bilayer-modifying drugs using a fluorescence-based approach to quantify changes in both KcsA function and lipid bilayer properties (using gramicidin channels as probes). All tested drugs inhibited KcsA activity, and the changes in the different gating steps varied with bilayer thickness, suggesting a coupling to the bilayer. Examining the correlations between changes in KcsA gating steps and bilayer properties reveals that drug-induced regulation of membrane protein function indeed involves bilayer-mediated mechanisms. Both direct, either specific or nonspecific, binding and bilayer-mediated mechanisms therefore are likely to be important whenever there is overlap between the concentration ranges at which a drug alters membrane protein function and bilayer properties. Because changes in bilayer properties will impact many diverse membrane proteins, they may cause indiscriminate changes in protein function.

Background Qingwei San (QWS), one of classic Chinese Medicine prescripts, has been widely used to treat stomach heat syndrome which manifests oral ulcer (OU), periodontitis and upper gastrointestinal bleeding for seven hundred years. However, the therapeutic effects of QWS on diabetic OU subjected to stomach heat syndrome are still ambiguous. In the study, we investigated the pharmacological mechanisms. Methods The main components of QWS aqueous extract were analyzed by LC–MS, and potential pathways of QWS targeting OU were predicted by network pharmacology. The db/db mice were administered with the decoction of dried Zingiber officinale Rosc. rhizome combined with NaOH cauterization to establish the model of diabetic OU subjected to stomach heat syndrome. Subsequently, the model mice were treated with QWS, and OU wound healing status were recorded. The pathological changes of gastric tissue and oral mucosa were evaluated using hematoxylin–eosin staining, and the morphology of collagen fibers in oral mucosa was assessed by Masson staining. The levels of thromboxane B2 (TXB2), 6-Keto-prostaglandin F1α (6-keto-PGF1α), interleukin-1 β (IL-1β), IL-2, IL-6, tumor necrosis factor-α (TNF-α), β-endorphin (β-EP) and 5-Hydroxytryptamine (5-HT) were determined by ELISA assay. The protein expressions of Toll-like receptor 4 (TLR4), TNF receptor associated factor 6 (TRAF6), myeloid differentiation factor 88 (MyD88), inhibitor of NF-κB alpha (IκΒα), p-IκΒα and nuclear factor kappa-B (NF-κB) p65 were measured by Western Blotting. Results A total of 183 compounds in QWS were identified by LC–MS, and identified 79 bioactive compounds corresponded to 269 targets and 59 pathways. QWS high-dose treatment significantly reduced the level of TXB2 and the ratio of TXB2/6-keto-PGF1α. Meanwhile, it improved mucosal pathological morphology, and reduced the area of OU and local edema. Simultaneously, the levels of TNF-α, IL-1β, IL-6, IL-2 and 5-HT, and the expressions of TLR4, TRAF6, MyD88, p-IκΒα and NF-κB p65 were decreased. Conclusion QWS treatment facilitates the healing of OU, ameliorates pathological morphologies of gastric and oral mucosa and decreases the levels of pro-inflammatory cytokines in db/db mice subjected to stomach heat syndrome, whose mechanism may be associated with the inhibition of TLR4/MyD88/NF-κB signaling pathway to exert anti-inflammatory effects.

Ionizing radiation (IR) has found widespread application in modern medicine. As a result, radiotherapy inevitably causes spermatogenic cell injury. Many Chinese herbal prescriptions or natural extracts have the potential to protect against radiation injury.We used GC-2spd cells to investigate the effects and potential mechanisms of YQJD decoction on protecting spermatogenic cells from ionizing radiation injury.Firstly, the GC-2spd cells were irradiated withAfter irradiation withTaken together, the protective effects of YQJD decoction on

A number of diverse G-protein signaling pathways have been shown to regulate insulin secretion from pancreatic β-cells. Accordingly, regulator of G-protein signaling (RGS) proteins have also been implicated in coordinating this process. One such protein, RGS4, is reported to show both positive and negative effects on insulin secretion from β-cells depending on the physiologic context under which it was studied. We here use an RGS4-deficient mouse model to characterize previously unknown G-protein signaling pathways that are regulated by RGS4 during glucose-stimulated insulin secretion from the pancreatic islets. Our data show that loss of RGS4 results in a marked deficiency in glucose-stimulated insulin secretion during both phase I and phase II of insulin release in intact mice and isolated islets. These deficiencies are associated with lower cAMP/PKA activity and a loss of normal calcium surge (phase I) and oscillatory (phase II) kinetics behavior in the RGS4-deficient β-cells, suggesting RGS4 may be important for regulation of both Gαi and Gαq signaling control during glucose-stimulated insulin secretion. Together, these studies add to the known list of G-protein coupled signaling events that are controlled by RGS4 during glucose-stimulated insulin secretion and highlight the importance of maintaining normal levels of RGS4 function in healthy pancreatic tissues.

There have been lingering controversies reported decompression and plus fusion. And the relative safety of fusion in addition to standard decompression remains unclear. This study aimed to assess the effectiveness and safety of decompression alone or combined with fusion in lumbar spinal stenosis (LSS) with degenerative spondylolisthesis (DS).In this systematic review and meta-analysis, we searched the databases of PubMed, Embase, Cochrane Library, and Web of Science for relevant literature from their inception to 28th December 2021. We identified the eligible studies based on the PICOS principles, populations (LSS with DS), interventions (decompression alone), controls (decompression combined with fusion), outcomes [overall reoperation rate, complications, Oswestry Disability Index (ODI), operative time, the amount of blood lost, length of stay (LOS), and visual analog scales (VAS)], study design (cohort studies). Quality assessment for individual study was performed with the Newcastle-Ottawa Scale (NOS).In all, 12 articles involving a total of 14,693 patients were finally included in the study, the majority of patients underwent decompression alone (DA group: n=11,598) and the rest underwent decompression associated with fusion (FU group: n=3,095). The quality of most of the included studies was regarded as high quality. The results indicated that the FU group had a higher rate of complication [relative risk (RR): 1.770, 95% confidence interval (CI): 1.485 to 2.110], longer operative time [weighted mean difference (WMD): 51.037, 95% CI: 13.743 to 88.330], and increased blood loss (WMD: 258.354, 95% CI: 150.468 to 366.239) than the DA group (all P0.05), with no significant differences for overall reoperation rate (RR: 0.879, 95% CI: 0.432 to 1.786), ODI (WMD: -2.569, 95% CI: -6.548 to 1.409), LOS (WMD: 3.838, 95% CI: -2.172 to 9.848), and VAS found between the two groups (P0.05).In patients with LSS + DS, the effectiveness and safety of decompression alone may be superior to decompression plus fusion in terms of complication rate, operative time, and the amount of bleeding. However, more high-quality literature is needed in the future to confirm the best treatment choice for patients with LSS + DS.

Ionizing radiation damage refers to acute, delayed, or chronic tissue damage associated with ionizing radiation. Specific or effective therapeutic options for systemic injuries induced by ionizing radiation have not been developed. Studies have shown that Chinese herbal Medicine or Chinese Herbal Prescription exhibit preventive properties against radiation damage. These medicines inhibit tissue injuries and promote repair with very minimal side effects. This study reviews traditional Chinese herbal medicines and prescriptions with radiation protective effects as well as their mechanisms of action. The information obtained will guide the development of alternative radioprotectants.

Secondary active transporters reside in cell membranes transporting polar solutes like amino acids against steep concentration gradients, using electrochemical gradients of ions as energy sources. Commonly, ensemble-based measurements of radiolabeled substrate uptakes or transport currents inform on kinetic parameters of transporters. Here we describe a fluorescence-based functional assay for glutamate and aspartate transporters that provides single-transporter, single-transport cycle resolution using an archaeal elevator-type sodium and aspartate symporter Glt(Ph) as a model system. We prepare proteo-liposomes containing reconstituted purified Glt(Ph) transporters and an encapsulated periplasmic glutamate/aspartate-binding protein, PEB1a, labeled with donor and acceptor fluorophores. We then surface-immobilize the proteo-liposomes and measure transport-dependent Fluorescence Resonance Energy Transfer (FRET) efficiency changes over time using single-molecule Total Internal Reflection Fluorescence (TIRF) microscopy. The assay provides a 10-100 fold increase in temporal resolution compared to radioligand uptake assays. It also allows kinetic characterization of different transport cycle steps and discerns kinetic heterogeneities within the transporter population.

Supplemental Digital Content is available in the text, Background: In recent years, some studies indicated that repetitive transcranial magnetic stimulation (rTMS) could relieve neuropathic pain (NP) following a spinal cord injury (SCI), whereas some studies showed no pain relief effect. In addition, some studies showed the analgesic effect of transcranial direct current stimulation (tDCS) on NP post SCI, whereas other studies showed no effect. Methods: We systematically searched on the PubMed, Web of Science, EMBASE, Medline, Google Scholar for studies exploring the analgesic effect of rTMS or tDCS on NP post SCI until November 2019. Meta-analysis was conducted to summarize results of these studies. Results: The present quantitative meta-analysis indicated no significant difference in the effect of treatment on NP following SCI between rTMS and sham rTMS over the motor cortex at about 1 week after the end of the rTMS period (standardized mean difference (SMD) = 2.89, 95% confidence interval (CI) = −0.27 to 6.04). However, the study indicated that rTMS showed significantly better pain relief of treatment compared with sham rTMS between 2 and 6 weeks after the end of the rTMS period (SMD = 3.81, 95%CI: 0.80–7.52). However, no sufficient evidence could be provided to make a meta-analysis for the analgesic effect of tDCS on NP following SCI over the primary motor area (M1). Conclusions: In conclusion, the present meta-analysis suggested that rTMS did not show early analgesic effect on NP after SCI, but showed better middle-term analgesic effect, compared with sham rTMS. More large scale, blinded randomized controlled trials (RCTs) were needed to explore the analgesic effect of rTMS and tDCS on NP following SCI.

Single-molecule detection of transport turnovers shows a distribution of rates with fast transporters favored by mutations., Kinetic properties of membrane transporters are typically poorly defined because high-resolution functional assays analogous to single-channel recordings are lacking. Here, we measure single-molecule transport kinetics of a glutamate transporter homolog from Pyrococcus horikoshii, GltPh, using fluorescently labeled periplasmic amino acid binding protein as a fluorescence resonance energy transfer–based sensor. We show that individual transporters can function at rates varying by at least two orders of magnitude that persist for multiple turnovers. A gain-of-function mutant shows increased population of the fast-acting transporters, leading to a 10-fold increase in the mean transport rate. These findings, which are broadly consistent with earlier single-molecule measurements of GltPh conformational dynamics, suggest that GltPh transport is defined by kinetically distinct populations that exhibit long-lasting “molecular memory.”

SUMMARY The kainate receptors (KARs) are members of the ionotropic glutamate receptor family and assemble into tetramers from a pool of five subunit types (GluK1–5). Each subunit confers distinct functional properties to a receptor, but the compositional and stoichiometric diversity of KAR tetramers is not well understood. To address this, we first solve the structure of the GluK1 homomer, which enables a systematic assessment of structural compatibility among KAR subunits. Next, we analyze single-cell RNA sequencing data, which reveal extreme diversity in the combinations of two or more KAR subunits co-expressed within the same cell. We then investigate the composition of individual receptor complexes using single-molecule fluorescence techniques and find that di-heteromers assembled from GluK1, GluK2, or GluK3 can form with all possible stoichiometries, while GluK1/K5, GluK2/K5, and GluK3/K5 can form 3:1 or 2:2 complexes. Finally, using three-color single-molecule imaging, we discover that KARs can form tri- and tetra-heteromers., Graphical Abstract, In brief Selvakumar et al. use cryo-electron microscopy, single-cell RNA sequencing analysis, and single-molecule fluorescence techniques to investigate the stoichiometric and assembly diversity of kainate receptors (KARs). The work gives insight into KAR molecular diversity and expands the potential KAR subunit combinations to include a variety of di-, tri-, and tetra-heteromers.

Objective Yiqi Jiedu (YQJD) decoction is a Chinese herbal prescription, based on an experienced expert of traditional Chinese medicine. It is used for the injuries caused by radiotherapy. The current study was designed to investigate the protective effects of YQJD decoction on radiation damage of testis in mice, and to explore its potential mechanisms. Methods Mice were randomly divided into blank control group (Ctrl), model group (IR), positive drug group (IRA), and YQJD decoction group (IRY). After 10-day period intervention, they were whole-body irradiated with 2 Gy 60Co γ-rays and sacrificed on 7th day after irradiation. The indicators including the index and histopathology examination of testis, spermatogenic cell types and apoptosis, and the expression of TLR5, MyD88, NF-κB, TNF-α, IL-6 and Bcl-2 in testis. Results The testis atrophied significantly on 7th day of exposure to radiation, while YQJD decoction promoted the recovery of testis index and structure. Moreover, spermatogenic cell types and apoptosis had significant changes after irradiation. YQJD decoction protected the testicular function of spermatogenesis, as while as reduced the apoptosis rate of spermatogenic cells. In addition, RT-PCR and immunohistochemical analysis showed that YQJD decoction up-regulated the expression of TLR5 in testis. The levels of TLR5's downstream factors were also up-regulated in YQJD decoction group, which indicated that TLR5 signaling pathway might play an important role in the protective effects of YQJD decoction. Conclusions The results showed that YQJD decoction attenuated irradiation induced testis injury in mice. Its potential mechanism was related to TLR5 signaling pathway.