Graft dysfunction in compassionate use of genetically engineered pig-to-human cardiac xenotransplantation: a case report.

BACKGROUND: A genetically engineered pig cardiac xenotransplantation was done on Jan 7, 2022, in a non-ambulatory male patient, aged 57 years, with end-stage heart failure, and on veno-arterial extracorporeal membrane oxygenation support, who was ineligible for an allograft. This report details our current understanding of factors important to the xenotransplantation outcome. METHODS: Physiological and biochemical parameters critical for the care of all heart transplant recipients were collected in extensive clinical monitoring in an intensive care unit. To ascertain the cause of xenograft dysfunction, we did extensive immunological and histopathological studies, including electron microscopy and quantification of porcine cytomegalovirus or porcine roseolovirus (PCMV/PRV) in the xenograft, recipient cells, and tissue by DNA PCR and RNA transcription. We performed intravenous immunoglobulin (IVIG) binding to donor cells and single-cell RNA sequencing of peripheral blood mononuclear cells. FINDINGS: After successful xenotransplantation, the graft functioned well on echocardiography and sustained cardiovascular and other organ systems functions until postoperative day 47 when diastolic heart failure occurred. At postoperative day 50, the endomyocardial biopsy revealed damaged capillaries with interstitial oedema, red cell extravasation, rare thrombotic microangiopathy, and complement deposition. Increased anti-pig xenoantibodies, mainly IgG, were detected after IVIG administration for hypogammaglobulinaemia and during the first plasma exchange. Endomyocardial biopsy on postoperative day 56 showed fibrotic changes consistent with progressive myocardial stiffness. Microbial cell-free DNA testing indicated increasing titres of PCMV/PRV cell-free DNA. Post-mortem single-cell RNA sequencing showed overlapping causes. INTERPRETATION: Hyperacute rejection was avoided. We identified potential mediators of the observed endothelial injury. First, widespread endothelial injury indicates antibody-mediated rejection. Second, IVIG bound strongly to donor endothelium, possibly causing immune activation. Finally, reactivation and replication of latent PCMV/PRV in the xenograft possibly initiated a damaging inflammatory response. The findings point to specific measures to improve xenotransplant outcomes in the future. FUNDING: The University of Maryland School of Medicine, and the University of Maryland Medical Center.

Electro-oxidative intermolecular CSP2-H amination of heteroarenes via proton-coupled electron transfer.

A new electrochemical proton-coupled electron transfer method for the intermolecular CSP2-H amination of heteroarenes without oxidants, metal catalysts and external electrolytes has been developed. Various new N-containing heteroarenes were prepared in medium to high yields, and the indole-containing product could be converted into practical 2-oxindole by simple basic hydrolysis. Mechanistic investigation indicated that ester sulfonyl-substituted N-radicals could be formed by the combination of 2,6-lutidine and electrochemical oxidation, which is the key to achieve the desired chemoselectivity.

Dissecting the Role of the Hydroxyl Moiety at C14 in (+)-Opioid-Based TLR4 Antagonists via Wet-Lab Experiments and Molecular Dynamics Simulations.

Toll-like receptor 4 (TLR4) is pivotal as an innate immune receptor, playing a critical role in mediating neuropathic pain and drug addiction through its regulation of the neuroinflammatory response. The nonclassical (+)-opioid isomers represent a unique subset of TLR4 antagonists known for their effective blood-brain barrier permeability. Despite growing interest in the structure-activity relationship of these (+)-opioid-based TLR4 antagonists, the specific impact of heteroatoms on their TLR4 antagonistic activities has not been fully explored. This study investigated the influence of the hydroxyl group at C14 in six (+)-opioid TLR4 antagonists (1-6) using wet-lab experiments and in silico simulations. The corresponding C14-deoxy derivatives (7-12) were synthesized, and upon comparison with their corresponding counterparts (1-6), it was discovered that their TLR4 antagonistic activities were significantly diminished. Molecular dynamics simulations showed that the (+)-opioid TLR4 antagonists (1-6) possessed more negative binding free energies to the TLR4 coreceptor MD2, which was responsible for ligand recognition. This was primarily attributed to the formation of a hydrogen bond between the hydroxyl group at the C-14 position of the antagonists (1-6) and the R90 residue of MD2 during the binding process. Such an interaction facilitated the entry and subsequent binding of these molecules within the MD2 cavity. In contrast, the C14-deoxy derivatives (7-12), lacking the hydroxyl group at the C-14 position, missed this crucial hydrogen bond interaction with the R90 residue of MD2, leading to their egression from the MD2 cavity during simulations. This study underscores the significant role of the C14 hydroxyl moiety in enhancing the effectiveness of (+)-opioid TLR4 antagonists, which provides insightful guidance for designing future (+)-isomer opioid-derived TLR4 antagonists.

Toxicological effects induced by two carbamates on earthworms (Eisenia fetida): Acute toxicity, arrested regeneration and underlying mechanisms.

Eisenia fetida is recognised as advantageous model species in ecotoxicological and regeneration investigations. The intensive utilization of carbamate pesticides (CARs) imposes heavy residue burdens and grave hazards on edaphic environments as well as soil fauna therein. However, precise mechanisms whereby the specific CAR exerted toxic effects on earthworms remain largely elusive, notably from regenerative perspective. Herein, acute responses and regenerative toxicity of two carbamates (metolcarb, MEB and fenoxycarb, FEB) against E. fetida were dissected using biochemical, histological as well as molecular approaches following OECD guidelines at the cellular, tissue and organismal level. The acute toxicity data implied that MEB/FEB were very toxic/medium to extremely toxic, respectively in filter paper contact test and low to medium toxic/low toxic, respectively in artificial soil test. Chronic exposure to MEB and FEB at sublethal concentrations significantly mitigated the soluble protein content, protein abundance while enhanced the protein carbonylation level. Moreover, severely retarded posterior renewal of amputated earthworms was noticed in MEB and FEB treatments relative to the control group, with pronouncedly compromised morphology, dwindling segments and elevated cell apoptosis of blastema tissues, which were mediated by the rising Sox2 and decreasing TCTP levels. Taken together, these findings not only presented baseline toxicity cues for MEB and FEB exposure against earthworms, but also yielded mechanistic insights into regenerative toxicity upon CAR exposure, further contributing to the environmental risk assessment and benchmark formulation of agrochemical pollution in terrestrial ecosystem.

Biomimetic Nanobomb for Synergistic Therapy with Inhibition of Cancer Stem Cells.

Cancer stem cells (CSCs), a type of cell with self-renewal, unlimited proliferation, and insensitivity to common physical and chemical factors, are the key to cancer metastasis, recurrence, and chemo-resistance. Available CSCs inhibition strategies are mainly based on small molecule drugs, yet are limited by their off-target toxicity. The link between CSCs and non-CSCs interconversion is difficult to sever. In this work, a nanotherapeutic strategy based on MnOx -loaded polydopamine (MnOx /PDA) nanobombs with chemodynamic, photodynamic, photothermal and biodegradation properties to inhibit CSCs and non-CSCs concurrently is reported. The MnOx /PDA nanobombs can directly disrupt the microenvironment and tumorigenic capacity of CSCs by generating hyperthermia, oxidative stress and alleviating hypoxia. The markers of CSCs are subsequently downregulated, leading to the clearance of CSCs. Meanwhile, the synergistic therapy mediated by MnOx /PDA nanobombs can directly ablate the bulk tumor cells, thus cutting off the supply of CSCs transformation. For tumor targeting, MnOx /PDA is coated with macrophage membrane. The final tumor inhibition rate of the synergistic therapy is 70.8% in colorectal cancer (CRC) model. Taken together, the present work may open up the exploration of nanomaterial-based synergistic therapy for the simultaneous elimination of therapeutically resistant CSCs and non-CSCs.

Increased human complement pathway regulatory protein gene dose is associated with increased endothelial expression and prolonged survival during ex-vivo perfusion of GTKO pig lungs with human blood.

INTRODUCTION: Expression of human complement pathway regulatory proteins (hCPRP's) such as CD46 or CD55 has been associated with improved survival of pig organ xenografts in multiple different models. Here we evaluate the hypothesis that an increased human CD46 gene dose, through homozygosity or additional expression of a second hCPRP, is associated with increased protein expression and with improved protection from injury when GTKO lung xenografts are perfused with human blood. METHODS: Twenty three GTKO lungs heterozygous for human CD46 (GTKO.heteroCD46), 10 lungs homozygous for hCD46 (GTKO.homoCD46), and six GTKO.homoCD46 lungs also heterozygous for hCD55 (GTKO.homoCD46.hCD55) were perfused with human blood for up to 4 h in an ex vivo circuit. RESULTS: Relative to GTKO.heteroCD46 (152 min, range 5-240; 6/23 surviving at 4 h), survival was significantly improved for GTKO.homoCD46 (>240 min, range 45-240, p = .034; 7/10 surviving at 4 h) or GTKO.homoCD46.hCD55 lungs (>240 min, p = .001; 6/6 surviving at 4 h). Homozygosity was associated with increased capillary expression of hCD46 (p < .0001). Increased hCD46 expression was associated with significantly prolonged lung survival (p = .048),) but surprisingly not with reduction in measured complement factor C3a. Hematocrit, monocyte count, and pulmonary vascular resistance were not significantly altered in association with increased hCD46 gene dose or protein expression. CONCLUSION: Genetic engineering approaches designed to augment hCPRP activity - increasing the expression of hCD46 through homozygosity or co-expressing hCD55 with hCD46 - were associated with prolonged GTKO lung xenograft survival. Increased expression of hCD46 was associated with reduced coagulation cascade activation, but did not further reduce complement activation relative to lungs with relatively low CD46 expression. We conclude that coagulation pathway dysregulation contributes to injury in GTKO pig lung xenografts perfused with human blood, and that the survival advantage for lungs with increased hCPRP expression is likely attributable to improved endothelial thromboregulation.

Expression of human thrombomodulin by GalTKO.hCD46 pigs modulates coagulation cascade activation by endothelial cells and during ex vivo lung perfusion with human blood.

Thrombomodulin is important for the production of activated protein C (APC), a molecule with significant regulatory roles in coagulation and inflammation. To address known molecular incompatibilities between pig thrombomodulin and human thrombin that affect the conversion of protein C into APC, GalTKO.hCD46 pigs have been genetically modified to express human thrombomodulin (hTBM). The aim of this study was to evaluate the impact of transgenic hTBM expression on the coagulation dysregulation that is observed in association with lung xenograft injury in an established lung perfusion model, with and without additional blockade of nonphysiologic interactions between pig vWF and human GPIb axis. Expression of hTBM was variable between pigs at the transcriptional and protein level. hTBM increased the activation of human protein C and inhibited thrombosis in an in vitro flow perfusion assay, confirming that the expressed protein was functional. Decreased platelet activation was observed during ex vivo perfusion of GalTKO.hCD46 lungs expressing hTBM and, in conjunction with transgenic hTBM, blockade of the platelet GPIb receptor further inhibited platelets and increased survival time. Altogether, our data indicate that expression of transgenic hTBM partially addresses coagulation pathway dysregulation associated with pig lung xenograft injury and, in combination with vWF-GP1b-directed strategies, is a promising approach to improve the outcomes of lung xenotransplantation.

Driver Injury from Vehicle Side Impacts When Automatic Emergency Braking and Active Seat Belts Are Used.

As an advanced driver assistance system, automatic emergency braking (AEB) can effectively reduce accidents by using high-precision and high-coverage sensors. In particular, it has a significant advantage in reducing front-end collisions and rear-end accidents. Unfortunately, avoiding side collisions is a challenging problem for AEB. To tackle these challenges, we propose active seat belt pretensioning on driver injury in vehicles equipped with AEB in unavoidable side crashes. Firstly, records of impact cases from China's National Automobile Accident In-Depth Investigation System were used to investigate a scenario in which a vehicle is impacted by an oncoming car after the vehicle's AEB system is triggered. The scenario was created using PreScan software. Then, the simulated vehicles in the side impact were devised using a finite element model of the Toyota Yaris and a moving barrier. These were constructed in HyperMesh software along with models of the driver's side seatbelt, side airbag, and side curtain airbag. Moreover, the models were verified, and driver out-of-position instances and injuries were evaluated in simulations with different AEB intensities up to 0.7 g for three typical side impact angles. Last but not least, the optimal combination of seatbelt pretensioning and the timing thereof for minimizing driver injury at each side impact angle was identified using orthogonal tests; immediate (at 0 ms) pretensioning at 80 N was applied. Our experiments show that our active seatbelt with the above parameters reduced the weighted injury criterion by 5.94%, 22.05%, and 20.37% at impact angles of 90°, 105°, and 120°, respectively, compared to that of a conventional seatbelt. The results of the experiment can be used as a reference to appropriately set the collision parameters of active seat belts for vehicles with AEB.

Advances in coastal ocean boundary layer detection technology and equipment in China.

Accurate and comprehensive knowledge of the atmospheric environment and its evolution within the coastal ocean boundary layer are necessary for understanding the sources, chemical mechanisms, and transport processes of air pollution in land, sea, and atmosphere. We present an overview of coastal ocean boundary layer detection technology and equipment in China and summarize the progress and main achievements in recent years. China has developed a series of coastal ocean boundary layer detection technologies, including Light Detection and Ranging (LIDAR), turbulent exchange analyzer, air-sea flux analyzer, stereoscopic remote sensing of air pollutants, and oceanic aerosol detection equipment to address the technical bottleneck caused by harsh environmental conditions in coastal ocean regions. Advances in these technologies and equipment have provided scientific assistance for addressing air pollution issues and understanding land-sea-atmosphere interactions over coastal ocean regions in China. In the future, routine atmospheric observations should cover the coastal ocean boundary layer of China.

Pseudohalioglobus sediminis sp. nov., isolated from coastal sediment.

A Gram-stain-negative, strictly aerobic, non-motile and rod-shaped bacterial strain, designated NY5T, was isolated from marine sediment collected from coastal area in Weihai, China (122°07' 38.80'' E, 37°33' 57.60'' N). Cells of strain NY5T were 0.6-0.7 µm width and 1.9-2.0 µm length, catalase-positive and oxidase-positive. Growth of NY5T was observed at 25-37 °C (optimum, 28 °C) and pH 6.5-9.5 (optimum, pH 7.5-8.0) and in the presence of 0.5-7.0% (w/v) NaCl (optimum, 2.0%). The isoprenoid quinone was Q-8 and the predominant fatty acids were summed feature 8 (C18:1 ω7c and/or C18:1 ω6c), summed feature 3 (C16:1 ω7c and/or C16:1 ω6c) and C17:1 ω8c. Diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol were the major polar lipids. The DNA G+C content of strain NY5T was 60.1%. Strain NY5T showed the highest 16S rRNA gene sequence similarity (98.2%) with Pseudohalioglobus lutimaris followed by Parahaliea aestuarii (96.9%), Parahaliea maris (96.7%), Parahaliea mediterranea (95.9%), and Halioglobus japonicus (94.9%). Given these phenotypic and chemotaxonomic properties and phylogenetic analyses, strain NY5T was considered to represent a novel species of the genus Pseudohalioglobus, for which the name Pseudohalioglobus sediminis sp. nov. is proposed. The type strain is NY5T (=KCTC 72416T=MCCC 1H00401T).

Progressive genetic modifications of porcine cardiac xenografts extend survival to 9 months.

We report orthotopic (life-supporting) survival of genetically engineered porcine cardiac xenografts (with six gene modifications) for almost 9 months in baboon recipients. This work builds on our previously reported heterotopic cardiac xenograft (three gene modifications) survival up to 945 days with an anti-CD40 monoclonal antibody-based immunosuppression. In this current study, life-supporting xenografts containing multiple human complement regulatory, thromboregulatory, and anti-inflammatory proteins, in addition to growth hormone receptor knockout (KO) and carbohydrate antigen KOs, were transplanted in the baboons. Selective "multi-gene" xenografts demonstrate survival greater than 8 months without the requirement of adjunctive medications and without evidence of abnormal xenograft thickness or rejection. These data demonstrate that selective "multi-gene" modifications improve cardiac xenograft survival significantly and may be foundational for paving the way to bridge transplantation in humans.

Diastereoselective Generation of C2-Azlactonized 2H-Chromenes via Brønsted Acid-Catalyzed Oxo-Cyclization of Propargyl Alcohols.

A new Brønsted acid-catalyzed oxo-cyclization of propargyl alcohols with azlactones to synthesize C2-azlactonized 2H-chromenes has been established that uses 1,1'-binaphthyl-2,2'-diyl hydrogen phosphate (BiNPO4H) as the catalyst and gives excellent diastereoselectivities (≥19:1 dr) in most cases. This protocol has a high compatibility with various substituents of substrates, offering a catalytic and useful entry to the fabrication of the synthetically important C2-functionalized 2H-chromene scaffold.

Cardiac Xenotransplantation: Progress in Preclinical Models and Prospects for Clinical Translation.

Survival of pig cardiac xenografts in a non-human primate (NHP) model has improved significantly over the last 4 years with the introduction of costimulation blockade based immunosuppression (IS) and genetically engineered (GE) pig donors. The longest survival of a cardiac xenograft in the heterotopic (HHTx) position was almost 3 years and only rejected when IS was stopped. Recent reports of cardiac xenograft survival in a life-sustaining orthotopic (OHTx) position for 6 months is a significant step forward. Despite these achievements, there are still several barriers to the clinical success of xenotransplantation (XTx). This includes the possible transmission of porcine pathogens with pig donors and continued xenograft growth after XTx. Both these concerns, and issues with additional incompatibilities, have been addressed recently with the genetic modification of pigs. This review discusses the spectrum of issues related to cardiac xenotransplantation, recent progress in preclinical models, and its feasibility for clinical translation.

Working memory capacity, mental rotation, and visual perspective taking: A study of the developmental cascade hypothesis.

The present study aimed to investigate the mechanism underlying the development of level 2 visual perspective taking (VPT2). Specifically, we examined the role of working memory capacity (WMC) and mental rotation (MR) in the developmental change of VPT2 among early school-aged children. Children aged between 6 and 8 years (N = 150) completed measures to assess WMC, MR, and VPT2. Results showed that WMC, the ability of MR, and VPT2 developed progressively from 6 to 8 years old. The ability of VPT2 was significantly correlated with WMC and MR, even when age was statistically controlled for. Mediation analyses further revealed that both age-related changes in WMC and MR partially mediated the development of VPT2. Furthermore, age-related development in MR mediated the relationship between changes of WMC and VPT2. Our findings suggest the importance of WMC and MR in the early development of VPT2 and provide preliminary support for the developmental cascade hypothesis. That is, as children grow up, their WMC increases, leading to better capability of MR, which in turn results in the improvement of VPT2.

Effects of insecticides on malacostraca when managing diamondback moth (Plutella xylostella) in combination planting-rearing fields.

The combination of crop planting and animal rearing in the same area is popular. However, if the methods of planting and rearing are not appropriate, it will result in losses and the disruption of pest management. The toxicities of 17 insecticides to Plutella xylostella, Eriocheir sinensis, and Procambarus clarkii were tested. The recommended maximum field doses were used in 2 d and 4 d bioassays, and the levels of resistance of P. xylostella to insecticides were determined. Of five insecticides that had relatively low toxicity to E. sinensis and P. clarkii, spinetoram and MbNPV showed the best control efficacy of P. xylostella, followed by tetrachlorantraniliprole, chlorantraniliprole, and avermectin. P. xylostella had relatively little resistance to spinetoram, MbNPV, chlorantraniliprole, and avermectin. Therefore, we concluded that the best insecticides suitable for combination planting and rearing fields (cauliflower-crab or cauliflower-crayfish) were spinetoram and MbNPV, followed by chlorantraniliprole and avermectin. Other insecticides, such as emamectin benzoate, indoxacarb, and chlorfenapyr were effective at controlling P. xylostella, but they were not suitable for use in combination planting and rearing fields because of their high toxicity to crabs and crayfish.

Influences and transmission mechanisms of financial agglomeration on environmental pollution.

The mechanism between financial agglomeration and environmental pollution is an important concern for both academia and policymaking. The main objective of this paper is to study the nonlinear impacts of financial agglomeration on environmental pollution. A theoretical framework was first constructed based on the scale effect, structure effect, and technology innovation effect of financial agglomeration and a Copeland-Taylor endogenous growth model. Using the panel data of 281 Chinese prefectural-level cities from 2003 to 2019, a panel threshold regression model was introduced to estimate the nonlinear association between financial agglomeration and environmental pollution. Industrial smoke (dust) emissions and industrial wastewater discharge were adopted to quantify current environmental pollution in China. The results show that financial agglomeration had a significant effect on improving the environment characterized by gradient thresholds; also notable is that 68.64% of the cities crossed the threshold value, entering the decelerating phase of financial agglomeration inhibiting environmental pollution. Both upgrading industrial structure and enhancing marketization could reduce environmental pollution, whereas increasing human capital, environmental regulation, and energy consumption had a deteriorating effect. The three channels for financial agglomeration to reduce environmental pollution were revealed to be financial scale, financial structure, and financial technology innovation. Our findings provide strong evidence for policymaking in sustainable development.

Distribution and transport characteristics of fine particulate matter in beijing with mobile lidar measurements from 2015 to 2018.

Accurately quantifying the concentration and transport flux of atmospheric fine particulate matter (PM2.5) is vital when attempting to thoroughly identify the pollution formation mechanism. In this study, the mobile lidar measurements in Beijing on heavily polluted days in December from 2015 to 2018 are presented. The lidar was mounted on a vehicle, which could perform measurements along designated routes. On the basis of mobile lidar measurements along closed circuits of the 6th Ring Road around Beijing, the spatial distribution and transport flux of PM2.5 in Beijing were determined with information of wind field. In the spatial distribution, both the concentration and transport of PM2.5 were revealed to be more significant in the southern section of Beijing. The regional transport layer at heights < 1.3 km plays an important role in pollution formation. The maximum transport flux reached 1600 µg/(m2*sec) on 11 December 2016. With the aerosol boundary layer height determined from the image edge detection (IED) method, the inter-annual variations of the aerosol boundary layer height (ABLH) were also analysed. The ABLH decreased from 0.73 to 0.46 km during the same heavy pollution period from 2015 to 2018. Increasingly adverse aerosol boundary layer (ABL) meteorological factors, including lower ABLH, light winds, temperature inversions, and accumulated moisture, have become necessary for pollution formation in Beijing.

Artemisinin inhibits TLR4 signaling by targeting co-receptor MD2 in microglial BV-2 cells and prevents lipopolysaccharide-induced blood-brain barrier leakage in mice.

Artemisinin and its derivatives have been the frontline drugs for treating malaria. In addition to the antiparasitic effect, accumulating evidence shows that artemisinins can alleviate neuroinflammatory responses in the central nervous system (CNS). However, the precise mechanisms underlying their anti-neuroinflammatory effects are unclear. Herein we attempted to delineate the molecule target of artemisinin in microglia. In vitro protein intrinsic fluorescence titrations and saturation transfer difference (STD)-NMR showed the direct binding of artemisinin to Toll-like receptor TLR4 co-receptor MD2. Cellular thermal shift assay (CETSA) showed that artemisinin binding increased MD2 stability, which implies that artemisinin directly binds to MD2 in the cellular context. Artemisinin bound MD2 showed much less collapse during the molecular dynamic simulations, which supports the increased stability of MD2 upon artemisinin binding. Flow cytometry analysis showed artemisinin inhibited LPS-induced TLR4 dimerization and endocytosis in microglial BV-2 cells. Therefore, artemisinin was found to inhibit the TLR4-JNK signaling axis and block LPS-induced pro-inflammatory factors nitric oxide, IL-1ß and TNF-α in BV-2 cells. Furthermore, artemisinin restored LPS-induced decrease of junction proteins ZO-1, Occludin and Claudin-5 in primary brain microvessel endothelial cells, and attenuated LPS-induced blood-brain barrier disruption in mice as assessed by Evans blue. In all, this study unambiguously adds MD2 as a direct binding target of artemisinin in its anti-neuroinflammatory function. The results also suggest that artemisinin could be repurposed as a potential therapeutic intervention for inflammatory CNS diseases.

Small-Molecule Modulators of Toll-like Receptors.

Toll-like receptors (TLRs) are the "gatekeepers" of the immune system in humans and other animals to protect the host from invading bacteria, viruses, and other microorganisms. Since TLR4 was discovered as the receptor for endotoxin in the late 1990s, significant progress has been made in exploiting an understanding of the function of TLRs. The TLR-signaling pathway is crucial for the induction and progression of various diseases. Dysregulation of TLR signaling contributes to numerous pathological conditions, including chronic inflammation, sepsis, cancers, asthma, neuropathic pain, drug addiction, and autoimmune diseases. Therefore, manipulation of TLR signaling is promising to halt their activity in inflammatory diseases, to enhance their signaling to fight cancers, to modulate their role in autoimmune diseases, and to suppress them to treat drug addiction. TLR agonists have demonstrated great potential as antimicrobial agents and vaccine adjuvants, whereas TLR antagonists are being developed as reagents and drugs to dampen immune responses. Because of their pivotal potential therapeutic applications, fruitful small-molecule compounds and peptide fragments have been discovered, and many of them have advanced to various stages of clinical trials (though only two have been approved by the Food and Drug Administration (FDA): MPLA as a TLR4 agonist and imiquimod as a TLR7 agonist).In this Account, we focus on the progress in developing TLR signaling pathway modulators (mainly focused on the Yin and Wang laboratories) over the past decade and highlight the accomplishments and currently existing challenges in the development of TLR modulators. First, we briefly describe the members of the human TLR family along with their natural modulators. Second, we illustrate our endeavors to discover TLR-targeted agents using comprehensive approaches. Specifically, a discussion of identification and characterization of new chemical entities, determination of modes of action, and further applications is presented. For instance, the TLR3 antagonist was first discovered through in silico screening, and the inhibitory activity was confirmed in murine cells. Considering the glycosylation on TLR3, a new direction for TLR3 modulator design was pointed out to target asparagine glycosylation. We have particularly focused on the discovery of TLR4 antagonists and have assessed their great potential in the clinical treatment of drug addiction and alcohol use disorders. In addition, we discuss multiple other popular and robust techniques for modulator discovery. Not only small organic modulators but also stapled peptides and peptidomimetics will attract more and more attention in the future. Finally, current challenges, opportunities, and future perspectives for TLR-targeted agents are also discussed.

Salibaculum halophilum gen. nov., sp. nov. and Salibaculum griseiflavum sp. nov., in the family Rhodobacteraceae.

Two Gram-stain-negative, moderately halophilic, non-motile, rod-shaped, pale yellow, and aerobic strains, designated WDS1C4T and WDS4C29T, were isolated from a marine solar saltern in Weihai, Shandong Province, PR China. Growth of strain WDS1C4T occurred at 10-45 °C (optimum, 37 °C), with 4-16 % (w/v) NaCl (optimum, 8 %) and at pH 6.5-9.0 (optimum, pH 7.5). Growth of strain WDS4C29T occurred at 10-45 °C (optimum, 40 °C), with 2-18 % (w/v) NaCl (optimum, 6 %) and at pH 6.5-9.0 (optimum, pH 7.5). Q-10 was the sole respiratory quinone of the two strains. The major polar lipids of strains WDS1C4T and WDS4C29T were phosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine. The major cellular fatty acid in strains WDS1C4T and WDS4C29T was C18 : 1 ω7c, and the genomic DNA G+C contents of strains WDS1C4T and WDS4C29T were 67.6 and 63.3 mol%, respectively. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strains WDS1C4T and WDS4C29T were members of the family Rhodobacteraceae and showed 94.3 and 95.3 % similarities to their closest relative, Celeribacter indicus, respectively. The similarity between WDS1C4T and WDS4C29T was 97.3 %. Differential phenotypic and genotypic characteristics of the two isolates from recognized genera showed that the two strains should be classified as representing two novel species in a new genus for which the names Salibaculum halophilum gen. nov., sp. nov. (type species, type strain WDS1C4T=MCCC 1H00179T=KCTC 52542T) and Salibaculum griseiflavum sp. nov. (WDS4C29T=MCCC 1H00175T=KCTC 52541T) are proposed.