Delta-6 desaturase (Fads2) deficiency alters triacylglycerol/fatty acid cycling in murine white adipose tissue

The Δ-6 desaturase (D6D) enzyme is not only critical for the synthesis of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from α-linolenic acid (ALA), but recent evidence suggests that it also plays a role in adipocyte lipid metabolism and body weight; however, the mechanisms remain largely unexplored. The goal of this study was to investigate if a D6D deficiency would inhibit triacylglycerol storage and alter lipolytic and lipogenic pathways in mouse white adipose tissue (WAT) depots due to a disruption in EPA and DHA production. Male C57BL/6J D6D knockout (KO) and wild-type (WT) mice were fed either a 7% w/w lard or flax (ALA rich) diet for 21 weeks. Energy expenditure, physical activity, and substrate utilization were measured with metabolic caging. Inguinal and epididymal WAT depots were analyzed for changes in tissue weight, fatty acid composition, adipocyte size, and markers of lipogenesis, lipolysis, and insulin signaling. KO mice had lower body weight, higher serum nonesterified fatty acids, smaller WAT depots, and reduced adipocyte size compared to WT mice without altered food intake, energy expenditure, or physical activity, regardless of the diet. Markers of lipogenesis and lipolysis were more highly expressed in KO mice compared to WT mice in both depots, regardless of the diet. These changes were concomitant with lower basal insulin signaling in WAT. Collectively, a D6D deficiency alters triacylglycerol/fatty acid cycling in WAT by promoting lipolysis and reducing fatty acid re-esterification, which may be partially attributed to a reduction in WAT insulin signaling.     

Heteroarylamide smoothened inhibitors: Discovery of N-[2,4-dimethyl-5-(1-methylimidazol-4-yl)phenyl]-4-(2-pyridylmethoxy)benzamide (AZD8542) and N-[5-(1H-imidazol-2-yl)-2,4-dimethyl-phenyl]-4-(2- pyridylmethoxy)benzamide (AZD7254)

Aberrant hedgehog (Hh) pathway signaling is implicated in multiple cancer types and targeting the Smoothened (SMO) receptor, a key protein of the Hh pathway, has proven effective in treating metastasized basal cell carcinoma. Our lead optimization effort focused on a series of heteroarylamides. We observed that a methyl substitution ortho to the heteroaryl groups on an aniline core significantly improved the potency of this series of compounds. These findings predated the availability of SMO crystal structure in 2013. Here we retrospectively applied quantum mechanics calculations to demonstrate the o-Me substitution favors the bioactive conformation by inducing a dihedral twist between the heteroaryl rings and the core aniline. The o-Me also makes favorable hydrophobic interactions with key residue side chains in the binding pocket. From this effort, two compounds (AZD8542 and AZD7254) showed excellent pharmacokinetics across multiple preclinical species and demonstrated in vivo activity in abrogating the Hh paracrine pathway as well as anti- tumor effects.     

Targeting histone acetylation/deacetylation in parasites: an update (2017–2020)

Histone modifying enzymes have vital roles in the growth and survival of both parasites and humans. Targeting the epigenome can be a new strategy for the treatment of parasitic diseases. Compounds modulating histone acetylation/deacetylation have recently been reported hampering Plasmodium, Schistosoma, Leishmania, and Trypanosoma infections. Beside new histone deacetylase inhibitors, PfGCN5 and bromodomain inhibitors have been recently described to inhibit Plasmodium proliferation. Sm histone deacetylase 8 and SmSIRT2, as well as Leishmania and Trypanosoma sirtuins (SIR2rps), seem to be the most reliable targets to effectively fight the related protozoan infections. The selectivity toward parasite over mammalian cells is still an open question, and significant optimization efforts of epidrugs are still required to improve potency/selectivity and decrease toxicity. Recent reports on the alteration of cellular signaling pathways provoked by parasite infection through changes in the host acetylation/deacetylation status at gene promoters may suggest novel therapeutic strategies to treat these diseases.     

Clinical Evaluation of BRCA1/2 Mutation in Mexican Ovarian Cancer Patients

Ovarian cancer (OC) is an important cause of gynecologic cancer-related deaths. In Mexico, around 4700 new cases of OC are diagnosed per year and it represents the second cause of gynecological cancer mortality with more than 2700 deaths. Germline mutations in BRCA1/2 genes are present in 13–18% of OC cases. Few studies have evaluated the presence of mutations in BRCA genes in a population of OC Mexican patients and their relationship with clinical response and survival rates.A total of 179 OC patients were studied by molecular testing for BRCA1/2 through next-generation sequencing and multiplex ligation-dependent probe amplification. Recurrence-free survival (RFS) was estimated by the Kaplan–Meier method. BRCA mutation was detected in 33% of patients. A percentage of 66.1% were BRCA1 mutated and 33.9% were BRCA2 mutated. BRCA1 mutation carriers had a worst RFS compared with BRCA2 mutation carriers (37.6 [29–46.2] vs 72.7 [38.4–107.2]; P = 0.030). The most common mutation for BRCA1 was ex9-12del (28.2%) (Mexican founder mutation). The Mexican founder mutation had a better RFS than other BRCA1 mutations (86.1 [37.2–135.1] vs 34.5 [20.7–48.2]; P = 0.033). The presence of BRCA2 mutations in the ovarian cancer cluster region (OCCR) had a significantly better RFS than mutations in breast cancer cluster regions (BCCR) and not-related risk region (NRR) (NR vs 72.8 [39–106.6] vs 25.8 [8.3–43.2]; P = 0.013). These results demonstrate that the prevalence of BRCA1/2 positive patients in OC Mexican patients are the highest reported. Patients with mutations in BRCA2 have a better prognosis than those mutated in BRCA1. The Mexican founder mutation has an important role in clinical outcomes. These results highlight the importance to test all the HGSP (high-grade serous papillary) OC patients with or without cancer family history (CFH) in Mexican population.     

Associations between insulin-like growth factor binding protein-2 and lipoprotein kinetics in men

Low circulating concentrations of insulin-like growth factor binding protein-2 (IGFBP-2) have been associated with dyslipidemia, notably with high triglyceride (TG) levels. However, the determinants by which IGFBP-2 influences lipoprotein metabolism, especially that of TG-rich lipoproteins (TRLs), are poorly understood. Here, we aimed to assess the relationships between IGFBP-2 levels and lipoprotein production and catabolism in human subjects. Fasting IGFBP-2 concentrations were measured in the plasma of 219 men pooled from previous lipoprotein kinetics studies. We analyzed production rate and fractional catabolic rates of TRLapoB-48, and LDL-, IDL-, and VLDLapoB-100 by multicompartmental modeling of l-[5,5,5-D3] leucine enrichment data after a 12 h primed constant infusion in individuals kept in a constant nutritional steady state. Subjects had an average BMI of 30 kg/m², plasma IGFBP-2 levels of 157 ng/ml, and TG of 2.2 mmol/l. After adjustments for age and BMI, IGFBP-2 levels were negatively associated with plasma TG (r = ?0.29; P < 0.0001) and positively associated with HDL-cholesterol (r = 0.26; P < 0.0001). In addition, IGFBP-2 levels were positively associated with the fractional catabolic rate of VLDLapoB-100 (r = 0.20; P < 0.01) and IDLapoB-100 (r = 0.19; P < 0.05) and inversely with the production rate of TRLapoB-48 (r = ?0.28; P < 0.001). These correlations remained statistically significant after adjustments for age, BMI, and the amount of fat given during the tracer infusion. These findings show that the association between low plasma IGFBP-2 and high TG concentrations could be due to both an impaired clearance of apoB-100-containing VLDL and IDL particles and an increased production of apoB-48-containing chylomicrons. Additional studies are necessary to investigate whether and how IGFBP-2 directly impacts the kinetics of TRL.     

Glutamine-Fructose-6-Phosphate Transaminase 2 (GFPT2) Is Upregulated in Breast Epithelial–Mesenchymal Transition and Responds to Oxidative Stress

Breast cancer cells that have undergone partial epithelial–mesenchymal transition (EMT) are believed to be more invasive than cells that have completed EMT. To study metabolic reprogramming in different mesenchymal states, we analyzed protein expression following EMT in the breast epithelial cell model D492 with single-shot LFQ supported by a SILAC proteomics approach. The D492 EMT cell model contains three cell lines: the epithelial D492 cells, the mesenchymal D492M cells, and a partial mesenchymal, tumorigenic variant of D492 that overexpresses the oncogene HER2. The analysis classified the D492 and D492M cells as basal-like and D492HER2 as claudin-low. Comparative analysis of D492 and D492M to tumorigenic D492HER2 differentiated metabolic markers of migration from those of invasion. Glutamine-fructose-6-phosphate transaminase 2 (GFPT2) was one of the top dysregulated enzymes in D492HER2. Gene expression analysis of the cancer genome atlas showed that GFPT2 expression was a characteristic of claudin-low breast cancer. siRNA-mediated knockdown of GFPT2 influenced the EMT marker vimentin and both cell growth and invasion in vitro and was accompanied by lowered metabolic flux through the hexosamine biosynthesis pathway (HBP). Knockdown of GFPT2 decreased cystathionine and sulfide:quinone oxidoreductase (SQOR) in the transsulfuration pathway that regulates H₂S production and mitochondrial homeostasis. Moreover, GFPT2 was within the regulation network of insulin and EGF, and its expression was regulated by reduced glutathione (GSH) and suppressed by the oxidative stress regulator GSK3-β. Our results demonstrate that GFPT2 controls growth and invasion in the D492 EMT model, is a marker for oxidative stress, and associated with poor prognosis in claudin-low breast cancer.     

Expression profiles and functional characterization of common carp (Cyprinus carpio) T2Rs

Bitter taste perception is mediated by a family of G protein-coupled receptors (T2Rs) in vertebrates. Common carp (Cyprinus carpio), which has experienced an additional round of whole genome duplication during the course of evolution, has a small number of T2R genes similar to zebrafish, a closely related cyprinid fish species, and their expression pattern at the cellular level or their cognate ligands have not been elucidated yet. Here, we showed through in situ hybridization experiments, that three common carp T2R (ccT2R) genes encoding ccT2R200-1, ccT2R202-1, and ccT2R202-2, were specifically expressed in the subsets of taste receptor cells in the lips and gill rakers. ccT2R200-1 was co-expressed with genes encoding downstream signal transduction molecules, such as PLC-β2 and Gαia. Heterologous expression system revealed that each ccT2R showed narrowly, intermediately, or broadly tuned ligand specificity, as in the case of zebrafish T2Rs. However, ccT2Rs showed different ligand profiles from their orthologous zebrafish T2Rs previously reported. Finally, we identified three ccT2Rs, namely ccT2R200-1, ccT2R200-2, and ccT2R203-1, to be activated by natural bitter compounds, andrographolide and/or picrotoxinin, which elicited no response to zebrafish T2Rs, in a dose-dependent manner. These results suggest that some ccT2Rs may have evolved to function in the oral cavity as taste receptors for natural bitter compounds found in the habitats in a species-specific manner.     

Identification of potent pyrazole based APELIN receptor (APJ) agonists

The apelinergic system comprises the apelin receptor and its cognate apelin and elabela peptide ligands of various lengths. This system has become an increasingly attractive target for pulmonary and cardiometabolic diseases. Small molecule regulators of this receptor with good drug-like properties are needed. Recently, we discovered a novel pyrazole based small molecule agonist 8 of the apelin receptor (EC₅₀ = 21.5 µM, K_i = 5.2 µM) through focused screening which was further optimized to initial lead 9 (EC₅₀ = 0.800 µM, K_i = 1.3 µM). In our efforts to synthesize more potent agonists and to explore the structural features important for apelin receptor agonism, we carried out structural modifications at N1 of the pyrazole core as well as the amino acid side-chain of 9. Systematic modifications at these two positions provided potent small molecule agonists exhibiting EC₅₀ values of <100 nM. Recruitment of β-arrestin as a measure of desensitization potential of select compounds was also investigated. Functional selectivity was a feature of several compounds with a bias towards calcium mobilization over β-arrestin recruitment. These compounds may be suitable as tools for in vivo studies of apelin receptor function.     

The angiogenic effects of exosomes secreted from retinal pigment epithelial cells on endothelial cells

Exosomes are informative microvesicles associated with intercellular communication via the transfer of many molecular constituents such as proteins, lipids, and nucleic acids; environmental changes and the cellular status around cells greatly affect exosome components. Cells of the retinal pigment epithelium (RPE) are key players in retinal homeostasis. Transforming growth factor (TGF)-β and tumour necrosis factor (TNF)-α are increased in the vitreous and retina in several retinal diseases and activate and undergo epithelial-mesenchymal transition (EMT) in RPE cells. EMT is closely associated with mechanisms of wound healing, including fibrosis and related angiogenesis; however, whether exosome components depend on the cell status, epithelium or mesenchyme and whether these exosomes have pro- or anti-angiogenic roles in the retina are unknown. We performed this study to investigate whether these EMT inducers affect the kinds of components in exosomes secreted from RPE cells and to assess their angiogenic effects. Exosomes were collected from culture media supernatants of a human RPE cell line (ARPE-19) stimulated with or without 10 ng/ml TNF-α and/or 5 ng/ml TGF-β2. NanoSight tracking analysis and immunoblot analysis using exosome markers were used to qualify harvested vesicles. Angiogenic factor microarray analysis revealed that exosomes derived from ARPE-19 cells cultured with TNF-α alone (Exo-TNF) and co-stimulated with TNF-α and TGF-β2 (Exo-CO) contained more angiogenic factors than exosomes derived from control cells (Exo-CTL) or ARPE-19 cells cultured with TGF-β2 alone (Exo-TGF). To assess the effect on angiogenesis, we performed chemotaxis, tube formation, and proliferation assays of human umbilical vein endothelial cells (HUVECs) stimulated with or without exosomes. HUVECs migrated to RPE-derived exosomes, and exosomes derived from ARPE-19 cells accelerated HUVEC tube formation. In contrast, Exo-TNF and Exo-CO reduced HUVEC proliferation. Our findings provide insight into the mechanisms underlying the relation between angiogenesis and exosomes derived from RPE cells.     

Identification of a Feed-Forward Loop Between 15(S)-HETE and PGE2 in Human Amnion at Parturition

Human parturition is associated with massive arachidonic acid (AA) mobilization in the amnion, indicating that large amounts of AA-derived eicosanoids are required for parturition. Prostaglandin E2 (PGE2) synthesized from the cyclooxygenase (COX) pathway is the best characterized AA-derived eicosanoid in the amnion which plays a pivotal role in parturition. The existence of any other pivotal AA-derived eicosanoids involved in parturition remains elusive. Here, we screened such eicosanoids in human amnion tissue with AA-targeted metabolomics and studied their role and synthesis in parturition by using human amnion fibroblasts and a mouse model. We found that lipoxygenase (ALOX) pathway-derived 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) and its synthetic enzymes ALOX15 and ALOX15B were significantly increased in human amnion at parturition. Although 15(S)-HETE is ineffective on its own, it potently potentiated the activation of NF-κB by inflammatory mediators including lipopolysaccharide, interleukin-1β, and serum amyloid A1, resulting in the amplification of COX-2 expression and PGE2 production in amnion fibroblasts. In turn, we determined that PGE2 induced ALOX15/15B expression and 15(S)-HETE production through its EP2 receptor-coupled PKA pathway, thereby forming a feed-forward loop between 15(S)-HETE and PGE2 production in the amnion at parturition. Our studies in pregnant mice showed that 15(S)-HETE injection induced preterm birth with increased COX-2 and PGE2 abundance in the fetal membranes and placenta. Conclusively, 15(S)-HETE is identified as another crucial parturition-pertinent AA-derived eicosanoid in the amnion, which may form a feed-forward loop with PGE2 in parturition. Interruption of this feed-forward loop may be of therapeutic value for the treatment of preterm birth.     

Ultrasound-Guided Radiofrequency Ablation for the Treatment of Primary Hyperparathyroidism: An Efficacy and Safety Study

ObjectiveTo evaluate the efficacy and safety of radiofrequency ablation (RFA) for the treatment of primary hyperparathyroidism (pHPT).MethodsOur study enrolled 25 patients who were treated with RFA for pHPT from September 2015 to January 2020. The serum intact parathyroid hormone (iPTH), calcium, and phosphate levels were tested within 1 week before RFA and at 1 day, 1 month, 3 months, 6 months, and 12 months after ablation. The ablation areas were evaluated using ultrasound at 1, 3, 6, and 12 months after RFA. Postoperative complications, including voice hoarseness, hematoma, postoperative pain, incision infections, hypoparathyroidism, and hypocalcemia, were recorded.ResultsA total of 25 patients with pHPT (mean age, 53.9 ± 10.9 years; 22 women and 3 men) with 29 enlarged parathyroid glands were treated with RFA. Of the 25 patients, 22 were treated in 1 session and 3 were treated in 2 sessions. Serum iPTH and calcium levels decreased significantly on day 1 after RFA (all P < .05). A total of 21 patients had normal levels of serum iPTH and calcium after RFA, with a cure rate of 84%. At the 12-month follow-up, 26 treated parathyroid glands exhibited a volume reduction rate of >70%. There were only a few minor complications, including 4 cases of postoperative pain (in 4 of the 25 patients [16%]) and 1 case of mild postoperative transient hypocalcemia (in 1 of the 25 patients [4%]).ConclusionUltrasound-guided RFA is an effective and safe technique for the treatment of carefully selected patients with pHPT. However, larger sample size and longer follow-up are still needed to further confirm its clinical value.     

Sulfatide with ceramide composed of phytosphingosine (t18:0) and 2-hydroxy FAs in renal intercalated cells

Diverse molecular species of sulfatide with differences in FA lengths, unsaturation degrees, and hydroxylation statuses are expressed in the kidneys. However, the physiological functions of specific sulfatide species in the kidneys are unclear. Here, we evaluated the distribution of specific sulfatide species in the kidneys and their physiological functions. Electron microscopic analysis of kidneys of Cst-deficient mice lacking sulfatide showed vacuolar accumulation in the cytoplasm of intercalated cells in the collecting duct, whereas the proximal and distal tubules were unchanged. Immunohistochemical analysis revealed that vacuolar H⁺-ATPase-positive vesicles were accumulated in intercalated cells in sulfatide-deficient kidneys. Seventeen sulfatide species were detected in the murine kidney by iMScope MALDI-MS analysis. The distribution of the specific sulfatide species was classified into four patterns. Although most sulfatide species were highly expressed in the outer medullary layer, two unique sulfatide species of m/z 896.6 (predicted ceramide structure: t18:0-C22:0h) and m/z 924.6 (predicted ceramide structure: t18:0-C24:0h) were dispersed along the collecting duct, implying expression in intercalated cells. In addition, the intercalated cell-enriched fraction was purified by fluorescence-activated cell sorting using the anti-vacuolar H⁺-ATPase subunit 6V0A4, which predominantly contained sulfatide species (m/z 896.6 and 924.6). The Degs2 and Fa2h genes, which are responsible for ceramide hydroxylation, were expressed in the purified intercalated cells. These results suggested that sulfatide molecular species with ceramide composed of phytosphingosine (t18:0) and 2-hydroxy FAs, which were characteristically expressed in intercalated cells, were involved in the excretion of NH₃ and protons into the urine.     

New Insights on Effects of Glucocorticoids in Patients With SARS-CoV-2 Infection

ObjectiveSince January 2020, the highly contagious novel coronavirus SARS-CoV-2 has caused a global pandemic. Severe COVID-19 leads to a massive release of proinflammatory mediators, leading to diffuse damage to the lung parenchyma, and the development of acute respiratory distress syndrome. Treatment with the highly potent glucocorticoid (GC) dexamethasone was found to be effective in reducing mortality in severely affected patients.MethodsTo review the effects of glucocorticoids in the context of COVID-19 we performed a literature search in the PubMed database using the terms COVID-19 and glucocorticoid treatment. We identified 1429 article publications related to COVID-19 and glucocorticoid published from 1.1.2020 to the present including 238 review articles and 36 Randomized Controlled Trials. From these studies, we retrieved 13 Randomized Controlled Trials and 86 review articles that were relevant to our review topics. We focused on the recent literature dealing with glucocorticoid metabolism in critically ill patients and investigating the effects of glucocorticoid therapy on the immune system in COVID-19 patients with severe lung injury.ResultsIn our review, we have discussed the regulation of the hypothalamic-pituitary-adrenal axis in patients with critical illness, selection of a specific GC for critical illness-related GC insufficiency, and recent studies that investigated hypothalamic-pituitary-adrenal dysfunction in patients with COVID-19. We have also addressed the specific activation of the immune system with chronic endogenous glucocorticoid excess, as seen in patients with Cushing syndrome, and, finally, we have discussed immune activation due to coronavirus infection and the possible mechanisms leading to improved outcomes in patients with COVID-19 treated with GCs.ConclusionFor clinical endocrinologists prescribing GCs for their patients, a precise understanding of both the molecular- and cellular-level mechanisms of endogenous and exogenous GCs is imperative, including timing of administration, dosage, duration of treatment, and specific formulations of GCs.     

Proteolysis targeting chimeras (PROTACs) for epigenetics research

Proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules and allow selective protein degradation by addressing the natural ubiquitin proteasome system. As this new strategy of chemically induced protein degradation can serve as a biological tool and provides new possibilities for drug discovery, it has been applied to a variety of targets including (nuclear) receptors, kinases, and epigenetic proteins. A lot of PROTACs have already been designed in the field of epigenetics, and their synthesis and characterization highly contributed to structural optimization and improved mechanistic understanding of these molecules. In this review, we will discuss and summarize recent advances in PROTAC discovery with focus on epigenetic targets.     

In-Depth Characterization of the Clostridioides difficile Phosphoproteome to Identify Ser/Thr Kinase Substrates

Clostridioides difficile is the leading cause of postantibiotic diarrhea in adults. During infection, the bacterium must rapidly adapt to the host environment by using survival strategies. Protein phosphorylation is a reversible post-translational modification employed ubiquitously for signal transduction and cellular regulation. Hanks-type serine/threonine kinases (STKs) and serine/threonine phosphatases have emerged as important players in bacterial cell signaling and pathogenicity. C. difficile encodes two STKs (PrkC and CD2148) and one phosphatase. We optimized a titanium dioxide phosphopeptide enrichment approach to determine the phosphoproteome of C. difficile. We identified and quantified 2500 proteins representing 63% of the theoretical proteome. To identify STK and serine/threonine phosphatase targets, we then performed comparative large-scale phosphoproteomics of the WT strain and isogenic ΔprkC, CD2148, Δstp, and prkC CD2148 mutants. We detected 635 proteins containing phosphorylated peptides. We showed that PrkC is phosphorylated on multiple sites in vivo and autophosphorylates in vitro. We were unable to detect a phosphorylation for CD2148 in vivo, whereas this kinase was phosphorylated in vitro only in the presence of PrkC. Forty-one phosphoproteins were identified as phosphorylated under the control of CD2148, whereas 114 proteins were phosphorylated under the control of PrkC including 27 phosphoproteins more phosphorylated in the ?stp mutant. We also observed enrichment for phosphothreonine among the phosphopeptides more phosphorylated in the Δstp mutant. Both kinases targeted pathways required for metabolism, translation, and stress response, whereas cell division and peptidoglycan metabolism were more specifically controlled by PrkC-dependent phosphorylation in agreement with the phenotypes of the ΔprkC mutant. Using a combination of approaches, we confirmed that FtsK was phosphorylated in vivo under the control of PrkC and that Spo0A was a substrate of PrkC in vitro. This study provides a detailed mapping of kinase–substrate relationships in C. difficile, paving the way for the identification of new biomarkers and therapeutic targets.     

Toll-like receptor (TLR) agonists as a driving force behind next-generation vaccine adjuvants and cancer therapeutics

Until recently, the development of new human adjuvants was held back by a poor understanding of their mechanisms of action. The field was revolutionized by the discovery of the toll-like receptors (TLRs), innate immune receptors that directly or indirectly are responsible for detecting pathogen-associated molecular patterns (PAMPs) and respond to them by activating innate and adaptive immune pathways. Hundreds of ligands targeting various TLRs have since been identified and characterized as vaccine adjuvants. This work has important implications not only for the development of vaccines against infectious diseases but also for immuno-therapies against cancer, allergy, Alzheimer's disease, drug addiction and other diseases. Each TLR has its own specific tissue localization and downstream gene signalling pathways, providing researchers the opportunity to precisely tailor adjuvants with specific immune effects. TLR agonists can be combined with other TLR or alternative adjuvants to create combination adjuvants with synergistic or modulatory effects. This review provides an introduction to the various classes of TLR adjuvants and their respective signalling pathways. It provides an overview of recent advancements in the TLR field in the past 2–3 years and discusses criteria for selecting specific TLR adjuvants based on considerations, such as disease mechanisms and correlates of protection, TLR immune biasing capabilities, route of administration, antigen compatibility, new vaccine technology platforms, and age- and species-specific effects.