Glucocorticoid-induced osteogenic dysfunction is the main pathologyical mechanism underlying the development of glucocorticoid-induced osteoporosis. Glucocorticoids promote adipogenic differentiation and osteoblast apoptosis through various pathways. Various ongoing studies are exploring the potential of natural products in preventing glucocorticoid-induced osteoporosis. Preclinical studies have consistently shown the bone protective effects of tocotrienol through its antioxidant and anabolic effects. This review aims to summarise the potential mechanisms of tocotrienol in preventing glucocorticoid-induced osteoporosis based on existing in vivo and in vitro evidence. The current literature showed that tocotrienol prevents oxidative damage on osteoblasts exposed to high levels of glucocorticoids. Tocotrienol reduces lipid peroxidation and increases oxidative stress enzyme activities. The reduction in oxidative stress protects the osteoblasts and preserves the bone microstructure and biomechanical strength of glucocorticoid-treated animals. In other animal models, tocotrienol has been shown to activate the Wnt/β-catenin pathway and lower the RANKL/OPG ratio, which are the targets of glucocorticoids. In conclusion, tocotrienol enhances osteogenic differentiation and bone formation in glucocorticoid-treated osteoblasts while improving structural integrity in glucocorticoid-treated rats. This is achieved by preventing oxidative stress and osteoblast apoptosis. However, these preclinical results should be validated in a randomised controlled trial. Osteoblasts are specialised bone-forming cells that play many roles in bone remodelling. Osteoblasts develop from multipotent mesenchymal cells under the influence of regulatory transcription factors runt-related transcription factor 2 (RUNX2) and osterix, which also can grow into other cell lineages such as adipocytes, myocytes and chondrocytes . OsteoblGCs are widely used as the treatment for inflammatory diseases and as chemotherapeutic agents. They are crucial for the induction of osteoblast differentiation and the formation of a mineralised extracellular matrix, as they influence the signals coordinating the differentiation of multipotent mesenchymal precursors cells into osteoblasts ,7,8. GCsThe current prevention for GC-induced osteoporosis (GIO) is ensuring an adequate intake of vitamin D and calcium among patients on GCs. The assessment of fall risk in patients taking high doses of GCs should be performed. Lifestyle modifications similar to those adopted by patients with postmenopausal osteoporosis, which include weight-bearing exercise and the elimination of other risk factors, such as smoking or alcohol, should also be implemented by chronic GC users . PatientVitamin E encompasses both tocotrienol and tocopherol, both of which are potent antioxidants. They could be divided into alpha (α), beta (β), gamma (γ) and delta (δ) isomers. Tocotrienol and tocopherol share a similar structure, which consists of a chromanol ring and a long carbon tail. Tocotrienols have three double bonds, whereas tocopherols have only single bonds on the carbon tail  17]. Th. Th17]. This review aims to summarise the preclinical evidence of the skeletal effects of tocotrienol in GIO models. The potential mechanisms of tocotrienol in achieving bone protection under the influence of GC are also discussed.Before appraising the effects of tocotrienol in preventing GIO, it is necessary to understand the actions of GCs on bone cells. The information will allow us to understand the avenues of intervention in GIO.GC-induced osteoblast dysfunction is one of the main pathological mechanisms underlying the development of GIO . GCs impThe actions of GCs on bone are also determined by variations in the expression and sensitivity of the GC receptors, the export of steroids from the cell by transmembrane transporters, and enzymatic metabolism of GCs to the more or less active metabolites . 11β-hyd−7M DEX), GCs promote uncommitted mesenchymal precursors cells to differentiate into osteoblasts [−7M DEX), GCs inhibit the proliferation of osteoblast-like cells in culture [GCs are important in the differentiation of osteoblasts, but their effects on osteoblast proliferation are inconsistent. At lower doses , transforming growth factors (TGFs), fibroblast growth factors (FGF), and platelet-derived growth factors are also other signalling pathways also targeted by GCs. GCs reduce the anabolic actions of TGFβ and suppress the expression of IGF-I and platelet-derived growth factors, which possess anabolic mitogenic actions in osteoblasts ,45,46. NGCs can cause oxidative stress through the production of reactive oxygen species (ROS), downregulation of cytoprotective antioxidant proteins and antioxidant enzyme activities . High ROA study by Zhang et al.  found thMost in vivo studies showed that GCs usually cause a decrease in bone formation, but some in vitro studies showed largely stimulatory effects of GCs actions. This observation may suggest that a low GC level possesses stimulatory effects, while a high GC level possesses inhibitory effects on bone . GCs exeGCs create an environment favouring osteoclast formation and bone resorption activities by increasing RANKL and suppressing OPG secretion by osteoblasts ,62,63,64Tocotrienol has been reported to demonstrate potential skeletal anabolic effects in in vivo and in vitro studies. In an earlier study, it was found that PTT preserved the bone calcium content in DEX-treated adrenalectomized rats. More recent studies on oestrogen and androgen-deficient rat models reported tocotrienol supplementation preserved the bone calcium content, and this was parallel to the observed cortical bone thickening at the midshaft caused, which also corresponded to the increased bone mechanical strength ,73. AnotThe efficacy of two different natural mixtures of tocotrienol, i.e., ATT and PTT, has been compared in adrenalectomised rats supplemented with DEX. Both tocotrienol mixtures were reported to maintain osteoblast surface in the femoral trabecular bone of GC-treated rats ,59. The The differentiation and proliferation of osteoblasts are regulated by cascades of genes ,75. GC wCorrecting oxidative stress induced by GCs using an antioxidant agent might be a viable method to prevent osteoporosis. The impact of vitamins and antioxidants on bone healing has been reported to have a positive effect on preventing osteoporosis. As oxidative stress is proven to be toxic to osteoblasts, agents that reduce oxidative stress may be beneficial in preserving bone formation. Tocotrienol has been shown to prevent oxidative stress-related diseases effectively ,20,21,79Apart from that, tocotrienol might enhance bone formation directly. PTT and ATT increased bone dynamic parameters  in rats with GIO ,59. The The RANKL/OPG and Wnt/β-catenin pathway are important targets of GCs. Apart from the positive effects of ATT and PTT in combating the effects of GCs on bone, they were also found to have protective effects in the oestrogen and androgen-deficient rat models as well as in osteoporosis induced by metabolic syndrome . ATT  rats ,83. AparThe direct effects of tocotrienol on GC-treated osteoclast formation and function have not been studied in depth. From other studies, tocotrienol isomers directly inhibited tartrate-resistant acid phosphatase-positive cell formation from peripheral blood mononuclear cells and bone marrow macrophages without affecting cell survival ,91. TocoIn summary, the research available so far suggests that tocotrienol protects bone from the effects of GCs by preventing oxidative stress and apoptosis and increasing the differentiation and maturation in osteoblasts .The effects of tocotrienol in GIO have not been examined in humans. There are only two studies on the protective effects of tocotrienol on bone health in humans. The effects of ATT in protecting bone health have been tested among postmenopausal osteopenic women. They were randomised into groups receiving placebo/olive oil, low-dose (430 mg) and high-dose ATT (860 mg) for 12 weeks. There was a significant reduction in urine N-telopeptides (NTX) levels, soluble RANKL, soluble RANKL/OPG ratio and urine 8-hydroxy-2′-deoxyguanosine and an improvement in bone ALP/NTX ratio. However, serum OPG and urinary calcium levels were not affected by the supplementation. No dose-dependent effects were observed between the two doses. BMD values were not evaluated in this study due to the short duration of action . In a reGiven the current evidence, there is a potential that tocotrienol will be able to reverse the negative effects of GCs on bone health by preserving osteoblasts. However, no randomized controlled trial has been conducted to validate the preclinical results currently. Efforts should be invested in bridging this gap so that tocotrienol could be used to reduce the skeletal outcomes among patients using GCs.


The chronic kidney disease-mineral bone disorder (CKD-MBD) is a complex multi-component syndrome occurring during kidney disease and its progression. Here, we update progress in the components of the syndrome, and synthesize recent investigations, which suggest a potential mechanism of the bone-vascular paradox. The discovery that calcified arteries in chronic kidney disease inhibit bone remodeling lead to the identification of factors produced by the vasculature that inhibit the skeleton, thus providing a potential explanation for the bone-vascular paradox. Among the factors produced by calcifying arteries, sclerostin secretion is especially enlightening. Sclerostin is a potent inhibitor of bone remodeling and an osteocyte specific protein. Its production by the vasculature in chronic kidney disease identifies the key role of vascular cell osteoblastic/osteocytic transdifferentiation in vascular calcification and renal osteodystrophy. Subsequent studies showing that inhibition of sclerostin activity by a monoclonal antibody improved bone remodeling as expected, but stimulated vascular calcification, demonstrate that vascular sclerostin functions to brake the Wnt stimulation of the calcification milieu. Thus, the target of therapy in the chronic kidney disease-mineral bone disorder is not inhibition of sclerostin function, which would intensify vascular calcification. Rather, decreasing sclerostin production by decreasing the vascular osteoblastic/osteocytic transdifferentiation is the goal. This might decrease vascular calcification, decrease vascular stiffness, decrease cardiac hypertrophy, decrease sclerostin production, reduce serum sclerostin and improve skeletal remodeling. Thus, the therapeutic target of the chronic kidney disease-mineral bone disorder may be vascular osteoblastic transdifferentiation, and sclerostin levels may be a useful biomarker for the diagnosis of the chronic kidney disease-mineral bone disorder and the progress of its therapy. The Chronic Kidney Disease–Mineral Bone Disorder (CKD-MBD) is an important contributor to the mortality associated with CKD. The finding that in the face of normal Ca, Pi, calcitriol, and PTH, CKD still produced renal osteodystrophy lead to the hypothesis that renal injury/repair factors released to the circulation produce systemic complications of CKD. Here, in providing updates on the CKD-MBD components, focusing on vascular calcification and the concept of kidney disease produced disruption of homeostasis; the bone vascular paradox is potentially explained. The synthesis of recent studies involving Wnt inhibitors produces the suggestion of the putative CKD-MBD therapeutic target and the identification of sclerostin levels as a useful biomarker for diagnosis and progress of therapy.Vascular calcification (VC) has been extensively studied since its addition to the CKD-MBD. In osteoblastic differentiation, Wnt/β-catenin signaling is critical for differentiation from mesenchymal stem cells, In early CKD, the increase in circulating Wnt inhibitors directly affects the skeleton through Wnt inhibition, In vitro and in vivo studies suggest that osteopontin not only inhibits mineral deposition but also actively promotes its dissolution by physically blocking hydroxyapatite crystal growth and inducing expression of carbonic anhydrase II in monocytic cells and promoting acidification of the extracellular milieu  and osteocalcin (OC). MGP and OC belong to the mineral-binding Gla-protein family. These proteins contain a variable number  of glutamic acid (Gla) residues. Gla residues undergo γ-carboxylation making the proteins active in calcium binding. γ-Carboxylation is vitamin K–dependent. Undercarboxylated Gla proteins are less active.OC is an abundant non-collagenous protein of bone. Its association with bone remodeling rates has made it a biomarker of renal osteodystrophy. OC is involved with the mineralization process through modulation of the shape and size of hydroxyapatite crystals. The mineralization function of OC requires activation through vitamin K–dependent carboxylation of Gla residues. Additionally, OC also modulates osteoblast and osteoclast activity, interacting with the G-protein–coupled receptor 6A (GPRC6A). Hormonal activity of OC was discovered by the finding that OC null mice develop a type 2 diabetes-like phenotype. The concept is that under-carboxylated OC (ucOC) acts as a hormone to increase pancreatic β-cell growth, insulin secretion, and insulin sensitivity. However, this concept is controversial and confirmatory research has not uniformly supported it. Besides bone, MGP is synthesized in several tissues, including VSMCs. Mice lacking MGP have intense medial calcification, demonstrating the role of MGP in VC , The mecCardiac hypertrophy is highly prevalent in CKD. We found in a cohort of patients with CKD stage 3 from a single center clinic and a mean eGFR of 50 mL/min/1.73m2, an 80% incidence of left ventricular hypertrophy (LVH) by CT scanning . This isFGF23 was discovered as the cause of autosomal dominant hypophosphatemic rickets in 2000 . Shortlyin vitro and in vivo that FGF23 stimulates cardiomyocyte hypertrophy through the FGFR4 independent of klotho , cardiovascular disease (CVD), abnormal phosphate and mineral regulation and frailty, similar to that of premature aging in humans . Klotho in vitro and in vivo support a protective role of klotho.  deficiency late in the course of CKD and promotes hyperparathyroidism. Calcitriol deficiency and secondary hyperparathyroidism were central components to the CKD-MBD, even before the syndrome was coined, and are critical to the development of renal osteodystrophy.The effects of calcitriol are protean. It is a steroid hormone regulating cellular function through the nuclear vitamin D receptor (VDR), and VDR response elements (VDRE) in the promotors of many genes. This includes the PTH gene in which the VDRE serves to decrease transcription. Its actions on vascular calcification are “U-shaped”, with both low and high levels promoting medial vascular calcification. In tubulopathies producing the Fanconi syndrome, calcitriol deficiency is associated with childhood osteomalacia.Calcitriol is thought to regulate cardiac hypertrophy, see Covic et al. for review.PTH is a central component of the pathogenesis of renal osteodystrophy and it is the major factor driving the high turnover remodeling state seen in late CKD and CKD treated with hemodialysis. The mechanism of transition from the early CKD low turnover state produced by Wnt inhibitors including sclerostin, to the high turnover state stimulated by PTH was discussed above in the skeletal section. PTH has also been implicated in vascular calcification, although the effects are complex. Several lines of evidence support a direct role of PTH. Elevated PTH is associated with higher rates of vascular atherosclerotic disease . Animal Treatment of hyperparathyroidism in CKD, with use of calcium-containing phosphate binders, can result in low bone turnover and reduced mineralization. This may potentiate vascular calcification . States The proximal tubule of the kidney regulates phosphate excretion. However, the balance of bone formation and resorption is also a contributor to serum levels . HormonaMultiple studies demonstrate the association between hyperphosphatemia and vascular calcification , especiain vitro demonstrated a process wherein exposure to elevated phosphate stimulated a dose- and time-dependent phenotypic change in vascular smooth muscle cells (VSMCs)  . Upregul (VSMCs)  (Villa-B (VSMCs) . The VSM (VSMCs) .Phosphate overload downregulates renal Klotho expression through inhibition of peroxisome proliferator-activated receptor gamma (PPARγ) and the activation of the Wnt/β-catenin signaling pathway, thus contributing to the development of vascular calcification and the alteration of the regulation of mineral metabolism . Failurein vitro showed that the calcified uremic aortic rings inhibited matrix mineralization by osteoblast cell lines compared to aortic rings from normal rats  in the CKD-MBD consists of the observation that as vascular calcification  is stimulated, bone formation  is inhibited . In thismal rats . The ureThe high levels of sclerostin secretion from the calcified uremic aortas are in agreement with studies from other investigators, showing that sclerostin is expressed in association with vascular calcification vasculature . This su−/− mice compared to adenine exposed wild type mice had much more CKD-induced cardiac vessel and renal artery calcification (Recent studies analyzing vascular calcification models in SOST deficient mice and sclerostin antibody treated mice confirm the expectations. De Mare et al., show that adenine-exposed Sostfication . They alfication . Thus, ification .The role of sclerostin in cardiovascular disease has been studied in other settings outside of CKD; see Golledge and Thanigaimani for an exhaustive review . DevelopGolledge and Thanigaimani state that “human studies report that genetic variants causing lower arterial sclerostin expression increase the prevalence of hypertension and diabetes and elevate the risk of cardiovascular events.” Other “human studies have associated high circulating sclerostin levels with the diagnosis of a variety of different cardiovascular diseases, surrogate markers of cardiovascular disease and high risk of cardiovascular events in some populations.” There are a number of possible explanations for the findings of human genetic studies, which suggest that low arterial sclerostin promotes cardiovascular disease, but human biomarker studies showing high serum sclerostin is associated with cardiovascular disease and events. One explanation is that the high blood sclerostin concentrations in participants with cardiovascular disease represent a response to the disease serving as a brake rather than a cause. The De Mare et al. studies discussed above support this concept .Romosozumab is an anti-sclerostin antibody approved for the treatment of osteoporosis, −/−model of atherosclerosis, inhibiting the sclerostin loop 3 did not increase vascular calcification, but did increase bone remodeling (The structural properties of the 24 kDa sclerostin protein reveal a tertiary structure consisting of three loops, a cysteine-knot motif with three disulfide bonds and N- and C-terminal spacer arms . Sclerosmodeling . These sThe idea that sclerostin might be a biomarker of the CKD-MBD was originally put forth by The CKD-MBD is a complex multifactorial syndrome. Recent progress continues to clarify its pathophysiology, and suggests that a target of therapy would be to reduce the vascular injury associated with CKD. Reduction of vascular osteoblastic/osteocytic transdifferentiation would decrease, possibly prevent, vascular calcification and stimulate skeletal homeostasis . Reducti


As the standard of living improves, chronic diseases and end-stage organ failure have been a regular occurrence in human beings. Organ transplantation has become one of the hopes in the fight against chronic diseases and end-stage organ failure. However, organs available for transplantation are far from sufficient to meet the demand, leading to a major organ shortage crisis. To solve this problem, researchers have turned to pigs as their target since pigs have many advantages as xenograft donors. Pigs are considered the ideal organ donor for human xenotransplantation, but direct transplantation of porcine organs to humans faces many obstacles, such as hyperacute rejection, acute humoral xenograft rejection, coagulation dysregulation, inflammatory response, coagulation dysregulation, and endogenous porcine retroviral infection. Many transgenic strategies have been developed to overcome these obstacles. This review provides an overview of current advances in genetically modified pigs for xenotransplantation. Future genetic engineering-based delivery of safe and effective organs and tissues for xenotransplantation remains our goal. Based on urgent clinical needs, replacing human organs with fully functional animal organs for xenotransplantation therapy is an effective method to address the shortage of donor organs.In recent years, the incidence of vital organ failure has increased. DiffereCompared with nonhuman primates, pigs have characteristics such as fast reproduction, easy breeding, lower cost, and closer anatomical characteristics and physiological indices to humans, and the use of pigs can avoid the ethical problems caused by the use of nonhuman primate organs . The useThe major carbohydrate antigen on porcine vascular endothelial cells has been identified as galactose-α1,3-galactose , to which humans and nonhuman primates have anti-pig antibodies. Activata produced by β4GalNT2 glycosyltransferase has been reported to prevent damage to pig cells by complement activation. A numbein vitro, such as heart, kidney, endothelial and fibroblast cells. Moreover, their results demonstrate that HO-1 plays a protective role in TNF-α-mediated apoptosis is a natural anticoagulant. TM inhibits thrombosis by suppressing direct prothrombinase activity through binding to prothrombinase and enhances its activation of protein C, which is an anticoagulant when activated. In 2014In addition, the expression of other human coagulation-regulatory proteins  has undergone extensive testing . It has In recent years, the application of gene editing technology has become increasingly common, which has led to prolonged survival of transplanted pig organs in nonhuman primates (NHPs) and a reduced risk of pathogen transfer in organs. Xenotransplantation has made breakthroughs in many fields, especially in heart see , liver , kidney In 1964, Hardy performed the first clinical orthotopic cardiac xenotransplantation (CXTx) when he implanted a chimpanzee’s heart into a 64-year-old male patient who died within 2 h of transplantation. Histopavia anti-CD154 antibodies significantly prolonged cardiac xenograft survival, but many coagulation disorders were observed with the use of anti-CD154 antibodies. In 2013, Mohiuddin’s group, the current obstacle to the clinical application of liver transplantation is severe thrombocytopenia. Burlak olleagues knocked olleagues producedOne of the possible approaches to address liver damage resulting from ASGR1 defects in pigs is to screen key amino acid functional loci of proteins at the individual level using base editing techniques. The approach aims to eliminate the liver damage caused by ASGR1 defects in pigs and to effectively alleviate thrombocytopenia in liver xenografts as much as possible.Shah’s group developeLiver xenotransplantation still has a long way to go before undergoing clinical trials, with thrombocytopenia and coagulation dysregulation remaining major hurdles. Gene edThe rapid development of genome editing technologies such as CRISPR‒Cas9 technology has led to significant progress in kidney transplantation from pigs to NHPs. To date, some groups have achieved more than 6 months of survival in life-supporting pig-to-baboon kidney transplants. RecentlPig islet xenotransplantation is a potential approach to patients with type 1 diabetes. In 1994, Although the current work has effectively reduced the occurrence of immune rejection, the cross-species infection of pathogens between pigs and humans remains a difficult problem to be solved. This difficulty arises from two aspects: first, overexpression of human genes may increase the risk of human pathogens infecting genetically engineered pigs; second, transplanting pig organs into human bodies may also increase the risk of infection by pig pathogens.Knock-in of certain human proteins in pigs may enhance the susceptibility of certain viruses to the organism. In engineering genetically modified pigs to overcome immune rejection, human CD46 was introduced into porcine cells to inhibit complement-mediated graft injury. CD46 noFurthermore, to further reduce the incidence of immune rejection that is still an issue in current xenotransplants, researchers could try to produce pigs with different genetic modifications using different gene-editing combinations, including knocking in human genes and knocking out pig immunogenicity-related genes, to testFrom the recent first-ever life-saving cardiac xenotransplantation, patients died of multiple organ failure, and organ grafts died from porcine virus infection. Therefo


Evolution and Core Processes in Gene Regulation met at the Stowers Institute in Kansas City, Missouri July 21 to 24, 2022. This symposium, sponsored by the American Society for Biochemistry and Molecular Biology (ASBMB), featured experts in gene regulation and evolutionary biology. Topic areas covered enhancer evolution, the cis-regulatory code, and regulatory variation, with an overall focus on bringing the power of deep learning (DL) to decipher DNA sequence information. DL is a machine learning method that uses neural networks to learn complex rules that make predictions about diverse types of data. When DL models are trained to predict genomic data from DNA sequence information, their high prediction accuracy allows the identification of impactful genetic variants within and across species. In addition, the learned sequence rules can be extracted from the model and provide important clues about the mechanistic underpinnings of the cis-regulatory code.After a COVID-related hiatus, the fifth biennial symposium on  Identifying these rules is done after model training and is not trivial; yet, a variety of interpretation tools already exist to obtain the important sequence features and their rules of interactions. In this manner, DL models reliably reveal the binding motifs of TFs and provide important clues as to how the motifs combine to produce an experimental outcome.A sought-after goal of the gene regulation field is to decode enhancer grammar . How do Alexander Stark  discussed his laboratory’s recent application of DL to their STARR-seq method to predict enhancer activity from DNA sequence (Julia Zeitlinger (Stowers Institute) described her laboratory’s work on applying DL to understand the cis-regulatory rules of enhancers during Drosophila embryogenesis. Using BPNet (versus low affinity motifs. Shaun Mahony (Pennsylvania State University) also explored the mechanistic basis of chromatin accessibility by studying the evolutionary impacts of FOX gene paralogs  talked about her approaches to identify sequence features important for determining chromatin states in diverse human immune cells. She discussed approaches to unlock elements in the DL algorithms that were informative and reproducible, including identifying the number of active motifs found at diverse enhancers  illustrated how the Kundaje laboratory extracts important DNA sequences from a DL model to identify the distance rules by which TF motifs cooperate in binding and opening chromatin  discussed Mave-NN, a computational framework for integration of diverse gene expression data to make DL accessible to a biological user base (Hernan Garcia (UC Berkeley) in the Drosophila embryo  used single-cell ATAC-seq and RNA-seq to identify activated enhancers during regeneration. Using DL, she uncovered a role for the Sox and Six TFs in coordinately regulating ear regeneration -sponsored conference. Evolution is a crucial trove of knowledge for understanding gene regulation, and conversely, an understanding of gene regulation is a key to unlocking evolutionary processes. Deep learning can therefore have a significant impact toward accelerating this dialogue between fields. One path forward is to integrate the sequence information extracted from DL models with other data modalities to construct gene regulatory networks (GRNs). For example, to characterize key players in zebrafish inner ear regeneration, neration . This anIrene Kaplow  by training a DL model on ATAC-seq data from several well-characterized mammalian brains  who applied the BPNet model to mouse liver TFs and showed how it could predict TF binding profiles in other mammalian species. In addition, his laboratory applied this model to interpret variations related to disease-causing alleles involved in blood coagulation and lipid regulation.Species- and population-level variation was also the subject of DL analysis by cis-regulatory sequence variation. Excellent examples were the talks from Drs Brem, Vierbuchen, Wunderlich, Fay, and Wittkopp. Using a mouse fibroblast senescence model, Rachel Brem’s laboratory at UC Berkeley used a classic F1 hybrid approach to identify cis-regulatory changes between two mouse species that explains their differential response to irradiation. Their analysis highlighted the TF USF2, which may play a role in senescence decision-making. Similarly, Thomas Vierbuchen  described the use of mouse hybrid cells to link cis-regulatory variation with TF binding and enhancer function in the context of mouse embryonic and brain development. Zeba Wunderlich (Boston University) studied distinct populations and hybrids of Drosophila melanogaster to understand how population-level variation impacts the innate immune response to infection. Her laboratory found that trans-acting alleles dominate the response to a Gram-negative infection, while cis-acting effects dominate in a Gram-positive infection. A similar population variation approach was used by the laboratory of Justin Fay (University of Rochester) to identify protein-based variation linked to thermal tolerance in different species of yeast. They identified numerous differences in protein stability and also an important role of the hybrid cellular environment. Patricia Wittkopp (University of Michigan) described her laboratory’s classical genetic approaches to empirically test the assumption that trans-acting variants are more pleiotropic than cis-acting variants in Saccharomyces cerevisiae. By comparing the impact of cis- and trans-acting mutations on fitness and gene expression, their highly quantitative assays revealed differences in the effects of these two classes of mutations that support the hypothesis that trans-regulatory mutations are more pleiotropic than cis (Such use of DL models is poised to influence and complement the current genetic approaches used to map and understand the impact of than cis .Nicolas Rohner (Stowers Institute) described work in his laboratory using Mexican cavefish, which independently underwent metabolic adaptation to the cave environment multiple times. His team combined ‘omics datasets from livers of surface and cave morphs to identify putative cis-regulatory changes that alter target genes and pathways directly involved in cave adaptation  using dung beetles. These beetles have sexually dimorphic horn development, and in some species, males develop horns in response to nutritional cues. Davidson explored the cis-regulatory basis of this developmental plasticity using ‘omics approaches and identified enhancers that may be responsible for nutritional and sex-responsive differential development. In these studies, identification of relevant cis-regulatory changes relied on current molecular biological tools including ATAC-seq. It is intriguing to consider how DL approaches may complement this objective in the future.The influence of genetic variation can also be studied at the organismal level. aptation . Such a Evgeny Kvon (UC Irvine) used chromatin conformation capture technology to map enhancer-promoter interactions for thousands of validated mouse enhancers  shared his work looking at global chromosomal architecture. These studies make inferences about critical 3D genome interactions that may differ between populations of cavefish. Since DL models can be trained to predict Hi-C data from sequence, these are areas that could benefit from an integrative approach using both DL and targeted experiments.Using DL models to obtain insights into the mechanisms of gene regulation should be a welcome addition to the current purely experimental approaches. For example, nhancers . They coMark Rebeiz (University of Pittsburgh) described elegant experiments dissecting evolutionary transformations at the ebony locus, where silencers have been systematically reshaped to impact pigmentation in specific Drosophila species. Likewise, Nicolas Gompel  used the Drosophila pigmentation system to revisit the notion of enhancer modularity at the yellow locus. Through analysis of wing spot pigmentation in a number of species, his laboratory showed how regulatory regions of this gene exhibit multifunctionality and partial redundancy that evolved over time. Dimple Notani  discussed her laboratory’s studies on estrogen-driven gene regulation, where clusters of enhancers appear to act in a cooperative fashion to drive gene expression. Some elements are prebound to the estrogen receptor prior to signaling. Others are induced and appear to require “driver” enhancers for activity. Notably, these elements are not functionally distinguishable based on previously measured chromatin properties.Several talks at the meeting leveraged the interplay between evolutionary changes and mechanistic insights into gene regulation. By taking evolutionary changes as a starting point, they used classical experimental approaches to uncover specific functions of enhancers, insulators, histone proteins, and TFs, sometimes at the level of a single locus. For instance, Pravrutha Raman’s work examines the variability in histone proteins over evolutionary time. She found that ancestral histone variants H2A.X and H2A.Z are found to have fused to form a composite gene, H2A.V in many Diptera. Interestingly, some Drosophila have duplicated the H2A.V variant, with the duplicates expressed in males, indicating that evolutionary innovations in histone proteins may drive biological novelties. David Arnosti’s talk about evolution of the C-terminal Binding Protein also investigated how this core component of the transcriptional apparatus has evolved in eukaryotes , David Arnosti (Michigan State University), and Pinar Onal  focused on particular TFs and their evolution. karyotes . Deep ph evolved .These focused individual studies demonstrate that learning general rules for gene regulation using DL models should inspire, but not replace, the focus on specific biological problems. Like individual works of art, biological systems need to be considered in their own right. Many aspects by which they operate represent highly specialized solutions to specific organismal challenges. As a result, highly complex combinations of molecular components, such as the HOX gene cluster, are often unique and may only be represented once in a genome . Thus, wThe meeting brought together experimental and computational biologists, whose goal is to uncover how gene expression is regulated in the context of evolution. In particular, there was a focus on how DL models can impact the study of gene regulation, development, and evolution . DL modeThe authors declare that they have no conflicts of interest with the contents of the article.


Caregiver neglect is common among older persons with dementia and is associated with significant morbidity and mortality. Despite this, little empirical research exists examining factors that contribute or prevent neglect. Our goal was to identify caregiving factors that may be associated with caregiver neglect. Data were drawn from the baseline information of 240 caregivers enrolled in the Caring for the Caregiver Network study who provided care to a family member with dementia. Caregiver depression, burden, social support, perceived mutuality with care-recipients, positive aspects of caregiving, and self-perceived caregiving preparedness, were measured using validated scales. To maximize sensitivity in our measure of caregiver neglect, we operationalized caregiver neglect as consisting of: 1) caregivers’ failure to meet care-recipients’ needs, using 8-item instrumental activities of daily living (IADL) and 6-item ADL scales, and 2) caregivers not receiving additional formal services  to address care-recipients’ unmet needs. Selection of independent variables into multivariate regression models examining predictors of neglect was based on significance in bivariate analysis (p < .10). Caregiver neglect was found in 29.2% of caregivers. The caregiver being male was significantly associated with greater risk of neglect. In the final adjusted model, only caregiver preparedness was found to be a significant risk factor for neglect . This analysis provides the first evidence of the association between caregiver preparedness and risk of caregiver neglect. Future research should examine possible psycho-social mechanism linking preparedness and neglect to inform neglect prevention and intervention programming.


Since the discovery of this cell population by His in 1850, the neural crest has been under intense study for its important role during vertebrate development. Much has been learned about the function and regulation of neural crest cell differentiation, and as a result, the neural crest has become a key model system for stem cell biology in general. The experiments performed in embryology, genetics, and cell biology in the last 150 years in the neural crest field has given rise to several big questions that have been debated intensely for many years: “How does positional information impact developmental potential? Are neural crest cells individually multipotent or a mixed population of committed progenitors? What are the key factors that regulate the acquisition of stem cell identity, and how does a stem cell decide to differentiate towards one cell fate versus another?” Recently, a marriage between single cell multi-omics, statistical modeling, and developmental biology has shed a substantial amount of light on these questions, and has paved a clear path for future researchers in the field. Since the invention of fate mapping technologies, the overall developmental repertoire of the neural crest lineage has been well-established. This long list includes melanocytes, peripheral sensory neurons, autonomic nervous system derivatives such as parasympathetic and sympathetic neurons, chromaffin cells of the adrenal medulla, enteric neurons, peripheral glia and other nerve-associated cells . In addition to this profound spectrum, the cranial neural crest gives rise to ectomesenchymal derivatives in the face, neck and teeth . In line with this, the evolutionary role of the neural crest is outstanding without doubt, as those cells enabled the articulated jaws, teeth, complex vertebrate heart, advanced neural and hormonal regulation, efficient sensory modalities, efficient camouflage and much more. In 1983, Gans and Northcutt suggested the hypothesis of a “new head”, in which the neural crest played a major role in the acquisition of the head compartment as we know it in vertebrates .–34.31–34The controversy about the degree of neural crest multipotency resulting from different experimental results ,34,35 ca2.The theory of epigenetic landscape presented by Conrad Waddington (1957) dictates that stem cells irreversibly decrease their developmental potential during development to produce distinct cell types . NowadayJoint analysis of datasets from distantly related species, acquired using different sequencing platforms, remains a massive challenge for future evolutionary studies that wish to take advantage of these data, although some steps are already being taken to minimize platform-dependent batch effects . Along tWhen applied to the neural crest, single cell studies made great strides towards cracking a few historically difficult questions about the structure of cell heterogeneity in the crest population and corresponding hierarchy of cell fate decisions ,53–55 F. These sSnai2 which are active only in the NCCs .,55.53,55identity .Krt19, Col2a1, MycN . Th. Th39]. Although the methodology of single cell applications is not new anymore, there remain notable caveats. We do not aim at detailing the importance of reducing cell isolation time, which can introduce a stress signature into transcriptomes, or other technical issues including tricky computational analysis, which has been reviewed recently . A great3.Neurog2, Pou4f1, Neurog1, NeuroD4, NeuroD1) from other lineages . Fo. Fo73]. Oct4/Nanog in the cranial neural crest represents an important biasing factor, which favors specification and expansion of various ectomesenchymal cell fates. The neural crest-specific ablation of Oct4 does not affect overall embryogenesis, except for the amount of ectomesenchymal cells giving rise to the frontonasal cell mass responsible for the formation of the face, without other detectable effects on other fates of the neural crest [The sum of these ideas raises an important question of how the proportionality of cell types is achieved in the neural crest lineage . Given tal crest . Thus, tbona fide bifurcation  is still present and needs to be considered as such. One would expect that experimental strengthening of this weak mesenchymal bias could result in mesenchymal progeny in the trunk. This was exactly achieved by the introduction of cranial crest-specific Twist2 into trunk neural crest, which is usually largely devoid of Twist2 expression [Twist2 in the chick trunk neural crest resulted in a burst of mesenchymal fates. In more natural settings, a similar bias control might be responsible for the remarkable and unusual mesenchymal contribution from the trunk neural crest to the developing turtle shell [Bcl11b, which desensitizes progenitors to the regulators of alternative fates [The mystery of whether NCC subpopulations use differential biases to navigate a single fate landscape, or are presented with fundamentally unique decisions on separate landscapes, may be approached by identifying “muted” bifurcations where multilineage priming occurs but one fate is never expressed. Accordingly, Soldatov et al. revealed a fate conflict between mesenchymal and autonomic pro-neuronal branches in the posterior mouse trunk, where mesenchymal fates do not normally form . This depression . As predle shell . Interesve fates .Not only extrinsic biasing molecules or region-specific patterning might be responsible for macro and micro-heterogeneity of the early neural crest population. Temporal fluctuation is another source of intrinsic heterogeneity , though Prrx2 and pro-autonomic Phox2b master regulators [Finally, pre-bifurcation heterogeneity in NCCs might affect the rate of differentiation, causing the decision-making of a cell population to appear rather like a dispersed area instead of a sharp point in pseudo-time. Indeed, in the cervical region of mouse embryos, cells undergoing fate bifurcation between mesenchymal and autonomic proneurogenic programs appear diffusely spread out in transcriptional space, compared to the primary sensory split. Notably, the corresponding neural crest streams generate the largest sympathetic ganglion in the body – the superior cervical ganglion, and, at the same time, contribute cells to the skeletogenic populations in the neck and cardiac neural crest. The fate conflict between the autonomic and mesenchymal fates pinnacles in co-activation of pro-mesenchymal gulators . Some cegulators ) or die.gulators . Therefogulators , for insgulators .5.Not all transitions proceed via bifurcation for progressing cell lineages. Many transitions proceed in a linear fashion and display transient intermediate states called “bridges” . Even inHtr3a and others [Fate transition via the “bridge” is observed when a continuum of cells is clustered connecting two cell types with or without their own unique gene expression programs. Therefore, such bridge states can correspond to a pure downregulation of the old state genes and an upregulation of the new state genes, showing a gradual mixture of two genetic programs along such transitions. Alternatively, the transitory state can reveal its own gene expression signature, rendering it closer to a true transitory cell type or less stable attractor with a specific gene expression program. One key example of such transition though the “bridge” in the neural crest lineage is represented by the differentiation of chromaffin neuroendocrine cells from SCPs . Chromafd others . The anad others  and exped others . Some ofd others .6.Fate convergence occurs when cell populations from different lineages, with different developmental competences, contribute to the same definitive or intermediate cell type ,48,82–84Mitf (in a combination with Sox10) becomes induced in various neural crest subpopulations, starting from the pre-migratory and delamination stages. Because the expression of melanocyte markers and master regulators of melanocyte fate does not achieve significant coordination and emerges in cells across multiple neural crest differentiation phases, a single and clear branch leading to the future melanocytes cannot be established during the phases of delamination and migration [In another example, the single cell transcriptomics analysis of murine neural crest did not result in identification of a melanocyte branch either in the trunk or in the cranial regions during the time of the neural crest formation and migration . This stigration . Furtherigration ,86–88. Tigration . Thus, tNot only melanocytes are jointly generated from the nerve-associated SCPs and the migratory crest. Another example includes the sympathetic neurons . The intin vitro and in vivo [The characterization of numerous fate convergence in the neural crest lineage raises an important caveat regarding clonal history. Although reconstructed trees of transcriptional events, including sequential bifurcations, might accurately describe the changes of the internal states of cells progressing along the Waddingtonian landscape, those changes and the transcriptionally defined fate splits do not necessarily correspond to actual lineage as per clonal genealogy. Indeed, Tracer-seq  revealed in vivo . Overall7.de facto gives rise to all downstream fates) or collective multipotency (when a cell de facto gives rise only to some of the possible downstream fates) can be resolved only by blending single cell transcriptomics with clonal analysis. This thesis is perfectly applied to neural crest multipotency, having been analyzed and challenged experimentally in multiple species.The preceding discussion hints that the question of whether stem cell populations utilize individual multipotency ” . AccordiUltimately, predictive understanding of neural crest multipotency requires a detailed study of the regulatory signaling mechanisms that produce spatiotemporally structured micro-heterogeneity in the dorsal neural tube, and how early-versus later-acting biasing factors drive specific clonal fate proportionality of NCCs at pre-migratory and delamination stages in the embryo. Future experiments with clonal labelling together with readouts of individual transcriptional states  will be 8.We conclude this review with a remark about the transformative power of single cell transcriptomics, and multi-omics in general, towards understanding micro-heterogeneity that drives the logic of cell fate decisions in the neural crest lineage. Beyond being simply descriptive when it comes to gene expression and chromatin accessibility states, single cell technologies lean now towards predicting the causal interactions, elucidating hierarchical structures of gene regulatory networks, and inferring dynamic aspects from seemingly static snapshots of individual cells. The existing datasets now predict a myriad of functional interactions between multiple cell types and functional connections between gene regulatory networks with associated signal transduction mechanisms. However, it is still unknown how exactly the extrinsic signals are converted into intracellular biases directing the development of heterogeneity and fate choices in the lineage of NCCs . Answeri


The present investigation evaluated the effect of the combination of different dental filling materials in Class I cavities under occlusal loading using three-dimensional finite elements analysis (FEA). Six computer-generated and restored models of a lower molar were created in the CAD software and compared according to the biomechanical response during chewing load condition. Two adhesively bonded bulk restorative materials [bulk-fill resin composite (BF) or Alkasite (Alk)] were evaluated with or without the presence of a base material below (flowable resin composite or glass ionomer cement). A food bolus was placed on the occlusal surface mimicking the compressive occlusal load (600 N) during the static linear analysis. The maximum principal stress (tensile) was calculated as stress criteria in enamel, dentin and restoration. All models showed high stresses along the enamel/restoration margin with a similar stress trend for models restored with the same upper-layer material. Stress values up to 12.04 MPa (Alk) or up to 11.12 MPa (BF) were recorded at the enamel margins. The use of flexible polymeric or ionic base material in combination with bulk-fill resin composite or Alk did not reduce the stress magnitude in dentine and enamel. Class I cavities adhesively restored with bulk-fill resin composite showed lighter stress concentration as well as Alk. Therefore, adhesively bonded Alk restoration showed a promising mechanical behavior when used with different base materials or as a bulk restoration for posterior Class I cavity.  Composite resin-based dental fillings are assumed to be advanced polymeric materials in adhesive dentistry due to their optimal properties that mechanically and aesthetically replace missing dental tissues ,2. TheseA greater clinical application of new and modified resin monomers as low stressing bulk dental materials is advocated to restore deep posterior cavities . LeakageAnother alternative to restore large cavities in molars has been advocated to be the use of a modified resin composite with alkaline filling . CentionCention N is an Alkasite [Alk] in a new category of filling material with setting reaction of four minutes, starting after the mixing of powder and liquid. Cention N contains Ivocerin as a photoinitiator and an acyl phosphine oxide initiator , as wellHowever, the mechanical behavior of bulk-fill restorations in resin composites or in Alk for molar cavities has not been investigated yet. Thus, is there any mechanical benefit that can result in the material clinical choice for Class I cavities in molars? In addition, what is the mechanical effect in the restoration when different base materials are used in combination with Alk?Finite element analysis has been widely used to investigate bulk-fill restorations behavior  and moreThe present study applied the computer-aided design and finite element method (CAD-FEM) as a bioengineering tool to calculate the stress distribution in molar Class I restorations. This method has been extensively applied to investigate the mechanical behavior in different dental fields ,18,19,20X-axis and Y-axis were used for the bucco-lingual direction, while the Z-axis was oriented upwards) [The previously created 3D CAD model of a sound molar ,9,21 wasTo simulate the masticatory loads variability that is affected by the contact between tooth and food bolus, a solid volumetric model of food on the occlusal surface was also designed D. This lThe response of the six restored models was assessed by the computer-aided engineering software . All models were discretized using 4-node tetrahedral elements with a total size extending from 0.08 mm to 0.18 mm. To minimalize the mesh effect in the stress results, caused by the small curvature radius and notch effects, mesh improvement techniques were used with a mesh convergence test considering von-Mises stress maximum values. The analyses were based on the loading during the maximum bite force at the chewing cycle [The present simulation did not consider the polymerization shrinkage stresses in the resin based materials due to the lack of data for Alk. Therefore, as a limitation, the kinetic stress relaxation was considered insignificant and only the elastic modulus and Poisson ratio were applied as the constant elastic properties for the stress calculation. The chemical formula for the monomers presented in the experimental material (Alk) are summarized in The physiological masticatory load (600N) was simulated during the occlusal static load applied to the food bolus. The lower surface of the models was constrained in all directions . StatistThe calculated stress magnitude for the models were qualitatively and quantitatively compared. Assuming that these materials exhibit brittle mechanical behavior , the firRegardless of the restorative material combination, the models exhibited a similar stress trend along the evaluated structures. For both enamel  and dentFor the restoration , color mThis study aimed to evaluate the mechanical behavior of different bulk-fill materials with and without the presence of a base material to restore a posterior Class I. Results showed differences between the materials behavior, rejecting the null hypothesis.When a single-increment restoration was simulated in the present study, similar biomechanical behavior was observed between the BF and the Alk groups, with a numerical difference of 0.92 MPa, not appreciated in Another study a found similar and higher bond strength, after 14 days, for Alk (14.38 MPa) and nano-hybrid composites compared to glass ionomer cement (5.96 MPa) . HoweverIn terms of flexural strength, self-cured and dual-cured Alk have also been compared to four conventional resin composites and two bulk-fill resin composites. Authors found lower and similar flexural strength for Tetric N-Ceram bulk-fill (103.7 MPa) and dual-cured Alk (96.4 MPa), i.e., higher than self-cured Alk (82.1 MPa). However, the strength significantly decreased after 12 months  . Other aIn addition to the colorimetric graphics, a quantitative analysis was carried out in order to compare the restorations by several authors ,29,30. LPrevious investigations with a similar methodology have applied the maximum principal stress as the analysis criteria for the failure of dental materials ,19,22. TThis study also simulated conditions with flexible base materials at the bottom of the cavity. The clinician often opts to keep it as part of the restorative treatment when cavities are large and deep. When the flowable composite was simulated, higher stress levels compared to glass ionomer cement at the enamel tissue were reported, as reported in With the limitations of a 3D finite element analysis, this study did not consider all the factors present in the oral medium , such asWith the limitations of the present study, the adhesively bonded Alkasite restorations showed a promising mechanical behavior with reduced stress magnitude when used above different base materials or as a bulk restoration for Class I posterior restorations.


Eltrombopag is a second-line therapy for refractory thrombocytopenia from immune thrombocytopenic purpura (ITP). The medication is generally well tolerated but can lead to adverse thromboembolic complications in rare instances. We present a case of lower extremity deep vein thrombosis with bilateral pulmonary embolism in an ITP patient receiving eltrombopag. The patient underwent catheter-directed thrombolysis for pulmonary embolism. Eltrombopag was stopped on discharge, considering the potential cause of venous thrombosis. Immune thrombocytopenic purpura (ITP) is an autoimmune disease characterized by the rapid peripheral destruction of platelets as well as impaired production of platelets from megakaryocytes within the bone marrow . The iniA 75-year-old male with a past medical history of immune thrombocytopenic purpura (ITP) presented to the emergency department (ED) with chief complaints of shortness of breath and palpitations for five days. He reported that the shortness of breath was mainly exertional and started almost a week ago but worsened over the last two days. He denied cough, orthopnea, chest pain, or fever. The patient also denied taking recent long flights, being bedbound, or having any history of cancer. Physical examination showed a non-toxic appearing man with vital signs: blood pressure of 168/80 mmHg, heart rate of 105 beats/min, respiratory rate of 23 breaths/min, and pulse oximetry oxygen saturation of 96% on 2L nasal cannula oxygen. CT pulmonary angiogram showed a bilateral pulmonary embolism  of the right femoral, popliteal, and tibioperoneal veins. The echocardiogram showed reduced right ventricular systolic function with TAPSE (tricuspid annular plane systolic excursion) of 0.8cm and RVSP (right ventricular systolic pressure) of 55 mmHg, while the left ventricular ejection fraction was 51%. The patient had a history of ITP and was initially treated with glucocorticoids and IVIG (intravenous immunoglobulins) with poor response. He subsequently underwent a splenectomy almost three months ago, but he continued to have persistent thrombocytopenia. He was subsequently started  on eltrombopag due to persistent thrombocytopenia even after the splenectomy. A review of previous records showed that his platelet count was 20-30 k/uL before starting eltrombopag. His platelet count on presentation was 173 k/uL and subsequently was noted to be as high as 940 k/uL within a week of hospitalization. The eltrombopag was not resumed. The patient was started on intravenous heparin and underwent successful catheter-directed thrombolysis (CDT) for pulmonary embolism. He was transitioned to warfarin with heparin bridge to target INR 2-3. Eltrombopag was stopped on discharge, and he was advised to follow up with his primary hematologist for close monitoring of platelets and to explore further options for long-term management of ITP.Eltrombopag is an oral thrombopoietin receptor agonist (TPO-RA) used in treating ITP, which is refractory to steroids, immunosuppressants, and splenectomy if they have an increased risk of bleeding . HoweverAlthough chronic ITP can be a risk factor for thrombosis, it has been seen that the use of TPO-RA in these patients can lead to adverse effects of thromboembolism, and monitoring for these complications is essential. This has been suggested by some studies where TPO-RAs were used instead of immunosuppressants in chronic ITP patients . A pooleThe introduction of TPO-RAs for the treatment of refractory ITP raised the issue of thrombosis. An interim analysis on the safety and efficacy of eltrombopag in 299 patients by Saleh et al. reported that 5% of the patients developed thromboembolic events . RodeghiThe exact pathogenesis of increased thrombosis with TPO-RA has yet to be well established. Although there has been reported an increase in many coagulation activation markers, for example, D-dimer, thrombin generation, prothrombin fragment, and plasminogen activator inhibitor-1 (PAI-1) in ITP patients, no further activation of coagulation markers has been observed after initiation of TPO-RAs . HoweverThe target platelet count for patients on eltrombopag should be 30-50 k/μL, which is an optimal balance between bleeding versus thrombotic risk . AchieviOur case adds to the existing literature of recorded thromboembolic events following the use of eltrombopag; it emphasizes the current underreporting of adverse events associated with TPO-RA use. We also wish to draw attention to the importance of close outpatient follow-up with regular monitoring of platelet counts and keeping a close eye on other thromboembolic risk factors on an individual case basis when prescribing these medications. The subsequent management of ITP in these patients who have had a thromboembolic episode while on TPO-RAs can be challenging.


Snake venom is an adaptive ecological trait that has evolved primarily as a form of prey subjugation. Thus, the selection pressure for toxin diversification is exerted by the prey’s physiological targets, with this pressure being particularly acute for specialist feeders, such as the King Cobra species, all of which are snake-prey specialists. However, while extensive research has been undertaken to elucidate key amino acids that guide toxin structure–activity relationships, reciprocal investigations into the specific sites guiding prey-lineage selective effects have been lacking. This has largely been due to the lack of assay systems amenable to systematic amino acid replacements of targeted proteins in the prey’s physiological pathways. To fill this knowledge gap, we used a recently described approach based upon mimotope peptides corresponding to the orthosteric site of nicotinic acetylcholine receptor alpha-1 subunits, a major binding site for snake venom neurotoxins that cause flaccid paralysis. We investigated the venoms of four different types of King Cobra . This approach allowed for the determination of the key amino acid positions in King Cobra snake prey that are selectively bound by the toxins, whereby replacing these amino acids in the snake-prey orthosteric site with those from lizards or rats resulted in a significantly lower level of binding by the venoms, while conversely replacing the lizard or rat amino acids with those from the snake at that position increased the binding. By doing such, we identified three negatively charged amino acids in the snake orthosteric site that are strongly bound by the positively charged neurotoxic three-finger toxins found in King Cobra venom. This study, thus, sheds light on the selection pressures exerted by a specialist prey item for the evolution of lineage-selective toxins. Predator–prey interactions amongst species are often considered major evolutionary driving forces; thus, traits that intensify such interactions are believed to provide positive selection pressures ,2. SnakeThis prey-specific targeting of some toxins has notably been found within three-finger toxins (3FTxs), which are ubiquitously found in venoms of the Elapidae family and also some members of the Colubridae and Lamprophiidae families ,17,18,19Oxybelis fulgidus (green vine snake) contains 3FTx named fulgimotoxin, which exhibits greater neurotoxic effects towards its natural prey items but no toxicity towards nonprey items [Boiga dendrophila (mangrove Snake), a snake that predates on birds, contains the bird-specific predatory toxin denmotoxin in its venom [Boiga irregularis (brown tree snake), showed a higher toxic potency towards birds and lizards but did not show significant mammalian toxicity [Spilotes sulphureus) is lethally toxic to both mammals and lizards yet contains individual 3FTx types, sulmotoxin 1 and sulditoxin, which have differential lethality towards these prey types. Sulmotoxin 1 exhibits high lethality towards mammals, but is nontoxic to lizards, while sulditoxin exhibits the reverse pattern  [Numerous studies have investigated diet-related adaptive evolutionary processes across different lineages of venomous snakes with the presence of taxon-specific crude venoms containing specific 3FTx isoforms that induce toxicity towards both natural and non-natural prey animals ,11,26,27ey items . Moreovets venom . Iridiottoxicity . The Amamammals) . Therefomammals) . These sOphiophagus hannah (King Cobra) venom has been shown to be highly selective for snake (Coelognathus radiata) alpha-1 [O. hannah is highly correlated with specific amino acids within the snake-prey alpha-1 orthosteric site [O. hannah. The novel mimotope-based biolayer interferometry assay offers a greater advantage in understanding receptor-toxin binding affinities in a taxon-specific manner, offering invaluable opportunities to explore the interactions of these incredible toxic constituents on biological targets, such as nAChRs. Since  alpha-1 , we hyporic site . To inveric site ,34 to scO. hannah venom to produce the previously noted snake selectivity, relative to the lower binding potency against lizard and rat homologous sites [To identify which amino acid residues of the alpha-1 binding site are being targeted by us sites , we consus sites ,34. MutaWhile the venoms displayed differences in relative potency against a particular mimotope, they displayed a high level of conservation of the relative binding affinity with all venoms being significantly more potent in binding native snake than native lizard  or nativAmino acid replacement mutants were enlightening in regard to which residues at particular positions were preferentially bound by the King Cobra venoms. For example, the lizard mutant mimotope 194L, which had the polar, uncharged amino acid threonine (T) replaced at position 194 by the corresponding snake amino acid, nonpolar, uncharged amino acid leucine (L), showed a dramatic increase in binding by all four King Cobra venoms , suggestAlso indicative of complex interactions between adjacent amino acids was the variable response to the introduction of negatively charged amino acids at positions 188, 191, and 195. Previous works have shown that negatively charged amino acids in the orthosteric site strongly guide binding by the positively charged neurotoxins . For theNegatively charged amino acids in a snake-prey orthosteric site exerting a selection pressure are further evidenced by an examination of all available alpha-neurotoxic three-finger peptides available in uniprot.org . A totalThese results suggest that the higher toxin susceptibility in snakes is not a direct consequence of a single amino acid residue, but multiple amino acids interacting with toxins to intensify the neurotoxin susceptibility of snakes to King Cobra venom. In addition, since the interactions are quite complex, it can be postulated that these residues might act synergistically. Moreover, it can be proposed that the dynamicity of amino acid targeting between the King Cobra venoms from different locales is a consequence of different King Cobra species that inhabit different ecological niches encompassing diverse venom arsenals comprising structurally divergent 3FTx isoforms.To test these theories further, we designed a series of reciprocal mimotopes . The aimSince the rodent mutant mimotope 194 L (position 194 of native rodent mimotope is substituted with snake leucine (L)) showed an increase in venom binding only with the King Cobra venom from the Javanese locale, we designed a snake mutant mimotope that has the rat proline at position 194 in place of leucine in the native snake and checked for the reduction of binding by the King Cobra venom from Java. The results showed a clear reduction of neurotoxin binding with the King Cobra venom from the Javanese locale. Therefore, our data further confirm that these amino acid residues in the orthosteric site of snake nAChR strongly influence the neurotoxin binding. Moreover, our data further suggest that the reduced neurotoxin susceptibility of rodents and lizards might be due to the lack of those critical amino acid residues in their orthosteric sequence.The double-mutant snake mimotope with the rat 195S (serine) and rat 195N (asparagine) instead of the aspartic acid (D) and glutamic acid (E) at both 191 and 195 positions showed a sharp drop of neurotoxin binding compared with the point-mutated mimotopes containing single mutations at either 191 or 195 positions. This can suggest that these two negatively charged residues might act synergistically to increase toxin susceptibility. This synergistic effect is particularly strongly observed in the King Cobra venom from the Javanese locale. However, there was no considerable decrease in neurotoxin binding identified with the snake mutant 188 V/194 T, where both the 188 (glutamic acid (E)) and 194 (leucine (L)) critical positions of the snake orthosteric site were substituted with corresponding lizard residues of 188 valine (V) and 194 threonine (T), respectively. Therefore, it can be proposed that the two amino acids, glutamic acid (E) and leucine (L), localized in positions 188 and 194, respectively, of the snake orthosteric site might not act synergistically to strengthen the neurotoxin binding. Further, the triple-mutant snake mimotope 191S/194P/195N, which was constructed via swapping key amino acids of the snake at positions 191 (aspartic acid (D)), 194 (leucine (L)), and 195 (glutamic acid (E)) with corresponding rodent residues (191 serine (S), 194 proline (P), and 195 asparagine (N)), was tested to identify that all three amino acids work together to intensify the venom susceptibility of the snake to the King Cobra venom of the Javanese locale . However, the results did not show a significant decrease in binding compared with the single and double mutants, thus indicating the nonexistence of a combined effect among those three residues.Conspicuously, our data propose that even though these critical negatively charged residues confer the increased susceptibility of snakes, the higher neurotoxin binding of snake receptors is directly or indirectly mediated by the other amino acid residues, which localize inside and outside the orthosteric site of snake nAChR; thus, the ligand–receptor molecular interactions are quite a complex process.Consistent with previous works suggesting that prey ecology is a major influencer in shaping predatory venom composition , the varIt is important to note that though the crude King Cobra venom sourced from all four geographic localities tested in the present study demonstrated a relatively similar binding pattern (with the snake native bound the strongest), a considerable divergence in overall neurotoxic binding was exhibited among Javanese and Malaysian locals. This supports the idea of the geographical variation of venom composition and venom toxicity. Recent quantitative proteomic studies on Malaysian and Javanese King Cobra venoms showed that the crude King Cobra venom of the Javanese locale contains a comparatively higher amount of alpha-neurotoxic 3FTxs, which accounts for 64.2% of total venom proteins with ≥20 subtypes . ConversIn summary, we identified key negatively charged amino acid residues  that guide the snake-prey preferential binding of positively charged alpha-neurotoxins in King Cobra venom. Changes made at those positions in snake receptors with corresponding rodent and lizard positions showed a drastic decrease in venom sensitivity, while lizard or rodent mutants containing these negatively charged amino acids had an increased rate of binding, confirming that these key structural residues might highly contribute to the neurotoxic sensitivity of snakes to King Cobra venom. Collectively, the findings of our study reinforce filling the gap in evolutionary biology knowledge by providing strong evidence on the diet-related adaptive evolution of snake venom systems and the selection pressure exerted by prey pathophysiological targets.Ophiophagus hannah) venom samples (pooled) from adult captive snakes spanning four geographic locales were sourced from the long-term cryogenic collection of the Venom Evolution Lab, University of Queensland, St Lucia, Australia. All the venom study protocols of this work were performed with University of Queensland Biosafety Approval #IBC134BSBS2015 and University of Queensland Animal Ethics Approval 2021/AE000075. The lyophilized crude venom samples were reconstituted with deionized water prior to the use. The centrifugation was performed at 14,000 RCF for 10 min with a temperature of 4 °C. The pellet, if any, was discarded, and the supernatant was used to make a working venom stock of 1 mg/mL in 50% of glycerol, which was used to preserve the enzymatic action while eliminating freezing upon storage at −20 °C. The concentrations of the prepared venom stocks were checked at 280 nm with a NanoDrop 2000 UV–VIS Spectrophotometer . King cobra (Coelognathus radiatus) was characterized in an earlier research work and was used for the present work. The conserved cysteine residues localized at positions 192 and 193 of each of the mimotope sequences were replaced with a pair of serine molecules to eliminate the uncontrollable postsynthetic thiol oxidation. Since the highly conserved cysteine residues do not directly contribute to receptor–ligand interactions, it is anticipated that the elimination of the two tandem cysteine residues in the orthosteric sequence does not produce a considerable effect on the ligand binding. However, it has been identified that the presence of cysteines plays an important role in the structural formation of the ligand-binding site of the overall receptor. Therefore, the ligand-binding kinetics of the synthetic peptide mimotopes and the entire nicotinic acetylcholine receptor are noncomparable or should be interpreted carefully. Working mimotope stock solutions of 50 µg/mL were made by dissolving each of the supplied mimotopes of dried pellet in 100% dimethyl sulfoxide (DMSO) following 1:10 dilution with deionized water. All prepared mimotope stock solutions were stored at −20 °C for future use. A series of 14-amino-acid-long native and mutated peptide mimotopes, which represent regions 187–200 of the alpha-1 subunit of muscle-type nAChR of snake, rodent, and lizard were designed following the previously validated protocol . The poiThe ligand–receptor binding affinities were measured utilizing a label-free, microfluidics-free novel biolayer interferometry assay, which was performed on an Octet HTX system . All the experimental procedures of the octet potency assay along with the data acquisition and data analysis were performed according to the previously validated protocol . The incAll the data acquired from biolayer interferometry on a Octet HTX  were processed in exact accordance with the previous validation protocol of the assay . The raw


Mycobacterium tuberculosis (Mtb) infection into active TB, this vaccine is ineffective in protecting adults with active TB manifestations, such as multiple-, extensive-, and total-drug-resistant (MDR/XDR/TDR) cases and the co-existence of TB with immune-compromising health conditions, such as HIV infection or diabetes. Moreover, BCG can cause disease in individuals with HIV infection or other immune compromises. Due to these limitations of BCG, novel strategies are urgently needed to improve global TB control measures. Since live vaccines elicit a broader immune response and do not require an adjuvant, developing recombinant BCG (rBCG) vaccine candidates have received significant attention as a potential replacement for the currently approved BCG vaccine for TB prevention. In this report, we aim to present the latest findings and outstanding questions that we consider worth investigating regarding novel mycobacteria-based live attenuated TB vaccine candidates. We also specifically discuss the important features of two key animal models, mice and rabbits, that are relevant to TB vaccine testing. Our review emphasizes that the development of vaccines that block the reactivation of latent Mtb infection (LTBI) into active TB would have a significant impact in reducing the spread and transmission of Mtb. The results and ideas discussed here are only based on reports from the last five years to keep the focus on recent developments.Vaccination is an excellent approach to stimulating the host immune response and reducing human morbidity and mortality against microbial infections, such as tuberculosis (TB). Bacillus Calmette–Guerin (BCG) is the most widely administered vaccine in the world and the only vaccine approved by the World Health Organization (WHO) to protect against TB. Although BCG confers “protective” immunity in children against the progression of  Despite the availability of effective antibiotics, treatment for active tuberculosis (TB) often has a poor prognosis. Suboptimal adherence to lengthy treatment regimens contributes to the emergence of drug-resistant TB, which poses a significant burden on the global economy and patients’ quality of life . The emeMycobacterium tuberculosis (Mtb)-infected immune-competent individuals, which amounts to 1.8 billion people globally, develop asymptomatic latent TB infection (LTBI). People with LTBI can reactivate with immune compromise and are thus a vast reservoir of potential new transmission sources upon reactivation [There were 10 million new cases and 1.5 million deaths due to TB in 2020, and it is estimated that roughly 85–90% of + T cells via immunodominant Mtb antigens confers adequate protection against TB. However, most new anti-TB vaccine candidates employing this strategy have not shown superiority over BCG, which includes, among other drawbacks and limitations, the risk of causing disease in individuals with HIV infection or other immune compromises, highlighting the need to identify novel approaches and/or to reinterpret existing concepts to guide the rational design of new TB vaccines [The primary strategy for new TB vaccines under development is based on data from human and animal studies, which suggest that the activation of Mtb-specific CD4vaccines ,4,5. In vaccines ,7,8. In Animal models are excellent tools to study the course of Mtb infection, including the pathological manifestations of acute and chronic active TB, as well as LTBI and reactivation, and to decipher the host and pathogen determinant(s) that are vital for pathogenesis. Knowledge gained from such studies can aid understanding of the pathogenesis of various forms of TB and the development of more effective intervention strategies to combat the disease. Several animal models, ranging from zebrafish to non-human primates (NHPs), have been explored to investigate their resemblance of biological manifestations during active TB and LTBI with corresponding human conditions . Each anThis article discusses the recent developments in novel recombinant BCG and Mtb-based vaccines and their pros and cons over conventional BCG vaccines that the World Health Organization currently approves for use in humans. We also highlight some novel mouse and rabbit models of TB as preclinical tools to screen potential TB vaccines.ureC::hly) is the most advanced vaccine candidate, which is based on BCG Danish. In a phase 2 clinical trial performed in South African newborn babies, VPM1002 was found to be better tolerated than BCG regarding side effects. These included 15-fold less grade 3–4 vaccine-related adverse reactions or lymphadenopathy of 10 mm or greater in diameter, 3-fold less scarring, and 10-fold less abscess formation in those who received VPM1002 compared with the BCG-administered group; however, ex vivo immune responses were higher in the group that received the BCG vaccine [ClinicalTrials.gov Identifier: NCT04351685) will inform about the immunogenicity afforded by VPM1002. Additionally, compared with wild-type BCG Danish, BCGΔureC::hlyΔnuoG (a VPM1002 variant with the deletion of the nuoG gene) reduced the replication of a laboratory strain (H37Rv) as well as a clinical Mtb strain (W/Beijing) in the lungs and spleens of infected mice, especially during chronic TB [The current TB vaccine, BCG, neither sterilizes Mtb infection nor effectively protects against pulmonary cavitary disease and the spread of infection in the human adult population, contributing the most to TB transmission in the community. Thus, the development of next-generation TB vaccines is of great urgency. In this regard, it was recently discussed that the development of efficacious rBCGs aimed at improving protection against TB often follows two strategies: (i) deleting genes from (knockouts) BCG or (ii) inserting mycobacterial antigen-coding genes into the BCG genome  . For theronic TB . HoweverBCG1419c). Compared with parental BCG, BCGΔBCG1419c induced different levels of TNF-α, IL-6, and IL-1β; was attenuated for intracellular replication in murine macrophages; and changed the expression of antigenic proteins when grown as surface pellicles [+ T cells with a similar CD4+ T cell immunogenicity was observed in C57BL/6 mice vaccinated with BCGΔBCG1419c compared with those that received BCG Pasteur; this differential immunogenicity also resulted in reduced pulmonary inflammation in the former group following infection with the Mtb M2 clinical strain [+ T helper (TH) cells and increased frequencies of IL10-producing T cells [Another BCG-based vaccine candidate that follows the gene-deletion strategy is BCGΔBCG1419c . Vaccination with BCG::phoPR was found to prolong the survival of Mtb-infected guinea pigs compared with those vaccinated with the parental BCG [In contrast to BCGΔBCG1419c, a modified vaccine candidate constructed by gene-insertion, rather than gene-deletion, is the BCG::phoPR strain, in which the ntal BCG . HoweverMtb, also known as BCG::RD1) is amongst the most effective rBCG strains ever developed, at least at the preclinical level. Although BCG::ESX1Mtb reduces the bacterial burden and is more immunogenic than BCG in animal models, the strain has been deemed unsafe due to its prolonged persistence and increased virulence in immunocompromised hosts [Mtb was mainly attributed to an improved T cell response to ESAT6 , an immunodominant protein secreted by the ESX1 locus of Mtb [Mtb, and a derivative of BCG::ESX1Mtb) and eight combinations of vaccine delivery, we recently demonstrated that the inclusion of ESX1-exported effector molecules was the overall most efficient way to improve BCG performance [esxA gene to the general secretion signal of the mycobacterial type VII secretion pathway [Mtb and parental BCG, the mucosal administration of BCG::ESAT6-PE25SS is associated with a more rapid clearance of Mtb from the infected mouse lungs [The modified BCG strain containing the “ESAT6 protein family secretion system-1” from Mtb  and devoid of the  strains . Conside strains . The L2- strains . cpnB or disA, involved in cyclic-di-adenosine monophosphate (c-di-AMP) metabolism were deleted from MTBVAC to produce MTBVACΔcnpB::Km and MTBVACΔdisA::Km [cnpB attenuated MTBVAC, the deletion of disA had no major effect on virulence in the immunized SCID mice [Recently, two genes, disA::Km . It was CID mice . FurtherCID mice .Clinical trial: NCT03536117). The advantages and limitations of some mycobacteria-based novel vaccine candidates are summarized in In a separate study performed in an advanced preclinical NHP model, rhesus macaques vaccinated with BCG or MTBVAC and challenged with an ultra-low dose of Mtb Erdman showed no difference in disease progression. However, at 16 weeks post-infection, the MTBVAC-vaccinated animals outperformed the BCG-vaccinated ones in reducing lung pathology and extrapulmonary bacterial loads . It shou+ T cell depletion has been considered the primary cause of the HIV-induced reactivation of LTBI, and this complex interaction was recently modeled in NHPs [+ T cells was reported [+ cells, the mice underwent the progressive, systemic spread of the bacteria accompanied by lung pathology. This model was used to determine that vaccination with BCGΔBCG1419c or BCG Pasteur prevented the systemic spread of Mtb from the infected lymph node to the spleen and lungs, which was a measure of the reactivation of LTBI, thus showing that lympho-centric LTBI mice could be a tractable and reproducible small animal model with potential application for TB vaccine testing [Progressive CD4 in NHPs . Despitereported . In this testing . The modSelecting a suitable small animal model that can develop cavitary TB, as well as spontaneous LTBI and the immunosuppression-mediated reactivation of LTBI, is crucial for evaluating novel therapeutic and prophylactic TB vaccine candidates. The rabbit model of pulmonary Mtb infection produces the range of pathologic manifestations seen in patients with asymptomatic and symptomatic cavitary TB ,64. In tIn a separate study, our group compared the response of BCG-vaccinated rabbits following central nervous system (CNS) infection with the Mtb strains HN878 or H37Rv . In thisRecently, several molecular tools have been developed to characterize the host immune responses in rabbits, including RNASeq analysis to decipher the genome-wide transcriptome profiling, functional classification, and the pathway analysis of differentially expressed genes in the granulomas; flow-cytometry methods have been established with additional antibodies for rabbits to create better immune cell phenotyping and functional assays than before. Furthermore, we developed an mRNA-based fluorescent in situ hybridization (mRNA-FISH) technique to map the spatial transcriptome of immune cells. In this technique, multiple gene transcripts can be probed at the site of infection  using a combination of multiple mRNA targets tagged with different fluorescent markers ,67,68.Considering that most novel TB vaccine candidates have been tested against the laboratory Mtb H37Rv strain challenge and their efficacy has mostly been evaluated in murine models of active TB post-infection in the short-term, we think that to produce more effective TB vaccines, it is worth addressing the following questions: (a) What is the efficacy of novel  vaccine candidates against multidrug-resistance (MDR) Mtb strains and how do these vaccine candidates work in adjunct antibiotic therapy? (b) Would they effectively reduce the TB caused by clinical Mtb isolates of variable virulence potential? (c) Would the routes of vaccine administration  impact the immunogenicity and protective efficacy of a given vaccine? (d) What is the comparative profile of the immunogenicity and protective efficacy conferred by the vaccine candidates in various preclinical animal models, including mice, guinea pigs, rabbits, ferrets, and NHPs? (e) What is the contribution of host genetics, sex, age, and other comorbidities to the efficacy of these vaccine candidates? (f) Would there be any benefit of combining multiple live attenuated mycobacteria-based vaccine candidates with subunit  vaccine candidates in terms of preventing or reducing TB? (g) Would strategies based on nanotechnology improve TB vaccine efficacy?In addition, it is crucial to explore new avenues that may result in alternative tools, other than additional drugs, to control the progression of Mtb infection into active TB disease, such as by promoting enhanced immune responses soon after primary infection to clear Mtb or reducing the pathological damage associated with primary TB. Furthermore, developing vaccines that block the recurrence of LTBI into active TB would significantly reduce the spread of Mtb strains in the community.In conclusion, novel and innovative vaccine candidates based on BCG and Mtb strains are emerging with superior safety, immunogenicity, and protective efficacy against TB compared with parental strains. Since these vaccine candidates are currently at various developmental stages, their intricate mechanisms of action are not fully understood and warrant more extensive in vitro, in vivo, ex vivo, and clinical evaluation studies. Such future studies should inform the suitability of these potential vaccine candidates to prevent TB in humans.


This cross-sectional study investigates rates of patients who left emergency departments without being seen from 2017 to 2021. These departures may have significant consequences for patients given the associated delayed or deferred care for acute conditions.4 Patients from minoritized communities are more likely to depart before evaluation, with problems relatively concentrated among hospitals serving low-income populations.5 This occurs despite the increasing added value of ED-based linkages to care for populations at increased risk, such as patients with opioid use disorder seeking medication treatment.6 We sought to characterize how often patients left the ED before clinical evaluation over time using a national sample of US hospitals.Acute care demands have increased dramatically alongside stagnant hospital capacity,STROBE reporting guidelines. The study used aggregated hospital measures available through a voluntary peer benchmarking service offered by Epic Systems Corporation, an electronic health record vendor. Measures were collected monthly from 2017 to 2021. Median (IQR) and 95th percentiles were reported for ED LWBS rates across hospitals each month. Statistical analyses were performed using R statistical software version 4.2.1  from January to March 2022.This cross-sectional study was classified as exempt from review and informed consent by the Yale University Institutional Review Board because it contained no patient data; all reporting adheres to There were 365 hospitals reporting benchmarking data in January 2017, increasing to 1769 hospitals by December 2021. Annual ED visit volumes and total hospital beds for participating sites are included in the Findings from this cross-sectional study demonstrate the failure of the emergency care system to maintain broad access in the context of pandemic demands, suggesting that existing regulatory and financial incentives may be inadequate to meet challenges of future pandemic waves and other disasters. Historically, LWBS was viewed as an ED management problem rather than a hospital- or systems-level issue. Thus, most solutions to date have relied on intradepartmental operational fixes to mitigate ED crowding; for example, doctor-in-triage or split-flow models offer more rapid medical screening evaluations, effectively bypassing traditional triage processes. These processes promote rapid but limited physician evaluations, often in the waiting room. Amid the current crisis, these ED-focused operational efforts may be inadequate to stem this growing problem.Our work is observational, and due to limitations of available data fields, we were not able to address hospital characteristics or local COVID-19 infections or address how the mix of participating hospitals changed over time. We hypothesize that system strain would be associated with increased differences in rates of LWBS for hospitals serving low-income and underinsured patient populations. Furthermore, while some work addresses the association of LWBS with patient outcomes, there is little contemporaneous work on this for patients during COVID-19.Access to emergency care cannot be considered universal until all patients presenting to EDs receive high-quality treatment for time-sensitive conditions. Given contributing system constraints, LWBS should be viewed as a failure to offer equitable access to acute care, understood in the context of other measures of care access.


Clinical studies suggest that the highly lipophilic, anti-inflammatory molecule, simvastatin, might be an ideal candidate for drug repurposing in the treatment of depression. The neuropsychological effects of simvastatin are not known, but their ascertainment would have significant translational value about simvastatin’s influence on mood and cognition.We aimed to investigate the effects of simvastatin on a battery of psychological tests and inflammatory markers in healthy volunteers.N = 27) or sucrose-based placebo (N = 26) given in a double-blind fashion. Then, participants were administered questionnaires measuring subjective rates of mood and anxiety, and a battery of tasks assessing emotional processing, reward learning, and verbal memory. Blood samples for C-reactive protein were also collected.Fifty-three healthy subjects were randomly assigned to 7 days of either simvastatin , but also an increase in anxiety scores . An exploratory analysis of the females’ subgroup (N = 27) showed lower number of misclassifications as sad facial expression in the simvastatin arm . No further statistically significant changes could be observed on any of the other outcomes measured.Compared to placebo, participants on simvastatin showed a higher number of positively valenced intrusions in the emotional recall task (We found limited evidence that 7-day simvastatin use in healthy volunteer induces a positive emotional bias while also being associated with an increase in anxiety, potentially reflecting the early effects of antidepressants in clinical practice. Such effect might be more evident in female subjects. Different drug dosages, treatment lengths, and sample selection need consideration in further experimental medicine and clinical studies.Clinicaltrials.gov: NCT04652089.The online version contains supplementary material available at 10.1007/s00213-022-06156-y. Statins are a class of molecules commonly prescribed to reduce peripheral cholesterol by inhibiting liver 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductases  effect of simvastatin on a battery of emotional processing tasks that are sensitive to the early effects of typical antidepressants, such as selective serotonin inhibitors (SSRIs), on emotional bias. Changes in emotional bias in healthy participants , are less affected by cognitive deficits oftentimes seen in depression, while retaining translational value for this disorder . The study protocol, which describes the methods in detail, was registered on Clinicaltrials.gov (NCT04652089).N) = 38 would give 90% power to detect changes of a comparable magnitude  ranging from 18 to 30] were recruited for this study. All provided full written informed consent.Inclusion and exclusion criteria were pre-specified in the study protocol and are reported in full in the Supplementary Material, Enrolled participants completed a battery of questionnaires including the Beck Depression Inventory . Accordingly, participants were randomised to either simvastatin 20 mg [a dose used in previous clinical trials of simvastatin in depression; Gougol et al. A randomisation code had been designed by a researcher uninvolved in the study using an online randomisation tool , and a second blood sample for hs-CRP .Finally, participants were administered a battery of neuropsychological tasks including the emotional test battery (ETB), the probabilistic instrumental learning task (PILT), and the auditory-verbal learning task (AVLT), described in detail in a previous similar study  and response bias   Grier  for thisThe ECAT involves thirty positively and thirty negatively valenced words Anderson  randomlyThe FDOT involves two faces, one neutral and one emotional (either happy or fearful), briefly displayed at the top and at the bottom of the computer screen and then replaced by a pair of dots with two different  orientations. Participants have to indicate whether the dots are vertically or horizontally aligned. Mean reaction times from trials when probes are in the same position as the emotional face  are subtracted from mean reaction times from trials when probes are in the opposite position to the emotional face  to calculate attentional vigilance scores.The PILT, adapted from Pessiglione et al. , assesseThe AVLT assesses verbal memory Rey,  through We used the IBM SPSS v27 statistical package IBM  for all Participants’ demographics, clinical characteristics, and baseline questionnaires were reported descriptively. Repeated questionnaires  and hs-CRP were analysed via repeated measures analysis of variance (ANOVA) with group (simvastatin versus placebo) as the between-subject factor and time (baseline versus testing visit) as the within-subject factor.η2) was reported for the main significant comparisons as a measure of effect size . On the basis of potentially diverse physiopathological effects of simvastatin, we further explored the main task  in specific subgroups of participants .We visually checked data distributions for all neuropsychological tasks using boxplots: extreme outliers  were excluded. The resulting data were then analysed using repeated measures ANOVA with group as the between-subject factor and emotion/valence as the within-subject factor. Significant interactions were followed up using simple main effect analyses. When assumptions of equality of variances were not fulfilled, the Greenhouse–Geisser correction was used. Partial eta squared  are reported in Table Participants’ repeated questionnaires  and hs-CRP are shown in Table F1,51 = 6.91, p = 0.01) and a group-time interaction  due to higher scores at the testing visit for the simvastatin group .The STAI-state showed a main effect of time   . This was driven by a reduced tendency to mislabel other facial expressions as sad in the simvastatin group amongst female participants . We therefore explored this effect within the subgroup of females . A group-emotion interaction for misclassifications was confirmed , which again was due to a lower number of misclassifications as sad facial expression for the simvastatin group . A further analysis clarified that females on simvastatin misclassified fewer positive facial expressions, when compared to negative emotions, as sad facial expressions . None of the other pre-specified variables .When we assessed the effect of gender on the FERT , where individuals on simvastatin recalled more positive intrusions compared to placebo . No effect on negatively valenced words was identified  .For emotional recall, we observed a group-valence interaction .There was no significant group-emotion interaction for attentional vigilance towards happy or fearful faces on any of the FDOT outcomes . The learning curves are reported in the Supplementary Material, For reward learning, there was no significant effect of simvastatin on any of the outcome measures .For verbal memory, no significant differences between the simvastatin and placebo groups were identified for any of the outcomes measured , duloxetine, led to an increased recall of false alarms for positive personality characteristics in the EREC, while also worsening anxiety and other subjective mood states  highlights intriguing, though preliminary, findings. There is evidence of sex differences at the interface between immunity and depression Below is the link to the electronic supplementary material.