Novel Corrector for Variants of SLC6A8: A Therapeutic Opportunity for Creatine Transporter Deficiency

Mutations in creatine transporter SLC6A8 cause creatine transporter deficiency (CTD), which is responsible for 2% of all cases of X-linked intellectual disability. CTD has no current treatments and has a high unmet medical need. Inspired by the transformational therapeutic impact of small molecule "correctors" for the treatment of cystic fibrosis, which bind to mutated versions of the CFTR ion channel to promote its trafficking to the cell surface, we sought to identify small molecules that could stabilize SLC6A8 as a potential treatment for CTD. We leveraged a novel chemoproteomic technology for ligand discovery, reactive affinity probe interaction discovery, to identify small-molecule fragments with photoaffinity handles that bind to SLC6A8 in a cellular environment. We synthesized a library of irreversible covalent analogs of these molecules to characterize in functional assays, which revealed molecules that could promote the trafficking of mutant SLC6A8 variants to the cell surface. Further medicinal chemistry was able to identify reversible drug-like small molecules that both promoted trafficking of the transporter and also rescued creatine uptake. When profiled across the 27 most prevalent SLC6A8 missense variants, we found that 10-20% of patient mutations were amenable to correction by our molecules. These results were verified in an endogenous setting using the CRISPR knock-in of selected missense alleles. We established in vivo proof-of-mechanism for correctors in a novel CTD mouse model with the P544L patient-defined variant knocked in to the SLC6A8 locus, where treatment with our orally bioavailable and brain penetrant tool corrector increased brain creatine levels in heterozygous female mice, validating correctors as a potential therapeutic approach for CTD

ISL1 Directly Regulates FGF10 Transcription during Human Cardiac Outflow Formation

The LIM homeodomain gene Islet-1 (ISL1) encodes a transcription factor that has been associated with the multipotency of human cardiac progenitors, and in mice enables the correct deployment of second heart field (SHF) cells to become the myocardium of atria, right ventricle and outflow tract. Other markers have been identified that characterize subdomains of the SHF, such as the fibroblast growth factor Fgf10 in its anterior region. While functional evidence of its essential contribution has been demonstrated in many vertebrate species, SHF expression of Isl1 has been shown in only some models. We examined the relationship between human ISL1 and FGF10 within the embryonic time window during which the linear heart tube remodels into four chambers. ISL1 transcription demarcated an anatomical region supporting the conserved existence of a SHF in humans, and transcription factors of the GATA family were co-expressed therein. In conjunction, we identified a novel enhancer containing a highly conserved ISL1 consensus binding site within the FGF10 first intron. ChIP and EMSA demonstrated its direct occupation by ISL1. Transcription mediated by ISL1 from this FGF10 intronic element was enhanced by the presence of GATA4 and TBX20 cardiac transcription factors. Finally, transgenic mice confirmed that endogenous factors bound the human FGF10 intronic enhancer to drive reporter expression in the developing cardiac outflow tract. These findings highlight the interest of examining developmental regulatory networks directly in human tissues, when possible, to assess candidate non-coding regions that may be responsible for congenital malformations

A united statement of the global chiropractic research community against the pseudoscientific claim that chiropractic care boosts immunity.

BACKGROUND: In the midst of the coronavirus pandemic, the International Chiropractors Association (ICA) posted reports claiming that chiropractic care can impact the immune system. These claims clash with recommendations from the World Health Organization and World Federation of Chiropractic. We discuss the scientific validity of the claims made in these ICA reports. MAIN BODY: We reviewed the two reports posted by the ICA on their website on March 20 and March 28, 2020. We explored the method used to develop the claim that chiropractic adjustments impact the immune system and discuss the scientific merit of that claim. We provide a response to the ICA reports and explain why this claim lacks scientific credibility and is dangerous to the public. More than 150 researchers from 11 countries reviewed and endorsed our response. CONCLUSION: In their reports, the ICA provided no valid clinical scientific evidence that chiropractic care can impact the immune system. We call on regulatory authorities and professional leaders to take robust political and regulatory action against those claiming that chiropractic adjustments have a clinical impact on the immune system

Cell-free production of a functional oligomeric form of a Chlamydia major outer-membrane protein (MOMP) for vaccine development.

Chlamydia is a prevalent sexually transmitted disease that infects more than 100 million people worldwide. Although most individuals infected with Chlamydia trachomatis are initially asymptomatic, symptoms can arise if left undiagnosed. Long-term infection can result in debilitating conditions such as pelvic inflammatory disease, infertility, and blindness. Chlamydia infection, therefore, constitutes a significant public health threat, underscoring the need for a Chlamydia-specific vaccine. Chlamydia strains express a major outer-membrane protein (MOMP) that has been shown to be an effective vaccine antigen. However, approaches to produce a functional recombinant MOMP protein for vaccine development are limited by poor solubility, low yield, and protein misfolding. Here, we used an Escherichia coli-based cell-free system to express a MOMP protein from the mouse-specific species Chlamydia muridarum (MoPn-MOMP or mMOMP). The codon-optimized mMOMP gene was co-translated with Δ49apolipoprotein A1 (Δ49ApoA1), a truncated version of mouse ApoA1 in which the N-terminal 49 amino acids were removed. This co-translation process produced mMOMP supported within a telodendrimer nanolipoprotein particle (mMOMP-tNLP). The cell-free expressed mMOMP-tNLPs contain mMOMP multimers similar to the native MOMP protein. This cell-free process produced on average 1.5 mg of purified, water-soluble mMOMP-tNLP complex in a 1-ml cell-free reaction. The mMOMP-tNLP particle also accommodated the co-localization of CpG oligodeoxynucleotide 1826, a single-stranded synthetic DNA adjuvant, eliciting an enhanced humoral immune response in vaccinated mice. Using our mMOMP-tNLP formulation, we demonstrate a unique approach to solubilizing and administering membrane-bound proteins for future vaccine development. This method can be applied to other previously difficult-to-obtain antigens while maintaining full functionality and immunogenicity

Metabolites of PPI-2458, a selective, irreversible inhibitor of methionine aminopeptidase-2: structure determination and in vivo activity

The natural product fumagillin exhibits potent antiproliferative and antiangiogenic properties. The semisynthetic analog PPI-2458, [(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-enyl)oxiran-2-yl]-1-oxaspiro[2.5]octan-6-yl] N-[(2R)-1-amino-3-methyl-1-oxobutan-2-yl]carbamate, demonstrates rapid inactivation of its molecular target, methionine aminopeptidase-2 (MetAP2), and good efficacy in several rodent models of cancer and inflammation with oral dosing despite low apparent oral bioavailability. To probe the basis of its in vivo efficacy, the metabolism of PPI-2458 was studied in detail. Reaction phenotyping identified CYP3A4/5 as the major source of metabolism in humans. Six metabolites were isolated from liver microsomes and characterized by mass spectrometry and nuclear resonance spectroscopy, and their structures were confirmed by chemical synthesis. The synthetic metabolites showed correlated inhibition of MetAP2 enzymatic activity and vascular endothelial cell growth. In an ex vivo experiment, MetAP2 inhibition in white blood cells, thymus, and lymph nodes in rats after single dosing with PPI-2458 and the isolated metabolites was found to correlate with the in vitro activity of the individual species. In a phase 1 clinical study, PPI-2458 was administered to patients with non-Hodgkin lymphoma. At 15 mg administered orally every other day, MetAP2 in whole blood was 80% inactivated for up to 48 hours, although the exposure of the parent compound was only ∼10% that of the summed cytochrome P450 metabolites. Taken together, the data confirm the participation of active metabolites in the in vivo efficacy of PPI-2458. The structures define a metabolic pathway for PPI-2458 that is distinct from that of TNP-470 ([(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-enyl)oxiran-2-yl]-1-oxaspiro[2.5]octan-6-yl] N-(2-chloroacetyl)carbamate). The high level of MetAP2 inhibition achieved in vivo supports the value of fumagillin-derived therapeutics for angiogenic diseases

Large-scale impacts of sea star wasting disease (SSWD) on intertidal sea stars and implications for recovery

Disease outbreaks can have substantial impacts on wild populations, but the often patchy or anecdotal evidence of these impacts impedes our ability to understand outbreak dynamics. Recently however, a severe disease outbreak occurred in a group of very well-studied organisms-sea stars along the west coast of North America. We analyzed nearly two decades of data from a coordinated monitoring effort at 88 sites ranging from southern British Columbia to San Diego, California along with 2 sites near Sitka, Alaska to better understand the effects of sea star wasting disease (SSWD) on the keystone intertidal predator, Pisaster ochraceus. Quantitative surveys revealed unprecedented declines of P. ochraceus in 2014 and 2015 across nearly the entire geographic range of the species. The intensity of the impact of SSWD was not uniform across the affected area, with proportionally greater population declines in the southern regions relative to the north. The degree of population decline was unrelated to pre-outbreak P. ochraceus density, although these factors have been linked in other well-documented disease events. While elevated seawater temperatures were not broadly linked to the initial emergence of SSWD, anomalously high seawater temperatures in 2014 and 2015 might have exacerbated the disease's impact. Both before and after the onset of the SSWD outbreak, we documented higher recruitment of P. ochraceus in the north than in the south, and while some juveniles are surviving (as evidenced by transition of recruitment pulses to larger size classes), post-SSWD survivorship is lower than during pre-SSWD periods. In hindsight, our data suggest that the SSWD event defied prediction based on two factors found to be important in other marine disease events, sea water temperature and population density, and illustrate the importance of surveillance of natural populations as one element of an integrated approach to marine disease ecology. Low levels of SSWD-symptomatic sea stars are still present throughout the impacted range, thus the outlook for population recovery is uncertain

Of Mice and Monkeys: Can Animal Models Be Utilized to Study Neurological Consequences of Pediatric HIV-1 Infection?

Pediatric human immunodeficiency virus (HIV-1) infection remains a global health crisis. Children are much more susceptible to HIV-1 neurological impairments than adults, which can be exacerbated by coinfections. Neurological characteristics of pediatric HIV-1 infection suggest dysfunction in the frontal cortex as well as the hippocampus; limited MRI data indicate global cerebral atrophy, and pathological data suggest accelerated neuronal apoptosis in the cortex. An obstacle to pediatric HIV-1 research is a human representative model system. Host-species specificity of HIV-1 limits the ability to model neurological consequences of pediatric HIV-1 infection in animals. Several models have been proposed including neonatal intracranial injections of HIV-1 viral proteins in rats and perinatal simian immunodeficiency virus (SIV) infection of infant macaques. Nonhuman primate models recapitulate the complexity of pediatric HIV-1, neuropathogenesis while rodent models are able to elucidate the role specific viral proteins exert on neurodevelopment. Nonhuman primate models show similar behavioral and neuropathological characteristics to pediatric HIV-1 infection and offer a stage to investigate early viral mechanisms, latency reservoirs, and therapeutic interventions. Here we review the relative strengths and limitations of pediatric HIV-1 model systems. [Image: see text