Hutchinson-Gilford Progeria Syndrome (HGPS) is a segmental premature aging disease causing patient death by early teenage years from cardiovascular dysfunction. Although HGPS does not totally recapitulate normal aging, it does harbor many similarities to the normal aging process, with patients also developing cardiovascular disease, alopecia, bone and joint abnormalities, and adipose changes. It is unsurprising, then, that as physicians and scientists have searched for treatments for HGPS, they have targeted many pathways known to be involved in normal aging, including inflammation, DNA damage, epigenetic changes, and stem cell exhaustion. Although less studied at a mechanistic level, severe metabolic problems are observed in HGPS patients. Interestingly, new research in animal models of HGPS has demonstrated impressive lifespan improvements secondary to metabolic interventions. As such, further understanding metabolism, its contribution to HGPS, and its therapeutic potential has far-reaching ramifications for this disease still lacking a robust treatment strategy.

While thrombin is a key enzyme in the coagulation cascade and is required for both normal hemostasis and pathologic thrombogenesis, it also participates in its own negative feedback via activation of protein C, which down-regulates thrombin generation by enzymatically inactivating factors Va and VIIIa. Our group and others have previously shown that thrombin's procoagulant and anticoagulant activities can be effectively disassociated to varying extents through site-directed mutagenesis. The thrombin mutant W215A/E217A (WE thrombin) has been one of the best characterized constructs with selective activity toward protein C. While animal studies have demonstrated that WE thrombin acts as an anticoagulant through activated protein C (APC) generation, the observed limited systemic anticoagulation does not fully explain the antithrombotic potency of this or other thrombin mutants. AB002 (E-WE thrombin) is an investigational protein C activator thrombin analog in phase 2 clinical development (clinicaltrials.gov, NCT03963895). Here we demonstrate that this molecule is a potent enzyme that is able to rapidly interrupt arterial-type thrombus propagation at exceedingly low doses (<2 µg/kg, IV), yet without substantial systemic anticoagulation in baboons. We demonstrate that AB002 produces APC on platelet aggregates and competitively inhibits TAFI (carboxypeptidase B2) activation, in vitro, which may contribute to the observed in vivo efficacy. We also describe its safety and activity in a phase 1 first-in-human clinical trial (www.clinicaltrials.gov; NCT03453060). Together, these results support further clinical evaluation of AB002 as a potentially safe and effective new approach for treating or preventing acute thrombotic and thromboembolic conditions.

The loss of estrogen (E ) initiates a rapid phase of bone loss leading to osteoporosis in half of postmenopausal women, but the mechanism is not fully understood. Here, we show for the first time how loss of E activates low-grade inflammation to promote the acute phase of bone catabolic activity in ovariectomized (OVX) mice. E regulates the abundance of dendritic cells (DC) that express IL-7 and IL-15 by inducing FasL and apoptosis of the DC. In the absence of E , DC become long-lived, leading to increased IL-7 and IL-15. We find that IL-7 and IL-15 together, but not alone, induced antigen-independent production of IL-17A and TNFα in a subset of memory T-cells (T ). OVX of mice with T-cell-specific ablation of IL15RA showed no IL-17A and TNFα expression, and no increase in bone resorption or bone loss, confirming the role IL-15 in activating the T and the need for inflammation. Our results provide a new mechanism by which E regulates the immune system, and how menopause leads to osteoporosis. The low-grade inflammation is likely to cause or contribute to other comorbidities observed postmenopause. This article is protected by copyright. All rights reserved.

There is an abundance of RNA sequence information available due to the efforts of sequencing projects. However, current techniques implemented to solve the tertiary structures of RNA, such as NMR and X-ray crystallography, are difficult and time-consuming. Therefore, biophysical techniques are not able to keep pace with the abundance of sequence information available. Because of this, there is a need to develop quick and efficient ways to predict RNA tertiary structure from sequence. One promising approach is to identify structural patterns within previously solved 3D structures and apply these patterns to new sequences. RNA tetraloops are one of the most common naturally occurring secondary structure motifs. Here, we use RNA Characterization of Secondary Structure Motifs (CoSSMos), Dissecting the Spatial Structure of RNA (DSSR), and a bioinformatic approach to search for and characterize tertiary structure patterns among tetraloops. Not surprising, we identified the well-known GNRA and UNCG tetraloops, as well as the previously identified RNYA tetraloop. However, some previously identified characteristics of these families were not observed in this data set, and some new characteristics were identified. In addition, we also identified and characterized three new tetraloop sequence families: YGAR, UGGU, and RMSA. This new structural information sheds light on the tertiary structure of tetraloops and contributes to the efforts of RNA tertiary structure prediction from sequence.

Activity in trypsin-like proteases is the result of proteolytic cleavage at R15 followed by an ionic interaction that ensues between the new N terminus of I16 and the side chain of the highly conserved D194. This mechanism of activation, first proposed by Huber and Bode, organizes the oxyanion hole and primary specificity pocket for substrate binding and catalysis. Using the clotting protease thrombin as a relevant model, we unravel contributions of the I16-D194 ionic interaction to Na binding, stability of the transition state and the allosteric E*-E equilibrium of the trypsin fold. The I16T mutation abolishes the I16-D194 interaction and compromises the architecture of the oxyanion hole. The D194A mutation also abrogates the I16-D194 interaction but, surprisingly, has no effect on the architecture of the oxyanion hole that remains intact through a new H-bond established between G43 and G193. In both mutants, loss of the I16-D194 ionic interaction compromises Na binding, reduces stability of the transition state, collapses the 215-217 segment into the primary specific pocket and abrogates the allosteric E*-E equilibrium in favor of a rigid conformation that binds ligand at the active site according to a simple lock-and-key mechanism. These findings refine the structural role of the I16-D194 ionic interaction in the Huber-Bode mechanism of activation and reveal a functional linkage with the allosteric properties of the trypsin fold like Na binding and the E*-E equilibrium.

Hutchinson Gilford progeria syndrome (HGPS) is a devastating accelerated aging disease caused by LMNA gene mutation. The truncated lamin A protein produced "progerin" has a dominant toxic effect in cells, causing disruption of nuclear architecture and chromatin structure, genomic instability, gene expression changes, oxidative stress, and premature senescence. It was previously shown that progerin-induced genomic instability involves replication stress (RS), characterized by replication fork stalling and nuclease-mediated degradation of stalled forks. RS is accompanied by activation of cGAS/STING cytosolic DNA sensing pathway and STAT1-regulated interferon (IFN)-like response. It is also found that calcitriol, the active hormonal form of vitamin D, rescues RS and represses the cGAS/STING/IFN cascade. Here, the mechanisms underlying RS in progerin-expressing cells and the rescue by calcitriol are explored. It is found that progerin elicits a marked downregulation of RAD51, concomitant with increased levels of phosphorylated-RPA, a marker of RS. Interestingly, calcitriol prevents RS and activation of the cGAS/STING/IFN response in part through maintenance of RAD51 levels in progerin-expressing cells. Thus, loss of RAD51 is one of the consequences of progerin expression that can contribute to RS and activation of the IFN response. Stabilization of RAD51 helps explain the beneficial effects of calcitriol in these processes.

can cause fatal meningoencephalitis in patients with AIDS or other immunocompromising conditions. Current antifungals are suboptimal to treat this disease; therefore, novel targets and new therapies are needed. Previously, we have shown that chitosan is a critical component of the cryptococcal cell wall and is required for survival in the mammalian host and that chitosan deficiency results in rapid clearance from the mammalian host. We had also identified several specific proteins that were required for chitosan biosynthesis, and we hypothesize that screening for compounds that inhibit chitosan biosynthesis would identify additional genes/proteins that influence chitosan biosynthesis. To identify these compounds, we developed a robust and novel cell-based flow cytometry screening method to identify small-molecule inhibitors of chitosan production. We screened the ICCB Known Bioactives library and identified 8 compounds that reduced chitosan in We used flow cytometry-based counterscreens and confirmatory screens, followed by a biochemical secondary screen to refine our primary screening hits to 2 confirmed hits. One of the confirmed hits that reduced chitosan content was the aminoalkylindole BML-190, a known inverse agonist of mammalian cannabinoid receptors. We demonstrated that BML-190 likely targets the G-protein-coupled receptor Gpr4 and, via the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway, contributes to an intracellular accumulation of cAMP that results in decreased chitosan. Our discovery suggests that this approach could be used to identify additional compounds and pathways that reduce chitosan biosynthesis and could lead to potential novel therapeutics against is a fungal pathogen that kills ∼200,000 people every year. The cell wall is an essential organelle that protects fungi from the environment. Chitosan, the deacetylated form of chitin, has been shown to be an essential component of the cryptococcal cell wall during infection of a mammalian host. In this study, we screened a set of 480 compounds, which are known to have defined biological activities, for activity that reduced chitosan production in Two of these compounds were confirmed using an alternative method of measuring chitosan, and one of these was demonstrated to impact the cAMP signal transduction pathway. This work demonstrates that the cAMP pathway regulates chitosan biosynthesis in and validates that this screening approach could be used to find potential antifungal agents.

Following publication of the original article [1], it was reported that Fig. 1c was not entirely readable due to overlapping Fig. 1d. The publishers apologise for this error.