Therapeutic targeting of HYPDH/PRODH2 with N-propargylglycine offers a Hyperoxaluria treatment opportunity.

N-propargylglycine prevents 4-hydroxyproline catabolism in mouse liver and kidney. N-propargylglycine is a novel suicide inhibitor of PRODH2 and induces mitochondrial degradation of PRODH2. PRODH2 is selectively expressed in liver and kidney and contributes to primary hyperoxaluria (PH). Preclinical evaluation of N-propargylglycine efficacy as a new PH therapeutic is warranted.

Brain transcriptomic, metabolic and mitohormesis properties associated with N-propargylglycine treatment: A prevention strategy against neurodegeneration.

INTRODUCTION: There is an urgent need for new or repurposed therapeutics that protect against or significantly delay the clinical progression of neurodegenerative diseases, such as Huntington's disease (HD), Parkinson's disease and Alzheimer's disease. In particular, preclinical studies are needed for well tolerated and brain-penetrating small molecules capable of mitigating the proteotoxic mitochondrial processes that are hallmarks of these diseases. We identified a unique suicide inhibitor of mitochondrial proline dehydrogenase (Prodh), N-propargylglycine (N-PPG), which has anticancer and brain-enhancing mitohormesis properties, and we hypothesize that induction of mitohormesis by N-PPG protects against neurodegenerative diseases. We carried out a series of mouse studies designed to: i) compare brain and metabolic responses while on oral N-PPG treatment (50 mg/kg, 9-14 days) of B6CBA wildtype (WT) and short-lived transgenic R6/2 (HD) mice; and ii) evaluate potential brain and systemwide stress rebound responses in WT mice 2 months after cessation of extended mitohormesis induction by well-tolerated higher doses of N-PPG (100-200 mg/kg x 60 days). WT and HD mice showed comparable global evidence of N-PPG induced brain mitohormesis characterized by Prodh protein decay and increased mitochondrial expression of chaperone and Yme1l1 protease proteins. Interestingly, transcriptional analysis (RNAseq) showed partial normalization of HD whole brain transcriptomes toward those of WT mice. Comprehensive metabolomic profiles performed on control and N-PPG treated blood, brain, and kidney samples revealed expected N-PPG-induced tissue increases in proline levels in both WT and HD mice, accompanied by surprising parallel increases in hydroxyproline and sarcosine. Two months after cessation of the higher dose N-PPG stress treatments, WT mouse brains showed robust rebound increases in Prodh protein levels and mitochondrial transcriptome responses, as well as altered profiles of blood amino acid-related metabolites. Our HD and WT mouse preclinical findings point to the brain penetrating and mitohormesis-inducing potential of the drug candidate, N-PPG, and provide new rationale and application insights supporting its further preclinical testing in various models of neurodegenerative diseases characterized by loss of mitochondrial proteostasis.

N6-Adenosine Methylation of SARS-CoV-2 5'-UTR Regulates Translation.

The coronavirus disease 2019 (COVID19) continues to spread despite global vaccination efforts (1). This, alongside the rapid emergence of vaccine resistant variants, creates a need for orthogonal therapeutic strategies targeting more conserved facets of severe acute respiratory syndrome coronavirus (SARS-CoV-2) (2-4). One conserved feature of all coronaviruses is their ability to undergo discontinuous transcription wherein individual open reading frames fuse with the 5'-UTR leader sequence during negative-strand RNA synthesis (5). As such all viral protein coding genes use the same 5'-UTR for translation (6). Using in vitro reporter assays, we demonstrate that the SARS-CoV-2 5'-UTR efficiently initiates protein translation despite its predicted structural complexity. Through a combination of bioinformatic and biochemical assays, we demonstrate that a single METTL3-dependent m6A methylation event in SARS-CoV-2 5'-UTR regulates the rate of translation initiation. We show that m6A likely exerts this effect by destabilizing secondary structure in the 5'-UTR, thereby facilitating access to the ribosomal pre-initiation complex. This discovery opens new avenues for novel therapeutic strategies aimed at controlling the ability of SARS-CoV-2 to replicate in host cells.

Directed Discovery of Tetrapeptide Emulsifiers.

Oil in water emulsions are an important class of soft material that are used in the food, cosmetic, and biomedical industries. These materials are formed through the use of emulsifiers that are able to stabilize oil droplets in water. Historically emulsifiers have been developed from lipids or from large biomolecules such as proteins. However, the ability to use short peptides, which have favorable degradability and toxicity profiles is seen as an attractive alternative. In this work, we demonstrate that it is possible to design emulsifiers from short (tetra) peptides that have tunability (i.e., the surface activity of the emulsion can be tuned according to the peptide primary sequence). This design process is achieved by applying coarse grain molecular dynamics simulation to consecutively reduce the molecular search space from the 83,521 candidates initially considered in the screen to four top ranking candidates that were then studied experimentally. The results of the experimental study correspond well to the predicted results from the computational screening verifying the potential of this screening methodology to be applied to a range of different molecular systems.

N-Propargylglycine: a unique suicide inhibitor of proline dehydrogenase with anticancer activity and brain-enhancing mitohormesis properties.

Proline dehydrogenase (PRODH) is a mitochondrial inner membrane flavoprotein critical for cancer cell survival under stress conditions and newly recognized as a potential target for cancer drug development. Reversible (competitive) and irreversible (suicide) inhibitors of PRODH have been shown in vivo to inhibit cancer cell growth with excellent host tolerance. Surprisingly, the PRODH suicide inhibitor N-propargylglycine (N-PPG) also induces rapid decay of PRODH with concordant upregulation of mitochondrial chaperones (HSP-60, GRP-75) and the inner membrane protease YME1L1, signifying activation of the mitochondrial unfolded protein response (UPRmt) independent of anticancer activity. The present study was undertaken to address two aims: (i) use PRODH overexpressing human cancer cells (ZR-75-1) to confirm the UPRmt inducing properties of N-PPG relative to another equipotent irreversible PRODH inhibitor, thiazolidine-2-carboxylate (T2C); and (ii) employ biochemical and transcriptomic approaches to determine if orally administered N-PPG can penetrate the blood-brain barrier, essential for its future use as a brain cancer therapeutic, and also potentially protect normal brain tissue by inducing mitohormesis. Oral daily treatments of N-PPG produced a dose-dependent decline in brain mitochondrial PRODH protein without detectable impairment in mouse health; furthermore, mice repeatedly dosed with 50 mg/kg N-PPG showed increased brain expression of the mitohormesis associated protease, YME1L1. Whole brain transcriptome (RNAseq) analyses of these mice revealed significant gene set enrichment in N-PPG stimulated neural processes (FDR p < 0.05). Given this in vivo evidence of brain bioavailability and neural mitohormesis induction, N-PPG appears to be unique among anticancer agents and should be evaluated for repurposing as a pharmaceutical capable of mitigating the proteotoxic mechanisms driving neurodegenerative disorders.

Dedicated spine nurses and scrub technicians improve intraoperative efficiency of surgery for adolescent idiopathic scoliosis.

STUDY DESIGN: Retrospective comparative study. OBJECTIVE: To determine how the use of dedicated spine surgical nurses and scrub technicians impacted surgical outcomes of posterior spinal fusions for adolescent idiopathic scoliosis (AIS). Dedicated team approaches to surgery have been shown to improve surgical outcomes. However, their study on orthopaedics and spine surgery is limited. METHODS: A retrospective review of all patients who underwent a primary posterior spinal fusion of seven or more levels for AIS at a tertiary care pediatric hospital with a minimum of 2 years of follow-up from 2006 to 2013 was conducted. Our institution had dedicated spine surgeons and anesthesiologists throughout the study period, but use of dedicated spine nurses and scrub technicians was variable. The relationship between the proportion of nurses and scrub technicians that were dedicated spine and surgical outcome variables was examined. A multiple regression was performed to control for the surgeon performing the case and the start time. RESULTS: A total of 146 patients met criteria. When teams were composed of < 60% dedicated spine nurses and scrub technicians, there was 34 min more total OR time (p = .008), 27 min more surgical time (p = .037), 7 min more nonsurgical OR time (p = .030), 30% more estimated blood loss (EBL) (p = .013), 27% more EBL per level instrumented (p = .020), 113% more allogeneic transfusion (p = .006), and 104% more allogeneic transfusion per level instrumented (p = .009). There was no significant difference in length of stay, unplanned staged procedures, surgical site infection, reoperation, or major medical complications. CONCLUSIONS: Performing posterior spinal fusions for AIS patients with dedicated spine nurses and scrub technicians is associated with a significant decrease in total OR time, blood loss, and transfusion rates. LEVEL OF EVIDENCE: III.

Targeting Mitochondrial Proline Dehydrogenase with a Suicide Inhibitor to Exploit Synthetic Lethal Interactions with p53 Upregulation and Glutaminase Inhibition.

Proline dehydrogenase (PRODH) is a p53-inducible inner mitochondrial membrane flavoprotein linked to electron transport for anaplerotic glutamate and ATP production, most critical for cancer cell survival under microenvironmental stress conditions. Proposing that PRODH is a unique mitochondrial cancer target, we structurally model and compare its cancer cell activity and consequences upon exposure to either a reversible (S-5-oxo: S-5-oxo-2-tetrahydrofurancarboxylic acid) or irreversible (N-PPG: N-propargylglycine) PRODH inhibitor. Unlike 5-oxo, the suicide inhibitor N-PPG induces early and selective decay of PRODH protein without triggering mitochondrial destruction, consistent with N-PPG activation of the mitochondrial unfolded protein response. Fly and breast tumor (MCF7)-xenografted mouse studies indicate that N-PPG doses sufficient to phenocopy PRODH knockout and induce its decay can be safely and effectively administered in vivo Among breast cancer cell lines and tumor samples, PRODH mRNA expression is subtype dependent and inversely correlated with glutaminase (GLS1) expression; combining inhibitors of PRODH (S-5-oxo and N-PPG) and GLS1 (CB-839) produces additive if not synergistic loss of cancer cell (ZR-75-1, MCF7, DU4475, and BT474) growth and viability. Although PRODH knockdown alone can induce cancer cell apoptosis, the anticancer potential of either reversible or irreversible PRODH inhibitors is strongly enhanced when p53 is simultaneously upregulated by an MDM2 antagonist (MI-63 and nutlin-3). However, maximum anticancer synergy is observed in vitro when the PRODH suicide inhibitor, N-PPG, is combined with both GLS1-inhibiting and a p53-upregulating MDM2 antagonist. These findings provide preclinical rationale for the development of N-PPG-like PRODH inhibitors as cancer therapeutics to exploit synthetic lethal interactions with p53 upregulation and GLS1 inhibition.

Small-molecule MDM2 antagonists attenuate the senescence-associated secretory phenotype.

Processes that have been linked to aging and cancer include an inflammatory milieu driven by senescent cells. Senescent cells lose the ability to divide, essentially irreversibly, and secrete numerous proteases, cytokines and growth factors, termed the senescence-associated secretory phenotype (SASP). Senescent cells that lack p53 tumor suppressor function show an exaggerated SASP, suggesting the SASP is negatively controlled by p53. Here, we show that increased p53 activity caused by small molecule inhibitors of MDM2, which promotes p53 degradation, reduces inflammatory cytokine production by senescent cells. Upon treatment with the MDM2 inhibitors nutlin-3a or MI-63, human cells acquired a senescence-like growth arrest, but the arrest was reversible. Importantly, the inhibitors reduced expression of the signature SASP factors IL-6 and IL-1α by cells made senescent by genotoxic stimuli, and suppressed the ability of senescent fibroblasts to stimulate breast cancer cell aggressiveness. Our findings suggest that MDM2 inhibitors could reduce cancer progression in part by reducing the pro-inflammatory environment created by senescent cells.

ERpS294 is a biomarker of ligand or mutational ERα activation and a breast cancer target for CDK2 inhibition.

ERα phosphorylation at hinge site S294 (pS294) was recently shown to be essential for ER-dependent gene transcription and mediated by an unknown cyclin-dependent kinase (CDK). This study was undertaken to identify the exact CDK pathway mediating pS294 formation, and to determine if this phosphorylation event occurs with, and can be targeted to treat, the ligand-independent growth of breast cancers expressing endocrine-refractory ESR1 mutations. Using a newly developed anti-pS294 monoclonal antibody, a combination of CDK specific siRNA knockdown studies and a broad panel of CDK selective inhibitors against ligand (E2)-stimulated MCF7 cells, we first identified CDK2 as the primary mediator of pS294 formation and showed that CDK2-selective inhibitors like Dinaciclib, but not CDK4/6 inhibitors like Palbociclib, can selectively prevent pS294 formation and repress ER-dependent gene expression. We then expressed the ER-activating mutations ERmut(Y537S) and ERmut(D538G) in MCF7 cells, and demonstrated their ability to induce ligand-independent and tamoxifen-resistant growth, associated with constitutive and CDK2-dependent pS294 expression. Following robust growth of E2-independent and TAM-resistant MCF7mutER(Y537S) tumors in vivo, nude mice were also treated with either Dinaciclib or Palbociclib at doses and injection schedules unable to retard tumor growth as single agents; the TAM plus Palbociclib combination arrested further tumor growth without affecting pS294 formation, while the TAM plus Dinaciclib combination produced tumor regression associated with loss of pS294 expression. These findings, and our proposed mechanistic model, provide new rationale for the clinical evaluation of CDK2 inhibitors given in combination with endocrine agents as a new treatment strategy against ESR1 mutation expressing breast cancers.

Design, Synthesis, and Characterization of N-Oxide-Containing Heterocycles with in Vivo Sterilizing Antitubercular Activity.

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), is the infectious disease responsible for the highest number of deaths worldwide. Herein, 22 new N-oxide-containing compounds were synthesized followed by in vitro and in vivo evaluation of their antitubercular potential against Mtb. Compound 8 was found to be the most promising compound, with MIC90 values of 1.10 and 6.62 µM against active and nonreplicating Mtb, respectively. Additionally, we carried out in vivo experiments to confirm the safety and efficacy of compound 8; the compound was found to be orally bioavailable and highly effective, leading to a reduction of Mtb to undetectable levels in a mouse model of infection. Microarray-based initial studies on the mechanism of action suggest that compound 8 blocks translation. Altogether, these results indicate that benzofuroxan derivative 8 is a promising lead compound for the development of a novel chemical class of antitubercular drugs.

Cooperative, ion-sensitive co-assembly of tripeptide hydrogels.

Peptide co-assembly is of interest for the development of functional supramolecular biomaterials. Herein, computational simulations were combined with experimental validation to aid the design and understanding of cooperative co-assembly of a structure-forming tripeptide (FFD) and a functional copper-binding tripeptide (GHK) leading to hydrogel formation in response to complexation with copper ions.

Polymeric peptide pigments with sequence-encoded properties.

Melanins are a family of heterogeneous polymeric pigments that provide ultraviolet (UV) light protection, structural support, coloration, and free radical scavenging. Formed by oxidative oligomerization of catecholic small molecules, the physical properties of melanins are influenced by covalent and noncovalent disorder. We report the use of tyrosine-containing tripeptides as tunable precursors for polymeric pigments. In these structures, phenols are presented in a (supra-)molecular context dictated by the positions of the amino acids in the peptide sequence. Oxidative polymerization can be tuned in a sequence-dependent manner, resulting in peptide sequence-encoded properties such as UV absorbance, morphology, coloration, and electrochemical properties over a considerable range. Short peptides have low barriers to application and can be easily scaled, suggesting near-term applications in cosmetics and biomedicine.

Featured Article: Pyruvate preserves antiglycation defenses in porcine brain after cardiac arrest.

Cardiac arrest (CA) and cardiocerebral resuscitation (CCR)-induced ischemia-reperfusion imposes oxidative and carbonyl stress that injures the brain. The ischemic shift to anaerobic glycolysis, combined with oxyradical inactivation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), provokes excessive formation of the powerful glycating agent, methylglyoxal. The glyoxalase (GLO) system, comprising the enzymes glyoxalase 1 (GLO1) and GLO2, utilizes reduced glutathione (GSH) supplied by glutathione reductase (GR) to detoxify methylglyoxal resulting in reduced protein glycation. Pyruvate, a natural antioxidant that augments GSH redox status, could sustain the GLO system in the face of ischemia-reperfusion. This study assessed the impact of CA-CCR on the cerebral GLO system and pyruvate's ability to preserve this neuroprotective system following CA. Domestic swine were subjected to 10 min CA, 4 min closed-chest CCR, defibrillation and 4 h recovery, or to a non-CA sham protocol. Sodium pyruvate or NaCl control was infused (0.1 mmol/kg/min, intravenous) throughout CCR and the first 60 min recovery. Protein glycation, GLO1 content, and activities of GLO1, GR, and GAPDH were analyzed in frontal cortex biopsied at 4 h recovery. CA-CCR produced marked protein glycation which was attenuated by pyruvate treatment. GLO1, GR, and GAPDH activities fell by 86, 55, and 30%, respectively, after CA-CCR with NaCl infusion. Pyruvate prevented inactivation of all three enzymes. CA-CCR sharply lowered GLO1 monomer content with commensurate formation of higher molecular weight immunoreactivity; pyruvate preserved GLO1 monomers. Thus, ischemia-reperfusion imposed by CA-CCR disabled the brain's antiglycation defenses. Pyruvate preserved these enzyme systems that protect the brain from glycation stress. Impact statement Recent studies have demonstrated a pivotal role of protein glycation in brain injury. Methylglyoxal, a by-product of glycolysis and a powerful glycating agent in brain, is detoxified by the glutathione-catalyzed glyoxalase (GLO) system, but the impact of cardiac arrest (CA) and cardiocerebral resuscitation (CCR) on the brain's antiglycation defenses is unknown. This study in a swine model of CA and CCR demonstrated for the first time that the intense cerebral ischemia-reperfusion imposed by CA-resuscitation disabled glyoxalase-1 and glutathione reductase (GR), the source of glutathione for methylglyoxal detoxification. Moreover, intravenous administration of pyruvate, a redox-active intermediary metabolite and antioxidant in brain, prevented inactivation of glyoxalase-1 and GR and blunted protein glycation in cerebral cortex. These findings in a large mammal are first evidence of GLO inactivation and the resultant cerebral protein glycation after CA-resuscitation, and identify novel actions of pyruvate to minimize protein glycation in postischemic brain.

Dentognathic remains of Australopithecus afarensis from Nefuraytu (Woranso-Mille, Ethiopia): Comparative description, geology, and paleoecological context.

Australopithecus afarensis is the best-known and most dimorphic species in the early hominin fossil record. Here, we present a comparative description of new fossil specimens of Au. afarensis from Nefuraytu, a 3.330-3.207 million-years-old fossil collection area in the Woranso-Mille study area, central Afar, Ethiopia. These specimens include NFR-VP-1/29, one of the most complete mandibles assigned to the species thus far and among the largest mandibles attributed to Au. afarensis, likely representing a male individual. NFR-VP-1/29 retains almost all of the distinctive archaic features documented for Au. afarensis. These features include a posteriorly sloping symphysis, a low and rounded basally set inferior transverse torus, anterosuperiorly opening mental foramen, a lateral corpus hollow bound anteriorly by the C/P3 jugae and posteriorly by the lateral prominence, and the ascending ramus arising high on the corpus. Dental morphology and metrics of the Nefuraytu specimens also falls within the range of Au. afarensis. The presence of this species at Woranso-Mille between 3.330 and 3.207 million years ago confirms the existence of this species in the area in close spatial and temporal proximity to other middle Pliocene hominin taxa such as the one represented by the Burtele foot (BRT-VP-2/73) and the recently named species Australopithecus deyiremeda. This has important implications for our understanding of middle Pliocene hominin diversity.

Antimycobacterial activity of pyrazinoate prodrugs in replicating and non-replicating Mycobacterium tuberculosis.

Tuberculosis (TB) is an important infectious disease caused by Mycobacterium tuberculosis (Mtb) and responsible for thousands of deaths every year. Although there are antimycobacterial drugs available in therapeutics, just few new chemical entities have reached clinical trials, and in fact, since introduction of rifampin only two important drugs had reached the market. Pyrazinoic acid (POA), the active agent of pyrazinamide, has been explored through prodrug approach to achieve novel molecules with anti-Mtb activity, however, there is no activity evaluation of these molecules against non-replicating Mtb until the present. Additionally, pharmacokinetic must be preliminary evaluated to avoid future problems during clinical trials. In this paper, we have presented six POA esters as prodrugs in order to evaluate their anti-Mtb activity in replicating and non-replicating Mtb, and these showed activity highly influenced by medium composition (especially by albumin). Lipophilicity seems to play the main role in the activity, possibly due to controlling membrane passage. Novel duplicated prodrugs of POA were also described, presenting interesting activity. Cytotoxicity of these prodrugs set was also evaluated, and these showed no important cytotoxic profile.

Chronology for the Cueva Victoria fossil site (SE Spain): Evidence for Early Pleistocene Afro-Iberian dispersals.

Cueva Victoria has provided remains of more than 90 species of fossil vertebrates, including a hominin phalanx, and the only specimens of the African cercopithecid Theropithecus oswaldi in Europe. To constrain the age of the vertebrate remains we used paleomagnetism, vertebrate biostratigraphy and (230)Th/U dating. Normal polarity was identified in the non-fossiliferous lowest and highest stratigraphic units (red clay and capping flowstones) while reverse polarity was found in the intermediate stratigraphic unit (fossiliferous breccia). A lower polarity change occurred during the deposition of the decalcification clay, when the cave was closed and karstification was active. A second polarity change occurred during the capping flowstone formation, when the upper galleries were filled with breccia. The mammal association indicates a post-Jaramillo age, which allows us to correlate this upper reversal with the Brunhes-Matuyama boundary (0.78 Ma). Consequently, the lower reversal (N-R) is interpreted as the end of the Jaramillo magnetochron (0.99 Ma). These ages bracket the age of the fossiliferous breccia between 0.99 and 0.78 Ma, suggesting that the capping flowstone was formed during the wet Marine Isotopic Stage 19, which includes the Brunhes-Matuyama boundary. Fossil remains of Theropithecus have been only found in situ ∼1 m below the B/M boundary, which allows us to place the arrival of Theropithecus to Cueva Victoria at ∼0.9-0.85 Ma. The fauna of Cueva Victoria lived during a period of important climatic change, known as the Early-Middle Pleistocene Climatic Transition. The occurrence of the oldest European Acheulean tools at the contemporaneous nearby site of Cueva Negra suggest an African dispersal into SE Iberia through the Strait of Gibraltar during MIS 22, when sea-level was ∼100 m below its present position, allowing the passage into Europe of, at least, Theropithecus and Homo bearing Acheulean technology.

Tripeptide Emulsifiers.

A series of tripeptides is shown to form emulsions with sequence tunable properties. Using a combination of simulations and experiments, it is shown that two types of oil-in-water emulsions may be produced, either forming stable interfacial nanofiber networks with remarkable stability, or more conventional surfactant-like monolayers.

FOXM1 cistrome predicts breast cancer metastatic outcome better than FOXM1 expression levels or tumor proliferation index.

FOXM1 is a key transcription factor regulating cell cycle progression, DNA damage response, and a host of other hallmark cancer features, but the role of the FOXM1 cistrome in driving estrogen receptor-positive (ER+) versus estrogen receptor-negative (ER-) breast cancer clinical outcomes remains undefined. Chromatin immunoprecipitation sequencing (ChIP-Seq) coupled with RNA sequencing (RNA-Seq) analyses was used to identify FOXM1 target genes in breast cancer cells (MCF-7) where FOXM1 expression was either induced by cell proliferation or repressed by p53 upregulation. The prognostic performance of these FOXM1 target genes was assessed relative to FOXM transcript levels and a 61-gene proliferation score (PS) for their ability to dichotomize a pooled cohort of 683 adjuvant chemotherapy-naïve, node-negative breast cancer cases (447 ER+, 236 ER-). Differences in distant metastasis-free survival (DMFS) between the dichotomized expression groups were determined by Cox proportional hazard modeling. Proliferation-associated FOXM1 upregulation induced a set of 145 differentially bound and expressed genes (direct targets), and these demonstrated minimal overlap with differentially bound and expressed genes following FOXM1 repression by p53 upregulation. This proliferation-associated FOXM1 cistrome was not only better at significantly predicting metastatic outcome of ER+ breast cancers (HR: 2.8 (2.0-3.8), p = 8.13E-10), but was the only parameter trending toward significance in predicting ER- metastatic outcome (HR: 1.6 (0.9-2.9), p = 0.087). Our findings demonstrate that FOXM1 target genes are highly dependent on the cellular context in which FOXM1 expression is modulated, and a newly identified proliferation-associated FOXM1 cistromic signature best predicts breast cancer metastatic outcome.