Insights into the regulation of malate dehydrogenase: inhibitors, activators, and allosteric modulation by small molecules.

Cellular metabolism comprises a complex network of biochemical anabolic and catabolic processes that fuel the growth and survival of living organisms. The enzyme malate dehydrogenase (MDH) is most known for its role in oxidizing malate to oxaloacetate (OAA) in the last step of the tricarboxylic acid (TCA) cycle, but it also participates in the malate-aspartate shuttle in the mitochondria as well as the glyoxylate cycle in plants. These pathways and the specific reactions within them are dynamic and must be carefully calibrated to ensure a balance between nutrient/energy supply and demand. MDH structural and functional complexity requires a variety of regulatory mechanisms, including allosteric regulation, feedback, and competitive inhibition, which are often dependent on whether the enzyme is catalyzing its forward or reverse reaction. Given the role of MDH in central metabolism and its potential as a target for therapeutics in both cancer and infectious diseases, there is a need to better understand its regulation. The involvement of MDH in multiple pathways makes it challenging to identify which effectors are critical to its activity. Many of the in vitro experiments examining MDH regulation were done decades ago, and though allosteric sites have been proposed, none to date have been specifically mapped. This review aims to provide an overview of the current knowledge surrounding MDH regulation by its substrate, products, and other intermediates of the TCA cycle while highlighting all the gaps in our understanding of its regulatory mechanisms.

External Collaboration Results in Student Learning Gains and Positive STEM Attitudes in CUREs.

The implementation of course-based undergraduate research experiences (CUREs) has made it possible to expose large undergraduate populations to research experiences. For these research experiences to be authentic, they should reflect the increasingly collaborative nature of research. While some CUREs have expanded, involving multiple schools across the nation, it is still unclear how a structured extramural collaboration between students and faculty from an outside institution affects student outcomes. In this study, we established three cohorts of students: 1) no-CURE, 2) single-institution CURE (CURE), and 3) external collaborative CURE (ec-CURE), and assessed academic and attitudinal outcomes. The ec-CURE differs from a regular CURE in that students work with faculty member from an external institution to refine their hypotheses and discuss their data. The sharing of ideas, data, and materials with an external faculty member allowed students to experience a level of collaboration not typically found in an undergraduate setting. Students in the ec-CURE had the greatest gains in experimental design; self-reported course benefits; scientific skills; and science, technology, engineering, and mathematics (STEM) importance. Importantly this study occurred in a diverse community of STEM disciplinary faculty from 2- and 4-year institutions, illustrating that exposing students to structured external collaboration is both feasible and beneficial to student learning.

Stereotactic Body Radiotherapy and Systemic Dose Chemotherapy for Locally Advanced Lung Cancer: Single Arm Phase 2 Study.

PURPOSE: This is a single arm phase 2 study (Clinical trials.gov NCT02568033) to examine the role of stereotactic body radiotherapy (SBRT) along with full dose systemic chemotherapy in the treatment of unresectable stage 2 and stage 3 nonsmall cell lung cancer. Primary endpoints are disease free survival and toxicity. MATERIALS: Patients were treated with SBRT to all sites of gross disease. Dosing consisted of 60 Gy in 3 fractions for peripheral lung tumors, 50 Gy in 5 fractions for central lung tumors, and 40 to 50 Gy in 5 fractions for hilar and mediastinal lymph nodes. Chemotherapy consisted of 4 cycles of pemetrexed and cisplatin or carboplatin and paclitaxel for nonsquamous histology and cisplatin and docetaxel or cisplatin and paclitaxel for squamous histology. SBRT was given in between the chemotherapy cycles. There was a 7 days break between chemotherapy and SBRT. Quality of life was measured using functional assessment of cancer therapy-lung. RESULTS: Twenty two patients were enrolled and analyzed. Seventeen (77%) were stage III and 19 (86%) had lymph node involvement. Median follow-up for all patients was 23.1 months. Median overall survival is 27.2 months. Overall survival at 1 year was 82% and overall survival at 2 years was 53%. Median disease free survival is 16.0 months with a 2-year regional failure rate of 19% and 2-year distant failure rate of 47.2%. There were 6 grade 3 acute toxicities and 2 late grade 3 or higher toxicities including 1 grade 5 hemoptysis. Quality of life scores were unchanged compared with baseline. CONCLUSION: A combination of SBRT and full dose chemotherapy appears to be a safe and effective treatment for locally advanced NSCLC and warrants further investigation.

Correction to: Behavioral Artistry: Examining the Relationship Between the Interpersonal Skills and Effective Practice Repertoires of Applied Behavior Analysis Practitioners.

The article Behavioral Artistry: Examining the Relationship Between the Interpersonal Skills and Effective Practice Repertoires of Applied Behavior Analysis Practitioners, written by Kevin Callahan, was originally published electronically on the publisher's internet portal (currently SpringerLink) on August 29, 2019 without open access. With the author(s)' decision to opt for Open Choice the copyright of the article changed on August, 2019 to © The Author(s) 2019 and the article is forthwith distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, duplication, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

Behavioral Artistry: Examining the Relationship Between the Interpersonal Skills and Effective Practice Repertoires of Applied Behavior Analysis Practitioners.

This study investigated interpersonal skills associated with the concept of behavioral artistry (BA), a repertoire of practitioner behaviors including care, attentiveness, and creativity, among others, associated with the effective delivery of applied behavior analysis (ABA) treatment. Survey results indicated parents of children with autism preferred BA descriptors for ABA therapists over non-BA descriptors. A separate survey of 212 university students on a standardized personality assessment revealed students majoring and/or working in the field of ABA had lower levels of BA than those in other human services professions. Practitioners with higher BA scores were observed and rated more positively in their delivery of ABA for children with autism. Implications for training/supervising effective ABA practitioners within a BA model are discussed.

Targeting aberrant glutathione metabolism to eradicate human acute myelogenous leukemia cells.

The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular, primitive leukemia cells, often termed leukemia stem cells, are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34(+)) leukemic versus normal specimens. Our data indicate that CD34(+) AML cells have elevated expression of multiple glutathione pathway regulatory proteins, presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation, CD34(+) AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34(+) cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise, we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34(+) AML cells. Importantly, these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34(+) cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism, which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1), as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism, an intrinsic property of primary human AML cells.

In Vivo RNAi screening identifies a leukemia-specific dependence on integrin beta 3 signaling.

We used an in vivo small hairpin RNA (shRNA) screening approach to identify genes that are essential for MLL-AF9 acute myeloid leukemia (AML). We found that Integrin Beta 3 (Itgb3) is essential for murine leukemia cells in vivo and for human leukemia cells in xenotransplantation studies. In leukemia cells, Itgb3 knockdown impaired homing, downregulated LSC transcriptional programs, and induced differentiation via the intracellular kinase Syk. In contrast, loss of Itgb3 in normal hematopoietic stem and progenitor cells did not affect engraftment, reconstitution, or differentiation. Finally, using an Itgb3 knockout mouse model, we confirmed that Itgb3 is dispensable for normal hematopoiesis but is required for leukemogenesis. Our results establish the significance of the Itgb3 signaling pathway as a potential therapeutic target in AML.

Monitoring Response and Resistance to the Novel Arsenical Darinaparsin in an AML Patient.

Acute myeloid leukemia (AML) with inversion of chromosome 3 is characterized by overexpression of EVI1 and carries a dismal prognosis. Arsenic-containing compounds have been described to be efficacious in malignancies overexpressing EVI1. Here, we describe a case of AML with inv(3)(q21q26.2) treated with the organic arsenical darinaparsin. Using a "personalized medicine approach," two different arsenicals were screened for anti-leukemic effect against the patient's cells ex vivo. The most promising compound, darinaparsin, was selected for in vivo treatment. Clinical effect was almost immediate, with a normalization of temperature, a stabilization of white blood cell (WBC) counts and an increased quality of life. Longitudinal monitoring of patient response and resistance incorporating significant correlative studies on patient-derived blood samples over the two cycles of darinaparsin given to this patient allowed us to evaluate potential mechanisms of response and resistance. The anti-leukemic effects of darinaparsin correlated with inhibition of the alternative NF-κB pathway and production of the inflammatory cytokine IL-8. Emergence of resistance was suspected during treatment cycle 2 and supported by xenograft studies in nude mice. Darinaparsin resistance correlated with an attenuation of the effect of treatment on the alternative NF-κB pathway. The results from this patient indicate that darinaparsin may be a good treatment option for inv(3) AML and that inhibition of the alternative NF-κB pathway may be predictive of response. Longitudinal monitoring of disease response as well as several correlative parameters allowed for the generation of novel correlations and predictors of response to experimental therapy in a heavily pretreated patient.

BCL-2 inhibition targets oxidative phosphorylation and selectively eradicates quiescent human leukemia stem cells.

Most forms of chemotherapy employ mechanisms involving induction of oxidative stress, a strategy that can be effective due to the elevated oxidative state commonly observed in cancer cells. However, recent studies have shown that relative redox levels in primary tumors can be heterogeneous, suggesting that regimens dependent on differential oxidative state may not be uniformly effective. To investigate this issue in hematological malignancies, we evaluated mechanisms controlling oxidative state in primary specimens derived from acute myelogenous leukemia (AML) patients. Our studies demonstrate three striking findings. First, the majority of functionally defined leukemia stem cells (LSCs) are characterized by relatively low levels of reactive oxygen species (termed "ROS-low"). Second, ROS-low LSCs aberrantly overexpress BCL-2. Third, BCL-2 inhibition reduced oxidative phosphorylation and selectively eradicated quiescent LSCs. Based on these findings, we propose a model wherein the unique physiology of ROS-low LSCs provides an opportunity for selective targeting via disruption of BCL-2-dependent oxidative phosphorylation.

TRAMP complex enhances RNA degradation by the nuclear exosome component Rrp6.

The RNA-processing exosome contains ribonucleases that degrade aberrant RNAs in archael and eukaryotic cells. In Saccharomyces cerevisiae, the nuclear/nucleolar 3'-5' exoribonuclease Rrp6 distinguishes the nuclear exosome from the cytoplasmic exosome. In vivo, the TRAMP complex enhances the ability of the nuclear exosome to destroy some aberrant RNAs. Previous reports showed that purified TRAMP enhanced RNA degradation by the nuclear exosome in vitro. However, the exoribonucleolytic component(s) of the nuclear exosome enhanced by TRAMP remain unidentified. We show that TRAMP does not significantly enhance RNA degradation by purified exosomes lacking Rrp6 in vitro, suggesting that TRAMP activation experiments with nuclear exosome preparations reflect, in part, effects on the activity of Rrp6. Consistent with this, we show that incubation of purified TRAMP with recombinant Rrp6 results in a 10-fold enhancement of the rate of RNA degradation. This increased activity results from enhancement of the hydrolytic activity of Rrp6 because TRAMP cannot enhance the activity of an Rrp6 mutant lacking a key amino acid side chain in its active site. We observed no ATP or polyadenylation dependence for the enhancement of Rrp6 activity by TRAMP, suggesting that neither the poly(A) polymerase activity of Trf4 nor the helicase activity of Mtr4 plays a role in the enhancement. These findings identify TRAMP as an exosome-independent enhancer of Rrp6 activity.

ABA versus TEACCH: the case for defining and validating comprehensive treatment models in autism.

The authors analyzed the results of a social validation survey to determine if autism service providers including special education teachers, parents, and administrators demonstrate a preference for the intervention components of Applied Behavior Analysis or Training and Education of Autistic and other Communication Handicapped Children. They also investigated the comprehensiveness of these treatment models for use in public school programs. The findings indicate no clear preference for either model, but a significantly higher level of social validity for components inherent in both approaches. The authors discuss the need for research to define what is meant by comprehensive programming in autism.

Evidence for core exosome independent function of the nuclear exoribonuclease Rrp6p.

The RNA exosome processes and degrades RNAs in archaeal and eukaryotic cells. Exosomes from yeast and humans contain two active exoribonuclease components, Rrp6p and Dis3p/Rrp44p. Rrp6p is concentrated in the nucleus and the dependence of its function on the nine-subunit core exosome and Dis3p remains unclear. We found that cells lacking Rrp6p accumulate poly(A)+ rRNA degradation intermediates distinct from those found in cells depleted of Dis3p, or the core exosome component Rrp43p. Depletion of Dis3p in the absence of Rrp6p causes a synergistic increase in the levels of degradation substrates common to the core exosome and Rrp6p, but has no effect on Rrp6p-specific substrates. Rrp6p lacking a portion of its C-terminal domain no longer co-purifies with the core exosome, but continues to carry out RNA 3'-end processing of 5.8S rRNA and snoRNAs, as well as the degradation of certain truncated Rrp6-specific rRNA intermediates. However, disruption of Rrp6p-core exosome interaction results in the inability of the cell to efficiently degrade certain poly(A)+ rRNA processing products that require the combined activities of Dis3p and Rrp6p. These findings indicate that Rrp6p may carry out some of its critical functions without physical association with the core exosome.

Lifting the veil on the transcriptome.

Inhibition of the cellular RNA surveillance system in Arabidopsis thaliana results in the accumulation of thousands of transcripts arising from annotated and unannotated regions of the genome. This normally hidden transcriptome is replete with noncoding RNAs with the potential to regulate wide-ranging physiological activities.

Social validation of evidence-based practices in autism by parents, teachers, and administrators.

Relatively little attention has been devoted to the social validation of potentially effective autism interventions. Thus, it is often difficult to identify and implement evidence-based practices, and programming is often inadequate. The authors identified autism intervention components with reported effectiveness for school settings. The results of a social validation survey completed by parents, teachers, and administrators indicate strong, consistent support for program components falling within five functional areas: (a) individualized programming, (b) data collection, (c) the use of empirically-based strategies, (d) active collaboration, and (e) a focus on long-term outcomes. These socially validated interventions can be used to evaluate existing autism curricula and develop training for professionals, parents, and students in order to improve public school autism programs.

Architecture of the yeast Rrp44 exosome complex suggests routes of RNA recruitment for 3' end processing.

The eukaryotic core exosome (CE) is a conserved nine-subunit protein complex important for 3' end trimming and degradation of RNA. In yeast, the Rrp44 protein constitutively associates with the CE and provides the sole source of processive 3'-to-5' exoribonuclease activity. Here we present EM reconstructions of the core and Rrp44-bound exosome complexes. The two-lobed Rrp44 protein binds to the RNase PH domain side of the exosome and buttresses the bottom of the exosome-processing chamber. The Rrp44 C-terminal body part containing an RNase II-type active site is anchored to the exosome through a conserved set of interactions mainly to the Rrp45 and Rrp43 subunit, whereas the Rrp44 N-terminal head part is anchored to the Rrp41 subunit and may function as a roadblock to restrict access of RNA to the active site in the body region. The Rrp44-exosome (RE) architecture suggests an active site sequestration mechanism for strict control of 3' exoribonuclease activity in the RE complex.

Effects of estrogen on platelet reactivity after transient forebrain ischemia in rats.

Estrogen's prothrombotic effects are of increasing concern, particularly in stroke risk and recovery. Using an ischemic rodent model, the authors sought to determine (a) if estrogen replacement increases postischemic platelet reactivity, (b) if changes in estrogen status alter intraplatelet endothelial nitric oxide synthase (eNOS) synthesis, and (c) if estrogen-mediated effects on platelets alter cerebral blood flow during reperfusion. Intact (I), ovariectomized (OVX), and OVX + 17 beta-estradiol (E50) rats were subjected to 30 min of forebrain ischemia and 60 min of reperfusion. Using the platelet activation marker P-selectin, postischemic platelet reactivity was quantified by flow cytometry. In a separate cohort (I, OVX, E50), the authors quantified platelet eNOS by Western blot. Another cohort (OVX, E50) was subjected to ischemia/reperfusion, and cerebral blood flow was determined using the iodoantipyrine technique. Collagen-stimulated platelet P-selectin expression was increased in the OVX rats at 60 min of reperfusion, and this effect was reversed by estrogen treatment. No differences in platelet eNOS expression were detected among groups. Cerebral blood flow at 60 min reperfusion was comparable between the OVX and the E50 rats. The authors conclude that during reper-fusion, estrogen deficiency increases postischemic platelet sensitivity to stimuli in estrogen-deficient rats. Estrogen treatment mitigates effects of estrogen loss on platelets, but this early effect is apparently not caused by intraplatelet eNOS depression. Neither estrogen deficiency nor estrogen treatment changes early postischemic regional brain blood flow. In this rodent global cerebral ischemic model, physiologic doses of estrogen are not deleterious to platelet reactivity and may initially reduce postischemic platelet reactivity.

Periocular injection of microspheres containing PKC412 inhibits choroidal neovascularization in a porcine model.

PURPOSE: Oral administration of PKC412, a kinase inhibitor that blocks several isoforms of protein kinase C (PKC) and receptors for vascular endothelial growth factor (VEGF), platelet-derived growth factor, and stem cell factor, inhibits ocular neovascularization in a murine model. The purpose of this study was to determine whether sustained local delivery of PKC412 in a human-sized eye inhibits choroidal neovascularization (CNV). METHODS: Laser photocoagulation was used to rupture Bruch's membrane in young domestic pigs, and then a periocular injection of control microspheres or microspheres containing 25% or 50% PKC412 was given. After 10 days the integrated area of CNV at Bruch's membrane rupture sites was measured by image analysis. The levels of PKC412 in choroid, retina, and vitreous were measured either 10 or 20 days after periocular injection of 50% PKC microspheres or at 20 days after injection of 25% PKC412 microspheres. RESULTS: The areas of CNV at Bruch's membrane rupture sites were significantly smaller in eyes that received a periocular injection of microspheres containing 25% (P=0.0042) or 50% (P=0.0012) PKC412 than those in eyes injected with control microspheres. Ten days after periocular injection of 50% PKC412 microspheres, PKC412 was detected in the choroid, but not in the retina or vitreous. Twenty days after periocular injection of 50% PKC412, high levels of PKC412 were measured in the choroid, vitreous, and retina. Levels were lower but still substantial in all three compartments 20 days after periocular injection of 25% microspheres. CONCLUSIONS: Sustained local delivery of PKC412 provides a promising approach for treatment of CNV.

Effect of tenecteplase versus alteplase on platelets during the first 3 hours of treatment for acute myocardial infarction: the Assessment of the Safety and Efficacy of a New Thrombolytic Agent (ASSENT-2) platelet substudy.

BACKGROUND: Platelets play a pivotal role in the pathogenesis of acute myocardial infarction, as well as in the occurrence of coronary artery reocclusion and bleeding events. Therefore, the success of fibrinolytic therapy may be dependent on its direct effects on platelets. METHODS AND RESULTS: We sought to determine how tenecteplase (TNK) and alteplase (tPA) affect platelets in vitro in human volunteers and ex vivo by use of patient data from the Assessment of the Safety and Efficacy of a New Thrombolytic Agent (ASSENT-2) trial. For the in vitro studies, whole blood from 9 healthy volunteers was incubated with 30 mg TNK and 60 mg tPA. Platelet function was measured by conventional aggregometry, bedside point-of-care devices, and sensitive flow cytometry techniques. For the ex vivo study, 41 patients were selected from the ASSENT-2 trial: 21 had received TNK and 20 had received tPA. Each patient underwent 7 serial blood draws every 30 minutes for 3 hours. Levels of platelet endothelial cell adhesion molecule-1 (PECAM-1), vascular cell adhesion molecule-1 (VCAM-1), P-selectin, beta-thromboglobulin, platelet factor 4, thromboxane, and prostacyclin were measured by enzyme-linked immunosorbent assay. Significant inhibition of conventional and whole blood aggregation and reduced platelet function by point-of-care analyzers were observed for both agents, but mostly in the TNK-treated samples. After incubation with TNK, flow cytometry revealed decreased expression of glycoprotein IIb/IIIa, PECAM-1, vitronectin receptor, CD151, and formation of the platelet-monocyte aggregates. Serial samples from patients in the ASSENT-2 trial showed a significant decrease of soluble platelet-endothelial biomarkers in the TNK group. There was a trend toward decreased platelet function characteristics with tPA; however, these differences were much smaller than those observed with TNK. CONCLUSIONS: Both tPA and TNK were shown to affect platelet function in human volunteers and in patients with AMI early after thrombolysis. The antiplatelet properties of TNK were shown to be more profound than those of tPA. These findings are relevant to ongoing investigations of combination therapy with fibrinolytic and antiplatelet agents in patients with AMI.

Effect of a single dose aspirin on platelets in humans with multiple risk factors for coronary artery disease.

We sought to assess how one tablet of non-enteric coated aspirin (325 mg) affects human platelets in subjects with risk factors for coronary artery disease. Data from 63 individuals with multiple cardiac risk factors were analyzed. Platelets were assessed twice at baseline (pre-aspirin), and after 3-4 h (post-aspirin). We employed 5 microM epinephrine-induced conventional aggregometry, closure time with epinephrine/collagen cartridge by PFA-100(R) (Dade-Behring), and aspirin response units (ARU) stimulated by propyl gallat with Ultegra (Accumetrics, San Diego, CA, USA) for measuring platelet function. In addition, the expression of platelet receptors was determined by using the following monoclonal antibodies: anti-CD31, CD41, CD42b, CD51/CD61, CD62p, CD63, CD107a, and CD151. Platelet-leukocyte formation was detected utilizing dual antibodies for a pan-platelet marker CD151, and CD14, a monocyte/macrophage marker. PAC-1 was used to measure fibrinogen-platelet binding. One pill of aspirin significantly decreased platelet-rich plasma (PRP) aggregation (74.18+/-16.75% vs. 24.92+/-8.64%; p<0.0001) and resulted in reduction of the aspirin response units (ARU) (662.24+/-65.65 vs. 451.05+/-69.31; p<0.0001). There was also prolongation of the closure time (194.4+/-25.3 vs. 258.63+/-55.61 s; p<0.0001). High correlation (r(2)=0.73-0.86) between platelet analyzer readings and aggregation was observed. One tablet of aspirin moderately inhibited expression of most surface platelet receptors measured, and such inhibition reached significance (p<0.05) for PAC-1, CD31, CD41, CD42, CD62p, and CD151. We conclude that a single dose of aspirin affects major platelet receptors, presumably directly or indirectly through the inhibition of prostanoids via platelet cyclooxygenase-1 blockade. The Ultegra Analyzer with a novel cartridge seems to be reliable in reflecting aspirins' effects on platelets and could be used in the future in clinical practice for monitoring aspirin therapy.