Synergistic interactions of cytarabine-adavosertib in leukemic cell lines proliferation and metabolomic endpoints.

Drug synergy allows reduced dosing, side effects and tolerance. Optimization of drug synergy chemotherapy is fundamental in acute lymphocytic leukemia and other cancers. This study aimed to analyze the pharmacodynamic synergy between the anti-metabolite cytarabine and WEE1 inhibitor adavosertib on acute leukemia cell lines CCRF-CEM and Jurkat. In both cell lines analysis of concentration-inhibition curves of adavosertib-cytarabine combinations and synergy matrixes supported mutually synergistic drug interactions. Overall mean ( ± SD) synergy scores were higher in Jurkat than CCRF-CEM: Jurkat, ZIP 22.51 ± 1.1, Bliss 22.49 ± 1.1, HSA 23.44 ± 1.0, Loewe 14.16 ± 1.2; and, CCRF-CEM, ZIP 9.17 ± 1.9, Bliss 8.13 ± 2.1, HSA 11.48 ± 1.9 and Loewe 4.99 ± 1.8. Jurkat also surpassed CCRF-CEM in high-degree synergistic adavosertib-cytarabine interactions with mean across-models synergy values of ∼89.1% ± 2.9 for 63 nM cytarabine-97 nM adavosertib (91.4% inhibition synergy barometer). Combination sensitivity scores scatter plots confirmed combination's synergy efficacy. This combined approach permitted identification and prioritization of 63 nM cytarabine-97 nM adavosertib for multiple endpoints analysis. This combination did not affect PBMC viability, while exhibiting Jurkat selective synergy. Immunoblots also revealed Jurkat selective synergistically increased γH2AX phosphorylation, while CDC2 phosphorylation effects were attributed to adavosertib's WEE1 inhibition. In conclusion, the high synergistic efficacy combination of cytarabine (63 nM) and adavosertib (97 nM) was associated with remarkable alterations in metabolites related to the Krebs cycle in Jurkat. The metabolic pathways and processes are related to gluconeogenesis, amino acids, nucleotides, glutathione, electron transport and Warburg effect. All above relate to cell survival, apoptosis, and cancer progression. Our findings could pave the way for novel biomarkers in treatment, diagnosis, and prognosis of leukemia and other cancers.

TEFM variants impair mitochondrial transcription causing childhood-onset neurological disease.

Mutations in the mitochondrial or nuclear genomes are associated with a diverse group of human disorders characterized by impaired mitochondrial respiration. Within this group, an increasing number of mutations have been identified in nuclear genes involved in mitochondrial RNA biology. The TEFM gene encodes the mitochondrial transcription elongation factor responsible for enhancing the processivity of mitochondrial RNA polymerase, POLRMT. We report for the first time that TEFM variants are associated with mitochondrial respiratory chain deficiency and a wide range of clinical presentations including mitochondrial myopathy with a treatable neuromuscular transmission defect. Mechanistically, we show muscle and primary fibroblasts from the affected individuals have reduced levels of promoter distal mitochondrial RNA transcripts. Finally, tefm knockdown in zebrafish embryos resulted in neuromuscular junction abnormalities and abnormal mitochondrial function, strengthening the genotype-phenotype correlation. Our study highlights that TEFM regulates mitochondrial transcription elongation and its defect results in variable, tissue-specific neurological and neuromuscular symptoms.

Two type I topoisomerases maintain DNA topology in human mitochondria.

Genetic processes require the activity of multiple topoisomerases, essential enzymes that remove topological tension and intermolecular linkages in DNA. We have investigated the subcellular localisation and activity of the six human topoisomerases with a view to understanding the topological maintenance of human mitochondrial DNA. Our results indicate that mitochondria contain two topoisomerases, TOP1MT and TOP3A. Using molecular, genomic and biochemical methods we find that both proteins contribute to mtDNA replication, in addition to the decatenation role of TOP3A, and that TOP1MT is stimulated by mtSSB. Loss of TOP3A or TOP1MT also dysregulates mitochondrial gene expression, and both proteins promote transcription elongation in vitro. We find no evidence for TOP2 localisation to mitochondria, and TOP2B knockout does not affect mtDNA maintenance or expression. Our results suggest a division of labour between TOP3A and TOP1MT in mtDNA topology control that is required for the proper maintenance and expression of human mtDNA.

The human mitochondrial genome contains a second light strand promoter.

The human mitochondrial genome must be replicated and expressed in a timely manner to maintain energy metabolism and supply cells with adequate levels of adenosine triphosphate. Central to this process is the idea that replication primers and gene products both arise via transcription from a single light strand promoter (LSP) such that primer formation can influence gene expression, with no consensus as to how this is regulated. Here, we report the discovery of a second light strand promoter (LSP2) in humans, with features characteristic of a bona fide mitochondrial promoter. We propose that the position of LSP2 on the mitochondrial genome allows replication and gene expression to be orchestrated from two distinct sites, which expands our long-held understanding of mitochondrial gene expression in humans.

Erratum: Tejeda-Bayron et al. Activation of Glutamate Transporter-1 (GLT-1) Confers Sex-Dependent Neuroprotection in Brain Ischemia. Brain Sci. 2021, 11, 76.

The authors wish to make the following corrections to this paper [...].

POLRMT mutations impair mitochondrial transcription causing neurological disease.

While >300 disease-causing variants have been identified in the mitochondrial DNA (mtDNA) polymerase γ, no mitochondrial phenotypes have been associated with POLRMT, the RNA polymerase responsible for transcription of the mitochondrial genome. Here, we characterise the clinical and molecular nature of POLRMT variants in eight individuals from seven unrelated families. Patients present with global developmental delay, hypotonia, short stature, and speech/intellectual disability in childhood; one subject displayed an indolent progressive external ophthalmoplegia phenotype. Massive parallel sequencing of all subjects identifies recessive and dominant variants in the POLRMT gene. Patient fibroblasts have a defect in mitochondrial mRNA synthesis, but no mtDNA deletions or copy number abnormalities. The in vitro characterisation of the recombinant POLRMT mutants reveals variable, but deleterious effects on mitochondrial transcription. Together, our in vivo and in vitro functional studies of POLRMT variants establish defective mitochondrial transcription as an important disease mechanism.

Activation of Glutamate Transporter-1 (GLT-1) Confers Sex-Dependent Neuroprotection in Brain Ischemia.

Stroke is one of the leading causes of long-term disability. During ischemic stroke, glutamate is released, reuptake processes are impaired, and glutamate promotes excitotoxic neuronal death. Astrocytic glutamate transporter 1 (GLT-1) is the major transporter responsible for removing excess glutamate from the extracellular space. A translational activator of GLT-1, LDN/OSU 0212320 (LDN) has been previously developed with beneficial outcomes in epileptic animal models but has never been tested as a potential therapeutic for ischemic strokes. The present study evaluated the effects of LDN on stroke-associated brain injury. Male and female mice received LDN or vehicle 24 h before or 2 h after focal ischemia was induced in the sensorimotor cortex. Sensorimotor performance was determined using the Rung Ladder Walk and infarct area was assessed using triphenyltetrazolium chloride staining. Males treated with LDN exhibited upregulated GLT-1 protein levels, significantly smaller infarct size, and displayed better sensorimotor performance in comparison to those treated with vehicle only. In contrast, there was no upregulation of GLT-1 protein levels and no difference in infarct size or sensorimotor performance between vehicle- and LDN-treated females. Taken together, our results indicate that the GLT-1 translational activator LDN improved stroke outcomes in young adult male, but not female mice.

Reaction time and response inhibition in autosomal dominant Alzheimer's disease.

OBJECTIVE: Subtle deficits in several cognitive domains characterize the neuropsychological profile of preclinical Alzheimer's disease (AD). Assessment of preclinical individuals with genes causing autosomal dominant AD (ADAD) provides a model for prodromal disease. We sought to sensitively evaluate attention and working memory using a computerized battery in non-demented persons carrying ADAD mutations. METHOD: A total of 71 non-demented Latinos at-risk for ADAD mutations were recruited [40 mutation carriers (MCs), 31 non-mutation carriers (NCs)] and completed a Spanish language chronometric battery of speeded decision and working memory tasks. RESULTS: On two complex reaction time (RT) tasks involving decision-making and response inhibition, MCs exhibited slower RTs than NCs as they approached their anticipated age of dementia diagnosis. Education moderated these effects, but only in younger MCs. APOE ε4 status was not associated with age-related slowing among NCs or MCs on any of the tests. CONCLUSIONS: Our findings indicate MCs respond more slowly as they approach the age of dementia onset on tasks with greater demands on executive function. Our results also suggest these effects were not explained by APOE ε4 status independently of ADAD mutation status. Computerized reaction time tests can provide sensitive measures of the earliest cognitive changes in AD.

Characterization of Cerebellum-Specific Ribosomal DNA Epigenetic Modifications in Alzheimer's Disease: Should the Cerebellum Serve as a Control Tissue After All?

Alzheimer's disease (AD) is a neurodegenerative disease, known as the most common form of dementia. In AD onset, abnormal rRNA expression has been reported to be linked in pathogenesis. Although region-specific expression patterns have previously been reported in AD, it is not until recently that the cerebellum has come under the spotlight. Specifically, it is unclear whether DNA methylation is the mechanism involved in rRNA expression regulation in AD. Hence, we sought to explore the rDNA methylation pattern of two different brain regions - auditory cortex and cerebellum - from AD and age-/sex-matched controls. Our results showed differential hypermethylation at an upstream CpG region to the rDNA promoter when comparing cerebellum controls to auditory cortex controls. This suggests a possible regulatory region from rDNA expression regulation. Moreover, when comparing between AD and control cerebellum samples, we observed hypermethylation of the rDNA promoter region as well as an increase in rDNA content. In addition, we also observed increased rRNA levels in AD compared to control cerebellum. Although still considered a pathology-free brain region, there are growing findings that continue to suggest otherwise. Indeed, cerebellum from AD has been recently described as affected by the disease, presenting a unique pattern of molecular alterations. Given that we observed that increased rDNA promoter methylation did not silence rDNA gene expression, we suggest that rDNA promoter hypermethylation is playing a protective role in rDNA genomic stability and, therefore, increasing rRNA levels in AD cerebellum.

Caveolin-1 Regulates P2Y2 Receptor Signaling during Mechanical Injury in Human 1321N1 Astrocytoma.

Caveolae-associated protein caveolin-1 (Cav-1) plays key roles in cellular processes such as mechanosensing, receptor coupling to signaling pathways, cell growth, apoptosis, and cancer. In 1321N1 astrocytoma cells Cav-1 interacts with the P2Y2 receptor (P2Y2R) to modulate its downstream signaling. P2Y2R and its signaling machinery also mediate pro-survival actions after mechanical injury. This study determines if Cav-1 knockdown (KD) affects P2Y2R signaling and its pro-survival actions in the 1321N1 astrocytoma cells mechanical injury model system. KD of Cav-1 decreased its expression in 1321N1 cells devoid of or expressing hHAP2Y2R by ~88% and ~85%, respectively. Cav-1 KD had no significant impact on P2Y2R expression. Post-injury densitometric analysis of pERK1/2 and Akt activities in Cav-1-positive 1321N1 cells (devoid of or expressing a hHAP2Y2R) revealed a P2Y2R-dependent temporal increase in both kinases. These temporal increases in pERK1/2 and pAkt were significantly decreased in Cav-1 KD 1321N1 (devoid of or expressing a hHAP2Y2R). Cav-1 KD led to an ~2.0-fold and ~2.4-fold decrease in the magnitude of the hHAP2Y2R-mediated pERK1/2 and pAkt kinases' activity, respectively. These early-onset hHAP2Y2R-mediated signaling responses in Cav-1-expressing and Cav-1 KD 1321N1 correlated with changes in cell viability (via a resazurin-based method) and apoptosis (via caspase-9 expression). In Cav-1-positive 1321N1 cells, expression of hHAP2Y2R led to a significant increase in cell viability and decreased apoptotic (caspase-9) activity after mechanical injury. In contrast, hHAP2Y2R-elicited changes in viability and apoptotic (caspase-9) activity were decreased after mechanical injury in Cav-1 KD 1321N1 cells expressing hHAP2Y2R. These findings support the importance of Cav-1 in modulating P2Y2R signaling during mechanical injury and its protective actions in a human astrocytoma cell line, whilst shedding light on potential new venues for brain injury or trauma interventions.

Correction: Differential expression of pathogenic genes of Entamoeba histolytica vs E. dispar in a model of infection using human liver tissue explants.

[This corrects the article DOI: 10.1371/journal.pone.0181962.].

Detailed Speciation of Intermediate Volatility and Semivolatile Organic Compound Emissions from Gasoline Vehicles: Effects of Cold-Starts and Implications for Secondary Organic Aerosol Formation.

Over the past two decades vehicle emission standards in the United States have been dramatically tightened with the goal of reducing urban air pollution. Secondary organic aerosol (SOA) is the dominant contributor to urban organic aerosol. Experiments were conducted at the California Air Resources Board Haagen-Smit Laboratory to characterize exhaust organics from 20 gasoline vehicles recruited from the California in-use fleet. The vehicles spanned a wide range of emission certification standards. We comprehensively characterized intermediate volatility and semivolatile organic compound emissions using thermal desorption two-dimensional gas-chromatography-mass-spectrometry with electron impact (GC × GC-EI-MS) and vacuum-ultraviolet (GC × GC-VUV-MS) ionization. Single-ring aromatic compounds with unsaturated C4 and C5 substituents contribute a large fraction of the intermediate volatility organic compound (IVOC) emissions in gasoline vehicle exhaust. The analyses of quartz filters used in GC × GC-VUV-MS show that primary organic aerosol emissions were dominated by motor oil. We combined our new emissions data with published SOA yield parametrizations to estimate SOA formation potential. After 24 h of oxidation, IVOC emissions contributed 45% of  SOA formation;  BTEX compounds (benzene, toluene, xylenes, and ethylbenzene), 40%;  other VOC aromatics, 15%. The composition of IVOC emissions was consistent across the test fleet, suggesting that future reductions in vehicular emissions will continue to reduce SOA formation and ambient particulate mass levels.

Characterization of the emissions impacts of hybrid excavators with a portable emissions measurement system (PEMS)-based methodology.

Hybrid engine technology is a potentially important strategy for reduction of tailpipe greenhouse gas (GHG) emissions and other pollutants that is now being implemented for off-road construction equipment. The goal of this study was to evaluate the emissions and fuel consumption impacts of electric-hybrid excavators using a Portable Emissions Measurement System (PEMS)-based methodology. In this study, three hybrid and four conventional excavators were studied for both real world activity patterns and tailpipe emissions. Activity data was obtained using engine control module (ECM) and global positioning system (GPS) logged data, coupled with interviews, historical records, and video. This activity data was used to develop a test cycle with seven modes representing different types of excavator work. Emissions data were collected over this test cycle using a PEMS. The results indicated the HB215 hybrid excavator provided a significant reduction in tailpipe carbon dioxide (CO2) emissions (from -13 to -26%), but increased diesel particulate matter (PM) (+26 to +27%) when compared to a similar model conventional excavator over the same duty cycle.

Investigation of associations between exposures to pesticides and testosterone levels in Thai farmers.

We conducted a cross-sectional study to assess the relationship between pesticide exposures and testosterone levels in 133 male Thai farmers. Urine and serum samples were collected concurrently from participants. Urine was analyzed for levels of specific and nonspecific metabolites of organophosphates (OPs), pyrethroids, select herbicides, and fungicides. Serum was analyzed for total and free testosterone. Linear regression analyses revealed significant negative relationships between total testosterone and the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) after controlling for covariates (eg, age, BMI, smoking status). Positive significant associations were found between some OP pesticides and total testosterone. Due to the small sample size and the observational nature of the study, future investigation is needed to confirm our results and to elucidate the biological mechanisms.

Effects of controlled whole-body vibration training in improving fall risk factors among individuals with multiple sclerosis: A pilot study.

PURPOSE: The purpose of this study was to systematically examine the effect of an 8-week controlled whole-body vibration training on improving fall risk factors and the bone mineral density among people with multiple sclerosis (PwMS). METHODS: This study adopted a single group pre-test-post-test design. Twenty-five PwMS (50.3 years SD 14.1) received vibration training on a side-alternating vibration platform. Each training session was repeated three times every week for 8 weeks. Prior to and following the 8-week training course, a battery of fall risk factors were evaluated: the body balance, functional mobility, muscle strength, range of motion, and fear of falling. Bone density at both calcanei was also assessed. RESULTS: Twenty-two participants completed the study. Compared with pre-test, almost all fall risk factors and the bone density measurement were significantly improved at post-test, with moderate to large effect sizes varying between 0.571 and 1.007. CONCLUSIONS: The 8-week vibration training was well accepted by PwMS and improved their fall risk factors. The important findings of this study were that vibration training may increase the range of motion of ankle joints on the sagittal plane, lower the fear of falling, and improve bone density. IMPLICATIONS FOR REHABILITATION An 8-week vibration training course could be well-accepted by people with multiple sclerosis (MS). Vibration training improves the risk factors of falls in people living with MS. Vibration training could be a promising rehabilitation intervention in individuals with MS.

Differential expression of pathogenic genes of Entamoeba histolytica vs E. dispar in a model of infection using human liver tissue explants.

We sought to establish an ex vivo model for examining the interaction of E. histolytica with human tissue, using precision-cut liver slices (PCLS) from donated organs. E. histolytica- or E. dispar-infected PCLS were analyzed at different post-infection times (0, 1, 3, 24 and 48 h) to evaluate the relation between tissue damage and the expression of genes associated with three factors: a) parasite survival (peroxiredoxin, superoxide dismutase and 70 kDa heat shock protein), b) parasite virulence (EhGal/GalNAc lectin, amoebapore, cysteine proteases and calreticulin), and c) the host inflammatory response (various cytokines). Unlike E. dispar (non-pathogenic), E. histolytica produced some damage to the structure of hepatic parenchyma. Overall, greater expression of virulence genes existed in E. histolytica-infected versus E. dispar-infected tissue. Accordingly, there was an increased expression of EhGal/GalNAc lectin, Ehap-a and Ehcp-5, Ehcp-2, ehcp-1 genes with E. histolytica, and a decreased or lack of expression of Ehcp-2, and Ehap-a genes with E. dispar. E. histolytica-infected tissue also exhibited an elevated expression of genes linked to survival, principally peroxiredoxin, superoxide dismutase and Ehhsp-70. Moreover, E. histolytica-infected tissue showed an overexpression of some genes encoding for pro-inflammatory interleukins (ILs), such as il-8, ifn-γ and tnf-α. Contrarily, E. dispar-infected tissue displayed higher levels of il-10, the gene for the corresponding anti-inflammatory cytokine. Additionally, other genes were investigated that are important in the host-parasite relationship, including those encoding for the 20 kDa heat shock protein (HSP-20), the AIG-1 protein, and immune dominant variable surface antigen, as well as for proteins apparently involved in mechanisms for the protection of the trophozoites in different environments (e.g., thioredoxin-reductase, oxido-reductase, and 9 hypothetical proteins). Some of the hypothetical proteins evidenced interesting overexpression rates, however we should wait to their characterization. This finding suggest that the present model could be advantageous for exploring the complex interaction between trophozoites and hepatocytes during the development of ALA, particularly in the initial stages of infection.

Comparison of Gasoline Direct-Injection (GDI) and Port Fuel Injection (PFI) Vehicle Emissions: Emission Certification Standards, Cold-Start, Secondary Organic Aerosol Formation Potential, and Potential Climate Impacts.

Recent increases in the Corporate Average Fuel Economy standards have led to widespread adoption of vehicles equipped with gasoline direct-injection (GDI) engines. Changes in engine technologies can alter emissions. To quantify these effects, we measured gas- and particle-phase emissions from 82 light-duty gasoline vehicles recruited from the California in-use fleet tested on a chassis dynamometer using the cold-start unified cycle. The fleet included 15 GDI vehicles, including 8 GDIs certified to the most-stringent emissions standard, superultra-low-emission vehicles (SULEV). We quantified the effects of engine technology, emission certification standards, and cold-start on emissions. For vehicles certified to the same emissions standard, there is no statistical difference of regulated gas-phase pollutant emissions between PFIs and GDIs. However, GDIs had, on average, a factor of 2 higher particulate matter (PM) mass emissions than PFIs due to higher elemental carbon (EC) emissions. SULEV certified GDIs have a factor of 2 lower PM mass emissions than GDIs certified as ultralow-emission vehicles (3.0 ± 1.1 versus 6.3 ± 1.1 mg/mi), suggesting improvements in engine design and calibration. Comprehensive organic speciation revealed no statistically significant differences in the composition of the volatile organic compounds emissions between PFI and GDIs, including benzene, toluene, ethylbenzene, and xylenes (BTEX). Therefore, the secondary organic aerosol and ozone formation potential of the exhaust does not depend on engine technology. Cold-start contributes a larger fraction of the total unified cycle emissions for vehicles meeting more-stringent emission standards. Organic gas emissions were the most sensitive to cold-start compared to the other pollutants tested here. There were no statistically significant differences in the effects of cold-start on GDIs and PFIs. For our test fleet, the measured 14.5% decrease in CO2 emissions from GDIs was much greater than the potential climate forcing associated with higher black carbon emissions. Thus, switching from PFI to GDI vehicles will likely lead to a reduction in net global warming.

Time Resolved Measurements of Speciated Tailpipe Emissions from Motor Vehicles: Trends with Emission Control Technology, Cold Start Effects, and Speciation.

Experiments were conducted at the California Air Resources Board Haagen-Smit Laboratory to understand changes in vehicle emissions in response to stricter emissions standards over the past 25 years. Measurements included a wide range of volatile organic compounds (VOCs) for a wide range of spark ignition gasoline vehicles meeting varying levels of emissions standards, including all certifications from Tier 0 up to Partial Zero Emission Vehicle. Standard gas chromatography (GC) and high performance liquid chromatography (HLPC) analyses were employed for drive-cycle phase emissions. A proton-transfer-reaction mass spectrometer measured time-resolved emissions for a wide range of VOCs. Cold-start emissions occur almost entirely in the first 30-60 s for newer vehicles. Cold-start emissions have compositions that are not significantly different across all vehicles tested and are markedly different from neat fuel. Hot-stabilized emissions have varying importance depending on species and may require a driving distance of 200 miles to equal the emissions from a single cold start. Average commute distances in the U.S. suggest the majority of in-use vehicles have emissions dominated by cold starts. The distribution of vehicle ages in the U.S. suggests that within several years only a few percent of vehicles will have significant driving emissions compared to cold-start emissions.

Transposase interaction with the ß sliding clamp: effects on insertion sequence proliferation and transposition rate.

Insertion sequences (ISs) are ubiquitous and abundant mobile genetic elements in prokaryotic genomes. ISs often encode only one protein, the transposase, which catalyzes their transposition. Recent studies have shown that transposases of many different IS families interact with the ß sliding clamp, a DNA replication factor of the host. However, it was unclear to what extent this interaction limits or favors the ability of ISs to colonize a chromosome from a phylogenetically-distant organism, or if the strength of this interaction affects the transposition rate. Here we describe the proliferation of a member of the IS1634 family in Acidiphilium over ~600 generations of cultured growth. We demonstrate that the purified transposase binds to the ß sliding clamp of Acidiphilium, Leptospirillum and E. coli. Further, we also demonstrate that the Acidiphilium IS1634 transposase binds to the archaeal sliding clamp (PCNA) from Methanosarcina, and that the transposase encoded by Methanosarcina IS1634 binds to Acidiphilium ß. Finally, we demonstrate that increasing the strength of the interaction between ß and transposase results in a higher transposition rate in vivo. Our results suggest that the interaction could determine the potential of ISs to be mobilized in bacterial populations and also their ability to proliferate within chromosomes.