Integrative structural and functional analysis of human malic enzyme 3: A potential therapeutic target for pancreatic cancer.

Malic enzymes (ME1, ME2, and ME3) are involved in cellular energy regulation, redox homeostasis, and biosynthetic processes, through the production of pyruvate and reducing agent NAD(P)H. Recent studies have implicated the third and least well-characterized isoform, mitochondrial NADP+-dependent malic enzyme 3 (ME3), as a therapeutic target for pancreatic cancers. Here, we utilized an integrated structure approach to determine the structures of ME3 in various ligand-binding states at near-atomic resolutions. ME3 is captured in the open form existing as a stable tetramer and its dynamic Domain C is critical for activity. Catalytic assay results reveal that ME3 is a non-allosteric enzyme and does not require modulators for activity while structural analysis suggests that the inner stability of ME3 Domain A relative to ME2 disables allostery in ME3. With structural information available for all three malic enzymes, the foundation has been laid to understand the structural and biochemical differences of these enzymes and could aid in the development of specific malic enzyme small molecule drugs.

Precision genetic cellular models identify therapies protective against ER stress.

Rare monogenic disorders often share molecular etiologies involved in the pathogenesis of common diseases. Congenital disorders of glycosylation (CDG) and deglycosylation (CDDG) are rare pediatric disorders with symptoms that range from mild to life threatening. A biological mechanism shared among CDG and CDDG as well as more common neurodegenerative diseases such as Alzheimer's disease and amyotrophic lateral sclerosis, is endoplasmic reticulum (ER) stress. We developed isogenic human cellular models of two types of CDG and the only known CDDG to discover drugs that can alleviate ER stress. Systematic phenotyping confirmed ER stress and identified elevated autophagy among other phenotypes in each model. We screened 1049 compounds and scored their ability to correct aberrant morphology in each model using an agnostic cell-painting assay based on >300 cellular features. This primary screen identified multiple compounds able to correct morphological phenotypes. Independent validation shows they also correct cellular phenotypes and alleviate each of the ER stress markers identified in each model. Many of the active compounds are associated with microtubule dynamics, which points to new therapeutic opportunities for both rare and more common disorders presenting with ER stress, such as Alzheimer's disease and amyotrophic lateral sclerosis.

Novel indicators to better monitor the collection and recovery of (critical) raw materials in WEEE: Focus on screens.

Currently, in the European Union (EU), e-waste chain performance is assessed by technical indicators that aim to ensure system compliance with collection and recovery targets set by the WEEE Directive. This study proposes indicators to improve WEEE flow monitoring beyond the current overall weight-based approach, including complementary flows and treatment performance. A case study focused on the screen category in France is presented. In 2017, the collection rate of cathode-ray tube screens (CRT) was 68%, while for flat panel display (FPD) generated only 14% was collected. CRT screens have less precious and critical materials than FDP. Thus, elements like cobalt and gold highly concentrated in FPD, have a collection rate two to four times lower than elements such as copper (37%) which represents a high proportion in CRTs. Recycling is the main treatment in France. Nevertheless, the recycling rate per element varies significantly due to the low collection, and also the lack of technology and/or secondary raw materials market. The elements with higher recycling rates are base metals such as copper (28%), followed by precious metals like silver (23%), and gold (13%). Except for palladium, the recycling rate of the critical raw materials targeted in the study ranged from 6% (cobalt) to 0% (e.g. neodymium and indium). The results stress the need for indicators to support the development of WEEE chain from waste management to secondary (critical) raw materials suppliers.

Immunological memory to hyperphosphorylated tau in asymptomatic individuals.

Several reports have described the presence of antibodies against Alzheimer's disease-associated hyperphosphorylated forms of tau in serum of healthy individuals. To characterize the specificities that can be found, we interrogated peripheral IgG+ memory B cells from asymptomatic blood donors for reactivity to a panel of phosphorylated tau peptides using a single-cell screening assay. Antibody sequences were recovered, cloned, and expressed as full-length IgGs. In total, 52 somatically mutated tau-binding antibodies were identified, corresponding to 35 unique clonal families. Forty-one of these antibodies recognize epitopes in the proline-rich and C-terminal domains, and binding of 26 of these antibodies is strictly phosphorylation dependent. Thirteen antibodies showed inhibitory activity in a P301S lysate seeded in vitro tau aggregation assay. Two such antibodies, CBTAU-7.1 and CBTAU-22.1, which bind to the proline-rich and C-terminal regions of tau, respectively, were characterized in more detail. CBTAU-7.1 recognizes an epitope that is similar to that of murine anti-PHF antibody AT8, but has different phospho requirements. Both CBTAU-7.1 and CBTAU-22.1 detect pathological tau deposits in post-mortem brain tissue. CBTAU-7.1 reveals a similar IHC distribution pattern as AT8, immunostaining (pre)tangles, threads, and neuritic plaques. CBTAU-22.1 shows selective detection of neurofibrillary changes by IHC. Taken together, these results suggest the presence of an ongoing antigen-driven immune response against tau in healthy individuals. The wide range of specificities to tau suggests that the human immune repertoire may contain antibodies that can serve as biomarkers or be exploited for therapy.

Skeletal light-scattering accelerates bleaching response in reef-building corals.

BACKGROUND: At the forefront of ecosystems adversely affected by climate change, coral reefs are sensitive to anomalously high temperatures which disassociate (bleaching) photosynthetic symbionts (Symbiodinium) from coral hosts and cause increasingly frequent and severe mass mortality events. Susceptibility to bleaching and mortality is variable among corals, and is determined by unknown proportions of environmental history and the synergy of Symbiodinium- and coral-specific properties. Symbiodinium live within host tissues overlaying the coral skeleton, which increases light availability through multiple light-scattering, forming one of the most efficient biological collectors of solar radiation. Light-transport in the upper ~200 µm layer of corals skeletons (measured as 'microscopic' reduced-scattering coefficient, µ'(S,m)), has been identified as a determinant of excess light increase during bleaching and is therefore a potential determinant of the differential rate and severity of bleaching response among coral species. RESULTS: Here we experimentally demonstrate (in ten coral species) that, under thermal stress alone or combined thermal and light stress, low-µ'(S,m) corals bleach at higher rate and severity than high-µ'(S,m) corals and the Symbiodinium associated with low-µ'(S,m) corals experience twice the decrease in photochemical efficiency. We further modelled the light absorbed by Symbiodinium due to skeletal-scattering and show that the estimated skeleton-dependent light absorbed by Symbiodinium (per unit of photosynthetic pigment) and the temporal rate of increase in absorbed light during bleaching are several fold higher in low-µ'(S,m) corals. CONCLUSIONS: While symbionts associated with low-[Formula: see text] corals receive less total light from the skeleton, they experience a higher rate of light increase once bleaching is initiated and absorbing bodies are lost; further precipitating the bleaching response. Because microscopic skeletal light-scattering is a robust predictor of light-dependent bleaching among the corals assessed here, this work establishes µ'(S,m) as one of the key determinants of differential bleaching response.

A stable trimeric influenza hemagglutinin stem as a broadly protective immunogen.

The identification of human broadly neutralizing antibodies (bnAbs) targeting the hemagglutinin (HA) stem revitalized hopes of developing a universal influenza vaccine. Using a rational design and library approach, we engineered stable HA stem antigens ("mini-HAs") based on an H1 subtype sequence. Our most advanced candidate exhibits structural and bnAb binding properties comparable to those of full-length HA, completely protects mice in lethal heterologous and heterosubtypic challenge models, and reduces fever after sublethal challenge in cynomolgus monkeys. Antibodies elicited by this mini-HA in mice and nonhuman primates bound a wide range of HAs, competed with human bnAbs for HA stem binding, neutralized H5N1 viruses, and mediated antibody-dependent effector activity. These results represent a proof of concept for the design of HA stem mimics that elicit bnAbs against influenza A group 1 viruses.

Evaluation of allele frequencies of inherited disease genes in subgroups of American Quarter Horses.

OBJECTIVE: To estimate allele frequencies of the hyperkalaemic periodic paralysis (HYPP), lethal white foal syndrome (LWFS), glycogen branching enzyme deficiency (GBED), hereditary equine regional dermal asthenia (HERDA), and type 1 polysaccharide storage myopathy (PSSM) genes in elite performance subgroups of American Quarter Horses (AQHs). DESIGN: Prospective genetic survey. ANIMALS: 651 elite performance AQHs, 200 control AQHs, and 180 control American Paint Horses (APHs). PROCEDURES: Elite performance AQHs successful in 7 competitive disciplines (barrel racing, cutting, halter, racing, reining, western pleasure, and working cow horse) were geno- typed for 5 disease-causing alleles. Age-matched control AQHs and APHs were used to establish comparative whole-breed estimates of allele frequencies. RESULTS: Highest allele frequencies among control AQHs were for type 1 PSSM (0.055) and GBED (0.054), whereas HERDA (0.021) and HYPP (0.008) were less prevalent. Control APHs uniquely harbored LWFS (0.107) and had high prevalence of HYPP (0.025), relative to AQHs. Halter horse subgroups had significantly greater allele frequencies for HYPP (0.299) and PSSM (0.155). Glycogen branching enzyme deficiency, HERDA, and PSSM were found broadly throughout subgroups; cutting subgroups were distinct for HERDA (0.142), and western pleasure subgroups were distinct for GBED (0.132). Racing and barrel racing subgroups had the lowest frequencies of the 5 disease genes. CONCLUSIONS AND CLINICAL RELEVANCE: Accurate estimates of disease-causing alleles in AQHs and APHs may guide use of diagnostic genetic testing, aid management of genetic diseases, and help minimize production of affected foals.

Whi5 regulation by site specific CDK-phosphorylation in Saccharomyces cerevisiae.

The Whi5 transcriptional repressor is a negative regulator of G1 cell cycle progression in Saccharomyces cerevisiae and is functionally equivalent to the Retinoblastoma (Rb) tumor suppressor protein in mammals. In early G1, Whi5 binds to and inhibits SBF (Swi4/Swi6) transcriptional complexes. At Start, Cln:Cdc28 kinases phosphorylate and inactivate Whi5, causing its dissociation from SBF promoters and nuclear export, allowing activation of SBF transcription and entry into late G1. In an analysis of Whi5 phosphorylation, we found that 10 of the 12 putative CDK phosphorylation sites on Whi5 were occupied in vivo in asynchronously growing cells. In addition, we identified 6 non-CDK Whi5 phosphorylation sites. Whi5 CDK and non-CDK phosphorylation mutants were functional and able to rescue the small cell size of whi5Delta cells. However, the Whi5 CDK mutant with all 12 putative CDK sites changed to alanine causes a dramatic cell cycle phenotype when expressed with a Swi6 CDK phosphorylation mutant. Mutational analysis of Whi5 determined that only four C-terminal CDK sites were necessary and sufficient for Whi5 inactivation when Swi6 CDK sites were also mutated. Although these four Whi5 CDK sites do not wholly determine Whi5 nuclear export, they do impact regulation of cell size. Taken together, these observations begin to dissect the regulatory role of specific phosphorylation sites on Whi5.

Assessment of the transformation of equine skin-derived fibroblasts to multinucleated skeletal myotubes following lentiviral-induced expression of equine myogenic differentiation 1.

OBJECTIVE: To develop a reliable method for converting cultured equine skin-derived fibroblasts into muscle cells. SAMPLE POPULATION: Equine skin-derived fibroblasts. PROCEDURES: The equine myogenic differentiation 1 (eqMyoD) genomic sequence was obtained by use of equine bacterial artificial chromosome screening and PCR sequencing. Total mRNA was extracted from foal skeletal muscle, and eqMyoD cDNA was cloned into a plasmid vector with an internal ribosomal entry site to express bicistronic eqMyoD or enhanced green fluorescent protein (EGFP). Transient expression was confirmed by immunocytochemical analysis and western immunoblots in equine fibroblasts and fibroblasts from National Institutes of Health Swiss mouse embryos, prior to generation of a lentiviral vector containing the same coding sequences. Transformation of equine skin-derived cells into skeletal myotubes was examined by use of immunohistochemical analysis, western immunoblotting, and periodic acid-Schiff staining. RESULTS: eqMyoD mRNA consists of 960 bp and shares high homology with myogenic differentiation 1 from other mammals. Transfection confirmed the expression of a 53-kd protein with mainly nuclear localization. Lentiviral transduction was efficient, with approximately 80% of EGFP-positive cells transformed into multinucleated myotubes during 15 days, as determined by expression of the muscle-specific proteins desmin, troponin-T, and sarcomeric myosin and by cytoplasmic storage of glycogen. CONCLUSIONS AND CLINICAL RELEVANCE: Equine primary fibroblasts were transformed by lentiviral transduction of eqMyoD into fusion-competent myoblasts. This may offer a preferable alternative to primary myoblast cultures for the investigation of cellular defects associated with muscle diseases of horses, such as recurrent exertional rhabdomyolysis and polysaccharide storage myopathy.

High-resolution gene maps of horse chromosomes 14 and 21: additional insights into evolution and rearrangements of HSA5 homologs in mammals.

High-resolution physically ordered gene maps for equine homologs of human chromosome 5 (HSA5), viz., horse chromosomes 14 and 21 (ECA14 and ECA21), were generated by adding 179 new loci (131 gene-specific and 48 microsatellites) to the existing maps of the two chromosomes. The loci were mapped primarily by genotyping on a 5000-rad horse x hamster radiation hybrid panel, of which 28 were mapped by fluorescence in situ hybridization. The approximately fivefold increase in the number of mapped markers on the two chromosomes improves the average resolution of the map to 1 marker/0.9 Mb. The improved resolution is vital for rapid chromosomal localization of traits of interest on these chromosomes and for facilitating candidate gene searches. The comparative gene mapping data on ECA14 and ECA21 finely align the chromosomes to sequence/gene maps of a range of evolutionarily distantly related species. It also demonstrates that compared to ECA14, the ECA21 segment corresponding to HSA5 is a more conserved region because of preserved gene order in a larger number of and more diverse species. Further, comparison of ECA14 and the distal three-quarters region of ECA21 with corresponding chromosomal segments in 50 species belonging to 11 mammalian orders provides a broad overview of the evolution of these segments in individual orders from the putative ancestral chromosomal configuration. Of particular interest is the identification and precise demarcation of equid/Perissodactyl-specific features that for the first time clearly distinguish the origins of ECA14 and ECA21 from similar-looking status in the Cetartiodactyls.

Randomized control trial of effects of a 10-week inspiratory muscle training program on measures of pulmonary function in persons with multiple sclerosis.

Pulmonary impairments have long been recognized as major causes of morbidity and mortality in individuals with advanced multiple sclerosis (MS). This study was designed to determine if a 10-week home exercise inspiratory training program in community-dwelling persons with MS improves pulmonary muscle strength and endurance. Forty-six ambulatory individuals with clinically diagnosed MS [Expanded Disability Status Scale (EDSS) 2.0-6.5, intervention group mean = 3.96 and control group mean = 3.36] were randomly assigned to an intervention group that received 10 weeks of inspiratory muscle strength training (IMT) or a nontreatment control group. Twenty-one subjects in the control group and 20 subjects in the intervention group completed the study. The intervention group demonstrated significantly greater improvement than the control group in maximal inspiratory pressure (P < 0.001). When compared to the control group, no significant differences were noted for maximal expiratory pressure or maximal ventilation volume after training in the intervention group. Baseline and postexercise training comparison of secondary pulmonary expiratory outcomes were significant in the intervention group for forced expiratory volume at one second (FEV1) (P = 0.014), forced vital capacity (FVC) (P = 0.041), and midexpiratory flow rate(FEF(25-75%)) (P = 0.011). No significant changes were noted for the control group. Thus, IMT significantly increased inspiratory muscle strength and resulted in generalized improvements in expiratory pulmonary function in persons with MS who have minimal to moderate disability. Future studies are needed that focus on the long-term effects of IMT with increased resistance and the impact it has on increasing pulmonary function and functional performance.

The role of histidine 200 in MndD, the Mn(II)-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenase from Arthrobacter globiformis CM-2, a site-directed mutagenesis study.

The manganese-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenase (MndD) from Arthrobacter globiformis CM-2 is an extradiol-cleaving catechol dioxygenase that catalyzes aromatic ring cleavage of 3,4-dihydroxyphenylacetate (DHPA). Based on the recent crystal structure of the MndD-DHPA complex, a series of site-directed mutations were made at a conserved second-sphere residue, histidine 200, to gain insight into and clarify the role this residue plays in the Mn(II)-dependent catalytic mechanism. In this study, we report the activities and spectroscopic data of these H200 variants and their DHPA and 4-nitrocatechol (4-NC) complexes. The data collected from wild-type and mutant MndDs are consistent with a role for H200 interacting with a manganese-bound dioxygen moiety and are inconsistent with other previously proposed roles involving proton transfer. Spectroscopic observations, including unique low-field EPR signals found when DHPA and 4-NC are bound to the Mn(II) center of MndD, are discussed and their relationship to dioxygen activation catalyzed in MndD is explored.

A high-resolution physical map of equine homologs of HSA19 shows divergent evolution compared with other mammals.

A high-resolution (1 marker/700 kb) physically ordered radiation hybrid (RH) and comparative map of 122 loci on equine homologs of human Chromosome 19 (HSA19) shows a variant evolution of these segments in equids/Perissodactyls compared with other mammals. The segments include parts of both the long and the short arm of horse Chromosome 7 (ECA7), the proximal part of ECA21, and the entire short arm of ECA10. The map includes 93 new markers, of which 89 (64 gene-specific and 25 microsatellite) were genotyped on a 5000-rad horse x hamster RH panel, and 4 were mapped exclusively by FISH. The orientation and alignment of the map was strengthened by 21 new FISH localizations, of which 15 represent genes. The approximately sevenfold-improved map resolution attained in this study will prove extremely useful for candidate gene discovery in the targeted equine chromosomal regions. The highlight of the comparative map is the fine definition of homology between the four equine chromosomal segments and corresponding HSA19 regions specified by physical coordinates (bp) in the human genome sequence. Of particular interest are the regions on ECA7 and ECA21 that correspond to the short arm of HSA19-a genomic rearrangement discovered to date only in equids/Perissodactyls as evidenced through comparative Zoo-FISH analysis of the evolution of ancestral HSA19 segments in eight mammalian orders involving about 50 species.

Expression of interferon-inducible RNA adenosine deaminase ADAR1 during pathogen infection and mouse embryo development involves tissue-selective promoter utilization and alternative splicing.

ADAR1 (adenosine deaminase acting on RNA) is widely expressed in adult mammals and has a critical role during embryogenesis. Two size forms of ADAR1 are known that possess adenosine-to-inosine editing activity: an interferon (IFN)-inducible approximately 150-kDa protein and a constitutively expressed N-terminally truncated approximately 110-kDa protein. We defined the structure of the 5'-flanking region of the mouse Adar1 gene, and we show here that mouse Adar1 transcripts possess alternative exon 1 structures (1A, 1B, and 1C) that initiate from unique promoters and are spliced to a common exon 2 junction. Exon 1A-containing transcripts encoding p150 were expressed in all tissues examined from adult mice (brain, cecum, heart, kidney, liver, lung, spleen, and Peyer's patches) and were elevated most significantly in liver but remained lowest in brain following oral infection with Salmonella. Exon 1B-containing RNA was most abundant in brain and was not increased in any tissue examined following infection. Exon 1C-containing RNA was very scarce. Exon 1A, but not exon 1B or 1C, expression was increased in fibroblast L cells treated with IFN, and a consensus ISRE element was present in the promoter driving exon 1A expression. Exon 1B, but not 1A, was detectable in embryonic day 10.5 embryos and was abundantly expressed in embryonic day 15 embryos. Furthermore, the ADAR1 p110 protein isoform was detected in embryonic tissue, whereas both p110 and the inducible p150 proteins were found in IFN-treated L cells. Finally, the presence of alternative exon 7a correlated with exon 1B-containing RNA, and alternative exon 7b correlated with exon 1A-containing RNA. These results establish that multiple promoters drive the expression of the Adar1 gene in adult mice, that the IFN inducible promoter and exon 1A-containing RNA are primarily responsible for the increased ADAR1 observed in Salmonella-infected mice, and that the constitutive exon 1B-containing transcript and encoded p110 protein product are abundantly expressed both in adult brain and during embryogenesis.

High-resolution RH map of horse chromosome 22 reveals a putative ancestral vertebrate chromosome.

High-resolution gene maps of individual equine chromosomes are essential to identify genes governing traits of economic importance in the horse. In pursuit of this goal we herein report the generation of a dense map of horse chromosome 22 (ECA22) comprising 83 markers, of which 52 represent specific genes and 31 are microsatellites. The map spans 831 cR over an estimated 64 Mb of physical length of the chromosome, thus providing markers at approximately 770 kb or 10 cR intervals. Overall, the resolution of the map is to date the densest in the horse and is the highest for any of the domesticated animal species for which annotated sequence data are not yet available. Comparative analysis showed that ECA22 shares remarkable conservation of gene order along the entire length of dog chromosome 24, something not yet found for an autosome in evolutionarily diverged species. Comparison with human, mouse, and rat homologues shows that ECA22 can be traced as two conserved linkage blocks, each related to individual arms of the human homologue-HSA20. Extending the comparison to the chicken genome showed that one of the ECA22 blocks that corresponds to HSA20q shares synteny conservation with chicken chromosome 20, suggesting the segment to be ancestral in mammals and birds.