The Lantern Vol. 35, No. 1, Winter 1969

• Industrialization • Convention • 86 Prof • Even Your Roommate • Specificity • Bo Jangles and Snowstorms in America • You Might Be • Election Night 1968 • Haiku • The Staff of Life • Wind • Brown Mills Blues • The Reunion • Ballad of the Lost Widow • Sunset • You - Revealed • Boredom? • Victim • I Owned A Tree • Days Bounce Along • Oblivion • Realityhttps://digitalcommons.ursinus.edu/lantern/1094/thumbnail.jp

Rational mutagenesis to support structure-based drug design: MAPKAP kinase 2 as a case study

<p>Abstract</p> <p>Background</p> <p>Structure-based drug design (SBDD) can provide valuable guidance to drug discovery programs. Robust construct design and expression, protein purification and characterization, protein crystallization, and high-resolution diffraction are all needed for rapid, iterative inhibitor design. We describe here robust methods to support SBDD on an oral anti-cytokine drug target, human MAPKAP kinase 2 (MK2). Our goal was to obtain useful diffraction data with a large number of chemically diverse lead compounds. Although MK2 structures and structural methods have been reported previously, reproducibility was low and improved methods were needed.</p> <p>Results</p> <p>Our construct design strategy had four tactics: <it>N</it>- and <it>C</it>-terminal variations; entropy-reducing surface mutations; activation loop deletions; and pseudoactivation mutations. Generic, high-throughput methods for cloning and expression were coupled with automated liquid dispensing for the rapid testing of crystallization conditions with minimal sample requirements. Initial results led to development of a novel, customized robotic crystallization screen that yielded MK2/inhibitor complex crystals under many conditions in seven crystal forms. In all, 44 MK2 constructs were generated, ~500 crystals were tested for diffraction, and ~30 structures were determined, delivering high-impact structural data to support our MK2 drug design effort.</p> <p>Conclusion</p> <p>Key lessons included setting reasonable criteria for construct performance and prioritization, a willingness to design and use customized crystallization screens, and, crucially, initiation of high-throughput construct exploration very early in the drug discovery process.</p

Mitochondrial DNA mutations affect calcium handling in differentiated neurons

Mutations in the mitochondrial genome are associated with a wide range of neurological symptoms, but many aspects of the basic neuronal pathology are not understood. One candidate mechanism, given the well-established role of mitochondria in calcium buffering, is a deficit in neuronal calcium homoeostasis. We therefore examined calcium responses in the neurons derived from various ‘cybrid’ embryonic stem cell lines carrying different mitochondrial DNA mutations. Brief (∼50 ms), focal glutamatergic stimuli induced a transient rise in intracellular calcium concentration, which was visualized by bulk loading the cells with the calcium dye, Oregon Green BAPTA-1. Calcium entered the neurons through N-methyl-d-aspartic acid and voltage-gated calcium channels, as has been described in many other neuronal classes. Intriguingly, while mitochondrial mutations did not affect the calcium transient in response to single glutamatergic stimuli, they did alter the responses to repeated stimuli, with each successive calcium transient decaying ever more slowly in mitochondrial mutant cell lines. A train of stimuli thus caused intracellular calcium in these cells to be significantly elevated for many tens of seconds. These results suggest that calcium-handling deficits are likely to contribute to the pathological phenotype seen in patients with mitochondrial DNA mutations

The Lantern Vol. 34, No. 2, May 1968

• The Man Without a System • A Medal for Malcolm • On Hearing That Tonya Will Be Married • The Black Sea • Odyssey \u2767 • Second Poem to Chris • Singularity • Period 5-A Began • Long and Aching Ride • Souvenirs • My Eschatological Epitaph • Discotheque • Some Borrowed Words • False Breakthrough • Shore Morning • The Beholder • Thursday Childless • A Most Prominent Role • It Ran Out • Shades of the Living • The Dark Night of the Mind II • One Step Beyond the Doors • A Note of Thanks to My Parents and Teachers • To a Dead Hippie • A Scrap • Love • Haiku No. 30 • Rachel • There Is No Present • Winter Woods • One Hundred Per Cent Genuine • Heaven • Silence Is Like God • I Soaked Up Silence • Opened Letter From Whistler Homer, Insaned Assailant • Sol Clutch Rides Tonight • I Have Seen Destruction • Upon That Night • That\u27s Weird • Alone • Kathy\u27s Tune • On Walking Home • The Wheel • Some Excuse, at Least • Freedom to Flap • Awareness • Okay, You Guys • You Say You Dream • Bacci Miahttps://digitalcommons.ursinus.edu/lantern/1093/thumbnail.jp

What is influencing the phenotype of the common homozygous polymerase-γ mutation p.Ala467Thr?

Polymerase-γ (POLG) is a major human disease gene and may account for up to 25% of all mitochondrial diseases in the UK and in Italy. To date, >150 different pathogenic mutations have been described in POLG. Some mutations behave as both dominant and recessive alleles, but an autosomal recessive inheritance pattern is much more common. The most frequently detected pathogenic POLG mutation in the Caucasian population is c.1399G>A leading to a p.Ala467Thr missense mutation in the linker domain of the protein. Although many patients are homozygous for this mutation, clinical presentation is highly variable, ranging from childhood-onset Alpers-Huttenlocher syndrome to adult-onset sensory ataxic neuropathy dysarthria and ophthalmoparesis. The reasons for this are not clear, but familial clustering of phenotypes suggests that modifying factors may influence the clinical manifestation. In this study, we collected clinical, histological and biochemical data from 68 patients carrying the homozygous p.Ala467Thr mutation from eight diagnostic centres in Europe and the USA. We performed DNA analysis in 44 of these patients to search for a genetic modifier within POLG and flanking regions potentially involved in the regulation of gene expression, and extended our analysis to other genes affecting mitochondrial DNA maintenance (POLG2, PEO1 and ANT1). The clinical presentation included almost the entire phenotypic spectrum of all known POLG mutations. Interestingly, the clinical presentation was similar in siblings, implying a genetic basis for the phenotypic variability amongst homozygotes. However, the p.Ala467Thr allele was present on a shared haplotype in each affected individual, and there was no correlation between the clinical presentation and genetic variants in any of the analysed nuclear genes. Patients with mitochondrial DNA haplogroup U developed epilepsy significantly less frequently than patients with any other mitochondrial DNA haplotype. Epilepsy was reported significantly more frequently in females than in males, and also showed an association with one of the chromosomal markers defining the POLG haplotype. In conclusion, our clinical results show that the homozygous p.Ala467Thr POLG mutation does not cause discrete phenotypes, as previously suggested, but rather there is a continuum of clinical symptoms. Our results suggest that the mitochondrial DNA background plays an important role in modifying the disease phenotype but nuclear modifiers, epigenetic and environmental factors may also influence the severity of disease

Loss of myelin-associated glycoprotein in kearns-sayre syndrome

OBJECTIVE: To explore myelin components and mitochondrial changes within the central nervous system in patients with well-characterized mitochondrial disorders due to nuclear DNA or mitochondrial DNA (mtDNA) mutations. DESIGN: Immunohistochemical analysis, histochemical analysis, mtDNA sequencing, and real-time and long-range polymerase chain reaction were used to determine the pathogenicity of mtDNA deletions. SETTING: Department of Clinical Pathology, Columbia University Medical Center, and Newcastle Brain Tissue Resource. PATIENTS: Seventeen patients with mitochondrial disorders and 7 controls were studied from August 1, 2009, to August 1, 2010. MAIN OUTCOME MEASURE: Regions of myelin-associated glycoprotein (MAG) loss. RESULTS: Myelin-associated glycoprotein loss in Kearns-Sayre syndrome was associated with oligodendrocyte loss and nuclear translocation of apoptosis-inducing factor, whereas inflammation, neuronal loss, and axonal injury were minimal. In a Kearns-Sayre syndrome MAG loss region, high levels of mtDNA deletions together with cytochrome- c oxidase–deficient cells and loss of mitochondrial respiratory chain subunits (more prominent in the white than gray matter and glia than axons) confirmed the pathogenicity of mtDNA deletions. CONCLUSION: Primary mitochondrial respiratory chain defects affecting the white matter, and unrelated to inflammation, are associated with MAG loss and central nervous system demyelination

Metabolic shift underlies recovery in reversible infantile respiratory chain deficiency.

Reversible infantile respiratory chain deficiency (RIRCD) is a rare mitochondrial myopathy leading to severe metabolic disturbances in infants, which recover spontaneously after 6-months of age. RIRCD is associated with the homoplasmic m.14674T>C mitochondrial DNA mutation; however, only ~ 1/100 carriers develop the disease. We studied 27 affected and 15 unaffected individuals from 19 families and found additional heterozygous mutations in nuclear genes interacting with mt-tRNAGlu including EARS2 and TRMU in the majority of affected individuals, but not in healthy carriers of m.14674T>C, supporting a digenic inheritance. Our transcriptomic and proteomic analysis of patient muscle suggests a stepwise mechanism where first, the integrated stress response associated with increased FGF21 and GDF15 expression enhances the metabolism modulated by serine biosynthesis, one carbon metabolism, TCA lipid oxidation and amino acid availability, while in the second step mTOR activation leads to increased mitochondrial biogenesis. Our data suggest that the spontaneous recovery in infants with digenic mutations may be modulated by the above described changes. Similar mechanisms may explain the variable penetrance and tissue specificity of other mtDNA mutations and highlight the potential role of amino acids in improving mitochondrial disease

Attosecond Delays in X-ray Molecular Ionization

The photoelectric effect is not truly instantaneous, but exhibits attosecond delays that can reveal complex molecular dynamics. Sub-femtosecond duration light pulses provide the requisite tools to resolve the dynamics of photoionization. Accordingly, the past decade has produced a large volume of work on photoionization delays following single photon absorption of an extreme ultraviolet (XUV) photon. However, the measurement of time-resolved core-level photoionization remained out of reach. The required x-ray photon energies needed for core-level photoionization were not available with attosecond tabletop sources. We have now measured the x-ray photoemission delay of core-level electrons, and here report unexpectedly large delays, ranging up to 700 attoseconds in NO near the oxygen K-shell threshold. These measurements exploit attosecond soft x-ray pulses from a free-electron laser (XFEL) to scan across the entire region near the K-shell threshold. Furthermore, we find the delay spectrum is richly modulated, suggesting several contributions including transient trapping of the photoelectron due to shape resonances, collisions with the Auger-Meitner electron that is emitted in the rapid non-radiative relaxation of the molecule, and multi-electron scattering effects. The results demonstrate how x-ray attosecond experiments, supported by comprehensive theoretical modelling, can unravel the complex correlated dynamics of core-level photoionization

Genome-Wide Association Analysis Identifies a Mutation in the Thiamine Transporter 2 (SLC19A3) Gene Associated with Alaskan Husky Encephalopathy

Alaskan Husky Encephalopathy (AHE) has been previously proposed as a mitochondrial encephalopathy based on neuropathological similarities with human Leigh Syndrome (LS). We studied 11 Alaskan Husky dogs with AHE, but found no abnormalities in respiratory chain enzyme activities in muscle and liver, or mutations in mitochondrial or nuclear genes that cause LS in people. A genome wide association study was performed using eight of the affected dogs and 20 related but unaffected control AHs using the Illumina canine HD array. SLC19A3 was identified as a positional candidate gene. This gene controls the uptake of thiamine in the CNS via expression of the thiamine transporter protein THTR2. Dogs have two copies of this gene located within the candidate interval (SLC19A3.2 – 43.36–43.38 Mb and SLC19A3.1 – 43.411–43.419 Mb) on chromosome 25. Expression analysis in a normal dog revealed that one of the paralogs, SLC19A3.1, was expressed in the brain and spinal cord while the other was not. Subsequent exon sequencing of SLC19A3.1 revealed a 4bp insertion and SNP in the second exon that is predicted to result in a functional protein truncation of 279 amino acids (c.624 insTTGC, c.625 C>A). All dogs with AHE were homozygous for this mutation, 15/41 healthy AH control dogs were heterozygous carriers while 26/41 normal healthy AH dogs were wild type. Furthermore, this mutation was not detected in another 187 dogs of different breeds. These results suggest that this mutation in SLC19A3.1, encoding a thiamine transporter protein, plays a critical role in the pathogenesis of AHE.University of California, Davis. School of Veterinary Medicine. Center for Companion Animal Healt

Microangiopathy in the cerebellum of patients with mitochondrial DNA disease

Neuropathological findings in mitochondrial DNA disease vary and are often dependent on the type of mitochondrial DNA defect. Many reports document neuronal cell loss, demyelination, gliosis and necrotic lesions in post-mortem material. However, previous studies highlight vascular abnormalities in patients harbouring mitochondrial DNA defects, particularly in those with the m.3243A>G mutation in whom stroke-like events are part of the mitochondrial encephalopathy lactic acidosis and stroke-like episodes syndrome. We investigated microangiopathic changes in the cerebellum of 16 genetically and clinically well-defined patients. Respiratory chain deficiency, high levels of mutated mitochondrial DNA and increased mitochondrial mass were present within the smooth muscle cells and endothelial cells comprising the vessel wall in patients. These changes were not limited to those harbouring the m.3243A>G mutation frequently associated with mitochondrial encephalopathy, lactic acidosis and stroke-like episodes, but were documented in patients harbouring m.8344A>G and autosomal recessive polymerase (DNA directed), gamma (POLG) mutations. In 8 of the 16 patients, multiple ischaemic-like lesions occurred in the cerebellar cortex suggestive of vascular smooth muscle cell dysfunction. Indeed, changes in vascular smooth muscle and endothelium distribution and cell size are indicative of vascular cell loss. We found evidence of blood–brain barrier breakdown characterized by plasma protein extravasation following fibrinogen and IgG immunohistochemistry. Reduced immunofluorescence was also observed using markers for endothelial tight junctions providing further evidence in support of blood–brain barrier breakdown. Understanding the structural and functional changes occurring in central nervous system microvessels in patients harbouring mitochondrial DNA defects will provide an important insight into mechanisms of neurodegeneration in mitochondrial DNA disease. Since therapeutic strategies targeting the central nervous system are limited, modulating vascular function presents an exciting opportunity to lessen the burden of disease in these patients