Posttranslational modification of proteins expands their structural and functional capabilities beyond those directly specified by the genetic code. However, the vast diversity of chemically plausible (including unnatural but functionally relevant) side chains is not readily accessible. We describe C (sp)-C (sp) bond-forming reactions on proteins under biocompatible conditions, which exploit unusual carbon free-radical chemistry, and use them to form Cβ-Cγ bonds with altered side chains. We demonstrate how these transformations enable a wide diversity of natural, unnatural, posttranslationally modified (methylated, glycosylated, phosphorylated, hydroxylated), and labeled (fluorinated, isotopically labeled) side chains to be added to a common, readily accessible dehydroalanine precursor in a range of representative protein types and scaffolds. This approach, outside of the rigid constraints of the ribosome and enzymatic processing, may be modified more generally for access to diverse proteins.

We describe biophysical and ultrastructural differences in genome release from adeno-associated virus (AAV) capsids packaging wild-type DNA, recombinant single-stranded DNA (ssDNA), or dimeric, self-complementary DNA (scDNA) genomes. Atomic force microscopy and electron microscopy (EM) revealed that AAV particles release packaged genomes and undergo marked changes in capsid morphology upon heating in physiological buffer (pH 7.2). When different AAV capsids packaging ss/scDNA varying in length from 72 to 123% of wild-type DNA (3.4 to 5.8 kb) were incrementally heated, the proportion of uncoated AAV capsids decreased with genome length as observed by EM. Genome release was further characterized by a fluorimetric assay, which demonstrated that acidic pH and high osmotic pressure suppress genome release from AAV particles. In addition, fluorimetric analysis corroborated an inverse correlation between packaged genome length and the temperature needed to induce uncoating. Surprisingly, scAAV vectors required significantly higher temperatures to uncoat than their ssDNA-packaging counterparts. However, externalization of VP1 N termini appears to be unaffected by packaged genome length or self-complementarity. Further analysis by tungsten-shadowing EM revealed striking differences in the morphologies of ssDNA and scDNA genomes upon release from intact capsids. Computational modeling and molecular dynamics simulations suggest that the unusual thermal stability of scAAV vectors might arise from partial base pairing and optimal organization of packaged scDNA. Our work further defines the biophysical mechanisms underlying adeno-associated virus uncoating and genome release.A deno-associated virus (AAV) is a small (25 nm) nonenveloped virus belonging to the family Parvoviridae and genus Dependovirus. The AAV capsid packages a single-stranded (ssDNA) genome approximately 4.7 kb in length (1). The wildtype genome consists of two open reading frames flanked by two inverted terminal hairpin repeats (ITRs). The ITRs, which are 145 nucleotides each, are the only cis element in the AAV genome required for successful packaging (2, 3). The AAV capsid is composed of 60 (T ϭ 1) viral protein subunits VP1, VP2, and VP3, in approximately the ratio 1:1:10. The three different subunits are generated from overlapping reading frames and interact within the capsid through the common VP3 subunit region. The largest VP1 subunit is known to possess a phospholipase A2 domain required for infectivity (4). Because of its broad tropism, lack of pathogenicity, and flexibility in genome content, AAV has become a promising candidate for therapeutic gene transfer applications. In the past 2 decades, AAV has been utilized as a gene transfer vector in a number of phase I and phase II clinical trials treating various genetic diseases (5).Different AAV serotypes infect cells by engaging a variety of cell surface glycans and coreceptors, followed by endocytic uptake (4, 6, 7). Viral particles are then thought to escape from the endosome and translocate to th...

Post-translational modifications (PTMs) greatly expand the structures and functions of proteins in nature 1,2 . Although synthetic protein functionalization strategies allow mimicry of PTMs 3,4 , as well as formation of unnatural protein variants with diverse potential functions, including drug carrying 5 , tracking, imaging 6 and partner crosslinking 7 , the range of functional groups that can be introduced remains limited. Here we describe the visible-light-driven installation of side chains at dehydroalanine residues in proteins through the formation of carbon radicals that allow C-C bond formation in water. Control of the reaction redox allows site-selective modification with good conversion efficiencies and reduced protein damage. In situ generation of boronic acid catechol ester derivatives generates RH2C • radicals that form the native (β-CH2-γ-CH2) linkage of natural residues and PTMs, whereas in situ potentiation of pyridylsulfonyl derivatives by Fe(II) generates RF2C • radicals that form equivalent β-CH2-γ-CF2 linkages bearing difluoromethylene labels. These reactions are chemically tolerant and incorporate a wide range of functionalities (more than 50 unique residues/side chains) into diverse protein scaffolds and sites. Initiation can be applied chemoselectively in the presence of sensitive groups in the radical precursors, enabling installation of previously incompatible side chains. The resulting protein function and reactivity are used to install radical precursors for homolytic on-protein radical

SummaryInterventions that extend lifespan in mice can show substantial sexual dimorphism. Here, we show that male‐specific lifespan extension with two pharmacological treatments, acarbose (ACA) and 17‐α estradiol (17aE2), is associated, in males only, with increased insulin sensitivity and improved glucose tolerance. Females, which show either smaller (ACA) or no lifespan extension (17aE2), do not derive these metabolic benefits from drug treatment. We find that these male‐specific metabolic improvements are associated with enhanced hepatic mTORC2 signaling, increased Akt activity, and phosphorylation of FOXO1a – changes that might promote metabolic health and survival in males. By manipulating sex hormone levels through gonadectomy, we show that sex‐specific changes in these metabolic pathways are modulated, in opposite directions, by both male and female gonadal hormones: Castrated males show fewer metabolic responses to drug treatment than intact males, and only those that are also observed in intact females, while ovariectomized females show some responses similar to those seen in intact males. Our results demonstrate that sex‐specific metabolic benefits occur concordantly with sexual dimorphism in lifespan extension. These sex‐specific effects can be influenced by the presence of both male and female gonadal hormones, suggesting that gonadally derived hormones from both sexes may contribute to sexual dimorphism in responses to interventions that extend mouse lifespan.

SUMMARY The classical 4:1 ketogenic diet, the medium‐chain triglyceride (MCT) diet and a modified MCT diet were used in the treatment of 55 children and four adults with intractable epilepsy. During a three‐month treatment period 51 of 63 studies (81 per cent) showed >50 per cent reduction in seizure frequency. This improvement was independent of diet used and type of seizure experienced by patients. The diets were found to be acceptable and of therapeutic benefit to the children, but treatment of the four adult patients with the MCT diet was unsuccessful. There was no correlation between EEG changes and clinical response. The three ketogenic diets were shown to be effective in the short‐term management of children whose epilepsy is poorly controlled with anticonvulsant drugs. RÉSUMÉ Régimes cétogéniques dans le traitement de I'épilepsie: effets cliniques à court terme Le régime cétogénique 4:1, le régime à triglycérides à chaines moyennes (MCT) et un régime MCT modifié, ont été utilisés dans le traitement de 55 enfants et quatre adultes présentant une épilepsie rebelle. Durant la Période de traitement de trois mois, 51 de 63 (81 pour cent) présentant une réduction, de >50 pour cent de la fréquence des crises. Cette amélioration était indépendante du type de régime et de la nature des crises présentées par les patients. Les régimes furent intéressants et bénéfiques pour les enfants mais le traitement par le régime MCT fut inefficace chez les quatre adultes. II n'y eu pas de correlation entre modifications EEG et réponse clinique. Les trois régimes cétogéniques se sont montrés efficaces dans le traitement d'enfants dont I'épilepsie répondait mal aux médications anticomitiales. ZUSAMMENFASUNG Ketogene Diäten bei der Behandlung der Epilepsie: klinische Kurzzeitbeobachtungen Die 4:1 ketogene Diät, die Diät mit mittelkettigen Triglyceriden (MCT) und die modifizierte MCT Diät wurden bei der Behandlung von 55 Kindern und vier Erwachsenen mit unbeeinflußbarer Epilepsie eingesetzt. Innerhalb einer dreimonatigen Behandlungsperiode zeigten 51 von 63 (81 Prozent) eine um 50 Prozent verminderte Anfallshäufigkeit. Diese Besserung war unabhangig von der Art der Diät und unabhängig von der Art der Anfälle. Die Diäten waren akzeptabel und von therpeutischem Nutzen für die Kinder, die Behandlung der vier Erwachsenen mit der MCT Diät jedoch war nicht erfolgreich. Es fand sich keine {Correlation zwischen den EEG‐Veränderungun und dem klinischen Befund. Die drei ketogenen Diäten waren bei einer Kurzzeitbehandlung von Kindern, bei denen die Epilepsie durch Anitkonvulsiva schlecht einzustellen war, erfolgreich. RESUMEN Dietas cetogénicas en el tratamiento de la epilepsia: effectos clínicos a corto plazo El el tratamiento de 55 niños y cuatro adultos con epilepsia resistente, se uso una dieta 4:1 cetogénica, una dieta con triglicéridos de cadena mediana (TCM) y una delta TCM modificada. Durante un tratamiento de tres meses, 51 de 63 (el 81 por ciento) mostraron una reducción del >50 por ciento en las convulsiones. Se vio que las diet...

SUMMARY Children with infantile spasms, 104 boys and 46 girls, were followed up; 105 received steroids. Thirty‐three children had died, nearly all of the rest were 5 yr old or more. Of children whose development was normal before the onset of spasms, (cryptogenic and immunization groups) 37% made a full recovery and attended normal schools; early therapy with steroids seemed to be of benefit in them. Other factors of importance were a short period of spasms (less than 10 mo) and absence of other types of fit. A bad prognosis – death or severe subnormality – was associated with early onset and long duration of spasms, severe retardation on presentation, the presence of other fits, abnormal development prior to the onset of spasms, and evidence of any cerebral damage, disorder or disease. RÉSUMÉ On a suivi ľévolution ďenfants ayant présenté des spasmes infantiles, 104 garcons et 46 filles. Trente‐trois enfants sont morts et la plupart des autres avaient 5 ans ou plus. Parmi les enfants dont le développement était normal ou seulement très légèrement retardé au début des spasmes (groupe sans étiologie et après vaccination) 37% ont guéri complètement et ont suivi une scolarité normale. Une thérapeutique précoce par des stéroïdes semble etre bénéfique chez eux. Ďautres facteurs importants sont une courte période de spasmes (moins de 10 mois) et ľabsence ďautres types de crises. Un mauvais pronostic – mort ou déficience mentale sévère – associait un début précoce et une longue durée des spasmes, un retard sévère initial ou au moment de ľobservation, la présence ďautres crises, ďun développement anormal avant le début des spasmes et la présence ďune souffrance cérébrale évidente de différente nature. ZUSAMMENFASSUNG Hundertvier Jungen und 46 Mädchen mit BNS‐Krämpfen wurden langjährig beobachtet; 105 wurden mit Steroid‐Präparaten behandelt. Dreiunddreissig Kinder starben, fast alle übrigen waren bei der Untersuchung mindestens 5 Jahre alt. Von den Kindern, deren Entwicklung bis zu Beginn der Krämpfe normal verlief (Gruppen der kryptogenetischen bzw. nach Impfungen auftretenden Krämpfe), erholten sich 37% völlig und besuchten normale Schulen; die frühzeitige Therapie mit Steroidal scheint bei ihnen erfolgreich gewesen zu sein. Wichtig für dieses Ergebnis waren eine kurze Krankheitsdauer (weniger als 10 Monate) und das Fehlen anderer Anfallsformen. Zeichen für eine schlechte Prognose – Tod oder schwere cerebrale Schädigung mit Intelligenzminderung – waren: früher Beginn der Krämpfe, lange Krankheitsdauer, eine pathologische Entwicklung bzw. schwere Rückstandigkeit bei Beginn der Behandlung, das Vorhandensein anderer Anfallsformen und Zeichen jeder cerebralen Schädigung oder Erkrankung. RESUMEN Ha sido seguida la evolución de 104 niños y 46 niñas con espasmos infantiles; 33 han fallecido, mientras que casi todos los restantes tienen actualmente 5 años de edad o más. En los niños cuyo desarrollo era normal o sólo ligeramente retrasado al inicio del síndrome (grupo criptogenético y de immunización), el 37% han llegado a una co...

We have increased the production of bovine chymosin in Aspergillus niger var. awamori to more than one gram per liter of secreted authentic enzyme by combining a mutagenesis protocol with a novel robotic screening program. Analysis of the superior chymosin producing strains indicated that they have enhanced capabilities to secrete extracellular proteins.

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