Time-restricted ketogenic diet in amyotrophic lateral sclerosis: a case study.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disorder. The most devastating variant is bulbar-onset ALS, which portends a median survival of 24 months from the onset of symptoms. Abundant evidence indicates that neuron metabolism and mitochondrial function are impaired in ALS. Metabolic strategies, particularly fasting and ketogenic diet protocols, alter neuron metabolism and mitochondria function in a manner that may mitigate the symptoms of this disorder. We report the case of a 64-year-old man with a 21-month history of progressive, deteriorating bulbar-onset ALS, with an associated pseudobulbar affect, who implemented a time-restricted ketogenic diet (TRKD) for 18 months. During this time, he improved in ALS-related function (7% improvement from baseline), forced expiratory volume (17% improvement), forced vital capacity (13% improvement), depression (normalized), stress levels (normalized), and quality of life (19% improvement), particularly fatigue (23% improvement). His swallowing impairment and neurocognitive status remained stable. Declines were measured in physical function, maximal inspiratory pressure, and maximal expiratory pressure. Weight loss was attenuated and no significant adverse effects occurred. This case study represents the first documented occurrence of a patient with ALS managed with either a fasting or ketogenic diet protocol, co-administered as a TRKD. We measured improved or stabilized ALS-related function, forced expiratory volume, forced vital capacity, swallowing, neurocognitive status, mood, and quality of life. Measurable declines were restricted to physical function, maximal inspiratory pressure, and maximal expiratory pressure. Now over 45 months since symptom onset, our patient remains functionally independent and dedicated to his TRKD.

The bristle patterning genes hairy and extramacrochaetae regulate the development of structures required for flight in Diptera.

The distribution of sensory bristles on the thorax of Diptera (true flies) provides a useful model for the study of the evolution of spatial patterns. Large bristles called macrochaetes are arranged into species-specific stereotypical patterns determined via spatially discrete expression of the proneural genes achaete-scute (ac-sc). In Drosophila ac-sc expression is regulated by transcriptional activation at sites where bristle precursors develop and by repression outside of these sites. Three genes, extramacrochaetae (emc), hairy (h) and stripe (sr), involved in repression have been documented. Here we demonstrate that in Drosophila, the repressor genes emc and h, like sr, play an essential role in the development of structures forming part of the flight apparatus. In addition we find that, in Calliphora vicina a species diverged from D. melanogaster by about 100 Myr, spatial expression of emc, h and sr is conserved at the location of development of those structures. Based on these findings we argue, first, that the role emc, h and sr in development of the flight apparatus preceded their activities for macrochaete patterning; second, that species-specific variation in activation and repression of ac-sc expression is evolving in parallel to establish a unique distribution of macrochaetes in each species.

Diversity of transposable elements and repeats in a 600 kb region of the fly Calliphora vicina.

BACKGROUND: Transposable elements (TEs) are a very dynamic component of eukaryotic genomes with important implications (e.g., in evolution) and applications (e.g., as transgenic tools). They also represent a major challenge for the assembly and annotation of genomic sequences. However, they are still largely unknown in non-model species. RESULTS: Here, we have annotated the repeats and transposable elements present in a 600 kb genomic region of the blowfly Calliphora vicina (Diptera: Calliphoridae) which contains most of the achaete-scute gene complex of this species. This is the largest genomic region to be sequenced and analyzed in higher flies outside the Drosophila genus. We find that the repeat content spans at least 24% of the sequence. It includes 318 insertions classified as 3 LTR retrotransposons, 21 LINEs, 14 cut-and-paste DNA transposons, 4 helitrons and 33 unclassified repeats. CONCLUSIONS: This is the most detailed description of TEs and repeats in the Calliphoridae to date. This contribution not only adds to our knowledge about TE evolution but will also help in the annotation of repeats on Dipteran whole genome sequences.

The kinase Sgg modulates temporal development of macrochaetes in Drosophila by phosphorylation of Scute and Pannier.

Evolution of novel structures is often made possible by changes in the timing or spatial expression of genes regulating development. Macrochaetes, large sensory bristles arranged into species-specific stereotypical patterns, are an evolutionary novelty of cyclorraphous flies and are associated with changes in both the temporal and spatial expression of the proneural genes achaete (ac) and scute (sc). Changes in spatial expression are associated with the evolution of cis-regulatory sequences, but it is not known how temporal regulation is achieved. One factor required for ac-sc expression, the expression of which coincides temporally with that of ac-sc in the notum, is Wingless (Wg; also known as Wnt). Wingless downregulates the activity of the serine/threonine kinase Shaggy (Sgg; also known as GSK-3). We demonstrate that Scute is phosphorylated by Sgg on a serine residue and that mutation of this residue results in a form of Sc with heightened proneural activity that can rescue the loss of bristles characteristic of wg mutants. We suggest that the phosphorylated form of Sc has reduced transcriptional activity such that sc is unable to autoregulate, an essential function for the segregation of bristle precursors. Sgg also phosphorylates Pannier, a transcriptional activator of ac-sc, the activity of which is similarly dampened when in the phosphorylated state. Furthermore, we show that Wg signalling does not act directly via a cis-regulatory element of the ac-sc genes. We suggest that temporal control of ac-sc activity in cyclorraphous flies is likely to be regulated by permissive factors and might therefore not be encoded at the level of ac-sc gene sequences.

Liberation and containment: re-visualising the eugenic and evolutionary ideal of the "Fizkul'turnitsa" in 1944.

In July 1944 cross-country races and parades of physical culturists were prominently used to celebrate Soviet liberation from German occupation. While journalistic accounts stressed the manly health and vigour of the victorious Red Army, press photographs in Pravda and Red Sport, and Aleksandr Deneika's monumental painting 'Liberation', emphasised images of the young female physical culturist. This essay explores what a contextualised analysis of these images may have to tell historians about the connections between women, physical culture and liberation being projected. The argument suggests that, on one level, the images straightforwardly symbolised and celebrated the liberation of the Soviet 'Motherland'. On another, more complex level, the images represented a particularly nuanced notion of constricted liberation for Soviet women deriving from 1920s eugenic and evolutionary discourse, inscribed into the contemporary imperative for engagement with physical culture as a necessary stage of healthful body discipline on the path to hygienic and successful motherhood.

An arthropod cis-regulatory element functioning in sensory organ precursor development dates back to the Cambrian.

BACKGROUND: An increasing number of publications demonstrate conservation of function of cis-regulatory elements without sequence similarity. In invertebrates such functional conservation has only been shown for closely related species. Here we demonstrate the existence of an ancient arthropod regulatory element that functions during the selection of neural precursors. The activity of genes of the achaete-scute (ac-sc) family endows cells with neural potential. An essential, conserved characteristic of proneural genes is their ability to restrict their own activity to single or a small number of progenitor cells from their initially broad domains of expression. This is achieved through a process called lateral inhibition. A regulatory element, the sensory organ precursor enhancer (SOPE), is required for this process. First identified in Drosophila, the SOPE contains discrete binding sites for four regulatory factors. The SOPE of the Drosophila asense gene is situated in the 5' UTR. RESULTS: Through a manual comparison of consensus binding site sequences we have been able to identify a SOPE in UTR sequences of asense-like genes in species belonging to all four arthropod groups (Crustacea, Myriapoda, Chelicerata and Insecta). The SOPEs of the spider Cupiennius salei and the insect Tribolium castaneum are shown to be functional in transgenic Drosophila. This would place the origin of this regulatory sequence as far back as the last common ancestor of the Arthropoda, that is, in the Cambrian, 550 million years ago. CONCLUSIONS: The SOPE is not detectable by inter-specific sequence comparison, raising the possibility that other ancient regulatory modules in invertebrates might have escaped detection.

Evolution of the achaete-scute complex in insects: convergent duplication of proneural genes.

Proneural genes encode transcriptional activators of the basic Helix-loop-helix class that are involved in neuronal specification and differentiation. We have used the recent availability of genome sequences of multiple distant insect species to study the evolution of a family of proneural genes, the achaete-scute genes, and to examine their genomic organization and evolution. We document independent evolution of multiple copies of achaete-scute homologues and argue that this might have contributed to morphological diversity in Diptera and Lepidoptera.

Redundant mechanisms mediate bristle patterning on the Drosophila thorax.

The thoracic bristle pattern of Drosophila results from the spatially restricted expression of the achaete-scute (ac-sc) genes in clusters of cells, mediated by the activity of many discrete cis-regulatory sequences. However, ubiquitous expression of sc or asense (ase) achieved with a heterologous promoter, in the absence of endogenous ac-sc expression, and the activity of the cis-regulatory elements, allows the development of bristles positioned at wild-type locations. We demonstrate that the products of the genes stripe, hairy, and extramacrochaetae contribute to rescue by antagonizing the activity of Sc and Ase. The three genes are expressed in specific but overlapping spatial domains of expression that form a prepattern that allows precise positioning of bristles. The redundant mechanisms might contribute to the robustness of the pattern. We discuss the possibility that patterning in trans by antagonism is ancestral and that the positional cis-regulatory sequences might be of recent origin.

Evolution of cis-regulatory sequences in Drosophila.

Altered expression of genes during development is one mechanism that might underlie morphological diversity in animals. Comparison has shown that differences in gene expression often correlate with differences in morphology between species. However, many of these examples involve slowly evolving traits between widely diverged taxa, making investigation of how such changes came about all but impossible. Changes in expression of a specific gene can be due to changes in the activity of trans-acting regulatory factors or to evolution of the cis-acting sequences of the gene itself. A number of studies indicate that cis-regulatory regions can undergo significant sequence turnover even when their function is maintained. In other cases, however, regulatory regions of considerable sequence similarity mediate a different gene expression pattern. These observations make it difficult to predict a change in transcriptional output from an examination of the sequence alone. Here we review recent observations on the evolution of cis-regulatory sequences between Drosophila species and some other species of dipteran flies. We look at specific cases that have been investigated in detail, where the function of the regulatory element is either maintained or has evolved to mediate a different transcriptional pattern of gene activity. The examples chosen illustrate the necessity to identify the interactions between the proteins that bind the element, and to verify binding sites through in vivo assays. Although the number of such studies is still small, they suggest that changes in gene regulation might be an important factor in the evolution of animal morphology.

NF-kappaB/Rel-mediated regulation of the neural fate in Drosophila.

Two distinct roles are described for Dorsal, Dif and Relish, the three NF-kappaB/Rel proteins of Drosophila, in the development of the peripheral nervous system. First, these factors regulate transcription of scute during the singling out of sensory organ precursors from clusters of cells expressing the proneural genes achaete and scute. This effect is possibly mediated through binding sites for NF-kappaB/Rel proteins in a regulatory module of the scute gene required for maintenance of scute expression in precursors as well as repression in cells surrounding precursors. Second, genetic evidence suggests that the receptor Toll-8, Relish, Dif and Dorsal, and the caspase Dredd pathway are active over the entire imaginal disc epithelium, but Toll-8 expression is excluded from sensory organ precursors. Relish promotes rapid turnover of transcripts of the target genes scute and asense through an indirect, post-transcriptional mechanism. We propose that this buffering of gene expression levels serves to keep the neuro-epithelium constantly poised for neurogenesis.

The stars and stripes of animal bodies: evolution of regulatory elements mediating pigment and bristle patterns in Drosophila.

Evolution has generated enormous morphological diversity in animals and one of the genetic processes that might have contributed to this is evolution of the cis-regulatory sequences responsible for the temporal and spatial expression of genes regulating embryonic development. This could be particularly relevant to pleiotropic genes with multiple independently acting regulatory modules. Loss or gain of modules enables altered expression without loss of other functions. Here I focus on recent studies correlating differences in morphological traits between related species of Drosophila to changes in cis-regulatory sequences. They show that ancestral regulatory modules have evolved to mediate different transcriptional outputs and suggest that evolution of cis-regulatory sequences might reflect a general mechanism driving evolutionary change.

Two or four bristles: functional evolution of an enhancer of scute in Drosophilidae.

Changes in cis-regulatory sequences are proposed to underlie much of morphological evolution. Yet, little is known about how such modifications translate into phenotypic differences. To address this problem, we focus on the dorsocentral bristles of Drosophilidae. In Drosophila melanogaster, development of these bristles depends on a cis-regulatory element, the dorsocentral enhancer, to activate scute in a cluster of cells from which two bristles on the posterior scutum arise. A few species however, such as D. quadrilineata, bear anterior dorsocentral bristles as well as posterior ones, a derived feature. This correlates with an anterior expansion of the scute expression domain. Here, we show that the D. quadrilineata enhancer has evolved, and is now active in more anterior regions. When used to rescue scute expression in transgenic D. melanogaster, the D. quadrilineata enhancer is able to induce anterior bristles. Importantly, these properties are not displayed by homologous enhancers from control species bearing only two posterior bristles. We also provide evidence that upstream regulation of the enhancer, by the GATA transcription factor Pannier, has been evolutionarily conserved. This work illustrates how, in the context of a conserved trans-regulatory landscape, evolutionary tinkering of pre-existing enhancers can modify gene expression patterns and contribute to morphological diversification.

Conservation of upstream regulators of scute on the notum of cyclorraphous Diptera.

Bristles on the notum of many cyclorraphous flies are arranged into species-specific stereotyped patterns. Differences in the spatial expression of the proneural gene scute correlate with the positions of bristles in those species looked at so far. However, the examination of a number of genes encoding trans-regulatory factors, such as pannier, stripe, u-shaped, caupolican and wingless, indicates that they are expressed in conserved domains on the prospective notum. This suggests that the function of a trans-regulatory network of genes is relatively unchanged in derived Diptera, and that many differences are likely to be due to changes in cis-regulatory sequences of scute. In contrast, in Anopheles gambiae, a basal species with no stereotyped bristle pattern, the expression patterns of pannier and wingless are not conserved, and expression of AgASH, the Anopheles proneural gene, does not correlate in a similar manner with the bristle pattern. We discuss the possibility that independently acting cis-regulatory sequences at the scute locus may have arisen in the lineage giving rise to cyclorraphous flies.

A conserved trans-regulatory landscape for scute expression on the notum of cyclorraphous Diptera.

Bristles on the notum of many cyclorraphous flies are arranged into species-specific stereotyped patterns. The positions of bristles correlate with differences in the spatial expression of the scute (sc) gene in those species examined so far. However, a major upstream activator of scute, Pannier (Pnr), is expressed in a conserved domain over the entire medial notum. Here we examine the expression patterns in Calliphora vicina of stripe (sr), u-shaped (ush), caupolican (caup) and wingless (wg), genes known to modify the activity of Pnr or to act downstream of Pnr in Drosophila. We find that, with minor differences, their expression patterns are conserved. This suggests that the function of a trans-regulatory network of genes is relatively unchanged in derived Diptera and that many differences are likely to be due to changes in cis-regulatory sequences of scute.

A major bristle QTL from a selected population of Drosophila uncovers the zinc-finger transcription factor poils-au-dos, a repressor of achaete-scute.

Traditional screens aiming at identifying genes regulating development have relied on mutagenesis. Here, we describe a new gene involved in bristle development, identified through the use of natural variation and selection. Drosophila melanogaster bears a pattern of 11 macrochaetes per heminotum. From a population initially sampled in Marrakech, a strain was selected for an increased number of thoracic macrochaetes. Using recombination and single nucleotide polymorphisms, the factor responsible was mapped to a single locus on the third chromosome, poils au dos, that encodes a zinc-finger-ZAD protein. The original, as well as new, presumed null, alleles of poils au dos, is associated with ectopic achaete-scute expression that results in the additional bristles. This suggests a possible role for Poils au dos as a repressor of achaete and scute. Ectopic expression appears to be independent of the activity of known cis-regulatory enhancer sequences at the achaete-scute complex that mediate activation at specific sites on the notum. The target sequences for Poils au dos activity were mapped to a 14 kb region around scute. In addition, we show that pad interacts synergistically with the repressor hairy and with Dpp signaling in posterior and anterior regions of the notum, respectively.

achaete, but not scute, is dispensable for the peripheral nervous system of Drosophila.

The achaete-scute complex of Drosophila has been the focus of extensive genetic and developmental analysis. Of the four genes at this locus, achaete and scute appear to act redundantly to specify the peripheral nervous system. They share cis-regulatory elements and are co-expressed at the same locations. A mutation removing scute activity has been previously described; it causes a loss of some sensory bristles. Thus, when Scute is absent, the activity of achaete allows formation of the remaining bristles. However, all existing achaete mutants are rearrangements affecting regulatory sequences common to both achaete and scute. To determine the level of redundancy between the two genes, we have used a P element approach to generate a null allele of achaete, which leaves scute and all cis-regulatory elements intact. We find that the peripheral nervous system of achaete null mutant larvae and imagos lacks any detectable phenotype. However, when the levels of Scute are limiting, then some sensory organs are missing in achaete mutant flies. achaete and scute are thought to have arisen from a duplication event about 100 Myr ago. The difference between achaete and scute null flies is surprising and raises the question of the retention of both genes during the course of evolution.

Evolution of early development of the nervous system: a comparison between arthropods.

Large numbers of cells with unique neuronal specificity are generated during development of the central nervous system of animals. Here we discuss the events that generate cell diversity during early development of the ventral nerve cord of different arthropod groups. Neural precursors are generated in a spatial array in the epithelium of each hemisegment over a period of time. Spatial cues within the epithelium are thought to evolve as embryogenesis proceeds. This spatiotemporal information might generate diversity among the neural precursors in all arthropod groups, although the mechanisms regulating the positioning of individual precursors have diverged. However, distinct strategies for the generation of neuronal diversity have evolved in the different arthropod lineages that appear to correlate with specific modes of ontogenesis. We hypothesize that an evolutionary trend towards reduced cell numbers and possibly rapid embryogenesis in insects has culminated in the appearance of stereotyped neuroblast lineages.

Differences in sensory projections between macro- and microchaetes in Drosophilid flies.

From examination of the central axonal projections of sensory bristles on the notum of several species of Drosophilidae, we demonstrate different features that may indicate different functions for macro- and microchaetes. The large macrochaetes have conserved arborizations that correlate with their conserved position. Nevertheless, we find evidence for only two discrete projection patterns for bristles in the dorsocentral (DC) row, even when there may be four or five bristles present. We show that the small microchaetes of Drosophila melanogaster display regional specificity and subsets of contiguous bristles project to a common region in the thoracic ganglion. Interestingly, the axons of each of these subsets also form a specific fasciculation group on the scutum before joining the axon of a particular macrochaete. The positions of microchaetes on the scutum and the shape of the fasciculation groups vary between closely related species. There is no correlation between body size, bristle patterns, and fasciculation patterns. Furthermore, none of these traits correlate with the phylogenetic relationships between the species studied. We discuss the possibility that macro- and microchaetes may have different functions and that these have implications for evolutionary constraints on bristle patterns.

Mutual exclusion of sensory bristles and tendons on the notum of dipteran flies.

BACKGROUND: Genes of the achaete-scute complex encode transcription factors whose activity regulates the development of neural cells. The spatially restricted expression of achaete-scute on the mesonotum of higher flies governs the development and positioning of the large sensory bristles. On the scutum the bristles are arranged into conserved patterns, based on an ancestral arrangement of four longitudinal rows. This pattern appears to date back to the origin of cyclorraphous flies about 100-140 million years ago. The origin of the four-row bauplan, which is independent of body size, and the reasons for its conservation, are not known. RESULTS: We report that tendons for attachment of the indirect flight muscles are invariably located between the bristle rows of the scutum throughout the Diptera. Tendon development depends on the activity of a transcription factor encoded by the gene stripe. In Drosophila, stripe and achaete-scute have separate expression domains, leading to spatial segregation of tendon precursors and bristle precursors. Furthermore the products of these genes act antagonistically: ectopic sr expression prevents bristle development and ectopic sc expression prevents normal muscle attachment. The product of stripe acts downstream of Achaete-Scute and interferes with the development of bristle precursors. CONCLUSIONS: The pattern of flight muscles has changed little throughout the Diptera and we argue that the sites of muscle attachment may have constrained the positioning of bristles during the course of evolution. This could account for the pattern of four bristle rows on the scutum.