Investigating the Synergistic Potential of Low-Dose HDAC3 Inhibition and Radiotherapy in Alzheimer's Disease Models.

We have previously shown that histone deacetylase (HDAC) inhibition and cranial radiotherapy (RT) independently improve molecular and behavioral Alzheimer's disease (AD)-like phenotypes. In the present study, we investigate the synergistic potential of using both RT and HDACi as a low-dose combination therapy (LDCT) to maximize disease modification (reduce neuroinflammation and amyloidogenic APP processing, increase neurotrophic gene expression) while minimizing the potential for treatment-associated side effects.LDCT consisted of daily administration of the HDAC3 inhibitor RGFP966 and/or bi-weekly cranial x-irradiation. Amyloid-beta precursor protein (APP) processing and innate immune response to LDCT were assessed in vitro and in vivo using human and murine cell models and 3xTg-AD mice. After 2 months of LDCT in mice, behavioral analyses as well as expression and modification of key AD-related targets (Aß, tau, Csf1r, Bdnf, etc.) were assessed in the hippocampus (HIP) and prefrontal cortex (PFC).LDCT induced a tolerant, anti-inflammatory innate immune response in microglia and increased non-amyloidogenic APP processing in vitro. Both RT and LDCT improved the rate of learning and spatial memory in the Barnes maze test. LDCT induced a unique anti-AD HIP gene expression profile that included upregulation of neurotrophic genes and downregulation of inflammation-related genes. RT lowered HIP Aß42/40 ratio and Bace1 protein, while LDCT lowered PFC p-tau181 and HIP Bace1 levels.Our study supports the rationale for combining complementary therapeutic approaches at low doses to target multifactorial AD pathology synergistically. Namely, LDCT with RGFP966 and cranial RT shows disease-modifying potential against a wide range of AD-related hallmarks.

Novel Lung Targeting Cell Penetrating Peptides as Vectors for Delivery of Therapeutics.

Cell penetrating peptides are unique, 5-30 amino acid long peptides that are able to breach cell membrane barriers and carry cargoes intracellularly in a functional form. Our prior work identified a synthetic, non-naturally occurring 12-amino acid long peptide that we termed cardiac targeting peptide (CTP: APWHLSSQYSRT) due to its ability to transduce cardiomyocytes in vivo. Studies looking into its mechanism of transduction identified two lung targeting peptides (LTPs), S7A (APWHLSAQYSRT) and R11A (APWHLSSQYSAT). These peptides robustly transduced human bronchial epithelial cell lines in vitro and mouse lung tissue in vivo. This uptake occurred independently of clathrin mediated endocytosis. Biodistribution studies of R11A showed peak uptake at 15 minutes with uptake in liver but not kidneys, indicating primarily a hepatobiliary mode of excretion. Cyclic version of both peptides was ~100-fold more efficient in permeating cells than their linear counterparts. As proof of principle, we conjugated anti-spike and anti-envelope SARS-CoV-2 siRNAs to cyclized R11A and demonstrate anti-viral efficacy in vitro. Our work presented here identifies two novel lung-specific cell penetrating peptides that could potentially deliver myriad therapeutic cargoes to lung tissue.

Thermal response of two sexually dimorphic Calopteryx (Odonata) over an ambient temperature range.

Organisms may internally or behaviorally regulate their body temperatures or conform to the ambient air temperatures. Previous evidence is mixed on whether wing pigmentation influences thermoregulation in various odonates.We investigated the thermal response of sympatric North American Calopteryx aequabilis and Calopteryx maculata with a thermal imaging study across a 25°C ambient temperature range.We found that regressions of thorax temperature on ambient temperature standardized by species had similar slopes for male and female C. maculata, but females were consistently 1.5°C warmer than males. In contrast, the sexes of C. aequabilis differed in slope, with C. aequabilis females having a slope less than 1.0 and males having a slope greater than 1.0.We found that regressions of thorax temperature on ambient temperature standardized by sex had similar slopes for males and females of both species, but C. maculata females were consistently 2.1°C warmer than C. aequabilis females.Given that C. aequabilis is strongly sexually dimorphic in pigment, but C. maculata is not, our findings suggest that wing pigmentation may influence thermal response rate in sympatric populations of both species.

Ligand Conjugated Multimeric siRNAs Enable Enhanced Uptake and Multiplexed Gene Silencing.

Small interfering RNAs (siRNAs) conjugated to N-acetylgalactosamine (GalNAc) ligands have been used to treat disease in patients. However, conjugates with other ligands deliver siRNA less efficiently, limiting the development of new targeted therapies. Most approaches to enhancing the potency of such conjugates have concentrated on increasing ligand effectiveness and/or the chemical stability of the siRNA drug. One complementary and unexplored alternative is to increase the number of siRNAs delivered per ligand. An ideal system would be a single chemical entity capable of delivering multiple copies of an oligonucleotide drug and/or several such drugs simultaneously. Here we report that siRNAs can be stably linked together under neutral aqueous conditions to form chemically defined siRNA "multimers," and that these multimers can be delivered in vivo by a GalNAc ligand. Conjugates containing multiple copies of the same siRNA showed enhanced activity per unit of ligand, whereas siRNAs targeting different genes linked to a single ligand facilitated multigene silencing in vivo; this is the first demonstration of silencing several genes simultaneously in vivo using ligand-directed multimeric siRNA. Multimeric oligonucleotides represent a powerful and practical new approach to improve intracellular conjugate delivery.

Clade age and not diversification rate explains species richness among animal taxa.

Animal taxa show remarkable variability in species richness across phylogenetic groups. Most explanations for this disparity postulate that taxa with more species have phenotypes or ecologies that cause higher diversification rates (i.e., higher speciation rates or lower extinction rates). Here we show that clade longevity, and not diversification rate, has primarily shaped patterns of species richness across major animal clades: more diverse taxa are older and thus have had more time to accumulate species. Diversification rates calculated from 163 species-level molecular phylogenies were highly consistent within and among three major animal phyla (Arthropoda, Chordata, Mollusca) and did not correlate with species richness. Clades with higher estimated diversification rates were younger, but species numbers increased with increasing clade age. A fossil-based data set also revealed a strong, positive relationship between total extant species richness and crown group age across the orders of insects and vertebrates. These findings do not negate the importance of ecology or phenotype in influencing diversification rates, but they do show that clade longevity is the dominant signal in major animal biodiversity patterns. Thus, some key innovations may have acted through fostering clade longevity and not by heightening diversification rate.

Evolution and the latitudinal diversity gradient: speciation, extinction and biogeography.

A latitudinal gradient in biodiversity has existed since before the time of the dinosaurs, yet how and why this gradient arose remains unresolved. Here we review two major hypotheses for the origin of the latitudinal diversity gradient. The time and area hypothesis holds that tropical climates are older and historically larger, allowing more opportunity for diversification. This hypothesis is supported by observations that temperate taxa are often younger than, and nested within, tropical taxa, and that diversity is positively correlated with the age and area of geographical regions. The diversification rate hypothesis holds that tropical regions diversify faster due to higher rates of speciation (caused by increased opportunities for the evolution of reproductive isolation, or faster molecular evolution, or the increased importance of biotic interactions), or due to lower extinction rates. There is phylogenetic evidence for higher rates of diversification in tropical clades, and palaeontological data demonstrate higher rates of origination for tropical taxa, but mixed evidence for latitudinal differences in extinction rates. Studies of latitudinal variation in incipient speciation also suggest faster speciation in the tropics. Distinguishing the roles of history, speciation and extinction in the origin of the latitudinal gradient represents a major challenge to future research.

On the elusiveness of enemy-free space: spatial, temporal, and host-plant-related variation in parasitoid attack rates on three gallmakers of goldenrods.

Host shifting by phytophagous insects may play an important role in generating insect diversity by initiating host-race formation and speciation. Models of the host shifting process often invoke reduced rates of natural enemy attack on a novel host in order to balance the maladaptation expected following the shift. Such "enemy-free space" has been documented for some insects, at some times and places, but few studies have assessed the occurrence of enemy-free space across years, among sites, or among insect species. We measured parasitoid attack rates on three insect herbivores of two goldenrods (Solidago altissima L. and Solidago gigantea Ait.), with data from multiple sites and multiple years for each herbivore. For each insect herbivore, there were times and sites at which parasitoid attack rates differed strongly and significantly between host plants (that is, enemy-free space existed on one host plant or the other). However, the extent and even the direction of the attack-rate difference varied strongly among sites and even among years at the same site. There was no evidence of consistent enemy-free space for any herbivore on either host plant. Our data suggest that enemy-free space, like many ecological and evolutionary forces, is likely to operate as a geographic and temporal mosaic, and that conceptual models of host shifting that include enemy-free space as a consequence of host novelty are likely too simple.

Simultaneous Quaternary radiations of three damselfly clades across the Holarctic.

If climate change during the Quaternary shaped the macroevolutionary dynamics of a taxon, we expect to see three features in its history: elevated speciation or extinction rates should date to this time, more northerly distributed clades should show greater discontinuities in these rates, and similar signatures of those effects should be evident in the phylogenetic and phylodemographic histories of multiple clades. In accordance with the role of glacial cycles, speciation rates increased in the Holarctic Enallagma damselflies during the Quaternary, with a 4.25x greater increase in a more northerly distributed clade as compared with a more southern clade. Finer-scale phylogenetic analyses of three radiating clades within the northern clade show similar, complex recent histories over the past 250,000 years to produce 17 Nearctic and four Palearctic extant species. All three are marked by nearly synchronous deep splits that date to approximately 250,000 years ago, resulting in speciation in two. This was soon followed by significant demographic expansions in at least two of the three clades. In two, these expansions seem to have preceded the radiations that have given rise to most of the current biodiversity. Each also produced species at the periphery of the clade's range. In spite of clear genetic support for reproductive isolation among almost all species, mtDNA signals of past asymmetric hybridization between species in different clades also suggest a role for the evolution of mate choice in generating reproductive isolation as species recolonized the landscape following deglaciation. These analyses suggest that recent climate fluctuations resulted in radiations driven by similar combinations of speciation processes acting in different lineages.

MITOCHONDRIAL DNA PHYLOGEOGRAPHY OF HOST RACES OF THE GOLDENROD BALL GALLMAKER, EUROSTA SOLIDAGINIS (DIPTERA: TEPHRITIDAE).

We determined the phylogenetic relationships and geographic distribution of mitochondrial haplotypes of two host races of the tephritid fly Eurosta solidaginis, a gallmaker that attacks species of goldenrod (Solidago). We performed a preliminary survey by sequencing 492 bp from the 3' ends of the mitochondrial cytochrome oxidase I and II subunits from a single individual from eight S. gigantea- and 10 S. altissima-associated populations across their range in eastern North America and from two outgroup species, Eurosta comma (two populations) and E. cribrata. Eurosta solidaginis haplotypes fell into two groups ("E" and "W" clades), which differed by four substitutions, one of which occurred within the recognition site of the DdeI restriction enzyme. We used the presence or absence of the restriction site to survey a total of 11 S. gigantea (20 individuals) and 20 S. altissima (43 individuals) host-race populations. All gigantea-fly haplotypes regardless of geographic origin carried the E-clade haplotype, whereas altissima-fly haplotypes were geographically partitioned. Altissima flies east of Michigan were of haplotype E, whereas those west of Michigan were of haplotype W, with mixed populations found in lower Michigan. These patterns confirm an earlier allozyme survey that suggested that S. altissima is the ancestral host for the gallmaker, but also suggest that the gigantea fly populations were derived from eastern U.S. altissima fly populations. The data support the conclusions of behavioral and ecological studies indicating that the shift to the derived host was facilitated by escape from natural enemies.