Delivering genes to and across the brain vasculature efficiently and specifically across species remains a critical challenge for addressing neurological diseases. We have evolved adeno-associated ...virus (AAV9) capsids into vectors that transduce brain endothelial cells specifically and efficiently following systemic administration in wild-type mice with diverse genetic backgrounds, and in rats. These AAVs also exhibit superior transduction of the CNS across non-human primates (marmosets and rhesus macaques), and in ex vivo human brain slices, although the endothelial tropism is not conserved across species. The capsid modifications translate from AAV9 to other serotypes such as AAV1 and AAV-DJ, enabling serotype switching for sequential AAV administration in mice. We demonstrate that the endothelial-specific mouse capsids can be used to genetically engineer the blood-brain barrier by transforming the mouse brain vasculature into a functional biofactory. We apply this approach to Hevin knockout mice, where AAV-X1-mediated ectopic expression of the synaptogenic protein Sparcl1/Hevin in brain endothelial cells rescued synaptic deficits.
Gene therapy offers great promise in addressing neuropathologies associated with the central and peripheral nervous systems (CNS and PNS). However, genetic access remains difficult, reflecting the ...critical need for the development of effective and non-invasive gene delivery vectors across species. To that end, we evolved adeno-associated virus serotype 9 (AAV9) capsid in mice and validated two capsids, AAV-MaCPNS1 and AAV-MaCPNS2, across rodent species (mice and rats) and non-human primate (NHP) species (marmosets and rhesus macaques). Intravenous administration of either AAV efficiently transduced the PNS in rodents and both the PNS and CNS in NHPs. Furthermore, we used AAV-MaCPNS1 in mice to systemically deliver the following: (1) the neuronal sensor jGCaMP8s to record calcium signal dynamics in nodose ganglia and (2) the neuronal actuator DREADD to dorsal root ganglia to mediate pain. This conclusively demonstrates the translatability of these two systemic AAVs across four species and their functional utility through proof-of-concept studies in mice.
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•Directed evolution in rodents identified AAV vectors targeting the PNS•Systemically administered MaCPNS1 and MaCPNS2 efficiently target the PNS in rodents•Systemic MaCPNS1 is used for functional readout and non-invasive modulation of the PNS•Systemic MaCPNS1 and MaCPNS2 transduce both the PNS and CNS in macaque and marmoset
Chen et al. evolved a family of AAV capsid variants, including MaCPNS1 and MaCPNS2, that efficiently transduced the PNS in rodents following systemic administration, enabling functional readout and non-invasive modulation of PNS. Both vectors could also enable efficient gene delivery to both PNS and CNS in macaque and marmoset.
The blood-brain barrier (BBB) presents a major challenge for delivering large molecules to study and treat the central nervous system. This is due in part to the scarcity of targets known to mediate ...BBB crossing. To identify novel targets, we leverage a panel of adeno-associated viruses (AAVs) previously identified through mechanism-agnostic directed evolution for improved BBB transcytosis. Screening potential cognate receptors for enhanced BBB crossing, we identify two targets: murine-restricted LY6C1 and widely conserved carbonic anhydrase IV (CA-IV). We apply AlphaFold-based in silico methods to generate capsid-receptor binding models to predict the affinity of AAVs for these identified receptors. Demonstrating how these tools can unlock target-focused engineering strategies, we create an enhanced LY6C1-binding vector, AAV-PHP.eC, that, unlike our prior PHP.eB, also works in
-deficient mouse strains such as BALB/cJ. Combined with structural insights from computational modeling, the identification of primate-conserved CA-IV enables the design of more specific and potent human brain-penetrant chemicals and biologicals, including gene delivery vectors.
Microbial communities are commonly studied using culture-independent methods, such as 16S rRNA gene sequencing. However, one challenge in accurately characterizing microbial communities is exogenous ...bacterial DNA contamination, particularly in low-microbial-biomass niches. Computational approaches to identify contaminant sequences have been proposed, but their performance has not been independently evaluated. To identify the impact of decreasing microbial biomass on polymicrobial 16S rRNA gene sequencing experiments, we created a mock microbial community dilution series. We evaluated four computational approaches to identify and remove contaminants, as follows: (i) filtering sequences present in a negative control, (ii) filtering sequences based on relative abundance, (iii) identifying sequences that have an inverse correlation with DNA concentration implemented in Decontam, and (iv) predicting the sequence proportion arising from defined contaminant sources implemented in SourceTracker. As expected, the proportion of contaminant bacterial DNA increased with decreasing starting microbial biomass, with 80.1% of the most diluted sample arising from contaminant sequences. Inclusion of contaminant sequences led to overinflated diversity estimates and distorted microbiome composition. All methods for contaminant identification successfully identified some contaminant sequences, which varied depending on the method parameters used and contaminant prevalence. Notably, removing sequences present in a negative control erroneously removed >20% of expected sequences. SourceTracker successfully removed over 98% of contaminants when the experimental environments were well defined. However, SourceTracker misclassified expected sequences and performed poorly when the experimental environment was unknown, failing to remove >97% of contaminants. In contrast, the Decontam frequency method did not remove expected sequences and successfully removed 70 to 90% of the contaminants.
The relative scarcity of microbes in low-microbial-biomass environments makes accurate determination of community composition challenging. Identifying and controlling for contaminant bacterial DNA are critical steps in understanding microbial communities from these low-biomass environments. Our study introduces the use of a mock community dilution series as a positive control and evaluates four computational strategies that can identify contaminants in 16S rRNA gene sequencing experiments in order to remove them from downstream analyses. The appropriate computational approach for removing contaminant sequences from an experiment depends on prior knowledge about the microbial environment under investigation and can be evaluated with a dilution series of a mock microbial community.
Most human infectious diseases, especially recently emerging pathogens, originate from animals, and ongoing disease transmission from animals to people presents a significant global health burden. ...Recognition of the epidemiologic circumstances involved in zoonotic spillover, amplification, and spread of diseases is essential for prioritizing surveillance and predicting future disease emergence risk. We examine the animal hosts and transmission mechanisms involved in spillover of zoonotic viruses to date, and discover that viruses with high host plasticity (i.e. taxonomically and ecologically diverse host range) were more likely to amplify viral spillover by secondary human-to-human transmission and have broader geographic spread. Viruses transmitted to humans during practices that facilitate mixing of diverse animal species had significantly higher host plasticity. Our findings suggest that animal-to-human spillover of new viruses that are capable of infecting diverse host species signal emerging disease events with higher pandemic potential in that these viruses are more likely to amplify by human-to-human transmission with spread on a global scale.
Global patterns in coronavirus diversity Anthony, Simon J; Johnson, Christine K; Greig, Denise J ...
Virus evolution,
01/2017, Letnik:
3, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Abstract
Since the emergence of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrom Coronavirus (MERS-CoV) it has become increasingly clear that bats are ...important reservoirs of CoVs. Despite this, only 6% of all CoV sequences in GenBank are from bats. The remaining 94% largely consist of known pathogens of public health or agricultural significance, indicating that current research effort is heavily biased towards describing known diseases rather than the ‘pre-emergent’ diversity in bats. Our study addresses this critical gap, and focuses on resource poor countries where the risk of zoonotic emergence is believed to be highest. We surveyed the diversity of CoVs in multiple host taxa from twenty countries to explore the factors driving viral diversity at a global scale. We identified sequences representing 100 discrete phylogenetic clusters, ninety-one of which were found in bats, and used ecological and epidemiologic analyses to show that patterns of CoV diversity correlate with those of bat diversity. This cements bats as the major evolutionary reservoirs and ecological drivers of CoV diversity. Co-phylogenetic reconciliation analysis was also used to show that host switching has contributed to CoV evolution, and a preliminary analysis suggests that regional variation exists in the dynamics of this process. Overall our study represents a model for exploring global viral diversity and advances our fundamental understanding of CoV biodiversity and the potential risk factors associated with zoonotic emergence.
Climate change is a defining challenge of the 21st century, and this decade is a critical time for action to mitigate the worst effects on human populations and ecosystems. Plant science can play an ...important role in developing crops with enhanced resilience to harsh conditions (e.g. heat, drought, salt stress, flooding, disease outbreaks) and engineering efficient carbon-capturing and carbon-sequestering plants. Here, we present examples of research being conducted in these areas and discuss challenges and open questions as a call to action for the plant science community.
Nerve transfers for peripheral nerve injuries can result in variable outcomes. We investigated the neuroprotective effect of epineurial lidocaine injection in the donor nerve prior to transection, ...with the hypothesis that proximal axon loss would be decreased with consequent increased neuroregeneration and functional recovery.
A rat sciatic nerve model was used with 4 intervention groups: (1) lidocaine; (2) lidocaine/calcium gluconate (CG); (3) CG; or (4) saline (control). Behavioral testing and qualitative and quantitative histological evaluation was performed at 8 and 12 weeks. Histological assays included transmission electron microscopy, retrograde fluorogold labeling, and whole mount immunostaining.
Functional assessments through the sciatic functional index and Basso, Beattie, and Bresnahan scale showed a statistically significant increase in recovery at 8 and 12 weeks with lidocaine treatment. Significantly higher axonal counts were obtained in the lidocaine-treated groups. Fragmentation and increased myelin damage was present in the CG and saline groups. Retrograde fluorogold labeling showed a statistically significant increase in the number of L4-6 dorsal root ganglion neurons in the lidocaine-treated groups. Whole mount immunostaining identified extension of the axonal growth cone past the nerve coaptation site in lidocaine-treated groups, but not in CG and saline groups.
Our results suggest that epineurial lidocaine injection prior to donor nerve transection for nerve transfer has a neuroprotective effect, resulting in increased proximal axon counts and improved functional recovery.
These findings may have direct clinical application because epineurial lidocaine can be used in surgery as a simple and inexpensive intervention for promoting improved clinical outcomes after nerve transfer.
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