Biology subjects

Moreno, A. M.

Publications and source records attributed to Moreno, A. M..

3 recordsLinked to original sources

bioRxiv2026

Mobilome of Enterococcus faecalis from healthy nursery pigs exposed to antibiotic pressure

In response to the comparatively sudden application of industrial scale levels of antibiotics to an ecosystem where naturally produced antibiotics are scarce, namely the ecologies within and around agricultural settings, animal-associated microbes have had to rapidly adapt, mostly relying on mobile genetic elements (MGEs) taken up due to loss of CRISPR functionality. Due to selection for resistance and other adaptive traits carried by dynamic and rapidly recombining MGEs, plasmids and transposons have rapidly accumulated in this human-proximal environment. Because of the occurrence of Enterococcus faecalis in a wide range of hosts up and down the food chain, and the fact that this species represents the greatest generalist of the genus, we comprehensively examined the mobilome of multidrug-resistant E. faecalis (ST330, ST591, ST710, and ST711) from healthy piglets raised on dispersed Brazilian farms, using long-read sequencing, analysis of plasmid pangenomes, and conjugation assays with these strains serving as donors. Genomes ranged from [~]2.8-3.1 Mb, with diverse MGEs constituting [~]7-15% of those genomes. Large modular antimicrobial resistance-encoding gene blocks ([~]40 Kb) were observed to be integrated into a [~]67 Kb chromosomal segment of the pathogenicity island AF454824. Prophages contributed up to 70% of the chromosomal mobile element content, integrating into both CRISPR-deficient genomes and those with intact type II-A CRISPR1 arrays, which were enriched with Caudoviricetes phage-targeting spacers across all strains. Plasmid content showed pronounced mosaicism driven by diverse insertion sequences, transposons, and related mobile elements, many directly implicated in AMR gene cluster acquisitions. RepA_N, Inc18, and Rep3 plasmids, mostly conjugative, also carried various persistence-related traits, suggesting they may actively enhance agricultural fitness rather than passively accumulate due to loss of CRISPR protection.

microbiology↗

bioRxiv2019

Long-lasting Analgesia via Targeted in vivo Epigenetic Repression of Nav1.7

Current treatments for chronic pain rely largely on opioids despite their unwanted side effects and risk of addiction. Genetic studies have identified in humans key targets pivotal to nociceptive processing, with the voltage-gated sodium channel, NaV1.7 (SCN9A), being perhaps the most promising candidate for analgesic drug development. Specifically, a hereditary loss-of-function mutation in NaV1.7 leads to insensitivity to pain without other neurodevelopmental alterations. However, the high sequence similarity between NaV subtypes has frustrated efforts to develop selective inhibitors. Here, we investigated targeted epigenetic repression of NaV1.7 via genome engineering approaches based on clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9 and zinc finger proteins as a potential treatment for chronic pain. Towards this end, we first optimized the efficiency of NaV1.7 repression in vitro in Neuro2A cells, and then by the lumbar intrathecal route delivered both genome-engineering platforms via adeno-associated viruses (AAVs) to assess their effects in three mouse models of pain: carrageenan-induced inflammatory pain, paclitaxel-induced neuropathic pain and BzATP-induced pain. Our results demonstrate: one, effective repression of NaV1.7 in lumbar dorsal root ganglia; two, reduced thermal hyperalgesia in the inflammatory state; three, decreased tactile allodynia in the neuropathic state; and four, no changes in normal motor function. We anticipate this genomically scarless and non-addictive pain amelioration approach enabling Long-lasting Analgesia via Targeted in vivo Epigenetic Repression of Nav1.7, a methodology we dub pain LATER, will have significant therapeutic potential, such as for preemptive administration in anticipation of a pain stimulus (pre-operatively), or during an established chronic pain state.\n\nOne sentence summaryIn situ epigenome engineering approach for genomically scarless, durable, and non-addictive management of pain.

genetics↗

bioRxiv2018

Exploring protein orthogonality in immune space: a case study with AAV and Cas9 orthologs

A major hurdle in protein-based therapeutics is the interaction with the adaptive immune system, which can lead to neutralization by circulating antibodies and clearance of treated cells by cytotoxic T-lymphocytes. One method of circumventing these issues is to use human or humanized proteins which avoid the immune response by self-recognition. However, this approach limits potential protein therapeutics to those of human origin, excluding many exciting effectors and delivery vehicles such as CRISPR-Cas9 and adeno-associated viruses (AAVs). To address this issue, we propose here the sequential use of orthologous proteins whose function is constrained by natural selection, but whose structure is subject to diversification by genetic drift. This would, in principle, allow for repeated treatments by immune orthogonal orthologs without reduced efficacy due to lack of immune cross-reactivity among the proteins. To explore and validate this concept we chose 91 Type II CRISPR-Cas9 orthologs and 167 AAV capsid protein orthologs, and developed a pipeline to compare total sequence similarity as well as predicted binding to class I and class II Major Histocompatibility Complex (MHC) proteins. Interestingly, MHC binding predictions revealed wide diversity among the set of Cas9 orthologs, with 83% of pairs predicted to have non cross-reacting immune responses, while no global immune orthogonality among AAV serotypes was observed. To confirm these findings we selected two Cas9 orthologs, from S. pyogenes and S. aureus, predicted to be orthogonal in immune space, and delivered them into mice via multiple AAV serotypes. We observed cross-reacting antibodies against AAV but not Cas9 orthologs in sera from immunized mice, validating the computationally predicted immune orthogonality among these proteins. Moving forward, we anticipate this framework can be applied to prescribe sequential regimens of immune orthogonal protein therapeutics to circumvent pre-existing or induced immunity, and eventually, to rationally engineer immune orthogonality among protein orthologs.

synthetic biology↗