Biology subjects

Boyd, H.

Publications and source records attributed to Boyd, H..

7 recordsLinked to original sources

bioRxiv2026

A Minimal Packaging Signal Enables Production of High-Purity Phage-Like Particles for CRISPR-Cas Antimicrobials in Staphylococcus aureus

Precision phage therapeutics provide a promising strategy to combat multidrug-resistant pathogens, including Staphylococcus aureus. Efficient, specific packaging of genetic cargoes remains challenging. Using modular design principles, we report a minimal phagemid packaging signal consisting of the phage terminase small subunit under its native promoter that significantly outperforms conventional packaging signals. The utility of this synthetic terS operon was demonstrated through production of highly concentrated and genetically pure CRISPR-Cas antimicrobials. To circumvent CRISPR-mediated self-targeting during antimicrobial generation, a terS-deficient strain was engineered to express the anti-CRISPR protein AcrIIA4, enabling titers above 1010 transducing units per milliliter (TRU/mL) with over 94% purity. With a high-copy origin of replication module, CRISPR-Cas phage-like particle titers could approach 1012 TRU/mL. We discovered that pure CRISPR-Cas antimicrobials are potent and can be amplified in hosts possessing prophages. Taken altogether, this study defines the minimal and optimal genetic requirements for efficient, specific creation of phage-based technologies. Technological ReadinessThe described system for engineering phage-like particles has reached a technological readiness level (TRL) of 4-5 based on the provided laboratory validation and strong literature support from other engineered phage therapies applied to in vivo models. Previous systems have demonstrated high-purity phage-like particle preparation, but always at the cost of severely reduced productivity. Therefore, our demarcation of a specific, efficient, and minimal system for packaging nucleic acid cargoes into phage vectors is a critical step toward real-world use. Despite this, low levels of contaminating host/phage DNA remain a key barrier to phage-based therapies. Protein and strain engineering efforts can help mitigate terminase nonspecificity, but care must be taken to not compromise productivity. More generally, widespread adoption will require deeper understanding of host-pathogen-phage interactions, development of scalable GMP manufacturing processes, and harmonized regulatory guidance that recognizes the dynamic nature of phage-derived technologies.

Synthetic Biology↗

bioRxiv2025

Cortical potentials evoked by stimulation of cervical vagus vs. auricular nerve: a comparative, parametric study in nonhuman primates

Stimulation of sensory vagal pathways is typically delivered via invasive, cervical vagus nerve stimulation (cVNS) or noninvasive, trans-auricular nerve stimulation (taNS). While both methods are investigated therapeutically, their effects on brain physiology remain poorly understood, hindering mechanistic understanding and stimulus optimization. In 6 awake nonhuman primates, we recorded cortical vagal-evoked potentials (VEPs) from subdural electrodes placed in prefrontal, sensorimotor and parietal cortical areas, in response to cVNS or taNS. Across 478 different taNS and cVNS protocols, we varied stimulation side, intensity, frequency, pulse count, and pulse width (PW) and assessed independent effects on amplitude and latency of early (EC; 30-100 ms), intermediate (IC; 101-200 ms) and late components (LC; 201-500 ms) of VEPs. Fixed and random effects of stimulation parameters and subjects, respectively, on VEP measurements, were assessed using a linear mixed-effects model. Overall, cVNS elicits more robust VEPs than taNS, with larger EC, IC and LC amplitudes in both hemispheres. cVNS-elicited ECs and LCs are largest in PFC and PC areas, whereas ICs are largest in SM areas. On the other hand, taNS generally does not elicit area-specific responses. cVNS-elicited ECs have slower latency than ta-NS elicited ECs. Higher stimulation frequencies and intensities and a longer pulse width elicit larger ECs and ICs for cVNS, and to some extent for taNS. Both short and long cVNS trains elicit stronger ECs, and long trains elicit slower ICs. Earlobe stimulation elicits VEPs that partially overlap with those from taNS. In conclusion, cVNS and taNS elicit cortical VEPs in a manner consistent with distinct engagement of ascending vagal pathways, and with similarities and differences in the effects of stimulation parameters on evoked responses.

neuroscience↗

bioRxiv2024

Glia multitask to compensate for neighboring glial cell dysfunction

As glia mature, they undergo glial tiling to abut one another without invading each others boundaries. Upon the loss of the secreted neurotrophin Spatzle3 (Spz3), Drosophila cortex glia transform morphologically and lose their intricate interactions with neurons and surrounding glial subtypes. Here, we reveal that all neighboring glial cell types (astrocytes, ensheathing glia, and subperineurial glia) react by extending processes into the previous cortex glial territory to compensate for lost cortex glial function and reduce the buildup of neuronal debris. However, the loss of Spz3 alone is not sufficient for glia to cross their natural borders, as blocking CNS growth via nutrient-restriction blocks the aberrant infiltration induced by the loss of Spz3. Surprisingly, even when these neighboring glia divert their cellular resources beyond their typical borders to take on new compensatory roles, they are able to multitask to continue to preserve their own normal functions to maintain CNS homeostasis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/611719v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@1a1a884org.highwire.dtl.DTLVardef@3ea222org.highwire.dtl.DTLVardef@676b7org.highwire.dtl.DTLVardef@5c21d6_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

Hierarchical structural organization in bioinspired peptide coacervate microdroplets

This study explores the dynamic and hierarchical structural organization of peptide coacervate microdroplets at the meso-to atomic-scale resolution using a combination of Transferred Nuclear Overhauser Effect Spectroscopy (TrNOESY), Small Angle Neutron Scattering (SANS), and confocal microscopy. Dynamic interactions driving the self-association of peptide clusters are revealed, highlighting the critical roles of interacting residues. These phase-separating model peptides form small oligomers at low pH, which aggregate into larger clusters at neutral pH. These clusters organize into a porous network within the droplets, facilitating size-selective cargo sequestration. The findings underscore the significance of the dynamic spatio-temporal properties of peptide-based coacervates, contributing to our understanding of phase separation at the atomic and molecular levels. Critically, this approach enables the investigation of coacervate structures in their native state, offering insights into the physical and dynamic interactions governing droplet formation and cargo encapsulation. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/602323v4_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@15aadb2org.highwire.dtl.DTLVardef@41be55org.highwire.dtl.DTLVardef@36f0d8org.highwire.dtl.DTLVardef@fe1af8_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Potent and pan-neutralization of SARS-CoV-2 variants of concern by DARPins

We report the engineering and selection of two synthetic proteins - FSR16m and FSR22 - for possible treatment of SARS-CoV-2 infection. FSR16m and FSR22 are trimeric proteins composed of DARPin SR16m or SR22 fused with a T4 foldon and exhibit broad spectrum neutralization of SARS-Cov-2 strains. The IC50 values of FSR16m against authentic B.1.351, B.1.617.2 and BA.1.1 variants are 3.4 ng/mL, 2.2 ng/mL and 7.4 ng/mL, respectively, comparable to currently used therapeutic antibodies. Despite the use of the spike protein from a now historical wild-type virus for design, FSR16m and FSR22 both exhibit increased neutralization against newly-emerged variants of concern (39- to 296-fold) in pseudovirus assays. Cryo-EM structures revealed that these DARPins recognize a region of the receptor binding domain (RBD, residues 455-456, 486-489) overlapping a critical portion of the ACE2-binding surface. K18-hACE2 transgenic mice inoculated with a B.1.617.2 variant and receiving intranasally-administered FSR16m were protected as judged by less weight loss and 10-100-fold reductions in viral burden in the upper and lower respiratory tracts. The strong and broad neutralization potency make FSR16m and FSR22 promising candidates for prevention and treatment of infection by current and potential future strains of SARS-CoV-2.

bioengineering↗

bioRxiv2022

Engineering SARS-CoV-2 cocktail antibodies into a bispecific format improves neutralizing potency and breadth

One major limitation of neutralizing antibody-based COVID-19 therapy is the requirement of costly cocktails to reduce antibody resistance. We engineered two bispecific antibodies (bsAbs) using distinct designs and compared them with parental antibodies and their cocktail. Single molecules of both bsAbs block the two epitopes targeted by parental antibodies on the receptor-binding domain (RBD). However, bsAb with the IgG-(scFv)2 design (14-H-06) but not the CrossMAb design (14-crs-06) increases antigen-binding and virus-neutralizing activities and spectrum against multiple SARS-CoV-2 variants including the Omicron, than the cocktail. X-ray crystallography and computational simulations reveal distinct neutralizing mechanisms for individual cocktail antibodies and suggest higher inter-spike crosslinking potentials by 14-H-06 than 14-crs-06. In mouse models of infections by SARS-CoV-2 and the Beta, Gamma, and Delta variants, 14-H-06 exhibits higher or equivalent therapeutic efficacy than the cocktail. Rationally engineered bsAbs represent a cost-effective alternative to antibody cocktails and a promising strategy to improve potency and breadth.

microbiology↗

bioRxiv2019

Cortical network mechanisms of anodal and cathodal transcranial direct current stimulation in awake primates

Transcranial direct current stimulation (tDCS) is a non-invasive neuromodulation technique that is widely used to stimulate the sensorimotor cortex, and yet the mechanism by which it influences the natural activity of cortical networks is still under debate. Here, we characterize the effects of anodal and cathodal tDCS on underlying neurons in active macaque sensorimotor cortex across a range of doses. We find changes in spike rates that are sensitive to both current intensity and polarity, behavioral state, and that are cell-type specific. At high currents, effects persist after the offset of stimulation, and the spatiotemporal activity associated with motor activity of the contralateral limb, measured by dynamics of neural ensembles, are altered. These data suggest that tDCS induces reproducible and noticeable changes in cortical neuron activity and support the theory that it affects brain activity through a combination of single neuron polarization and network interactions.

neuroscience↗