Biology subjects

Ahmed, U.

Publications and source records attributed to Ahmed, U..

6 recordsLinked to original sources

bioRxiv2026

Cas12a cleavage and trimming kinetics reveal mismatches as a tool to steer editing

Gene knockouts by CRISPR-Cas nucleases rely on targeted DNA cleavage and error-prone DNA repair: end-joining pathways can introduce insertions and deletions that assist in disrupting the coding sequence. However, only a fraction of edits achieves this, and an unfavorable array of repair outcomes typically requires switching to another editing technology. Key factors that influence repair are the types and lengths of DNA ends following cleavage. Here, we investigated Cas12a's ability to produce different ends and if they can be used to redistribute editing outcomes. We determined the sites and rates of target cleavage by Cas12a in vitro by combining kinetic modeling with nucleotide-resolution assays. Here, we show that trimming - repeated cleavage of an already cut target - occurs about 4x faster than initial cleavage; it also presents alternative DNA end structures for cellular repair. We also introduced specific mismatches to the gRNA: Cas12a maintained fast target cleavage, but changed where the target was cleaved and how quickly it was trimmed, compared to matched gRNA. We exploited the differences in cleavage kinetics between matched and mismatched gRNAs to develop reprogrammed gRNAs, i.e. rpgRNAs. Intentionally-mismatched rpgRNAs retained the high editing efficiency observed with traditional gRNAs. However, they shifted editing between in-frame and out-of-frame outcomes to enhance gene knockout success across genes. Reprogrammed gRNAs offer an efficient way to steer editing toward such preferred outcomes, while retaining the simplicity of gene editing with CRISPR-Cas nucleases.

biochemistry↗

bioRxiv2023

Ultrasound neuromodulation of an anti-inflammatory pathway at the spleen produces sustained improvement of experimental pulmonary hypertension

BackgroundInflammation is pathogenically implicated in pulmonary arterial hypertension (PAH); however, it has not been adequately targeted therapeutically. We investigated whether neuromodulation of an anti-inflammatory neuroimmune pathway involving the splenic nerve using noninvasive, focused ultrasound stimulation of the spleen (sFUS) can improve experimental pulmonary hypertension (PH). MethodsPH was induced in rats either by SU5416 (20 mg/kg SQ) injection, followed by 21 (or 35) days of hypoxia (SuHx model), or by monocrotaline (60 mg/kg IP) injection (MCT model). Animals were randomized to receive either daily, 12-min-long sessions of sFUS or sham stimulation, for 14 days. Catheterizations, echocardiography, indices of autonomic function, lung and heart histology and immunohistochemistry, spleen flow cytometry and lung single-cell-RNA sequencing were performed after treatment to assess the effects of sFUS. ResultsSplenic denervation right before induction of PH results in a more severe phenotype. In both SuHx and MCT models of PH, sFUS treatment reduces right ventricular (RV) systolic pressure by 25-30% compared to sham therapy, without affecting systemic pressure, and improves RV function and autonomic indices. sFUS reduces wall thickness, apoptosis, and proliferation in small pulmonary arterioles, suppresses CD3+ and CD68+ cell infiltration in lungs and RV fibrosis and hypertrophy and lowers brain natriuretic peptide. Beneficial effects persist for weeks after sFUS discontinuation and are more robust with early and longer treatment. Splenic denervation abolishes sFUS therapeutic benefits. sFUS partially normalizes CD68+ and CD8+ T-cells cell counts in the spleen and downregulates several inflammatory genes and pathways in nonclassical and classical monocytes, and macrophages in the lung. Differentially expressed genes in those cell types are significantly enriched for human PAH-associated genes. ConclusionssFUS causes dose-dependent, sustained improvement of hemodynamic, autonomic, laboratory and pathological manifestations in two models of experimental PH. Mechanistically, sFUS normalizes immune cell populations in the spleen and downregulates inflammatory genes and pathways in the lung, many of which are relevant in human disease.

neuroscience↗

bioRxiv2022

Organ- and function-specific organization and bioelectronic modulation of vagus fascicles and fibers

Vagal fibers travel inside fascicles and form branches to innervate organs and regulate organ functions. Vagus nerve stimulation (VNS) therapies activate fibers non-selectively, often resulting in reduced efficacy and side effects from non-targeted organs. Transverse and longitudinal arrangement of fibers according to functions they mediate and organs they innervate is unknown, however it is crucial for selective VNS. Using microcomputed tomography, we found that, in swine, fascicles are arranged in 2 overlapping axes, with sensory and motor fascicles separated cephalad and merging caudad, and larynx-, heart- and lung-specific fascicles separated caudad and progressively merging cephalad. Using immunohistochemistry, we found that the distribution of single fibers is highly nonuniform: myelinated afferents and efferents occupy separate fascicles, unmyelinated efferents co-localize with myelinated afferents, and small unmyelinated afferents are widely distributed. Using a multi-contact cuff electrode, we delivered fascicular cervical VNS in anesthetized and awake swine. Compound action potentials, from distinct fiber types, and organ responses, including laryngeal muscle, cough, breathing, heart rate and blood pressure responses are elicited in a radially asymmetric manner, with consistent angular separations. These results indicate that vagal fibers are anatomically organized according to functions they mediate and organs they innervate and can be asymmetrically activated by fascicular cervical VNS.

neuroscience↗

bioRxiv2021

Manipulation of stimulus waveform and frequency permits targeted fiber activation during vagus nerve stimulation in 2 rodent species

Afferent and efferent vagal fibers mediate bidirectional communication between the brain and visceral organs. Small, unmyelinated C-afferents constitute the majority of vagal fibers, play critical roles in numerous interoceptive circuits and autonomic reflexes in health and disease and may contribute to the efficacy and safety of vagus nerve stimulation (VNS). Selective engagement of C-afferents with electrical stimuli has not been feasible, due to the default fiber recruitment order: larger fibers first, smaller fibers last. Here, we determine and optimize an electrical stimulus that selectively engages vagal C-afferents. Intermittent KHz-frequency electrical stimulation (KES) activates motor and, preferentially, sensory vagal neurons in the brainstem. During KES, asynchronous activity of C-afferents increases, while that of larger fibers remains largely unchanged. In parallel, KES effectively blocks excitability of larger fibers while moderately suppressing excitability of C-afferents. By compiling selectivity indices in individual animals, we find that optimal KES parameters for C-afferents are >5KHz frequency and 7-10 times engagement threshold (xT) intensity in rats, 15-25xT in mice. These effects can be explained in computational models by how sodium channel responses to KES are shaped by axonal size and myelin. Our results indicate that selective engagement of vagal C-afferents is attainable by intermittent KES.

bioengineering↗

bioRxiv2021

Implant- and anesthesia-related factors affecting threshold intensities for vagus nerve stimulation

Vagus nerve stimulation (VNS) is used as therapy in epilepsy and depression and is tested as a potential treatment for several chronic disorders. Typically, VNS is delivered at increasing stimulus intensity until a response is observed (threshold intensity). Factors that affect threshold intensities for engagement of different fiber types and concomitant physiological responses have not been studied. We determined neural and physiological responses to increasing stimulus intensities of VNS in anesthetized and awake animals, and examined the effect of implant- and anesthesia-related factors on threshold intensities in a rodent model of VNS. In acute and long-term cervical vagus nerve implants (53 and 14 rats, respectively) VNS was delivered under isoflurane, ketamine-xylazine, or awake at different intensities. Stimulus-evoked compound action potentials (eCAPs) were recorded, elicited physiological responses were registered, including changes heart rate (HR), breathing, and blood pressure (BP), and threshold intensities were determined. The intensity that elicits eCAPs ("neural threshold") is significantly lower than what elicits a physiological response ("physiological threshold", PT) (25 A {+/-}1.8 vs. 70 A {+/-}5.2, respectively; Mean {+/-}SEM). Changes in BP occur at the lowest stimulus intensities (80 A {+/-}7), followed by changes in HR (105 A {+/-}8.4) and finally in breathing (310 A {+/-}32.5). PT is lower with than without electrode insulation (60 A {+/-}12, vs. 700 A {+/-}123). PT and electrode impedance are correlated in long-term (r=0.47; p<0.001) but not in acute implants (r=-0.34; p NS); both PT and impedance increase with implant age (Pearson correlation r=0.44; p<0.001 and r=0.64; p<0.001, respectively). PT is lowest when animals are awake (210 A {+/-}33; Mean {+/-}SEM), followed by ketamine-xylazine (630 A {+/-}154), and isoflurane (1075 A {+/-}131). The sequence of physiological responses with increasing VNS intensity is similar in both anesthetized and awake states. Implant age, electrical impedance and the type of anesthesia affect VNS threshold and should be accounted for when determining stimulation dose.

bioengineering↗

bioRxiv2019

GIT2 is dispensable for normal learning and memory function due to a predominant brain GIT2 splice variant that evades GIT/PIX complexes.

G protein-coupled receptor kinase-interacting protein 2 (GIT2) and GIT1 are highly similar, sharing the same domain structure and many binding partners. The most important GIT partners are the p21-activated protein kinase-interacting exchange factor (PIX) proteins, since through homomeric and heteromeric interactions, GIT and PIX proteins form oligomeric GIT/PIX complexes. Oligomeric GIT/PIX complexes function both as regulators of small GTP-binding proteins and as scaffolds for signalling molecules, including p21-activated protein kinases (PAKs). Deficits in learning and memory have been demonstrated in GIT1 knockout mice, and it has been assumed that GIT2 also would affect learning and memory. Unexpectedly, we find that GIT2-deficient mice respond normally in multiple tests of learning and memory, and have normal hippocampal long-term potentiation. Further, we find no evidence that GIT2 regulates ADHD-like phenotypes. To investigate why GIT2 and GIT1 differ so markedly in the brain, we identified the major isoform of GIT2 in the brain as a previously uncharacterized splice variant, GIT2({Delta}BCE). This variant cannot dimerize or form oligomeric complexes with PIX proteins, and is thus incapable of regulating PAK in synapses, compared to oligomeric GIT1/PIX complexes. Because localized activation of PAK in synapses is required for structural plasticity underlying cognitive performance, loss of monomeric GIT2({Delta}BCE) in the brain does not influence these responses.

animal behavior and cognition↗