Biology subjects

Gandhi, N.

Publications and source records attributed to Gandhi, N..

5 recordsLinked to original sources

bioRxiv2024

Breaking Free: Development of Circular AAV Cargos for Targeted Seamless Integration in the Liver

Recent advancements in gene insertion have shifted from DNA repair-dependent mechanisms to more precise approaches, enhancing safety and predictability for editing outcomes. Integrase-mediated programmable genomic integration (I-PGI) utilizes a DNA cargo to insert transgenes in a targeted, unidirectional manner. In vivo, where nuclear delivery of DNA is challenging, adeno-associated virus (AAV) can act as the cargo vector. While I-PGI does not require DNA double-stranded breaks (DSBs) for activity, linear DNA cargo, like AAV, stimulates DNA end joining activity after integration. To mitigate potential risks from DSBs with linear viral cargo, we developed two circular genome types capable of seamless gene insertion in non-dividing cells. We first harnessed the orthogonal property of large serine integrases to produce circle-AAV (cAAV) from linear viral genomes in cells. cAAV demonstrated faithful seamless cargo integration in primary human hepatocytes (PHH) and robust DSB-free insertion structures in vivo. We then investigated the delivery of packaged circular AAV cargo (AAV.AD), which eliminates the need for enzymatic manipulation in the cell. AAV.AD proved to be a viable cargo for I-PGI, exhibiting functional integration in PHH and in vivo, that resulted in seamless insertion structures. Together, these findings provide the first reported evidence of DSB-free programmable genomic integration using integrase and AAV cargo, addressing a previously unrecognized challenge in the field.

bioengineering↗

bioRxiv2022

A transcriptomic signature for single pancreatic islet leader beta cells

AimsSpatially-organised increases in cytosolic Ca2+ within pancreatic beta cells in the pancreatic islet underlie the stimulation of insulin secretion by high glucose. Recent data have revealed the existence of subpopulations of beta cells including "leaders" which initiate Ca2+ waves. Whether leader cells possess unique molecular features, or localisation, is unknown. Main methodsHigh speed confocal Ca2+ imaging was used to identify leader cells and connectivity analysis, running under MATLAB and Python, to identify highly connected "hub" cells. To explore transcriptomic differences between beta cell sub-groups, individual leaders or followers were labelled by photo-activation of the cryptic fluorescent protein PA-mCherry and subjected to single cell RNA sequencing ("Flash-Seq"). Key findingsDistinct Ca2+ wave types were identified in individual islets, with leader cells present in 73 % (28 of 38 islets imaged). Scale-free, power law-adherent behaviour was also observed in 29% of islets, though "hub" cells in these islets did not overlap with leaders. Transcripts differentially expressed (295; padj<0.05) between leader and follower cells included genes involved in cilium biogenesis and transcriptional regulation. Functionally validating these findings, cilia number and length tended to be lower in leader vs follower cells. Leader cells were also located significantly closer to delta cells in Euclidian space than were follower cells. SignificanceThe existence of both a discrete transcriptome and unique localisation implies a role for these features in defining the specialized function of leaders. Specifically, these data raise the possibility of altered signalling from delta cells towards somatostatin receptors present on leader cell cilia.

cell biology↗

bioRxiv2021

MicroRNA775 and target Galactosyltransferase (GALT9) module regulates recovery from submergence induced senescence by modulating SAGs in Arabidopsis thaliana

Submergence induced hypoxic condition is one of the abiotic stresses which negatively affects the plant growth and development, and causes early onset of senescence. Hypoxic conditions ateres the expression of a number of non-coding microRNAs (miRNAs), besides protein-coding genes. However, the molecular function of stress-induced miRNA in submergence induced physiological or developmental changes and recovery remains to be understood. The expression of miR775 is highly induced under hypoxic stress conditions. Here, we show that miR775 is a potential post-transcriptional regulator number of targets, including Galactosyltransferase (GALT9). The expression of miR775 and target GALT9 was significantly induced and reduced respectively at 24 hours of submergence. The overexpression of miR775 (miR775-Oe) confers enhanced recovery from submergence stress and reduced accumulation of ROS, in contrast to wild type and endogenous target mimic of miR775 (MIM775) Arabidopsis plants. We observed a similar recovery phenotype in case of target galt9 mutant plants, indicating the role of miR775-GALT9 module in recovery from submergence. Further, we showed that the expression of SENESCENCE ASSOCIATED GENES (SAGs), such as SAG12, SAG29, and ORE1. was increased in MIM775 and reduced in miR775-Oe and galt9 plants. Thus, our results suggest that miR775-GALT9 module plays a crucial role in the recovery from submergence by modulating the expression of SAGs through differential accumulation of ROS.

plant biology↗

bioRxiv2017

Spatial distribution of extensively drug-resistant tuberculosis (XDR-TB) patients in KwaZulu-Natal, South Africa

BackgroundKwaZulu-Natal province, South Africa, has among the highest burden of XDR-TB worldwide with the majority of cases occurring due to transmission. Poor access to health facilities can be a barrier to timely diagnosis and treatment of TB, which can contribute to ongoing transmission. We sought to determine the geographic distribution of XDR-TB patients and proximity to health facilities in KwaZulu-Natal.\n\nMethodsWe recruited adults and children with XDR-TB diagnosed in KwaZulu-Natal. We calculated distance and time from participants home to the closest hospital or clinic, as well as to the actual facility that diagnosed XDR-TB, using tools within ArcGIS Network analyst. Speed of travel was assigned to road classes based on Department of Transport regulations. Results were compared to guidelines for the provision of social facilities in South Africa: 5km to a clinic and 30km to a hospital.\n\nResultsDuring 2011-2014, 1027 new XDR-TB cases were diagnosed throughout all 11 districts of KwaZulu-Natal, of whom 404 (39%) were enrolled and had geospatial data collected. Participants would have had to travel a mean distance of 2.9 km (CI 95%: 1.8-4.1) to the nearest clinic and 17.6 km (CI 95%: 11.4-23.8) to the nearest hospital. Actual distances that participants travelled to the health facility that diagnosed XDR-TB ranged from <10 km (n=143, 36%) to >50 km (n=109, 27%). The majority (77%) of participants travelled farther than the recommended distance to a clinic (5 km) and 39% travelled farther than the recommended distance to a hospital (30 km). Nearly half (46%) of participants were diagnosed at a health facility in eThekwini district, of whom, 36% resided outside the Durban metropolitan area.\n\nConclusionsXDR-TB cases are widely distributed throughout KwaZulu-Natal province with a denser focus in eThekwini district. Patients travelled long distances to the health facility where they were diagnosed with XDR-TB, suggesting a potential role for migration or transportation in the XDR-TB epidemic.

epidemiology↗

bioRxiv2017

The Superior Colliculus And The Steering Of Saccades Toward A Moving Visual Target

Following the suggestion that a command encoding the expected here-and-now target location feeds the oculomotor system during interceptive saccades, we tested whether this command originates in the deep superior colliculus (SC). Monkeys generated saccades to targets that were static or moving along the preferred axis, away from (outward) or toward a fixated target (inward) with a constant speed (20{degrees}/s). Vertical and horizontal motions were also tested. Extracellular activity of 57 saccade-related neurons was recorded in 3 monkeys. The movement field (MF) parameters (boundaries, center and firing rate) were estimated after spline fitting the relation between the saccade amplitude and the average firing rate of the motor burst. During radial motion, the inner MF boundary shifted in the same direction as the target motion for some neurons, not all. During vertical motion, both lower and upper boundaries were shifted upward during upward motion whereas the upper boundary only shifted during downward motions. For horizontal motions, the medial boundaries were not changed. The MF center was shifted only for outward motion. Regardless of the motion direction, the average firing rate was consistently reduced during interceptive saccades. Our study shows an involvement of the saccade-related burst of SC neurons in steering the gaze toward a moving target. When observed, the shifts of MF boundary in the direction of target motion correspond to commands related to antecedent target locations. The absence of shift in the opposite direction shows that SC activity does not issue predictive commands related to the future target location.\n\nSIGNIFICANCE STATEMENTBy comparing the movement field (MF) of saccade-related neurons between saccades toward static and moving targets, we show that the motor burst issued by neurons in the superior colliculus does not convey commands related to the future location of a moving target. During interceptive saccades, the active population consists of a continuum of neurons, ranging from cells exhibiting a shift in the center or boundary of their MF to cells which exhibit no change. The shifts correspond to residual activity related to the fact that the active population does not change as fast as the target in the visual field. By contrast, the absence of shift indicates commands related to the current target location, as if it were static.

neuroscience↗