Biology subjects

Padilla, J.

Publications and source records attributed to Padilla, J..

4 recordsLinked to original sources

bioRxiv2024

Accelerating CAR T cell manufacturing with an automated next-day process

The traditional method of CAR T cell production involves lengthy ex vivo culture times which can result in the reduction of crucial naive T cell subsets. Moreover, traditional CAR T cell therapy manufacturing processes can prolong time-to-patient and contribute to disease progression. In this study, we describe an innovative and automated 24-hour CAR T manufacturing process that yields a higher percentage of naive/stem-cell like T cells which have increased cytotoxic activity and cytokine release. The data supports the feasibility of implementing this streamlined manufacturing process in clinics. This approach also has the potential to enhance CAR T therapy efficacy and improve patient access to therapy.

bioengineering↗

bioRxiv2023

qsmR encoding an IclR-family transcriptional factor is a core pathogenic determinant of Burkholderia glumae beyond the acyl-homoserine lactone-mediated quorum-sensing system

The plant pathogenic bacterium Burkholderia glumae causes bacterial panicle blight (BPB) in rice-growing areas worldwide. It has been widely accepted that an acyl-homoserine lactone (AHL)-type quorum sensing (QS) system encoded by tofI and tofR genes (TofIR QS) is a key regulatory mechanism underlying the bacterial pathogenesis of B. glumae. In addition, qsmR, which encodes an IclR-family regulatory protein, has been considered an important part of TofIR QS. However, the present study with three strains of B. glumae representing different pathogenic strains revealed that this currently accepted paradigm should be modified. We characterized the regulatory function of TofIR QS and qsmR in three different strains of B. glumae, 336gr-1 (virulent), 411gr-6 (hypervirulent) and 257sh-1 (avirulent). In 336gr-1, both TofIR QS and qsmR were critical for the pathogenesis, being consistent with previous studies. However, in the hypervirulent strain 411gr-6, TofIR QS only partially contributes to the virulence, whereas qsmR was critical for pathogenesis like in 336gr-1. Furthermore, we found that a single nucleotide polymorphism causing T50K substitution in the qsmR coding sequence was the cause of the non-pathogenic trait of the naturally avirulent strain 257sh-1. Subsequent analyses of gene expression and transcriptome revealed that TofIR QS is partially controlled by qsmR at the transcriptional level in both virulent strains. Further genetic tests of additional B. glumae strains showed that 11 out of 20 virulent strains retained the ability to produce toxoflavin even after removing the tofI/tofM/tofR QS gene cluster like 411gr-6. In contrast, all the virulent strains tested lost the function almost completely upon deletion of the qsmR gene. Taking these results together, qsmR, rather than TofIR QS, is a master regulator that determines the pathogenic trait of B. glumae thus a more appropriate pathogen target for successful management of BPB. Author summaryBacterial cell-to-cell communication, called quorum-sensing, systems mediated by homoserine lactone (HSL)-type signaling molecules play pivotal roles in the virulence-related functions of diverse plant and animal pathogenic Gram-negative bacteria. Burkholderia glumae is the chief causal agent for bacterial panicle blight, a devastating rice disease worldwide. In this pathogen, the HSL-type quorum-sensing system dependent on the tofI and tofR genes had long been thought to be the core regulatory/signaling system essential for virulence. However, we discovered in this study that a highly virulent strain of B. glumae, 411gr-6, retained its virulence even after the tofI/tofR-dependent quorum-sensing function was disabled. We also found that more than a half of the natural strains of this pathogen species tested in this study also exhibited their virulence function in the absence of the tofI/tofR-dependent quorum-sensing system. Then we characterized the qsmR gene as an essential regulatory/signaling element for the bacterial pathogenesis regardless of the genetic variations among the strains of B. glumae. This study indicates that the qsmR gene, rather than the tofI/tofR-dependent quorum-sensing system, should be considered as an essential master regulatory/signaling factor for the virulence of B. glumae and, thus, a promising target for suppressing bacterial panicle blight.

microbiology↗

bioRxiv2022

Genomic deletions and rearrangements in monkeypox virus from the 2022 outbreak, USA

Genomic surveillance of monkeypox virus (MPXV) during the 2022 outbreak has been mainly focused on single nucleotide polymorphism (SNP) changes. DNA viruses, including MPXV, have a lower SNP mutation rate than RNA viruses due to higher fidelity replication machinery. We identified a large genomic rearrangement in a MPXV sequence from a 2022 case in the state of Minnesota (MN), USA, from an abnormal, uneven MPXV read mapping coverage profile in whole-genome sequencing (WGS) data. We further screened WGS data of 206 U.S. MPXV samples and found seven (3.4 percent) sequenced genomes contained similar abnormal read coverage profiles that suggested putative large deletions or genomic rearrangements. Here, we present three MPXV genomes containing deletions ranging from 2.3 to 15 kb and four genomes containing more complex rearrangements. Five genomic changes were each only seen in one sample, but two sequences from linked cases shared an identical 2.3 kb deletion in the 3 terminal region. All samples were positive using VAC1 and Clade II (formerly West African)-specific MPXV diagnostic tests; however, large deletions and genomic rearrangements like the ones reported here have the potential to result in viruses in which the target of a PCR diagnostic test is deleted. The emergence of genomic rearrangements during the outbreak may have public health implications and highlight the importance of continued genomic surveillance.

genomics↗

bioRxiv2020

Long-read sequencing resolves structural variants in SERPINC1 causing antithrombin deficiency and identifies a complex rearrangement and a retrotransposon insertion not characterized by routine diagnostic methods

The identification and characterization of structural variants (SVs) in clinical genetics have remained historically challenging as routine genetic diagnostic techniques have limited ability to evaluate repetitive regions and SVs. Long-read whole-genome sequencing (LR-WGS) has emerged as a powerful approach to resolve SVs. Here, we used LR-WGS to study 19 unrelated cases with type I Antithrombin Deficiency (ATD), the most severe thrombophilia, where routine molecular tests were either negative, ambiguous, or not fully characterized. We developed an analysis workflow to identify disease-associated SVs and resolved 10 cases. For the first time, we identified a germline complex rearrangement involved in ATD previously misclassified as a deletion. Additionally, we provided molecular diagnoses for two unresolved individuals that harbored a novel SINE-VNTR-Alu retroelement insertion that we fully characterized by de novo assembly and confirmed by PCR amplification in all affected relatives. Finally, the nucleotide-level resolution achieved for all the SVs allowed breakpoint analysis, which revealed a replication-based mechanism for most of the cases. Our study underscores the utility of LR-WGS as a complementary diagnostic method to identify, characterize, and unveil the molecular mechanism of formation of disease-causing SVs, and facilitates decision making about long-term thromboprophylaxis in ATD patients.

genomics↗