Biology subjects

Kalita, M.

Publications and source records attributed to Kalita, M..

3 recordsLinked to original sources

bioRxiv2025

Novel insights into the genome organization of Rhizobiaceae: identification of linear plasmids

2.Members of the family Rhizobiaceae typically have multipartite genomes, that are split between two or more replicons, including the chromosome and a variable number of extrachromosomal replicons (chromids and plasmids). Nearly all Rhizobiaceae replicons sequenced and described to date have a circular topology, with the exception of the linear chromid found in the genomes of most Agrobacterium spp. In this study, genomes of five nonpathogenic Agrobacterium strains and one plant tumorigenic Allorhizobium strain were fully sequenced. Surprisingly, genome analysis revealed that these six strains each carry an 80-kbp linear plasmid. Linear plasmids were so far not identified in this bacterial family or other bacteria within the class Alphaproteobacteria. The ends of all six plasmids identified in this study have a hairpin structure with covalently closed ends. The plasmid sequences showed a high degree of homology, clearly indicating their common ancestry. Database searches led to the identification of additional linear plasmids in previously published Rhizobiaceae genome assemblies that were not previously recognized to have linear plasmids, suggesting that these replicons may be more widespread. Most likely, linear plasmids may be even more widely distributed than anticipated. Although the biological functions of the linear plasmids identified in this study remain unknown, they are associated with both nonpathogenic and plant tumorigenic Rhizobiaceae strains. 3. Impact statementThe family Rhizobiaceae includes some remarkable and important representatives, such as plant symbiotic bacteria (rhizobia) and plant pathogenic bacteria associated with neoplasia (agrobacteria). In this study, the complete genome sequences of six Rhizobiaceae strains were generated and their genome organizations were examined. Strikingly, our results showed that these six strains harbor a linear plasmid. Moreover, GenBank searches suggested that linear plasmids may be even more widespread in the family Rhizobiaceae. Linear plasmids may go undetected in genome sequencing studies if the assemblies are not specifically examined for linear plasmid. Overall, this study provides further evidence for the extraordinary genome plasticity of members of the family Rhizobiaceae and expands the taxonomic range in which linear plasmids have been identified. To the best of our knowledge, this is the first report of linear plasmids in the family Rhizobiaceae or the class Alphaproteobacteria. 4. Data summaryThe whole-genome sequences have been deposited at DDBJ/ENA/GenBank under the accessions CP192696-CP192701 (Av2), CP000000-CP000000 (rho-7.1), CP000000-CP000000 (rho-8.1), CP000000-CP000000 (rho-11.1), CP000000-CP000000 (rho-13.3), and CP000000-CP000000 (rho-14.1), within the BioProjects PRJNA557463 and PRJNA1009994. The raw sequencing reads were deposited in the Sequence Read Archive (SRA) under the same BioProjects PRJNA557463 and PRJNA1009994: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA557463 and https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1009994. NCBI submission for five genome sequences is undergoing processing and accession numbers will be added when available; in https://figshare.com/s/29a9e621adc1b66d0957

genomics↗

bioRxiv2024

In vivo imaging of T-cell coregulator B7-H4 reveals protumor macrophage status in prostate cancer

BackgroundB7-H4 is a cell surface ligand overexpressed by tumors to inhibit T cell functions and evade the immune system. B7-H4 is minimally expressed in normal tissues but is highly expressed by various cancer cells and tumor-associated macrophages (TAM). Despite its importance as an immune checkpoint inhibitor, no imaging techniques specifically targeting B7-H4 have been established. To close this gap, we sought to assess the ability of a monoclonal antibody (mAb) based immunoPET radiotracer to visualize B7-H4 in human and murine prostate cancer models. MethodsAnti-B7-H4 mAb clone 2H9 was functionally characterized for binding to the human and mouse B7-H4 protein. The antibody was conjugated with chelator p-SCN-Bn-Deferoxamine (DFO) and labeled with radioisotope Zirconium-89 (89Zr) to obtain immunoPET tracer 89Zr-2H9-mAb. The biolayer interferometry method was used to test the binding kinetics of DFO-2H9-mAb compared to that of parental 2H9 mAb. A group of six athymic nude mice with human DU145 prostate tumor xenograft underwent MicroPET imaging after tail vein injection of [~]150{micro}Ci 89Zr-2H9-mAb or non-binding 89Zr-Isotype-mAb. Next, immunocompetent C57BL/6J mice with TRAMP-C2 tumors each were injected with either PBS (n=8), cold 2H9 mAb (10mg/kg) to block B7-H4 (n=6), or chlodronate liposome (15mg/kg) to cause total macrophage depletion (n=6), followed by 89Zr-2H9-mAb MicroPET imaging. An ex vivo biodistribution assay was performed after 144 hr post radiotracer injection. Tumor radiotracer binding, quantified as a percentage injected dose per gram (%ID/g), was compared between different experimental groups using two-way ANOVA with Bonferroni or Tukey corrections. ResultsImmunoconjugation yielded a 2.59 {+/-} 0.08 chelator-to-antibody ratio, and the binding of DFO conjugated 2H9-mAb was similar to that of parental 2H9 mAb, with unaffected affinity in targeting B7-H4 protein moiety. The radiochemical purity of 89Zr-2H9-mAb tracer was yielded >95% with an average specific activity of 5{micro}Ci/{micro}g antibody. DU145 tumor xenografts demonstrated significantly stronger radiotracer binding at 24, 48, 72, 96, and 120 hr than the non-binding isotype control group. In TRAMP-C2 tumor xenografts, the radiotracer binding in B7-H4 blocked tumors was significantly lower than in the non-blocked PBS-injected group. Macrophage depletion resulted in a significant decrease in tumor binding compared to the control group. 89Zr-2H9-mAb could efficiently distinguish tumors with high sensitivity, showing a high correlation between PET imaging and bio-distribution. More importantly, the immunohistochemistry of the harvested tumor revealed no significant difference between the three groups, as discernible through in vivo PET imaging. ConclusionThis study highlights the potential of B7-H4 immunoPET imaging for monitoring immunotherapy response. With the emerging potential of B7-H4 blocking as an immunotherapeutic, immunoPET imaging could be readily expanded to patient stratification and therapy monitoring. B7-H4 imaging could augment our understanding of B7-H4 dynamics in response to various therapeutic interventions in clinical trials. The new B7-H4 immunoPET probe is, in principle, clinically translatable.

immunology↗

bioRxiv2022

Antigen-dependent inducible T cell reporter system for PET imaging of breast cancer and glioblastoma

For the past several decades, chimeric antigen receptor T cell (CAR T) therapies have shown promise in the treatment of cancers. These treatments would greatly benefit from companion imaging biomarkers to follow the trafficking of T cells in vivo. Using synthetic biology, we engineered T cells with a chimeric receptor SyNthetic Intramembrane Proteolysis Receptor (SNIPR) that induces overexpression of an exogenous reporter gene cassette upon recognition of specific tumor markers. We then applied a SNIPR-based positron emission tomography (PET) reporter system to two cancer-relevant antigens, human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor variant III (EGFRvIII), commonly expressed in breast and glial tumors respectively. Antigen-specific reporter induction of the SNIPR-PET T cells was confirmed in vitro using GFP fluorescence, luciferase luminescence, and the HSV-TK PET reporter with [18F]FHBG. T cells associated with their target antigens were successfully imaged using PET in dual xenograft HER2+/HER2- and EGFRvIII+/EGFRvIII-animal models, with > 10-fold higher [18F]FHBG signals seen in antigen-expressing tumors versus the corresponding controls. The main innovation described is therefore PET detection of T cells via specific antigen-induced signals, in contrast to reporter systems relying on constitutive gene expression.

synthetic biology↗