Assembly of genes into operons is generally viewed as an important process during the continual adaptation of microbes to changing environmental challenges. However, the genome reorganization events that drive this process are also the roots of instability for existing operons. We have determined that there exists a statistically significant trend that correlates the proportion of genes encoded in operons in archaea to their phylogenetic lineage. We have further characterized how microbes deal with operon instability by mapping and comparing transcriptome architectures of four phylogenetically diverse extremophiles that span the range of operon stabilities observed across archaeal lineages: a photoheterotrophic halophile (Halobacterium salinarum NRC-1), a hydrogenotrophic methanogen (Methanococcus maripaludis S2), an acidophilic and aerobic thermophile (Sulfolobus solfataricus P2), and an anaerobic hyperthermophile (Pyrococcus furiosus DSM 3638). We demonstrate how the evolution of transcriptional elements (promoters and terminators) generates new operons, restores the coordinated regulation of translocated, inverted, and newly acquired genes, and introduces completely novel regulation for even some of the most conserved operonic genes such as those encoding subunits of the ribosome. The inverse correlation (r = -0.92) between the proportion of operons with such internally located transcriptional elements and the fraction of conserved operons in each of the four archaea reveals an unprecedented view into varying stages of operon evolution. Importantly, our integrated analysis has revealed that organisms adapted to higher growth temperatures have lower tolerance for genome reorganization events that disrupt operon structures.

Retinoic acid (RA) is used in differentiation therapy to treat a variety of cancers including neuroblastoma. The contributing factors for its therapeutic efficacy are poorly understood. However, mitochondria (MT) have been implicated as key effectors in RA-mediated differentiation process. Here we utilize the SH-SY5Y human neuroblastoma cell line as a model to examine how RA influences MT during the differentiation process. We find that RA confers an approximately 6-fold increase in the oxygen consumption rate while the rate of glycolysis modestly increases. RA treatment does not increase the number of MT or cause measurable changes in the composition of the electron transport chain. Rather, RA treatment significantly increases the mitochondrial spare respiratory capacity. We propose a competition model for the therapeutic effects of RA. Specifically, the high metabolic rate in differentiated cells limits the availability of metabolic nutrients for use by the undifferentiated cells and suppresses their growth. Thus, RA treatment provides a selective advantage for the differentiated state.

Werner syndrome (WS) is a rare progeroid disorder characterized by genomic instability, increased cancer incidence, and early onset of a variety of aging pathologies. WS is unique among early aging syndromes in that affected individuals are developmentally normal, and phenotypic onset is in early adulthood. The protein defective in WS (WRN) is a member of the large RecQ family of helicases but is unique among this family in having an exonuclease. RecQ helicases form multimers, but the mechanism and consequence of multimerization remain incompletely defined. Here, we identify a novel heptad repeat coiled coil region between the WRN nuclease and helicase domains that facilitates multimerization of WRN. We mapped a novel and unique DNA-dependent protein kinase phosphorylation site proximal to the WRN multimerization region. However, phosphorylation at this site affected neither exonuclease activity nor multimeric state. We found that WRN nuclease is stimulated by DNA-dependent protein kinase independently of kinase activity or WRN nuclease multimeric status. In addition, WRN nuclease multimerization significantly increased nuclease processivity. We found that the novel WRN coiled coil domain is necessary for multimerization of the nuclease domain and sufficient to multimerize with full-length WRN in human cells. Importantly, correct homomultimerization is required for WRN function in vivo as overexpression of this multimerization domain caused increased sensitivity to camptothecin and 4-nitroquinoline 1-oxide similar to that in cells lacking functional WRN protein.

Thermoanaerobacter thermohydrosulfuricus BSB-33 is a thermophilic gram positive obligate anaerobe isolated from a hot spring in West Bengal, India. Unlike other T. thermohydrosulfuricus strains, BSB-33 is able to anaerobically reduce Fe(III) and Cr(VI) optimally at 60 °C. BSB-33 is the first Cr(VI) reducing T. thermohydrosulfuricus genome sequenced and of particular interest for bioremediation of environmental chromium contaminations. Here we discuss features of T. thermohydrosulfuricus BSB-33 and the unique genetic elements that may account for the peculiar metal reducing properties of this organism. The T. thermohydrosulfuricus BSB-33 genome comprises 2597606 bp encoding 2581 protein genes, 12 rRNA, 193 pseudogenes and has a G + C content of 34.20 %. Putative chromate reductases were identified by comparative analyses with other Thermoanaerobacter and chromate-reducing bacteria.

Purpose: There is a critical need in immunotherapy drug development to enable focused and sustained immune cell modulation within a tumor to induce and propagate a system-wide anti-tumor response. We have developed a novel immunotherapy platform that could be used to generate geographically focused cancer cell growth inhibition or immune cell activation, thereby stimulating an anti-tumor immune response against primary solid tumors that can also travel to secondary metastases. Methods: Using published methods, we synthesized multivalent protein (MVP) conjugates by conjugating multiple copies (i.e. valency) of immune stimulating proteins, checkpoint inhibitors or anti-tumor antibodies to soluble, long-chain biopolymers. We verified that we can reproducibly generate MVP valencies ranging from 20-120 protein copies (±10%) per polymer backbone. We determined the binding affinity of these MVPs to their respective targets using biolayer interferometry and cell bioassays, and we measured the hydrodynamic radius of these immunotherapies using dynamic light scattering. Then, we injected fluorescently modified MVPs or their unconjugated counterparts directly into a variety of solid tumor models in mice. By taking longitudinal in vivo fluorescence measurements of the intratumoral (IT) drug signal over multiple days, we measured the IT half-life of each treatment. Results: Based on binding affinity measurements, we found that MVP potency increased directly with protein valency, and at high valency, the potency of MVPs were substantially greater than the unconjugated protein controls. Multivalent conjugation also increased the hydrodynamic radius of the MVPs to at least ten times larger than the unconjugated therapeutics. This large size was sufficient to slow the diffusion of MVP immunotherapies through dense tissues, such as solid tumors, as demonstrated by our in vivo studies. MVPs exhibited a higher IT drug signal with a more durable gradient within the tumor compared to the unconjugated controls, resulting in an extension of their IT half-lives by >5X in mouse solid tumors. Conclusions: The MVP platform can be used to modulate the potency and therapeutic durability for a wide range of immunotherapy targets. Further, the MVPs stay focused within the tumor after IT injection where they could generate a sustained anti-tumor immune response with minimal systemic exposure. Therefore, we expect MVP immunotherapies to have a better safety profile than IT or systemic delivery of an unconjugated therapeutic. We will continue to develop our internal MVP pipeline to finalize a candidate for IND-enabling studies. We are also seeking collaborations for co-development of additional immunotherapies that could benefit from the extended IT exposure and potency modulation enabled by the MVP platform. Citation Format: Livia Brier, Amy A. Twite, Adam Barnebey, Mavish Mahomed, Wesley M. Jackson. Using a multivalent immunotherapy platform to extend intratumoral therapeutic durability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4157.