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Many solid tumors present with perineural invasion (PNI), and innervation correlates with worsened prognosis. The effects that commonly administered therapies such as radiation therapy (RT) have on PNI status remain unknown. We investigated the contribution of RT on the nervous system and elucidated the implications that increased nerve signaling can have on tumor burden using our previously developed orthotopic murine model of rectal cancer (RC) and our targeted and clinically relevant short-course RT (SCRT) regimen.Medical charts for patients with RC treated at the Wilmot Cancer Institute were obtained and PNI status was analyzed. Human data were accompanied by an orthotopic murine model of RC. Briefly, luciferase-expressing murine colon-38 (MC38-luc) tumor cells were injected orthotopically into the rectal wall of C57BL6 mice. Targeted SCRT (5 gray (Gy) per fraction for 5 consecutive fractions) was administered to the tumor. Intratumoral innervation was determined by immunohistochemistry (IHC), local norepinephrine (NE) concentration was quantified by enzyme-linked immunosorbent assay (ELISA), and β2-adrenergic receptor (B2AR) expression was assessed by flow cytometry. Chronic NE signaling was mirrored by daily isoproterenol treatment, and the effect on tumor burden was determined by overall survival, presence of metastatic lesions, and tumor size. Isoproterenol signaling was inhibited by administration of propranolol.Human RC patients with PNI have decreased overall survival compared with patients without PNI. In our mouse model, SCRT induced the expression of genes involved in neurogenesis, increased local NE secretion, and upregulated B2AR expression. Treating mice with isoproterenol resulted in decreased overall survival, increased rate of metastasis, and reduced SCRT efficacy. Interestingly, the isoproterenol-induced decrease in SCRT efficacy could be abrogated by blocking the BAR through the use of propranolol, suggesting a direct role of BAR stimulation on impairing SCRT responses.Our results indicate that while SCRT is a valuable treatment, it is accompanied by adverse effects on the nervous system that may impede the efficacy of therapy and promote tumor burden. Therefore, we could speculate that therapies aimed at targeting this signaling cascade or impairing nerve growth in combination with SCRT may prove beneficial in future cancer treatment.

Accurate quantification of proteomics data is essential for revealing and understanding biological signaling processes. We have recently developed a chemical proteomic strategy termed phosphatase inhibitor beads and mass spectrometry (PIB-MS) to investigate endogenous phosphoprotein phosphatase (PPP) dephosphorylation signaling. Here, we compare the robustness and reproducibility of status quo quantification methods for optimal performance and ease of implementation. We then apply PIB-MS to an array of breast cancer cell lines to determine differences in PPP signaling between subtypes. Breast cancer, a leading cause of cancer death in women, consists of three main subtypes: estrogen receptor-positive (ER+), human epidermal growth factor receptor two positive (HER2+), and triple-negative (TNBC). Although there are effective treatment strategies for ER+ and HER2+ subtypes, tumors become resistant and progress. Furthermore, TNBC has few targeted therapies. Therefore, there is a need to identify new approaches for treating breast cancers. Using PIB-MS, we distinguished TNBC from non-TNBC based on subtype-specific PPP holoenzyme composition. In addition, we identified an increase in PPP interactions with Hippo pathway proteins in TNBC. These interactions suggest that phosphatases in TNBC play an inhibitory role on the Hippo pathway and correlate with increased expression of YAP/TAZ target genes both in TNBC cell lines and in TNBC patients.

Small-molecule inhibitors of PD-L1 are postulated to control immune evasion in tumors similar to antibodies that target the PD-L1/PD-1 immune checkpoint axis. However, the identity of targetable PD-L1 inducers is required to develop small-molecule PD-L1 inhibitors. In this study, using chromatin immunoprecipitation (ChIP) assay and siRNA, we demonstrate that vitamin D/VDR regulates PD-L1 expression in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) cells. We have examined whether a VDR antagonist, MeTC7, can inhibit PD-L1. To ensure that MeTC7 inhibits VDR/PD-L1 without off-target effects, we examined competitive inhibition of VDR by MeTC7, utilizing ligand-dependent dimerization of VDR-RXR, RXR-RXR, and VDR-coactivators in a mammalian 2-hybrid (M2H) assay. MeTC7 inhibits VDR selectively, suppresses PD-L1 expression sparing PD-L2, and inhibits the cell viability, clonogenicity, and xenograft growth of AML cells. MeTC7 blocks AML/mesenchymal stem cells (MSCs) adhesion and increases the efferocytotic efficiency of THP-1 AML cells. Additionally, utilizing a syngeneic colorectal cancer model in which VDR/PD-L1 co-upregulation occurs in vivo under radiation therapy (RT), MeTC7 inhibits PD-L1 and enhances intra-tumoral CD8+T cells expressing lymphoid activation antigen-CD69. Taken together, MeTC7 is a promising small-molecule inhibitor of PD-L1 with clinical potential.

Lung infections caused by antibiotic resistant strains of Pseudomonas aeruginosa are difficult to eradicate in immunocompromised hosts such as those with cystic fibrosis. We previously demonstrated that extracellular vesicles (EVs) secreted by primary human airway epithelial cells (AEC) deliver miRNA let-7b-5p to P. aeruginosa to suppress biofilm formation and increase sensitivity to beta-lactam antibiotics. In this study, we show that EVs secreted by AEC transfer multiple distinct sRNA fragments to P. aeruginosa that are predicted to target the three subunits of the fluoroquinolone efflux pump MexHI-OpmD, thus increasing antibiotic sensitivity. Exposure of P. aeruginosa to EVs resulted in a significant reduction in the protein levels of MexH (-48%), MexI (-50%) and OpmD (-35%). Moreover, EVs reduced planktonic growth of P. aeruginosa in the presence of the fluoroquinolone antibiotic ciprofloxacin by 20%. A mexGHI-opmD deletion mutant of P. aeruginosa phenocopied this increased sensitivity to ciprofloxacin. Finally, we found that a fragment of an 18S rRNA external transcribed spacer that was transferred to P. aeruginosa by EVs reduced planktonic growth of P. aeruginosa in the presence of ciprofloxacin, reduced the minimum inhibitory concentration (MIC) of P. aeruginosa for ciprofloxacin by over 50%, and significantly reduced protein levels of both MexH and OpmD. In conclusion, an rRNA fragment secreted by AEC in EVs that targets the fluoroquinolone efflux pump MexHI-OpmD down-regulated these proteins and increased the ciprofloxacin sensitivity of P. aeruginosa. A combination of rRNA fragments and ciprofloxacin packaged in nanoparticles or EVs may benefit patients with antibiotic-resistant P. aeruginosa infections.

The epithelial-mesenchymal transition (EMT) is a developmental program co-opted by tumor cells that aids the initiation of the metastatic cascade. Tumor cells that undergo EMT are relatively chemoresistant, and there are currently no therapeutic avenues specifically targeting cells that have acquired mesenchymal traits. We show that treatment of mesenchymal-like triple-negative breast cancer (TNBC) cells with the microtubule-destabilizing chemotherapeutic eribulin, which is FDA-approved for the treatment of advanced breast cancer, leads to a mesenchymal-epithelial transition (MET). This MET is accompanied by loss of metastatic propensity and sensitization to subsequent treatment with other FDA-approved chemotherapeutics. We uncover a novel epigenetic mechanism of action that supports eribulin pretreatment as a path to MET induction that curtails metastatic progression and the evolution of therapy resistance.SummaryWhile the advent of targeted therapy has led to vast improvements in outcomes for certain types of breast cancer, a mainstay for triple-negative breast cancer (TNBC) remains cytotoxic chemotherapy. A major clinical hurdle in successfully managing this disease is the eventual development of therapeutic resistance and disease relapse in more aggressive forms. Our data reveal that epigenetic modulation of EMT state using the FDA-approved therapeutic eribulin curtails metastatic propensity of breast tumors and, when administered in the treatment-naïve setting, sensitizes to subsequent treatment with other chemotherapeutics.

Pancreatic ductal adenocarcinoma (PDAC) continues to have a dismal prognosis, in part, due to ineffective treatment strategies. The efficacy of some chemotherapies and especially radiotherapy is mediated partially by the immune system. Therefore, we hypothesized that profiling the immune response following chemotherapy and/or irradiation can be used as a readout for treatment efficacy but also to help identify optimal therapeutic schedules for PDAC. Using murine models of PDAC, we demonstrated that concurrent administration of stereotactic body radiotherapy (SBRT) and a modified dose of FOLFIRINOX (mFX) resulted in superior tumor control when compared with single or sequential treatment groups. Importantly, this combined treatment schedule enhanced the magnitude of immunogenic cell death, which in turn amplified tumor antigen presentation by dendritic cells and intratumoral CD8+ T-cell infiltration. Concurrent therapy also resulted in systemic immunity contributing to the control of established metastases. These findings provide a rationale for pursuing concurrent treatment schedules of SBRT with mFX in PDAC.

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Supplemental figure legends and supplemental tables

Supplementary Data from Modulation of the Human Pancreatic Ductal Adenocarcinoma Immune Microenvironment by Stereotactic Body Radiotherapy

Purpose:Stereotactic body radiotherapy (SBRT) is an emerging treatment modality for pancreatic ductal adenocarcinoma (PDAC), which can effectively prime cytotoxic T cells by inducing immunogenic tumor cell death in preclinical models. SBRT effects on human PDAC have yet to be thoroughly investigated; therefore, this study aimed to characterize immunomodulation in the human PDAC tumor microenvironment following therapy.Experimental Design:Tumor samples were obtained from patients with resectable PDAC. Radiotherapy was delivered a median of 7 days prior to surgical resection, and sections were analyzed by multiplex IHC (mIHC), RNA sequencing, and T-cell receptor sequencing (TCR-seq).Results:Analysis of SBRT-treated tumor tissue indicated reduced tumor cell density and increased immunogenic cell death relative to untreated controls. Radiotherapy promoted collagen deposition; however, vasculature was unaffected and spatial analyses lacked evidence of T-cell sequestration. Conversely, SBRT resulted in fewer tertiary lymphoid structures and failed to lessen or reprogram abundant immune suppressor populations. Higher percentages of PD-1+ T cells were observed following SBRT, and a subset of tumors displayed more clonal T-cell repertoires.Conclusions:These findings suggest that SBRT augmentation of antitumor immunogenicity may be dampened by an overabundance of refractory immunosuppressive populations, and support the continued development of SBRT/immunotherapy combination for human PDAC.

Vitamin-D receptor (VDR) mRNA is overexpressed in neuroblastoma and carcinomas of lung, pancreas, and ovaries and predicts poor prognoses. VDR antagonists may be able to inhibit tumors that overexpress VDR. However, the current antagonists are arduous to synthesize and are only partial antagonists, limiting their use. Here, we show that the VDR antagonist MeTC7 (

Introduction: Two major hurdles faced in clinical cancer therapy are metastatic progression that results in the majority of cancer deaths, and resistance to therapy that fuels tumor relapse. The epithelial-mesenchymal transition (EMT) is a reversible cellular program that contributes to the intratumoral heterogeneity of carcinoma cells and confers traits that promote metastatic progression and resistance to chemotherapy. The standard of care for triple negative breast cancer (TNBC) remains neoadjuvant chemotherapy where cyclophosphamide, doxorubicin and paclitaxel are administered in combination. In case of advanced/metastatic disease, third-line eribulin is administered as a single agent. In some cases of advanced disease, vinorelbine has also demonstrated efficacy. Eribulin, vinorelbine, and paclitaxel are microtubule inhibitors that act via mitotic blockade. Knowledge gap: Despite major advances in our understanding, the contributions of EMT research to improvements in cancer therapy have been minimal and yielded no clinically viable strategies to selectively modulate EMT in human tumors. Results: Our new data reveals that eribulin, in addition to blocking tubulin polymerization, utilizes microtubule-independent mechanism to epigenetically induce MET. Using thermal proteome profiling and mass spectrometry analyses, we identify candidate target proteins of eribulin that enable it to act in an epigenetic fashion to induce MET.Treating TNBC patient-derived xenograft tumors with eribulin induces a significant reduction in metastatic burden, with the remaining metastatic colonies exhibiting a well-differentiated histopathology. Eribulin efficiently induce MET in cells that have received no prior chemotherapy; in cells pretreated with anthracyclines and taxanes, eribulin is less efficient at MET induction. Additionally, cells that were resistant to first-line taxane treatment were also resistant to eribulin-induced cell death. Using patient-derived xenograft (PDX) models, we have determined the optimal sequence of eribulin administration to maximize its MET-inducing and cytotoxic effects. Conclusions: We uncover that, working through altering the chromatin landscape and transcriptional profile of cells, eribulin induces MET and primes cells for subsequent chemotherapy. Additionally, the efficacy of eribulin is highest when administered in the treatment-naïve setting. By developing an understanding of eribulin’s mechanism of action, treatment strategies can be optimized to maximize therapeutic potential by exploiting its effects in both mitotic blockade as well as MET. Upon completion of this study, we will better understand how MET-induced tumor differentiation would work and the impact that it would have on current conventional therapeutic strategies. Acknowledgements: Eribulin studies funded by Eisai Inc. Citation Format: Diwakar R Pattabiraman, Meisam Bagheri, Nevena B Ognjenovic, Ian S LaCroix, Scott A Gerber. Eribulin alters the chromatin landscape to induce MET, attenuating metastatic progression and sensitizing breast tumors to subsequent chemotherapy [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-06-01.

Purpose: Stereotactic body radiotherapy (SBRT) is an emerging treatment modality for pancreatic ductal adenocarcinoma (PDAC), which can effectively prime cytotoxic T cells by inducing immunogenic tumor cell death in preclinical models. SBRT effects on human PDAC have yet to be thoroughly investigated; therefore, this study aimed to characterize immunomodulation in the human PDAC tumor microenvironment following therapy. Experimental Design: Tumor samples were obtained from patients with resectable PDAC. Radiotherapy was delivered a median of 7 days prior to surgical resection, and sections were analyzed by multiplex IHC (mIHC), RNA sequencing, and T-cell receptor sequencing (TCR-seq). Results: Analysis of SBRT-treated tumor tissue indicated reduced tumor cell density and increased immunogenic cell death relative to untreated controls. Radiotherapy promoted collagen deposition; however, vasculature was unaffected and spatial analyses lacked evidence of T-cell sequestration. Conversely, SBRT resulted in fewer tertiary lymphoid structures and failed to lessen or reprogram abundant immune suppressor populations. Higher percentages of PD-1+ T cells were observed following SBRT, and a subset of tumors displayed more clonal T-cell repertoires. Conclusions: These findings suggest that SBRT augmentation of antitumor immunogenicity may be dampened by an overabundance of refractory immunosuppressive populations, and support the continued development of SBRT/immunotherapy combination for human PDAC.

PurposeDelirium presents a significant healthcare burden. It complicates post-operative care in up to 50% of cardiac surgical patients with worse hospital outcomes, longer hospital stays and higher overall cost of care. Moreover, the nature of delirium following cardiac surgery with cardiopulmonary bypass (CPB) remains unclear, the underlying pathobiology is poorly understood, status quo diagnostic methods are subjective, and diagnostic biomarkers are currently lacking.ObjectiveTo identify diagnostic biomarkers of delirium and for insights into possible neuronal pathomechanisms.Experimental designComparative proteomic analyses were performed on plasma samples from a nested matched cohort of patients who underwent cardiac surgery on CPB. A targeted proteomics strategy was used for validation in an independent set of samples. Biomarkers were assessed for biological functions and diagnostic accuracy.Results47% of subjects demonstrated delirium. Of 3803 total proteins identified and quantified from patient plasma samples by multiplexed quantitative proteomics, 16 were identified as signatures of exposure to CPB, and 11 biomarkers distinguished delirium cases from non-cases (AuROC = 93%). Notable among these biomarkers are C-reactive protein, serum amyloid A-1 and cathepsin-B.Conclusions and clinical relevanceThe interplay of systemic and central inflammatory markers shed new light on delirium pathogenesis. This work suggests that accurate identification of cases may be achievable using a panel of biomarkers.Statement of Clinical RelevanceThe acute implication of delirium is well-documented, yet the true extent of the consequences beyond the immediate post-operative period has yet to be fully known. Despite its impact on the geriatric population, delirium remains underdiagnosed. Correctly identifying cases remain a challenge in clinical practice: the arbitrary and subjective nature of current diagnostic tools, such as the confusion assessment method, underscores the urgent need for diagnostic biomarkers. The clinical usefulness of delirium biomarkers extent beyond the objective identification of cases. Delirium biomarkers will also be useful for risk stratification, long-term follow-up of patients and may offer insights into possible etiologies that underpin the condition. In this report, we found systemic markers of inflammation with well-established association with delirium, as well as new biomarkers that shed new light on the condition. Although validation in a larger cohort is the necessary next step, our efforts lay the groundwork for future studies and highlight new frontiers in delirium research yet to be explored.

BackgroundStereotactic body radiotherapy (SBRT) induces immunogenic cell death, leading to subsequent antitumor immune response that is in part counterbalanced by activation of immune evasive processes, for example, upregulation of programmed cell death-ligand 1 (PD-L1) and adenosine generating enzyme, CD73. CD73 is upregulated in pancreatic ductal adenocarcinoma (PDAC) compared with normal pancreatic tissue and high expression of CD73 in PDACs is associated with increased tumor size, advanced stage, lymph node involvement, metastasis, PD-L1 expression and poor prognosis. Therefore, we hypothesized that blockade of both CD73 and PD-L1 in combination with SBRT might improve antitumor efficacy in an orthotopic murine PDAC model.MethodsWe assessed the combination of systemic blockade of CD73/PD-L1 and local SBRT on tumor growth in primary pancreatic tumors, and investigated systemic antitumor immunity using a metastatic murine model bearing both orthotopic primary pancreatic tumor and distal hepatic metastases. Immune response was quantified by flow cytometric and Luminex analyses.ResultsWe demonstrated that blockade of both CD73 and PD-L1 significantly amplified the antitumor effect of SBRT, leading to superior survival. The triple therapy (SBRT+anti-CD73+anti-PD-L1) modulated tumor-infiltrating immune cells with increases of interferon-γ+CD8+T cells. Additionally, triple therapy reprogramed the profile of cytokines/chemokines in the tumor microenvironment toward a more immunostimulatory phenotype. The beneficial effects of triple therapy are completely abrogated by depletion of CD8+T cells, and partially reversed by depletion of CD4+T cells. Triple therapy promoted systemic antitumor responses illustrated by: (1) potent long-term antitumor memory and (2) enhanced both primaryandliver metastases control along with prolonged survival.

Background Skeletal muscle wasting (SMW) in cancer patients is associated with increased morbidity, mortality, treatment intolerance and discontinuation, and poor quality of life. This is particularly true for patients with pancreatic ductal adenocarcinoma (PDAC), as over 85% experience SMW, which is responsible for ~30% of patient deaths. While the established paradigm to explain SMW posits that muscle catabolism from systemic inflammation and nutritional deficiencies, the cause of death, and the cellular and molecular mechanisms responsible remain to be elucidated. To address this, we investigated the relationship between tumour burden and survival in the KCKO murine PDAC model. Methods Female C57BL/6J mice 6–8 weeks of age underwent orthotopic injection with KCKO‐luc tumour cells. Solid tumour was verified on Day 5, post‐tumour inoculation. In vivo, longitudinal lean mass and tumour burden were assessed via dual‐energy X‐ray absorptiometry and IVIS imaging, respectively, and total body weight was assessed, weekly. Animals were sacrificed at a designated end point of ‘failure to thrive’. After sacrifice, lower limb hind muscles were harvested for histology and RNA extraction. Results We found a strong correlation between primary tumour size and survival (r 2 = 0.83, P

Kinetochore protein phosphorylation promotes the correction of erroneous microtubule attachments to ensure faithful chromosome segregation during cell division. Determining how phosphorylation executes error correction requires an understanding of whether kinetochore substrates are completely (i.e., all-or-none) or only fractionally phosphorylated. Using quantitative mass spectrometry (MS), we measured phospho-occupancy on the conserved kinetochore protein Hec1 (NDC80) that directly binds microtubules. None of the positions measured exceeded ∼50% phospho-occupancy, and the cumulative phospho-occupancy changed by only ∼20% in response to changes in microtubule attachment status. The narrow dynamic range of phospho-occupancy is maintained, in part, by the ongoing phosphatase activity. Further, both Cdk1–Cyclin B1 and Aurora kinases phosphorylate Hec1 to enhance error correction in response to different types of microtubule attachment errors. The low inherent phospho-occupancy promotes microtubule attachment to kinetochores while the high sensitivity of kinetochore–microtubule attachments to small changes in phospho-occupancy drives error correction and ensures high mitotic fidelity.

BackgroundNeurologic and neurobehavioural complications are common after cardiac surgery with cardiopulmonary bypass (CPB). Exposure to the artificial bypass surface, conversion to laminar flow and hypothermia likely contribute to systemic inflammation observed after CPB. To ensure adequate systemic oxygenation, the CPB patient is often exposed to supraphysiologic levels of oxygen. Relative to normoxia, perioperative hyperoxia during CPB has not been shown to impact neurocognition in the long-term. Whether this holds true for the immediate post-operative neurocognitive function is the question of this nested case-control study.Methods46 age-and sex-matched subjects, aged ≥65 years, selected for this study were randomized to receive normoxia or hyperoxia during CABG with CPB in the parent trial. Levels of four neuroinflammatory biomarkers (S100B, ENO2, CHI3L1, UCHL1) were measured at baseline and at post-bypass. Baseline neurocognition was established with the Montreal Cognitive Assessment tool and patients were assessed on each post-operative day for delirium using the confusion assessment method. Mediation analyses was conducted for the conditional effect of perioperative oxygen treatment on the occurrence of delirium, assuming mediation effect from change in biomarker levels.Results26 subjects (n = 12) demonstrated delirium. Of the four biomarkers, only S100B levels were differentially abundant post-bypass regardless of treatment (8.18 versus 10.15pg/mL, p value < 0.001). We found significant direct effects of treatment on the occurrence of delirium (effect size = -2.477, p = 0.004). There was no statistically significant mediating effect by S100B.ConclusionWhile perioperative hyperoxia may not be associated with neurocognitive dysfunction in the long-term, its immediate effects may contribute significantly to the occurrence of post-operative delirium. Taken together, our findings suggest a dose-response-time relationship between hyperoxia and neurocognitive function.

Delirium represents a significant health care burden, diagnosed in more than 2 million elderly Americans each year. In the surgical population, delirium remains the most common complication among elderly patients, and is associated with longer hospital stays, higher costs of care, increased mortality, and functional impairment. The pathomechanism of disease is poorly understood, with current diagnostic approaches somewhat subjective and arbitrary, and definitive diagnostic biomarkers are currently lacking. Despite the recent interest in delirium research, biomarker discovery for it remains new. Most attempts to discover biomarkers are targeted studies that seek to assess the involvement of one or more members of a focused panel of candidates in delirium. For a more unbiased, system-biology view, we searched literature from Medical Literature Analysis and Retrieval System Online (MEDLINE), Cochrane Central, Web of Science, SCOPUS, and Dimensions between 2016 and 2021 for untargeted proteomic discovery studies for biomarkers of delirium conducted on human geriatric subjects. Two reviewers conducted an independent review of all search results and resolved discordance by consensus. From an overall search of 1172 publications, 8 peer-reviewed studies met our defined inclusion criteria. The 370 unique perioperative biomarkers identified in these reports are enriched in pathways involving activation of the immune system, inflammatory response, and the coagulation cascade. The most frequently identified biomarker was interleukin-6 (IL-6). By reviewing the distribution of protein biomarker candidates from these studies, we conclude that a panel of proteins, rather than a single biomarker, would allow for discriminating delirium cases from noncases. The paucity of hypothesis-generating studies in the peer-reviewed literature also suggests that a system-biology view of delirium pathomechanisms has yet to fully emerge.

Lung infection by antibiotic resistant strains of Pseudomonas aeruginosa is a well-known concern for immunocompromised hosts including people with lung diseases such as cystic fibrosis. We have previously demonstrated that extracellular vesicles (EVs) secreted by primary human airway epithelial cells (AEC) deliver miRNA let-7b-5p to P. aeruginosa where it suppresses biofilm formation and increases sensitivity to beta-lactam antibiotics. In this study we used RNA-seq to characterize the small RNA (sRNA) content of EVs secreted by AEC and demonstrate transfer of multiple distinct RNA fragments from EVs to P. aeruginosa. Bioinformatic predictions reveal that several sRNAs may target all three subunits of the fluoroquinolone efflux pump MexHI-OpmD, an effect predicted to increase antibiotic sensitivity to fluoroquinolone antibiotics. Exposure of P. aeruginosa to EVs resulted in a significant reduction in the protein levels of MexH (−48%), MexI (−50%) and OpmD (−35%). Moreover, EVs reduced planktonic growth of P. aeruginosa in the presence of the fluoroquinolone antibiotic ciprofloxacin by 20%. A mexGHI-opmD deletion mutant of P. aeruginosa phenocopied this increased sensitivity to ciprofloxacin. Finally, we found that a fragment of an 18S rRNA external transcribed spacer that was transferred to P. aeruginosa by EVs was sufficient to reduce planktonic growth of P. aeruginosa in the presence of ciprofloxacin, to reduce the minimum inhibitory concentration (MIC) of P. aeruginosa for ciprofloxacin by over 50%, and to significantly reduce protein levels of MexH and OpmD. In conclusion, an rRNA fragment secreted by AEC in EVs increases the ciprofloxacin sensitivity of P. aeruginosa by targeting and down-regulating the fluoroquinolone efflux pump MexHI-OpmD. A combination of rRNA fragments and ciprofloxacin packaged in nanoparticles or EVs may benefit patients with antibiotic-resistant P. aeruginosa infections.Author SummaryAccording to the World Health Organization and the U.S. Centers for Disease Control and Prevention the development of antibiotic resistant strains of bacteria, including Pseudomonas aeruginosa, are a significant global threat to human health. Thus, development of new approaches to eliminate antibiotic resistant infections is required. In this study, we report that lung epithelial cells secrete extracellular vesicles (EVs) that fuse with and deliver small rRNAs to P. aeruginosa, and that the rRNAs increase the sensitivity of P. aeruginosa to the antibiotic ciprofloxacin by reducing protein levels of the drug efflux pump MexHI-OpmD. We identified one rRNA fragment that by itself significantly reduced the protein levels of MexH and OpmD and increased the ability of ciprofloxacin to kill P. aeruginosa. We propose that developing synthetic vesicles containing a combination of the rRNA that inhibits antibiotic efflux pumps and ciprofloxacin would benefit patients with antibiotic resistant P. aeruginosa infections.

2020 marks the 400th anniversary of the planting of Plymouth Colony. Although the literature about Plymouth is voluminous, the discussion about law and religion has been inappropriately superficial to date. This article addresses the Pilgrims’ conception of law on matters of religion and the new insights into the Pilgrims’ story that can be ascertained by focusing on law. “Law” has been defined in many different ways by many different people throughout history. Aristotle, Cicero, Thomas Aquinas, and other proponents of natural law argued that law is the exercise of reason to deduce binding rules of moral behavior from nature’s or God’s creation. The renowned English positivist John Austin, in contrast, maintained that law is the command of the sovereign. To Karl von Savigny and other proponents of the so-called historical school, law is the unconscious embodiment of the common will of the people. To the philosophical school, law is the expression of idealized ethical custom. The dominant contemporary view seems to be that law is the reflection of social, political, and economic interests. For the Pilgrims of Plymouth Colony, law was both the memorialization of their commitment to the Word of God and an instrument for exercising social control so as to effectuate that commitment. The Pilgrims, of course, used law to regulate the more mundane aspects of life as well. Indeed, quantitatively speaking, more laws were enacted by the Pilgrims that addressed the day-to-day activities of life in Plymouth Colony than memorialized the Pilgrims’ commitment to eternal glory in the afterlife, but the latter was unquestionably more important, qualitatively speaking, than the former. In the oft-quoted words of a young William Bradford, “to keep a good conscience, and walk in such a way as God has prescribed in his Word, is a thing which I must prefer before you all, and above life itself.”

Significance More than 2 billion people are iron deficient, because their plant-based diets are not a rich source of this essential nutrient. Despite progress in tracing how iron moves throughout the plant, we still do not fully understand how plants sense and respond to iron availability. Here, we identify an essential basic helix–loop–helix transcription factor, Upstream Regulator of IRT1 (URI), and describe its central role as an Fe-dependent switch. The phosphorylated form of URI accumulates under Fe-deficient growth conditions and activates most known Fe deficiency-induced genes. Under Fe-replete growth conditions, the phosphorylated URI undergoes proteasomal degradation dependent on the E3 ligase BTS. Our study provides a molecular mechanism for Fe-dependent regulation of Fe deficiency signaling in plants., Iron is an essential nutrient for plants, but excess iron is toxic due to its catalytic role in the formation of hydroxyl radicals. Thus, iron uptake is highly regulated and induced only under iron deficiency. The mechanisms of iron uptake in roots are well characterized, but less is known about how plants perceive iron deficiency. We show that a basic helix–loop–helix (bHLH) transcription factor Upstream Regulator of IRT1 (URI) acts as an essential part of the iron deficiency signaling pathway in Arabidopsis thaliana. The uri mutant is defective in inducing Iron-Regulated Transporter1 (IRT1) and Ferric Reduction Oxidase2 (FRO2) and their transcriptional regulators FER-like iron deficiency-induced transcription factor (FIT) and bHLH38/39/100/101 in response to iron deficiency. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) reveals direct binding of URI to promoters of many iron-regulated genes, including bHLH38/39/100/101 but not FIT. While URI transcript and protein are expressed regardless of iron status, a phosphorylated form of URI only accumulates under iron deficiency. Phosphorylated URI is subject to proteasome-dependent degradation during iron resupply, and turnover of phosphorylated URI is dependent on the E3 ligase BTS. The subgroup IVc bHLH transcription factors, which have previously been shown to regulate bHLH38/39/100/101, coimmunoprecipitate with URI mainly under Fe-deficient conditions, suggesting that it is the phosphorylated form of URI that is capable of forming heterodimers in vivo. We propose that the phosphorylated form of URI accumulates under Fe deficiency, forms heterodimers with subgroup IVc proteins, and induces transcription of bHLH38/39/100/101. These transcription factors in turn heterodimerize with FIT and drive the transcription of IRT1 and FRO2 to increase Fe uptake.

Rab40c is a SOCS box–containing protein which binds Cullin5 to form a ubiquitin E3 ligase complex (Rab40c/CRL5) to regulate protein ubiquitylation. However, the exact functions of Rab40c remain to be determined, and what proteins are the targets of Rab40c-Cullin5–mediated ubiquitylation in mammalian cells are unknown. Here we showed that in migrating MDA-MB-231 cells Rab40c regulates focal adhesion’s number, size, and distribution. Mechanistically, we found that Rab40c binds the protein phosphatase 6 (PP6) complex and ubiquitylates one of its subunits, ankyrin repeat domain 28 (ANKRD28), thus leading to its lysosomal degradation. Furthermore, we identified that phosphorylation of FAK and MOB1 is decreased in Rab40c knock-out cells, which may contribute to focal adhesion site regulation by Rab40c. Thus, we propose a model where Rab40c/CRL5 regulates ANKRD28 ubiquitylation and degradation, leading to a decrease in PP6 activity, which ultimately affects FAK and Hippo pathway signaling to alter focal adhesion dynamics.

Phosphorylation signaling is an essential post-translational regulatory mechanism that governs almost all eukaryotic biological processes and is controlled by an interplay between protein kinases and phosphatases. Knowledge of direct substrates of kinases provides evidence of mechanisms that relate activity to biological function. Linking kinases to their protein substrates can be achieved by inhibiting or reducing kinase activity and quantitative comparisons of phosphoproteomes in the presence and absence of kinase activity. Unfortunately, most of the human kinases lack chemical inhibitors with selectivity required to unambiguously assign protein substrates to their respective kinases. Here, we develop and validate a chemical proteomics strategy for linking kinase activities to protein substrates via targeted protein degradation and quantitative phosphoproteomics and apply it to the well-studied, essential mitotic regulator polo-like kinase 1 (Plk1). We leveraged the Tir1/auxin system to engineer HeLa cells with endogenously homozygous auxin-inducible degron (AID)-Plk1). We used HeLa cells and determined the impact of AID-tagging on Plk1 activity, localization, protein interactors, and substrate motifs. Using quantitative proteomics, we show that of over 8,000 proteins quantified, auxin addition is highly selective for degrading AID-Plk1 in mitotic cells. Comparison of phosphoproteome changes in response to chemical Plk1 inhibition to auxin-induced degradation revealed a striking degree of correlation. Finally, we explored basal protein turnover as a potential basis for clonal differences in auxin-induced degradation rates for AID-Plk1 cells. Taken together, our work provides a roadmap for the application of AID technology as a general strategy for the kinome-wide discovery of kinase-substrate relationships.

Purpose: Orthotopic tumors more closely recapitulate human cancers than do ectopic models; however, precision targeting of such internal tumors for radiation therapy (RT) without inducing systemic toxicity remains a barrier. We developed an innovative murine orthotopic rectal tumor model where the insertion of clinical grade titanium fiducial clips on opposing sides of the rectal tumor allowed for targeted administration of short-course radiation therapy (SCRT). With this novel approach, clinically relevant RT regimens can be administered to orthotopic tumors to explore the biology and efficacy of radiation alone or as a combination therapy in a murine model that closely recapitulates human disease. Methods and Materials: Murine Colon 38-luciferase tumor cells were injected into the rectal wall of syngeneic mice, and fiducial clips were applied to demarcate the tumor. An SCRT regimen consisting of 5 consecutive daily doses of 5 Gy delivered by an image-guided conformal small animal irradiator was administered 9 days after implantation. Tumor burden and survival were monitored along with histological and flow cytometric analyses on irradiated versus untreated tumors at various time points. Results: SCRT administered to orthotopic rectal tumors resulted in a reduction in tumor burden and enhanced overall survival with no apparent signs of systemic toxicity. This treatment paradigm resulted in significant reductions in tumor cellularity and increases in fibrosis and hyaluronic acid production, recapitulating the SCRT-induced effects observed in human cancers. Conclusions: We have established a means to target murine orthotopic rectal tumors using fiducial markers with a fractionated and clinically relevant SCRT schedule that results in an RT response similar to what is observed in human rectal cancer. We also validated our model through examining various parameters associated with human cancer that are influenced by irradiation. This model can be used to further explore RT doses and scheduling, and to test combinatorial therapies.