Hypervirulent Klebsiella pneumoniae employs genomic island encoded toxins against bacterial competitors in the gut.

The hypervirulent lineages of Klebsiella pneumoniae (HvKp) cause invasive infections such as Klebsiella-liver abscess. Invasive infection often occurs after initial colonization of the host gastrointestinal tract by HvKp. Over 80% of HvKp isolates belong to the clonal group 23 sublineage I that has acquired genomic islands (GIs) GIE492 and ICEKp10. Our analysis of 12 361 K. pneumoniae genomes revealed that GIs GIE492 and ICEKp10 are co-associated with the CG23-I and CG10118 HvKp lineages. GIE492 and ICEKp10 enable HvKp to make a functional bacteriocin microcin E492 (mccE492) and the genotoxin colibactin, respectively. We discovered that GIE492 and ICEKp10 play cooperative roles and enhance gastrointestinal colonization by HvKp. Colibactin is the primary driver of this effect, modifying gut microbiome diversity. Our in vitro assays demonstrate that colibactin and mccE492 kill or inhibit a range of Gram-negative Klebsiella species and Escherichia coli strains, including Gram-positive bacteria, sometimes cooperatively. Moreover, mccE492 and colibactin kill human anaerobic gut commensals that are similar to the taxa found altered by colibactin in the mouse intestines. Our findings suggest that GIs GIE492 and ICEKp10 enable HvKp to kill several commensal bacterial taxa during interspecies interactions in the gut. Thus, acquisition of GIE492 and ICEKp10 could enable better carriage in host populations and explain the dominance of the CG23-I HvKp lineage.

Control line failure in Angiostrongylus vasorum point-of-care serology test in dogs with angiostrongylosis due to suspected hook effect.

OBJECTIVES: Angiostrongylosis is a significant differential for a diverse range of clinical signs in dogs, many of whom present acutely and sometimes with fatal consequences. Point-of-care diagnostic assays include a commercially available Angiostrongylus vasorum qualitative direct lateral flow assay. MATERIALS AND METHODS: Case records from one referral centre from dogs with an invalid A. vasorum lateral flow assay, comprising an absent control line alongside a visible test line, were reviewed. As control line failure was hypothesised to be due to antigen excess; where available the A. vasorum lateral flow assay was repeated using dilutions of the original serum. RESULTS: Six dogs had an invalid A. vasorum lateral flow assay result. Five dogs had presented with acute-onset, severe clinical disease consistent with angiostrongylosis, and one dog was a clinically healthy in-contact. Clinical suspicion of angiostrongylosis was confirmed using alternative diagnostic testing and/or response to treatment. Repetition of the A. vasorum lateral flow assay, in four cases, using diluted plasma (10% to 12.5% v/v) resulted in the appearance of a control line alongside the visible test line. CLINICAL SIGNIFICANCE: A heavy burden of A. vasorum infection resulting in angiostrongylosis should be suspected in dogs with compatible clinical signs and an invalid A. vasorum lateral flow assay result due to control failure alongside a visible test line. Repetition of the test with a diluted serum may be considered to account for the hook effect, also known as the postzone phenomenon, as a possible cause.

Peritoneal metastases in patients with neuroendocrine neoplasms: a challenging site of metastases with clinical and prognostic implications.

PURPOSE: Peritoneal metastases (PM) of neuroendocrine neoplasm (NEN) origin are identified with increasing frequency and exert a significant effect on quality of life and clinical status of the patients. The aim of this study was to identify the characteristics and the prognostic significance of PM in patients with NENs. METHODS: A retrospective analysis of the data of patients from two tertiary referral centers was performed. We defined a control group of age- and gender-matched NEN patients with comparable stage IV disease but no PM. RESULTS: We analysed 70 patients (41 females) with PM. Small intestine was the most common primary NEN site (87.1%). PM prevalence was 10.3%. Forty-four patients presented with synchronous PM, whereas 26 developed metachronous PM. The majority of patients had other concomitant metastases (50 hepatic, 6 lung and 12 bone metastases). Twelve patients developed intestinal obstruction. After PM diagnosis, 76% of patients received treatment with somatostatin analogues while six patients (8.6%) were treated with peptide receptor radionuclide therapy (PRRT). The median progression-free survival (PFS) in the PRRT-treated group was 15 months (95% CI 2-28). Median overall survival (OS) in the PM group was 142 months [95% CI 71-213] while it was not reached in the control group. CONCLUSION: Peritoneal metastases show low prevalence among NEN patients and are most likely to develop in patients with small intestinal NENs and advanced metastatic disease. The presence of PM does seem to be associated with a negative prognostic impact on OS of NEN patients and their identification and prompt treatment is of major importance.

Tunable PhenoCycler imaging of the murine pre-clinical tumour microenvironments.

BACKGROUND: The tumour microenvironment (TME) consists of tumour-supportive immune cells, endothelial cells, and fibroblasts. PhenoCycler, a high-plex single cell spatial biology imaging platform, is used to characterize the complexity of the TME. Researchers worldwide harvest and bank tissues from mouse models which are employed to model a plethora of human disease. With the explosion of interest in spatial biology, these panoplies of archival tissues provide a valuable resource to answer new questions. Here, we describe our protocols for developing tunable PhenoCycler multiplexed imaging panels and describe our open-source data analysis pipeline. Using these protocols, we used PhenoCycler to spatially resolve the TME of 8 routinely employed pre-clinical models of lymphoma, breast cancer, and melanoma preserved as FFPE. RESULTS: Our data reveal distinct TMEs in the different cancer models that were imaged and show that cell-cell contacts differ depending on the tumour type examined. For instance, we found that the immune infiltration in a murine model of melanoma is altered in cellular organization in melanomas that become resistant to αPD-1 therapy, with depletions in a number of cell-cell interactions. CONCLUSIONS: This work presents a valuable resource study seamlessly adaptable to any field of research involving murine models. The methodology described allows researchers to address newly formed hypotheses using archival materials, bypassing the new to perform new mouse studies.

CXCR2 antagonist navarixin in combination with pembrolizumab in select advanced solid tumors: a phase 2 randomized trial.

C-X-C motif chemokine receptor 2 (CXCR2) has a role in tumor progression, lineage plasticity, and reduction of immune checkpoint inhibitor efficacy. Preclinical evidence suggests potential benefit of CXCR2 inhibition in multiple solid tumors. In this phase 2 study (NCT03473925), adults with previously treated advanced or metastatic castration-resistant prostate cancer (CRPC), microsatellite-stable colorectal cancer (MSS CRC), or non-small-cell lung cancer (NSCLC) were randomized 1:1 to the CXCR2 antagonist navarixin 30 or 100 mg orally once daily plus pembrolizumab 200 mg intravenously every 3 weeks up to 35 cycles. Primary endpoints were investigator-assessed objective response rate (RECIST v1.1) and safety. Of 105 patients (CRPC, n=40; MSS CRC, n=40; NSCLC, n=25), 3 had a partial response (2 CRPC, 1 MSS CRC) for ORRs of 5%, 2.5%, and 0%, respectively. Median progression-free survival was 1.8-2.4 months without evidence of a dose-response relationship, and the study was closed at a prespecified interim analysis for lack of efficacy. Dose-limiting toxicities occurred in 2/48 patients (4%) receiving navarixin 30 mg and 3/48 (6%) receiving navarixin 100 mg; events included grade 4 neutropenia and grade 3 transaminase elevation, hepatitis, and pneumonitis. Treatment-related adverse events occurred in 70/105 patients (67%) and led to treatment discontinuation in 7/105 (7%). Maximal reductions from baseline in absolute neutrophil count were 44.5%-48.2% (cycle 1) and 37.5%-44.2% (cycle 2) and occurred within 6-12 hours postdose in both groups. Navarixin plus pembrolizumab did not demonstrate sufficient efficacy in this study. Safety and tolerability of the combination were manageable. (Trial registration: ClinicalTrials.gov , NCT03473925).

Divarasib plus cetuximab in KRAS G12C-positive colorectal cancer: a phase 1b trial.

KRAS G12C mutation is prevalent in ~4% of colorectal cancer (CRC) and is associated with poor prognosis. Divarasib, a KRAS G12C inhibitor, has shown modest activity as a single agent in KRAS G12C-positive CRC at 400 mg. Epidermal growth factor receptor has been recognized as a major upstream activator of RAS-MAPK signaling, a proposed key mechanism of resistance to KRAS G12C inhibition in CRC. Here, we report on divarasib plus cetuximab (epidermal growth factor receptor inhibitor) in patients with KRAS G12C-positive CRC (n = 29) from arm C of an ongoing phase 1b trial. The primary objective was to evaluate safety. Secondary objectives included preliminary antitumor activity. The safety profile of this combination was consistent with those of single-agent divarasib and cetuximab. Treatment-related adverse events led to divarasib dose reductions in four patients (13.8%); there were no treatment withdrawals. The objective response rate was 62.5% (95% confidence interval: 40.6%, 81.2%) in KRAS G12C inhibitor-naive patients (n = 24). The median duration of response was 6.9 months. The median progression-free survival was 8.1 months (95% confidence interval: 5.5, 12.3). As an exploratory objective, we observed a decline in KRAS G12C variant allele frequency associated with response and identified acquired genomic alterations at disease progression that may be associated with resistance. The manageable safety profile and encouraging antitumor activity of divarasib plus cetuximab support the further investigation of this combination in KRAS G12C-positive CRC.ClinicalTrials.gov identifier: NCT04449874.

Corridor-Level Impacts of Battery-Electric Heavy-Duty Trucks and the Effects of Policy in the United States.

Electrifying freight trucks will be key to alleviating air pollution burdens on disadvantaged communities and mitigating climate change. The United States plans to pursue this aim by adding vehicle charging infrastructure along specific freight corridors. This study explores the coevolution of the electricity grid and freight trucking landscape using an integrated assessment framework to identify when each interstate and drayage corridor becomes advantageous to electrify from a climate and human health standpoint. Nearly all corridors achieve greenhouse gas emission reductions if electrified now. Most can reduce health impacts from air pollution if electrified by 2040 although some corridors in the Midwest, South, and Mid-Atlantic regions remain unfavorable to electrify from a human health standpoint, absent policy support. Recent policy, namely, the Inflation Reduction Act, accelerates this timeline to 2030 for most corridors and results in net human health benefits on all corridors by 2050, suggesting that near-term investments in truck electrification, particularly drayage corridors, can meaningfully reduce climate and health burdens.

Chemical crosslinking and ligation methods for in vivo analysis of RNA structures and interactions.

RNA structures and interactions in living cells drive a variety of biological processes and play critical roles in physiology and disease states. However, studies of RNA structures and interactions have been challenging due to limitations in available technologies. Direct determination of structures in vitro has been only possible to a small number of RNAs with limited sizes and conformations. We recently introduced two chemical crosslink-ligation techniques that enabled studies of transcriptome-wide secondary and tertiary structures and their dynamics. In a dramatically improved version of the psoralen analysis of RNA interactions and structures (PARIS2) method, we detailed the synthesis and use of amotosalen, a highly soluble psoralen analogue, and enhanced enzymology for higher efficiency duplex capture. We also introduced spatial 2'-hydroxyl acylation reversible crosslinking (SHARC) with exonuclease (exo) trimming, a method which utilizes a novel crosslinker class that targets the 2'-OH to capture three-dimensional (3D) structures. Both are powerful orthogonal approaches for solving in vivo RNA structure and interactions, integrating crosslinking, exo trimming, proximity ligation, and high throughput sequencing. In this chapter, we present a detailed protocol for the methods and highlight steps that outperform existing crosslink-ligation approaches.

Two Cases of Durable and Deep Responses to Immune Checkpoint Inhibition-Refractory Metastatic Melanoma after Addition of Camu Camu Prebiotic.

Camu camu (CC) is a prebiotic that selectively stimulates growth and activity of beneficial gut microbiota. Work in murine models demonstrated that castalagin, the active compound in CC, preferentially binds to beneficial gut microbiome bacteria, promoting a stronger CD8+T cell anti-cancer response. We present two patients with metastatic melanoma whose cancer progressed on immune checkpoint inhibitors (ICIs) and developed clinically significant immune-related adverse events (irAEs). They were rechallenged with ICIs in combination with CC. The first patient is a 71-year-old woman with metastatic melanoma, whose ICI treatment was complicated by immune-related pneumonitis and colitis. Upon progression on maintenance nivolumab, CC was added to nivolumab, leading to a near complete response (CR). The second patient is a 90-year-old man with recurrent unresectable melanoma, treated with nivolumab, complicated by immune-related rash and diabetes. He developed new subcutaneous calf lesions and a metastatic popliteal lymph node. CC was added to nivolumab. One month later, the patient experienced a CR. Both patients have been on nivolumab and CC with durable responses for more than a year, with minimal irAEs. These two cases suggest that CC may modulate the microbiome, synergizing with ICIs to produce deep, durable responses with minimal irAEs.

Peroxisome disruption alters lipid metabolism and potentiates antitumor response with MAPK-targeted therapy in melanoma.

Melanomas reprogram their metabolism to rapidly adapt to therapy-induced stress conditions, allowing them to persist and ultimately develop resistance. We report that a subpopulation of melanoma cells tolerate MAPK pathway inhibitors (MAPKis) through a concerted metabolic reprogramming mediated by peroxisomes and UDP-glucose ceramide glycosyltransferase (UGCG). Compromising peroxisome biogenesis, by repressing PEX3 expression, potentiated the proapoptotic effects of MAPKis via an induction of ceramides, an effect limited by UGCG-mediated ceramide metabolism. Cotargeting PEX3 and UGCG selectively eliminated a subset of metabolically active, drug-tolerant CD36+ melanoma persister cells, thereby sensitizing melanoma to MAPKis and delaying resistance. Increased levels of peroxisomal genes and UGCG were found in patient-derived MAPKi-relapsed melanomas, and simultaneously inhibiting PEX3 and UGCG restored MAPKi sensitivity in multiple models of therapy resistance. Finally, combination therapy consisting of a newly identified inhibitor of the PEX3-PEX19 interaction, a UGCG inhibitor, and MAPKis demonstrated potent antitumor activity in preclinical melanoma models, thus representing a promising approach for melanoma treatment.

Acquisition of regulator on virulence plasmid of hypervirulent Klebsiella allows bacterial lifestyle switch in response to iron.

Hypervirulent Klebsiella pneumoniae causes liver abscess and potentially devastating metastatic complications. The majority of Klebsiella-induced liver abscess are caused by the CG23-I sublineage of hypervirulent Klebsiella pneumoniae. This and some other lineages possess a >200-kb virulence plasmid. We discovered a novel protein IroP nestled in the virulence plasmid-encoded salmochelin operon that cross-regulates and suppresses the promoter activity of chromosomal type 3 fimbriae (T3F) gene transcription. IroP is itself repressed by iron through the ferric uptake regulator. Iron-rich conditions increase T3F and suppress capsule mucoviscosity, leading to biofilm formation and cell adhesion. Conversely, iron-poor conditions cause a transcriptional switch to hypermucoid capsule production and T3F repression. The likely acquisition of iroP on mobile genetic elements and successful adaptive integration into the genetic circuitry of a major lineage of hypervirulent K. pneumoniae reveal a powerful example of plasmid chromosomal cross talk that confers an evolutionary advantage. Our discovery also addresses the conundrum of how the hypermucoid capsule that impedes adhesion could be regulated to facilitate biofilm formation and colonization. The acquired ability of the bacteria to alternate between a state favoring dissemination and one that favors colonization in response to iron availability through transcriptional regulation offers novel insights into the evolutionary success of this pathogen. IMPORTANCE Hypervirulent Klebsiella pneumoniae contributes to the majority of monomicrobial-induced liver abscess infections that can lead to several other metastatic complications. The large virulence plasmid is highly stable in major lineages, suggesting that it provides survival benefits. We discovered a protein IroP encoded on the virulence plasmid that suppresses expression of the type 3 fimbriae. IroP itself is regulated by iron, and we showed that iron regulates hypermucoid capsule production while inversely regulating type 3 fimbriae expression through IroP. The acquisition and integration of this inverse transcriptional switch between fimbriae and capsule mucoviscosity shows an evolved sophisticated plasmid-chromosomal cross talk that changes the behavior of hypervirulent K. pneumoniae in response to a key nutrient that could contribute to the evolutionary success of this pathogen.

Assessing Skin Cancer Risk Factors, Sun Safety Behaviors and Melanoma Concern in Atlantic Canada: A Comprehensive Survey Study.

BACKGROUND: The incidence of cutaneous melanoma (CM) is increasing at an alarming rate in Canada and elsewhere around the world. Significant regional differences in CM incidence have been identified in Atlantic provinces. The goal of this study is to compare ultraviolet exposure, sun protective behaviours, level of worry and baseline CM knowledge in provinces with a high versus low incidence of CM as well, as between various demographic groups. METHODS: A cross-sectional survey study was conducted in Atlantic provinces between July 2020 and August 2022. All participants aged ≥ 16 years with a completed survey were eligible. Survey responses were summarized using frequency counts, percentages, and means. Two-sided Z-tests for equality of proportions and logistic regression models were used to compare the survey results between geographic and demographic groups. RESULTS: In total, 7861 participants were included (28.0% men; mean age 61.3 years; response rate 28%). Our results (gender- and age-adjusted odds ratio, 95% confidence interval) show that high-incidence provinces for CM (Prince Edward Island and Nova Scotia) had significantly more sunburns (OR 2.00, 1.72-2.31), total sun exposure (OR 2.05, 1.68-2.50), recreational sun exposure (OR 1.95, 1.61-2.35) and tans (OR 1.77, 1.53-2.05) than individuals in low-incidence provinces (Newfoundland and Labrador). However, individuals in high-incidence provinces displayed more protective behaviors: there were less tanning bed users (OR 0.82, 0.71-0.95), they checked their skin more frequently for new moles (OR 1.26, 1.06-1.51) and practiced more sun protection overall. Additional analyses are presented based on education, income, sexual orientation and gender. DISCUSSION: These findings suggest that future efforts aimed at reducing the CM burden in Atlantic Canada should be tailored for target geographic and/or demographic groups. LIMITATIONS: the study participants are not representative of the population in Atlantic Canada due to recruitment strategies.

Single-Agent Divarasib (GDC-6036) in Solid Tumors with a KRAS G12C Mutation.

BACKGROUND: Divarasib (GDC-6036) is a covalent KRAS G12C inhibitor that was designed to have high potency and selectivity. METHODS: In a phase 1 study, we evaluated divarasib administered orally once daily (at doses ranging from 50 to 400 mg) in patients who had advanced or metastatic solid tumors that harbor a KRAS G12C mutation. The primary objective was an assessment of safety; pharmacokinetics, investigator-evaluated antitumor activity, and biomarkers of response and resistance were also assessed. RESULTS: A total of 137 patients (60 with non-small-cell lung cancer [NSCLC], 55 with colorectal cancer, and 22 with other solid tumors) received divarasib. No dose-limiting toxic effects or treatment-related deaths were reported. Treatment-related adverse events occurred in 127 patients (93%); grade 3 events occurred in 15 patients (11%) and a grade 4 event in 1 patient (1%). Treatment-related adverse events resulted in a dose reduction in 19 patients (14%) and discontinuation of treatment in 4 patients (3%). Among patients with NSCLC, a confirmed response was observed in 53.4% of patients (95% confidence interval [CI], 39.9 to 66.7), and the median progression-free survival was 13.1 months (95% CI, 8.8 to could not be estimated). Among patients with colorectal cancer, a confirmed response was observed in 29.1% of patients (95% CI, 17.6 to 42.9), and the median progression-free survival was 5.6 months (95% CI, 4.1 to 8.2). Responses were also observed in patients with other solid tumors. Serial assessment of circulating tumor DNA showed declines in KRAS G12C variant allele frequency associated with response and identified genomic alterations that may confer resistance to divarasib. CONCLUSIONS: Treatment with divarasib resulted in durable clinical responses across KRAS G12C-positive tumors, with mostly low-grade adverse events. (Funded by Genentech; ClinicalTrials.gov number, NCT04449874.).

Fecal microbiota transplantation plus anti-PD-1 immunotherapy in advanced melanoma: a phase I trial.

Fecal microbiota transplantation (FMT) represents a potential strategy to overcome resistance to immune checkpoint inhibitors in patients with refractory melanoma; however, the role of FMT in first-line treatment settings has not been evaluated. We conducted a multicenter phase I trial combining healthy donor FMT with the PD-1 inhibitors nivolumab or pembrolizumab in 20 previously untreated patients with advanced melanoma. The primary end point was safety. No grade 3 adverse events were reported from FMT alone. Five patients (25%) experienced grade 3 immune-related adverse events from combination therapy. Key secondary end points were objective response rate, changes in gut microbiome composition and systemic immune and metabolomics analyses. The objective response rate was 65% (13 of 20), including four (20%) complete responses. Longitudinal microbiome profiling revealed that all patients engrafted strains from their respective donors; however, the acquired similarity between donor and patient microbiomes only increased over time in responders. Responders experienced an enrichment of immunogenic and a loss of deleterious bacteria following FMT. Avatar mouse models confirmed the role of healthy donor feces in increasing anti-PD-1 efficacy. Our results show that FMT from healthy donors is safe in the first-line setting and warrants further investigation in combination with immune checkpoint inhibitors. ClinicalTrials.gov identifier NCT03772899 .

'Nesting networks': Women's experiences of social network support in high-risk pregnancy.

OBJECTIVE: Social support, an individual's social relationships (both online and offline), may provide protection against adverse mental health outcomes, such as anxiety and depression, which are high in women who have been hospitalised with high-risk pregnancy. This study explored the social support available to women at higher risk of preeclampsia during pregnancy by examining personal social networks. DESIGN: Semi-structured interviews were accompanied by social network mapping using the web-based social networking tool GENIE. SETTING: England. PARTICIPANTS: Twenty-one women were recruited, of whom 18 were interviewed both during pregnancy and postnatally between April 2019 and April 2020. Nineteen women completed maps pre-natally, 17 women completed maps pre-natally and post-natally. Women were taking part in the BUMP study, a randomised clinical trial that included 2441 pregnant individuals at higher risk of preeclampsia and recruited at a mean of 20 weeks' gestation from 15 hospital maternity units in England between November 2018 and October 2019. RESULTS: Women's social networks tightened during pregnancy. The inner network changed most dramatically postnatally with women reporting fewer network members. Interviews revealed networks were primarily 'real-life' rather than online social networks, with members providing emotional, informational, and practical support. Women with a high-risk pregnancy valued the relationships they developed with health professionals during pregnancy, and would like their midwife to have a more central role in their networks by providing informational and, where needed, emotional support. The social network mapping data supported the qualitative accounts of changing networks across high-risk pregnancy. CONCLUSION: Women with a high-risk pregnancy seek to build "nesting networks" to support them through pregnancy into motherhood. Different types of support are sought from trusted sources. Midwives can play a key role. PRACTICE IMPLICATIONS: As well as highlighting other potential needs during pregnancy and the ways in which they can be met, support from midwives has a key role. Through talking to women early in their pregnancy, signposting information and explaining ways to contact health professionals regarding informational or emotional support would fill a gap that currently is met by other aspects of their network.

Inhibition of the MNK1/2-eIF4E Axis Augments Palbociclib-Mediated Antitumor Activity in Melanoma and Breast Cancer.

Aberrant cell-cycle progression is characteristic of melanoma, and CDK4/6 inhibitors, such as palbociclib, are currently being tested for efficacy in this disease. Despite the promising nature of CDK4/6 inhibitors, their use as single agents in melanoma has shown limited clinical benefit. Herein, we discovered that treatment of tumor cells with palbociclib induces the phosphorylation of the mRNA translation initiation factor eIF4E. When phosphorylated, eIF4E specifically engenders the translation of mRNAs that code for proteins involved in cell survival. We hypothesized that cancer cells treated with palbociclib use upregulated phosphorylated eIF4E (phospho-eIF4E) to escape the antitumor benefits of this drug. Indeed, we found that pharmacologic or genetic disruption of MNK1/2 activity, the only known kinases for eIF4E, enhanced the ability of palbociclib to decrease clonogenic outgrowth. Moreover, a quantitative proteomics analysis of melanoma cells treated with combined MNK1/2 and CDK4/6 inhibitors showed downregulation of proteins with critical roles in cell-cycle progression and mitosis, including AURKB, TPX2, and survivin. We also observed that palbociclib-resistant breast cancer cells have higher basal levels of phospho-eIF4E, and that treatment with MNK1/2 inhibitors sensitized these palbociclib-resistant cells to CDK4/6 inhibition. In vivo we demonstrate that the combination of MNK1/2 and CDK4/6 inhibition significantly increases the overall survival of mice compared with either monotherapy. Overall, our data support MNK1/2 inhibitors as promising drugs to potentiate the antineoplastic effects of palbociclib and overcome therapy-resistant disease.