Antibiotic tolerance among clinical isolates: mechanisms, detection, prevalence, and significance.

SUMMARYAntibiotic treatment failures in the absence of resistance are not uncommon. Recently, attention has grown around the phenomenon of antibiotic tolerance, an underappreciated contributor to recalcitrant infections first detected in the 1970s. Tolerance describes the ability of a bacterial population to survive transient exposure to an otherwise lethal concentration of antibiotic without exhibiting resistance. With advances in genomics, we are gaining a better understanding of the molecular mechanisms behind tolerance, and several studies have sought to examine the clinical prevalence of tolerance. Attempts have also been made to assess the clinical significance of tolerance through in vivo infection models and prospective/retrospective clinical studies. Here, we review the data available on the molecular mechanisms, detection, prevalence, and clinical significance of genotypic tolerance that span ~50 years. We discuss the need for standardized methodology and interpretation criteria for tolerance detection and the impact that methodological inconsistencies have on our ability to accurately assess the scale of the problem. In terms of the clinical significance of tolerance, studies suggest that tolerance contributes to worse outcomes for patients (e.g., higher mortality, prolonged hospitalization), but historical data from animal models are varied. Furthermore, we lack the necessary information to effectively treat tolerant infections. Overall, while the tolerance field is gaining much-needed traction, the underlying clinical significance of tolerance that underpins all tolerance research is still far from clear and requires attention.

Comparison of the effects of normothermic machine perfusion and cold storage preservation on porcine intestinal allograft regenerative potential and viability.

Intestinal transplantation (IT) is the final treatment option for intestinal failure. Static cold storage (CS) is the standard preservation method used for intestinal allografts. However, CS and subsequent transplantation induce ischemia-reperfusion injury (IRI). Severe IRI impairs epithelial barrier function, including loss of intestinal stem cells (ISC), critical to epithelial regeneration. Normothermic machine perfusion (NMP) preservation of kidney and liver allografts minimizes CS-associated IRI; however, it has not been used clinically for IT. We hypothesized that intestine NMP would induce less epithelial injury and better protect the intestine's regenerative ability when compared with CS. Full-length porcine jejunum and ileum were procured, stored at 4 °C, or perfused at 34 °C for 6 hours (T6), and transplanted. Histology was assessed following procurement (T0), T6, and 1 hour after reperfusion. Real-time quantitative reverse transcription polymerase chain reaction, immunofluorescence, and crypt culture measured ISC viability and proliferative potential. A greater number of NMP-preserved intestine recipients survived posttransplant, which correlated with significantly decreased tissue injury following 1-hour reperfusion in NMP compared with CS samples. Additionally, ISC gene expression, spheroid area, and cellular proliferation were significantly increased in NMP-T6 compared with CS-T6 intestine. NMP appears to reduce IRI and improve graft regeneration with improved ISC viability and proliferation.

A LGR5 reporter pig model closely resembles human intestine for improved study of stem cells in disease.

Intestinal epithelial stem cells (ISCs) are responsible for intestinal epithelial barrier renewal; thereby, ISCs play a critical role in intestinal pathophysiology research. While transgenic ISC reporter mice are available, advanced translational studies lack a large animal model. This study validates ISC isolation in a new porcine Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (LGR5) reporter line and demonstrates the use of these pigs as a novel colorectal cancer (CRC) model. We applied histology, immunofluorescence, fluorescence-activated cell sorting, flow cytometry, gene expression quantification, and 3D organoid cultures to whole tissue and single cells from the duodenum, jejunum, ileum, and colon of LGR5-H2B-GFP and wild-type pigs. Ileum and colon LGR5-H2B-GFP, healthy human, and murine biopsies were compared by mRNA fluorescent in situ hybridization (FISH). To model CRC, adenomatous polyposis coli (APC) mutation was induced by CRISPR/Cas9 editing in porcine LGR5-H2B-GFP colonoids. Crypt-base, green fluorescent protein (GFP) expressing cells co-localized with ISC biomarkers. LGR5-H2B-GFPhi cells had significantly higher LGR5 expression (p < .01) and enteroid forming efficiency (p < .0001) compared with LGR5-H2B-GFPmed/lo/neg cells. Using FISH, similar LGR5, OLFM4, HOPX, LYZ, and SOX9 expression was identified between human and LGR5-H2B-GFP pig crypt-base cells. LGR5-H2B-GFP/APCnull colonoids had cystic growth in WNT/R-spondin-depleted media and significantly upregulated WNT/ß-catenin target gene expression (p < .05). LGR5+ ISCs are reproducibly isolated in LGR5-H2B-GFP pigs and used to model CRC in an organoid platform. The known anatomical and physiologic similarities between pig and human, and those shown by crypt-base FISH, underscore the significance of this novel LGR5-H2B-GFP pig to translational ISC research.

Emergency Department Pediatric Readiness: A Trauma Center Quality Improvement Initiative.

BACKGROUND: Most pediatric patients present to general emergency departments, yet maintaining pediatric equipment, skilled staff, and resources remains a challenge for many hospitals. Pediatric readiness assessment is now a requirement for trauma center verification. OBJECTIVE: This study aims to assess the impact of a quality improvement initiative to improve emergency department pediatric readiness. METHODS: A pre- and poststudy design was used to evaluate a quality improvement initiative to improve the National Pediatric Readiness assessment survey results conducted at a Southwestern United States adult Level I trauma center from September 2022 to April 2023. The multicomponent initiative included implementing a pediatric emergency care coordinator, pediatric-specific policies and procedures, identifying pediatric-specific quality and performance indicators, and educating pediatric-specific staff. Study inclusion criteria were all patients younger than 18 years who presented to the emergency department. The primary outcome measure was the improvement in the weighted Pediatric Readiness Score. Secondary outcomes were throughput, nursing documentation of vital signs, and pain scores. RESULTS: A total of N = 2,356 patients met inclusion, of which n = 1,158 (49.2%) were in the preintervention group and n = 1,198 (50.8%) postintervention group. The weighted Pediatric Readiness Score improved by 45.4%. Transfers to a pediatric hospital increased from 4.1% to 8.6% (p = .016). Blood pressure documentation improved slightly from 88.3% to 88.6%. Pain score documentation decreased from 83.9% to 63.1% (p = .008). Pain medication and administration improved from 19.8% to 26.7% (p = .046). CONCLUSION: We found that participation in the quality improvement initiative was associated with emergency department pediatric readiness improvements.

Evaluation of a complex intervention for prisoners with common mental health problems, near to and after release: the Engager randomised controlled trial.

BACKGROUND: Many male prisoners have significant mental health problems, including anxiety and depression. High proportions struggle with homelessness and substance misuse. AIMS: This study aims to evaluate whether the Engager intervention improves mental health outcomes following release. METHOD: The design is a parallel randomised superiority trial that was conducted in the North West and South West of England (ISRCTN11707331). Men serving a prison sentence of 2 years or less were individually allocated 1:1 to either the intervention (Engager plus usual care) or usual care alone. Engager included psychological and practical support in prison, on release and for 3-5 months in the community. The primary outcome was the Clinical Outcomes in Routine Evaluation Outcome Measure (CORE-OM), 6 months after release. Primary analysis compared groups based on intention-to-treat (ITT). RESULTS: In total, 280 men were randomised out of the 396 who were potentially eligible and agreed to participate; 105 did not meet the mental health inclusion criteria. There was no mean difference in the ITT complete case analysis between groups (92 in each arm) for change in the CORE-OM score (1.1, 95% CI -1.1 to 3.2, P = 0.325) or secondary analyses. There were no consistent clinically significant between-group differences for secondary outcomes. Full delivery was not achieved, with 77% (108/140) receiving community-based contact. CONCLUSIONS: Engager is the first trial of a collaborative care intervention adapted for prison leavers. The intervention was not shown to be effective using standard outcome measures. Further testing of different support strategies for prison with mental health problems is needed.

Promising Practices Observed in High-Throughput COVID-19 Vaccination Sites in the United States, February-May 2021.

Objectives. To identify promising practices for implementing COVID-19 vaccination sites. Methods. The Centers for Disease Control and Prevention (CDC) and Federal Emergency Management Agency (FEMA) assessed high-throughput COVID-19 vaccination sites across the United States, including Puerto Rico, after COVID-19 vaccinations began. Site assessors conducted site observations and interviews with site staff. Qualitative data were compiled and thematically analyzed. Results. CDC and FEMA conducted 134 assessments of high-throughput vaccination sites in 25 states and Puerto Rico from February 12 to May 28, 2021. Promising practices were identified across facility, clinical, and cross-cutting operational areas and related to 6 main themes: addressing health equity, leveraging partnerships, optimizing site design and flow, communicating through visual cues, using quick response codes, and prioritizing risk management and quality control. Conclusions. These practices might help planning and implementation of future vaccination operations for COVID-19, influenza, and other vaccine-preventable diseases. Public Health Implications. These practices can be considered by vaccination planners and providers to strengthen their vaccination site plans and implementation of future high-throughput vaccination sites. (Am J Public Health. 2023;113(8):909-918. https://doi.org/10.2105/AJPH.2023.307331).

CLAVATA modulates auxin homeostasis and transport to regulate stem cell identity and plant shape in a moss.

The CLAVATA pathway is a key regulator of stem cell function in the multicellular shoot tips of Arabidopsis, where it acts via the WUSCHEL transcription factor to modulate hormone homeostasis. Broad-scale evolutionary comparisons have shown that CLAVATA is a conserved regulator of land plant stem cell function, but CLAVATA acts independently of WUSCHEL-like (WOX) proteins in bryophytes. The relationship between CLAVATA, hormone homeostasis and the evolution of land plant stem cell functions is unknown. Here we show that in the moss, Physcomitrella (Physcomitrium patens), CLAVATA affects stem cell activity by modulating hormone homeostasis. CLAVATA pathway genes are expressed in the tip cells of filamentous tissues, regulating cell identity, filament branching, plant spread and auxin synthesis. The receptor-like kinase PpRPK2 plays the major role, and Pprpk2 mutants have abnormal responses to cytokinin, auxin and auxin transport inhibition, and show reduced expression of PIN auxin transporters. We propose a model whereby PpRPK2 modulates auxin gradients in filaments to determine stem cell identity and overall plant form. Our data indicate that CLAVATA-mediated auxin homeostasis is a fundamental property of plant stem cell function, probably exhibited by the last shared common ancestor of land plants.

Culture of equine intestinal epithelial stem cells after delayed tissue storage for future applications.

BACKGROUND: Equine intestinal epithelial stem cells (ISCs) serve as potential targets to treat horses with severe intestinal injury. The ability to isolate and store ISCs from intestinal biopsies creates an opportunity for both in vitro experiments to study ISC dynamics in a variety of intestinal diseases, and, in the future, utilize these cells as a possible therapy. If biopsies could be successfully stored prior to processing for ISCs, this would increase the availability of sample repositories for future experimental and therapeutic use. However, delayed culture of equine ISCs following prolonged sample storage has not been described. The objective of this study was to describe the isolation and culture of equine ISCs following delayed tissue storage. Small intestinal full thickness biopsies were collected post euthanasia. Fresh tissue was immediately processed or stored at 4 °C for 24, 48 and 72 h (H) before processing. Intestinal stem cells (crypts) were dissociated and cultured. Size, growth efficiency and proliferation potential were compared between resultant enteroids ("mini-guts") derived from each storage timepoint. In a separate study, growth efficiency of cryopreserved crypts was compared to cryopreserved enteroid fragments to investigate prolonged storage techniques. RESULTS: Intestinal crypts were successfully isolated and cultured from all timepoints. At 72H post initial collection, the intestine was friable with epithelial sloughing; resultant dissociation yielded more partial crypts. Enteroids grown from crypts isolated at 72H were smaller with less proliferative potential (bud units, (median 6.5, 3.75-14.25)) than control (median 25, 15-28, p < 0.0001). No statistical differences were noted from tissues stored for 24H compared to control. Following cryopreservation, growth efficiency improved when cells were stored as enteroid fragments (median 81.6%, 66.2-109) compared to crypts (median 21.2%, 20-21.5, p = 0.01). The main limitations included a small sample size and lack of additional functional assays on enteroids. CONCLUSIONS: Equine ISCs can be isolated and cultured after prolonged tissue storage. Resultant enteroids had minimal differences even after 24-48H of whole tissue storage. This suggests that ISCs could be isolated for several days from samples properly stored after procedures, including surgery or necropsy, and used to create ISC repositories for study or therapy of equine intestinal diseases.

Determining a threshold measurement of endometrial thickness for asymptomatic postmenopausal women: A tertiary centre case series.

BACKGROUND: An incidental finding of a thickened endometrium on ultrasound in the postmenopausal patient without bleeding is a common presentation to gynaecological services; however there is limited evidence to guide clinical practice as to when hysteroscopic evaluation and endometrial sampling is required. AIMS: To determine the endometrial thickness at which endometrial sampling is indicated in asymptomatic postmenopausal women referred with thickened endometrium on ultrasound. MATERIALS AND METHODS: A single-centre retrospective case series of postmenopausal women without bleeding undergoing hysteroscopy was conducted. Logistic regression was used to examine the association between a range of variables and pre-malignant or malignant pathology and endometrial thickness. The optimal endometrial thickness threshold was identified to maximise model sensitivity. RESULTS: A total of 404 postmenopausal women were included in this study, having undergone a hysteroscopy at the study site between 1 July 2008 and 30 June 2018. The mean (SD) age of patients at presentation was 65 (9.09) years and the mean body mass index was 29.86 kg/m2 (6.52). Of these women, nine (2.2%) were diagnosed with endometrial carcinoma and seven (1.7%) had endometrial hyperplasia with atypia. The most common histopathological finding was of a benign endometrial polyp (153: 37.9%). When including hyperplasia with or without atypia in histopathology of interest, a cut-off of ≥9 mm provides the greatest sensitivity (83.3%) and specificity (63.8%) for a diagnosis of pre-malignant or malignant pathology (classification accuracy of 64.8%; area under the receiver operating characteristic: 0.7358, 95% CI: 0.6439, 0.8278) in this cohort. CONCLUSIONS: Using an endometrial thickness of ≥9 mm can be used as a cut-off for endometrial sampling in postmenopausal women without bleeding.

HOPX+ injury-resistant intestinal stem cells drive epithelial recovery after severe intestinal ischemia.

Intestinal ischemia is a life-threatening emergency with mortality rates of 50%-80% due to epithelial cell death and resultant barrier loss. Loss of the epithelial barrier occurs in conditions including intestinal volvulus and neonatal necrotizing enterocolitis. Survival depends on effective epithelial repair; crypt-based intestinal epithelial stem cells (ISCs) are the source of epithelial renewal in homeostasis and after injury. Two ISC populations have been described: 1) active ISC [aISC; highly proliferative; leucine-rich-repeat-containing G protein-coupled receptor 5 (LGR5+)-positive or sex-determining region Y-box 9 -antigen Ki67-positive (SOX9+Ki67+)] and 2) reserve ISC [rISC; less proliferative; homeodomain-only protein X positive (HOPX+)]. The contributions of these ISCs have been evaluated both in vivo and in vitro using a porcine model of mesenteric vascular occlusion to understand mechanisms that modulate ISC recovery responses following ischemic injury. In our previously published work, we observed that rISC conversion to an activated state was associated with decreased HOPX expression during in vitro recovery. In the present study, we wanted to evaluate the direct role of HOPX on cellular proliferation during recovery after injury. Our data demonstrated that during early in vivo recovery, injury-resistant HOPX+ cells maintain quiescence. Subsequent early regeneration within the intestinal crypt occurs around 2 days after injury, a period in which HOPX expression decreased. When HOPX was silenced in vitro, cellular proliferation of injured cells was promoted during recovery. This suggests that HOPX may serve a functional role in ISC-mediated regeneration after injury and could be a target to control ISC proliferation.NEW & NOTEWORTHY This paper supports that rISCs are resistant to ischemic injury and likely an important source of cellular renewal following near-complete epithelial loss. Furthermore, we have evidence that HOPX controls ISC activity state and may be a critical signaling pathway during ISC-mediated repair. Finally, we use multiple novel methods to evaluate ISCs in a translationally relevant large animal model of severe intestinal injury and provide evidence for the potential role of rISCs as therapeutic targets.

Pharmacy stakeholder reports on ethical and logistical considerations in anti-opioid vaccine development.

BACKGROUND: As opioid use disorder (OUD) incidence and its associated deaths continue to persist at elevated rates, the development of novel treatment modalities is warranted. Recent strides in this therapeutic area include novel anti-opioid vaccine approaches. This work compares logistical and ethical considerations surrounding currently available interventions for opioid use disorder with an anti-opioid vaccine approach. METHODS: The opinions of student pharmacists and practicing pharmacists assessing knowledge, perceptions, and attitudes toward current and future OUD management strategies were characterized using a staged, multi-modal research approach incorporating a focus group, pilot survey development and refinement, and final survey deployment. Survey responses were assessed using one- and two-way parametric and non-parametric analyses where appropriate, and multi-dimensional matrix profiles were compared using z-tests following an exhaustive combinatorial sum of differences calculation between items within each compared matrix. RESULTS: Focus group content analysis revealed a high level of agreeableness among participants regarding anti-opioid vaccine technology and a sense of shared ownership regarding solutions to the opioid epidemic at large. Pilot survey results demonstrated subject ability to consider both pragmatic and ethical considerations related to current therapeutics and novel interventions in a single instrument, with high endurance amongst engaged subjects. Access inequality was the most concerning ethical consideration identified for anti-opioid vaccines. Support for anti-opioid vaccine implementation across various clinical scenarios was strongest for voluntary use amongst individuals in recovery, and lowest for mandatory use in at-risk individuals. CONCLUSIONS: Ethical and logistical concerns surrounding anti-opioid vaccines were largely similar to those for current OUD therapeutics overall. Anti-opioid vaccines were endorsed as helpful potential additions to current OUD therapeutic approaches, particularly for voluntary use in the later stages of clinical progression.

Development of a screening tool to identify patients likely to benefit from clinical pharmacist review in a home-based primary care population.

OBJECTIVE: To create a novel screening tool that identified patients who were most likely to benefit from pharmacist in-home medication reviews. DESIGN: Single-center, retrospective study. SETTING AND PARTICIPANTS: A total of 25 homebound patients in Forsyth County, NC, aged 60 years or older with physical or cognitive impairments and enrolled in home-based primary care or transitional and supportive care programs participated in the study. Pharmacy resident-provider pairs conducted home visits for all patients in the study. Pharmacy residents assessed the subjective risk (high, medium, low) of medication nonadherence using information obtained from home visits (health literacy, support network, medications, and detection of something unexpected related to medications). An electronic medical record-based risk score was simultaneously calculated using screening tool components (i.e., electronic frailty index score, LACE+ index [length of stay in the hospital, acuity of admission, comorbidity, emergency department utilization in the 6 months before admission], and 2015 American Geriatric Society Beers Criteria). OUTCOME MEASURES: The electronic medical record-based screening tool numerical risk scores were compared with pharmacy resident subjective risk assessments using tree-based classification models to determine screening tool components that best predicted pharmacy residents' subjective assessment of patients' likelihood of benefit from in-home pharmacist medication review. Following the study, satisfaction surveys were given to providers and pharmacy residents. RESULTS: The best predictor of high-risk patients was an electronic frailty index score greater than 0.32 (indicating very frail) or LACE+ index greater than or equal to 59 (at high risk for hospital readmission). Pharmacy residents and providers agreed that homebound patients at high-risk for medication noncompliance benefited from pharmacist time and attention in home visits. CONCLUSION: In homebound older persons, this screening tool allowed for the identification of patients at high-risk for medication nonadherence through targeted in-home pharmacist medication reviews. Further studies are needed to validate the accuracy of this tool internally and externally.

Preservation of reserve intestinal epithelial stem cells following severe ischemic injury.

Intestinal ischemia is an abdominal emergency with a mortality rate >50%, leading to epithelial barrier loss and subsequent sepsis. Epithelial renewal and repair after injury depend on intestinal epithelial stem cells (ISC) that reside within the crypts of Lieberkühn. Two ISC populations critical to epithelial repair have been described: 1) active ISC (aISC; highly proliferative; leucine-rich-repeat-containing G protein-coupled receptor 5 positive, sex determining region Y-box 9 positive) and 2) reserve ISC [rISC; less proliferative; homeodomain only protein X (Hopx)+]. Yorkshire crossbred pigs (8-10 wk old) were subjected to 1-4 h of ischemia and 1 h of reperfusion or recovery by reversible mesenteric vascular occlusion. This study was designed to evaluate whether ISC-expressing biomarkers of aISCs or rISCs show differential resistance to ischemic injury and different contributions to the subsequent repair and regenerative responses. Our data demonstrate that, following 3-4 h ischemic injury, aISC undergo apoptosis, whereas rISC are preserved. Furthermore, these rISC are retained ex vivo in spheroids in which cell populations are enriched in the rISC biomarker Hopx. These cells appear to go on to provide a proliferative pool of cells during the recovery period. Taken together, these data indicate that Hopx+ cells are resistant to injury and are the likely source of epithelial renewal following prolonged ischemic injury. It is therefore possible that targeting reserve stem cells will lead to new therapies for patients with severe intestinal injury. NEW & NOTEWORTHY The population of reserve less-proliferative intestinal epithelial stem cells appears resistant to injury despite severe epithelial cell loss, including that of the active stem cell population, which results from prolonged mesenteric ischemia. These cells can change to an activated state and are likely indispensable to regenerative processes. Reserve stem cell targeted therapies may improve treatment and outcome of patients with ischemic disease.

Use of Medical Countermeasures in Small-Scale Emergency Responses.

It is well documented that long-standing focus on public health emergency preparedness medical countermeasures (MCMs) distribution and mass dispensing capabilities for mitigation of bioterrorism incidents and a lack of real-world opportunities to test national preparedness for large-scale emergencies has hindered development of a body of evidence-based practices in the United States. To encourage jurisdictions seeking innovative opportunities for continuous improvement, we describe instances when the MCM capabilities were used to address smaller-scale, more-frequent public health emergencies such as disease outbreaks, natural disasters, or routine influenza vaccination. We argue that small-scale events represent a critical opportunity that state, local, tribal, and territorial entities can utilize for greater gains in MCM operational readiness than through exercises or planned reviews. By using and evaluating MCM capabilities during a real response, jurisdictions can advance preparedness science and support the translation of research into practice, thereby increasing their capacity to scale up for larger, rarer, higher-consequence emergencies.

Medication Reconciliation Makes Transitions Safer.

A thoughtfully designed and strictly followed review process can spare patients avoidable hospital readmissions.

Determining Staff Competencies.

CMS' requirements of participation for skilled nursing facilities highlight the level of each staff member's skill set.

Variations in organism-specific severe sepsis mortality in the United States: 1999-2008.

OBJECTIVE: Recent studies have reported decreased overall severe sepsis mortality, but associations with organism trends have not yet been investigated. This study explored organism-specific severe sepsis mortality trends from 1999 to 2008 in a large hospital-based administrative database. DESIGN: Secondary data analysis using the Nationwide Inpatient Sample. SETTING: United States hospitals sampled in the Nationwide Inpatient Sample dataset. PATIENTS: This sample approximates a stratified 20% sample of all nonfederal, short-term, general, and specialty hospitals serving adults in the United States. Severe sepsis hospitalizations and organism-specific causes were identified using predetermined International Classification of Diseases, 9th Revision, Clinical Modification codes. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Analysis was conducted using descriptive Cox proportional hazards and linear regression trend analysis. Adjustments were made for the influence of demographics, comorbidities, number of organisms, and number of organ failures on hospital mortality. The data for 5,033,257 severe sepsis hospitalizations were examined and revealed decreased in-hospital mortality from 40.0% to 27.8% during the study period. The leading cause of severe sepsis was 51.5% Gram-negative bacteria, followed by 45.6% Gram-positive, 1.7% anaerobic, and 1.2% fungal species. The most common Gram-negative organisms were 39.9% Escherichia coli and 17.6% Pseudomonas. Staphylococcus species (62.2% methicillin-sensitive Staphylococcus aureus and 22.6% Streptococcus) were the most commonly reported Gram-positive organisms. Crude mortality estimates were higher for anaerobic and fungal organisms, 34.5% and 31.4%, respectively. Among Gram-positive bacteria, mortality was highest for methicillin-sensitive S. aureus, 30.9%, whereas Pseudomonas was associated with the highest mortality for Gram-negative septicemia cases, 29.5%. After adjusting for covariates, anaerobes were associated with the highest mortality hazard of 1.31 (95% CI, 1.23-1.40). Methicillin-resistant S. aureus had the highest mortality hazard of 1.38 (1.33-1.44) for Gram-positive organisms, whereas all Gram-negative bacteria had decreased mortality hazards. CONCLUSIONS: We not only confirmed an overall decline in severe sepsis mortality from 1999 to 2008 but also identified previously unreported variations in organism-specific severe sepsis mortality. Gram-negative organisms predominate, whereas anaerobes and methicillin-resistant S. aureus are significant predictors of mortality. Future clinical trials exploring new treatments in severe sepsis should incorporate individual organism trends to elucidate potential effect on mortality.

A Novel Dual ATM/DNA-PK Inhibitor, XRD-0394, Potently Radiosensitizes and Potentiates PARP and Topoisomerase I Inhibitors.

A majority of patients with cancer receive radiotherapy as part of their treatment regimens whether using external beam therapy or locally-delivered radioisotopes. While often effective, some tumors are inadequately controlled with radiation and radiotherapy has significant short-term and long-term toxicities for cancer survivors. Insights into molecular mechanisms involved in cellular responses to DNA breaks introduced by radiation or other cancer therapies have been gained in recent years and approaches to manipulate these responses to enhance tumor cell killing or reduce normal tissue toxicity are of great interest. Here, we report the identification and initial characterization of XRD-0394, a potent and specific dual inhibitor of two DNA damage response kinases, ATM and DNA-PKcs. This orally bioavailable molecule demonstrates significantly enhanced tumor cell kill in the setting of therapeutic ionizing irradiation in vitro and in vivo. XRD-0394 also potentiates the effectiveness of topoisomerase I inhibitors in vitro. In addition, in cells lacking BRCA1/2 XRD-0394 shows single-agent activity and synergy in combination with PARP inhibitors. A phase Ia clinical trial (NCT05002140) with XRD-0394 in combination with radiotherapy has completed. These results provide a rationale for future clinical trials with XRD-0394 in combination with radiotherapy, PARP inhibitors, and targeted delivery of topoisomerase I inhibitors.

Developing an Injury-Free 15 Hour Preservation Protocol of Donor Porcine Kidneys Using Normothermic Machine Perfusion.

Normothermic machine perfusion (NMP) offers a superior alternative to hypothermic preservation but is currently time limited. Extending this time could electivise transplantation and enable physiologic assessments of functionality. Porcine kidneys were retrieved, stored on ice for 3.5 hours before being placed onto a NMP circuit for 12 hours. Hemodynamics, biochemistry, and urine output were assessed. After 12 hours, kidneys were scored using the clinical assessment score. Biopsies were collected for histological assessment. Kidneys demonstrated continual improvements in hemodynamics. Perfusate sodium concentrations remained within physiologic parameters. Sodium bicarbonate increased over-time with corresponding decreases in lactate, demonstrating active renal gluconeogenesis and Cori cycle processes. Urine production began immediately and was sustained, indicating renal functionality. Under the clinical perfusion assessment score, all kidneys received a score of 1 and would be considered suitable for transplantation. Histological assessment revealed kidneys were injury free. Our NMP protocol safely preserves kidneys for over 15 hours. Successful perfusion was achieved with stable hemodynamics and biochemistry, with maintained urination. Importantly, kidneys remained in optimal health, with no evidence of injury. This may enable electivisation of transplantation, while reducing hypothermic injury.

Mediator kinase inhibition impedes transcriptional plasticity and prevents resistance to ERK/MAPK-targeted therapy in KRAS-mutant cancers.

Acquired resistance remains a major challenge for therapies targeting oncogene activated pathways. KRAS is the most frequently mutated oncogene in human cancers, yet strategies targeting its downstream signaling kinases have failed to produce durable treatment responses. Here, we developed multiple models of acquired resistance to dual-mechanism ERK/MAPK inhibitors across KRAS-mutant pancreatic, colorectal, and lung cancers, and then probed the long-term events enabling survival against this class of drugs. These studies revealed that resistance emerges secondary to large-scale transcriptional adaptations that are diverse and cell line-specific. Transcriptional reprogramming extends beyond the well-established early response, and instead represents a dynamic, evolved process that is refined to attain a stably resistant phenotype. Mechanistic and translational studies reveal that resistance to dual-mechanism ERK/MAPK inhibition is broadly susceptible to manipulation of the epigenetic machinery, and that Mediator kinase, in particular, can be co-targeted at a bottleneck point to prevent diverse, cell line-specific resistance programs.