Short-Term Outcomes, Functional Status, and Risk Factors for Requiring Extracorporeal Life Support After Norwood Operation: A Single-Center Retrospective Study.

Patients requiring extracorporeal life support (ECLS) post-Norwood operation constitute an extremely high-risk group. We retrospectively described short-term outcomes, functional status, and assessed risk factors for requiring ECLS post-Norwood operation between January 2010 and December 2020 in a high-volume center. During the study period, 269 patients underwent a Norwood procedure of which 65 (24%) required ECLS. Of the 65 patients, 27 (41.5%) survived to hospital discharge. Mean functional status scale (FSS) score at discharge increased from 6.0 on admission to 8.48 (p < 0.0001). This change was primary in feeding (p < 0.0001) and respiratory domains (p = 0.017). Seven survivors (26%) developed new morbidity, and two (7%) developed unfavorable functional outcomes. In the regression analysis, we showed that patients with moderate-severe univentricular dysfunction on pre-Norwood transthoracic echocardiogram (odds ratio [OR] = 6.97), modified Blalock Taussig Thomas (m-BTT) shunt as source of pulmonary blood flow (OR = 2.65), moderate-severe atrioventricular valve regurgitation on transesophageal echocardiogram (OR = 8.50), longer cardiopulmonary bypass time (OR = 1.16), longer circulatory arrest time (OR = 1.20), and delayed sternal closure (OR = 3.86), had higher odds of requiring ECLS (p < 0.05). Careful identification of these risk factors is imperative to improve the care of this high-risk cohort and improve overall outcomes.

Understanding and overcoming resistance to immunotherapy in genitourinary cancers.

The introduction of novel immunotherapies has significantly transformed the treatment landscape of genitourinary (GU) cancers, even becoming the standard of care in some settings. One such type of immunotherapy, immune checkpoint inhibitors (ICIs) like nivolumab, ipilimumab, pembrolizumab, and atezolizumab play a pivotal role by disturbing signaling pathways that limit the immune system's ability to fight tumor cells. Despite the profound impact of these treatments, not all tumors are responsive. Recent research efforts have been focused on understanding how cancer cells manage to evade the immune response and identifying the possible mechanisms behind resistance to immunotherapy. In response, ICIs are being combined with other treatments to reduce resistance and attack cancer cells through multiple cellular pathways. Additionally, novel, targeted strategies are currently being investigated to develop innovative methods of overcoming resistance and treatment failure. This article presents a comprehensive overview of the mechanisms of immunotherapy resistance in GU cancers as currently described in the literature. It explores studies that have identified genetic markers, cytokines, and proteins that may predict resistance or response to immunotherapy. Additionally, we review current efforts to overcome this resistance, which include combination ICIs and sequential therapies, novel insights into the host immune profile, and new targeted therapies. Various approaches that combine immunotherapy with chemotherapy, targeted therapy, vaccines, and radiation have been studied in an effort to more effectively overcome resistance to immunotherapy. While each of these combination therapies has shown some efficacy in clinical trials, a deeper understanding of the immune system's role underscores the potential of novel targeted therapies as a particularly promising area of current research. Currently, several targeted agents are in development, along with the identification of key immune mediators involved in immunotherapy resistance. Further research is necessary to identify predictors of response.

Immunotherapy has transformed the treatment landscape for many cancer types, including genitourinary malignancies such as renal and bladder cancers.However, not all patients or tumor types, such as prostate cancer, respond to this type of treatment.Understanding the mechanisms of immunotherapy resistance is critical for developing strategies to overcome these challenges.Primary resistance, which is present at the onset of treatment, can bedue to genetic abnormalities or immune system dysregulation. These factors alter the interactions between host cells and cancer cells.Adaptive resistance develops during therapy due to dynamic changes in the levels of growth factors, cytokines, and the tumor microenvironment (TME).Acquired resistance mainly occurs at the genetic and translational levels, involving the downregulation of critical human leukocyte antigen (HLA) molecules and interference with mutational repair.Future therapies may focus on detailed genetic profiling of patients to guide treatment selection and on the use of immune profile monitoring to assist in assessing responsiveness, alongside developing novel targeted therapies and ICIs.Further research is needed to identify predictors of response to ICIs.

Case Report: Exceptional response to nivolumab plus cabozantinib in a patient with extrarenal clear cell renal cell carcinoma.

Extrarenal clear cell renal cell carcinoma (eccRCC) is a rare type of RCC that arises in areas other than the kidney. Given its rarity, consensus guidelines for optimal treatment of eccRCC have not been established, and the literature is lacking any reports of patient response to systemic therapy and any reports of administration of immunotherapy to patients with ecRCC. Here, we present the case of a patient in their 60s with eccRCC arising in the spleen. The patient underwent splenic resection and then received systemic therapy, due to disease recurrence, with a combination of immunotherapy (IO) and tyrosine kinase inhibitor targeted therapy (VEGF-TKI). The patient had an excellent and durable response to this therapeutic regimen with minimal adverse effects, completing 2 years of therapy of nivolumab and cabozantinib. At the time of this report, the disease remains stable. This case demonstrates that combination therapy with IO+VEGF-TKI represents a reasonable and well-tolerated treatment option with activity in eccRCC and reveals interesting correlative data, including nests of stem-like CD8+T-cell infiltration in tumor tissue, which provide important biological context to this patient's exceptional therapeutic response.

The role of immune checkpoint inhibition in triple negative breast cancer.

INTRODUCTION: Immunotherapy has revolutionized cancer treatment, including TNBC, which has limited options of treatment and poor prognosis. ICIs studied in TNBC include pembrolizumab, nivolumab, atezolizumab, and durvalumab. Initial studies exploring ICI monotherapy demonstrated promising yet limited responses. Subsequent studies, KEYNOTE 522 and KEYNOTE 355, which combined ICI with chemotherapy, have resulted in the FDA approval of pembrolizumab in the early-stage and metastatic setting, respectively. AREAS COVERED: This article provides a comprehensive review of the role of ICI in the treatment of TNBC. We reviewed the trials that have evaluated ICI monotherapy, dual therapy, ICI in combination with chemotherapy, targeted therapy, vaccines and radiation. Additionally, we reviewed potential biomarkers of response and immune-related adverse events (irAEs). A literature search was conducted via PubMed and ClinicalTrials.gov as of 5 June 2023. EXPERT OPINION: Various approaches combining immunotherapy with chemotherapy, targeted therapy, vaccines and radiation have been assessed. Pembrolizumab remains the only ICI approved in both the early stage and mTNBC. The role of adjuvant pembrolizumab in those who achieved pCR after neoadjuvant therapy is being investigated. Combining ICI with PARP inhibitors and radiation shows promise. More research is needed in identifying predictors of response. Monitoring of irAEs remains crucial.

The What and the How: A pilot study of feedback from service providers and consumers about service development in new urban mental health hub.

OBJECTIVE: An inner-city hospital purchased a local hotel to provide support, digital engagement, skill development groups and referrals to partner agencies. Being aware of the hotel's importance to the local community, we sought feedback on a model of care relevant to perceived gaps and needs in support for mental health. METHODS: Four online focus groups included healthcare professionals, nominated opinion leaders from local Non-Government Organisations (NGOs), consumers and carers to reflect a range of views. Focus group data were qualitatively analysed. RESULTS: Participants made useful suggestions about WHAT educational, preventative and therapeutic services were needed. They emphasised the importance of HOW people engage with the service, the balance between accessibility and security. Other themes included targeting people with limited health literacy, integration with existing services, building on site heritage and ongoing evaluation of objectives and needs. CONCLUSIONS: This pilot study demonstrated clear support for providing safe and welcoming access to services, with resources and access to services to improve their health and wellbeing built on principles of social justice and inclusion. Participants had constructive ideas of what was needed, and ongoing patient and public research is anticipated.

Sirolimus-induced pulmonary hemorrhage of an infant with massive cervicofacial lymphatic malformation: A case report.

Lymphatic malformations (LMs) are uncommon congenital abnormalities of the lymphatic system. As more than half of these lesions develop in the head and neck, LMs can be life-threatening if associated with airway involvement. LMs necessitate a multidisciplinary treatment approach, frequently including surgery and sclerotherapy. We present a case report of a 32-week pre-term male infant with a massive cervicofacial LM necessitating delivery via ex-utero intrapartum treatment (EXIT). The patient was treated with numerous rounds of sclerotherapy, systemic sirolimus, and surgical debulking, but ultimately died at 4 months of age due to acute pulmonary hemorrhage, which may have been related to sirolimus due to the absence of any other associable organ involvement or derangement. We document the patient's clinical course and treatment regimen, highlighting the myriad modalities employed to treat these challenging lesions, and describe a potentially lethal complication of sirolimus therapy not previously described in the treatment of pediatric LM.

Incidence and Risk Factors Associated with Respiratory Compromise in Planned PICU Admissions Following Tonsillectomy.

OBJECTIVES: Review the incidence and factors associated with respiratory compromise requiring intensive care unit level interventions in children with planned admission to the pediatric intensive care unit (PICU) following tonsillectomy or adenotonsillectomy (T/AT). STUDY DESIGN: Retrospective cohort study. METHODS: Review of all patients with PICU admissions following T/AT from 2015 to 2020 at a tertiary care pediatric hospital. Patient demographics, underlying comorbidities, operative data, and respiratory complications during PICU admission were extracted. RESULTS: Seven hundred and seventy-two patients were admitted to the PICU following T/AT, age 6.1 ± 4.6 years. All children were diagnosed with obstructive sleep apnea or sleep-disordered breathing (mean pre-operative apnea-hypopnea index 29 ± 26.5 and O2 nadir 77.1% ± 11.1). Neuromuscular disease, enteral feed dependence, and obesity were common findings (N = 240 (31%), N = 106 (14%), and N = 209 (27%) respectively). Overall, 29 patients (3.7%) developed respiratory compromise requiring PICU-level support, defined as new-onset continuous or bilevel positive airway pressure support (n = 25) or reintubation (n = 9). Three patients were diagnosed with pulmonary edema. Multivariable regression analysis demonstrated pre-operative oxygen nadir and enteral feed dependence were associated with respiratory compromise (OR = 0.97, 95% CI 0.94-0.99, P = .04; OR = 6.3, 95% CI 2.36-52.6, P = .001 respectively). CONCLUSIONS: Our study found respiratory compromise in 3.7% of patients with planned PICU admissions following T/AT. Oxygen nadir and enteral feeds were associated with higher respiratory compromise rates. Attention should be given to these factors in planning for post-operative disposition.

Virtually Assisted Personalized Tracheostomy Tube Design in Pediatric Complex Airway Anomalies.

We sought to assess the feasibility of virtually assisted personalized tracheostomy tube (vapTT) implementation for patients with congenital airway anomalies (CAAs) and persistent tracheostomy tube (TT)-related respiratory failure at a tertiary pediatric hospital. Three patients (0-18 years) with CAAs and recurrent TT-related respiratory complications were managed with vapTT over 5 years. Patients underwent airway computed tomography acquisition with 3-dimensional reconstruction and TT virtual modeling for shape customization. Models were transferred to Bivona for fabrication based on industry-standard materials and processes. Clinical information and tracheoscopies assessing position, obstruction, and granulation were reviewed. Patients demonstrated resolution of visualized TT-related obstruction, granulation, or ulceration and de-escalation of respiratory support. Clinical events requiring urgent tracheoscopy decreased in all 3 patients. Sufficient relief of critical airway obstruction allowed progression of medical care and/or discharge. VapTTs are feasible for patients with CAA. This new frontier in personalized devices may serve uniquely challenging patient populations for whom standard treatments have failed.

Urine exosome mRNA-based test for monitoring kidney allograft rejection: Effects of sample transportation and storage, and interference substances.

BACKGROUND: Exosomes are 30-150 nm nanovesicles with sophisticated nucleic acids cargo, actively secreted by all cells within human body, and found in abundance in all body fluids, including urine. These extracellular vesicles have tremendous potential for next generation diagnostics, theoretically enabling noninvasive assessment of organ and tissue function via liquid biopsy analysis. AIM: Recently, feasibility of an exosomal molecular test was demonstrated for post-organ transplant monitoring: Analysis of urine-derived exosomal mRNA cargo allowed early detection of kidney allograft rejection. Here, we further studied urine-derived exosomes and their mRNA content as a highly promising diagnostic modality. This included stability studies of urine samples and exosomal mRNA upon transportation from the point of collection to a centralized testing facility, short-term storage of urine at different conditions upon receipt till the point molecular assay is performed, and effects of various potentially interfering substances on the downstream quantitative polymerase chain reaction (qPCR) assay. METHODS: The urine specimens were stored at various conditions and pre-processed in different ways. Next, samples were passed through the columns to capture all extracellular vesicles, the vesicles were lysed to release their content and the exosomal RNA was purified on the mini-columns, reverse transcription was performed, next pre-amplification, followed by a qPCR analysis for a panel of mRNA markers. RESULTS: To ensure exosomal RNA integrity, the harvested urine specimens should be shipped refrigerated, by overnight delivery. Urine can next be stored at the test site for up to 1 wk at 4 °C, and long term should be frozen at -80 °C. Urine specimens must be centrifuge at low G-force to deplete cells and debris, to ensure consistent top results in downstream molecular assays. All commonly used medications (tacrolimus, cyclosporin A, mycophenolic acid, everolimus, sirolimus, ascomycin, teriflunomide) were tested and confirmed that they do not cause assay interference. CONCLUSION: mRNA from urine-derived exosomes was shown to be stable across a broad range of conditions and produced accurate results when analyzed via qPCR assay for detection of kidney allograft rejection. We identified the most optimal conditions for every step of the process, ensuring pre-analytical sample integrity and robust qPCR results.

Virtual interviewing for graduate medical education recruitment and selection: A BEME systematic review: BEME Guide No. 80.

BACKGROUND: The COVID-19 pandemic caused graduate medical education (GME) programs to pivot to virtual interviews (VIs) for recruitment and selection. This systematic review synthesizes the rapidly expanding evidence base on VIs, providing insights into preferred formats, strengths, and weaknesses. METHODS: PubMed/MEDLINE, Scopus, ERIC, PsycINFO, MedEdPublish, and Google Scholar were searched from 1 January 2012 to 21 February 2022. Two authors independently screened titles, abstracts, full texts, performed data extraction, and assessed risk of bias using the Medical Education Research Quality Instrument. Findings were reported according to Best Evidence in Medical Education guidance. RESULTS: One hundred ten studies were included. The majority (97%) were from North America. Fourteen were conducted before COVID-19 and 96 during the pandemic. Studies involved both medical students applying to residencies (61%) and residents applying to fellowships (39%). Surgical specialties were more represented than other specialties. Applicants preferred VI days that lasted 4-6 h, with three to five individual interviews (15-20 min each), with virtual tours and opportunities to connect with current faculty and trainees. Satisfaction with VIs was high, though both applicants and programs found VIs inferior to in-person interviews for assessing 'fit.' Confidence in ranking applicants and programs was decreased. Stakeholders universally noted significant cost and time savings with VIs, as well as equity gains and reduced carbon footprint due to eliminating travel. CONCLUSIONS: The use of VIs for GME recruitment and selection has accelerated rapidly. The findings of this review offer early insights that can guide future practice, policy, and research.

Evaluating obesity as a risk factor for complications after pediatric adenotonsillectomy.

OBJECTIVES: To evaluate associations between childhood obesity and post-adenotonsillectomy complications, informing guidelines for postoperative management. METHODS: The retrospective review assessed outpatient pediatric tonsillectomy/adenoidectomy cases performed at 2 ambulatory surgery centers in 2020. Complications in the recovery unit and within 2 weeks of surgical discharge were reviewed along with clinical and demographic variables. Obesity was defined as sex-specific body mass index-for-age, or weight-for-age if height data were unavailable, at/above the 95th percentile. The 99th percentile served as the threshold for severe obesity. Analyses used Chi-square/Fisher's exact tests and independent-samples t-tests with relative risk or effect sizes. RESULTS: The review included 707 cases (180 patients with obesity). Overall incidence of complications in the recovery unit was 9.1%. Patients with obesity were significantly more likely to require supplemental blow-by oxygen (P = .02); relative risk was 1.65 (95% CI: 1.16-2.35) times greater in the cohort with obesity. Obesity had a small effect on postoperative oxygen saturation nadirs, which were significantly lower among patients with obesity (d = -0.34; P < .001). No differences emerged between cohorts with and without obesity in the incidence of any other complications before or after surgical discharge. Overall incidence of post-discharge returns was 7.9%. Incidence of complications did not vary by obesity severity. CONCLUSION: From this cohort, childhood obesity without other significant comorbidities may not warrant routine inpatient care following adenotonsillectomy. Patients with obesity should receive additional monitoring for oxygen desaturation events during the first hours of recovery. Further prospective studies should continue to address this important topic.

Learning Outcomes in a Live Virtual versus In-Person Curriculum for Medical and Pharmacy Students.

Background: The coronavirus disease (COVID-19) pandemic has been a source of disruption, changing the face of medical education. In response to infection control measures at the University of California, San Diego, the hybrid in-person and recorded preclerkship curriculum was converted to a completely virtual format. The impact of this exclusive virtual teaching platform on the quality of trainee education is unknown. Objective: To determine the efficacy of a virtual course, relative to traditional hybrid in-person and recorded teaching, and to assess the impact of supplementary educational material on knowledge acquisition. Methods: A retrospective observational cohort study was performed to assess an introductory course, held mostly in person in 2019 versus completely virtual in 2020, for first-year medical students and second-year pharmacy students at the University of California, San Diego, School of Medicine and Skaggs School of Pharmacy and Pharmaceutical Sciences. Results: The midterm and final examination scores were similar for the hybrid and virtual courses. There was no association between the hours of recorded lectures watched and final examination scores for either course. In the 2019 in-person and recorded course, students who demonstrated consistent on-time use of practice quizzes scored statistically higher on the final examination (P = 0.0066). In the 2020 virtual course, students who downloaded quizzes regularly had statistically higher scores on the midterm examination (P < 0.0001). Conclusion: The similar examination scores for the hybrid in-person and recorded and exclusively virtual courses suggest that the short-term knowledge acquired was equivalent, independent of the modality with which the content was delivered. Consistent on-time use of practice quizzes was associated with higher examination scores. Future studies are needed to assess the difference between a completely in-person versus virtual curriculum.

Vaccine breakthrough infections with SARS-CoV-2: Why older adults need booster vaccinations.

Objectives: COVID-19 vaccinations are highly efficacious in preventing severe illness that can lead to hospitalizations and death, but incidents of vaccine breakthrough (VBT) infections persist. We examined VBT infections within a congregate setting to help guide public health practices. Study design: This is a retrospective cohort study of VBT infections identified via polymerase chain reaction (PCR) testing between 2/1/2021-11/1/2021. Methods: A VBT infection was defined as the detection of SARS-CoV-2 collected from a person ≥14 days after all recommended doses of a COVID-19 vaccine. VBT infections were examined in five California psychiatric inpatient hospitals with a workforce of more than 10,000 hospital staff and approximately 5500 patients. Results: 415 VBT infections out of 14,101 fully vaccinated individuals within our system (2.9%) were identified. Days since final vaccine date ranged from 16 to 291 days. Kruskal-Wallis nonparametric test revealed a statistically significant difference in age between individuals with VBT infections versus all other vaccinated individuals [U = 6.47, p = .01]. A chi-square test of independence revealed no significant sex differences between individuals with VBT infections (58.8% male and 41.2% female) versus all other vaccinated individuals (59.6% male and 40.4% female; X2 (3, N = 14101) = 5.059, p = .167). Out of 415 VBT cases, 65.1% received the Moderna vaccine, 33.2% received Pfizer, and 1.7% received J&J; and 38.1% were asymptomatic at time of VBT infection, 24.1% were symptomatic, while 37.8% were missing symptom data. Conclusions: Vaccination campaigns, including boosters and continued surveillance, are important complimentary strategies for reducing the proliferation of COVID-19 VBT cases and severity of symptoms associated with COVID-19.

Combination Immune Checkpoint Blockade Regimens for Previously Untreated Metastatic Renal Cell Carcinoma: The Winship Cancer Institute of Emory University Experience.

Introduction: There are three combination immune checkpoint inhibitor (ICI)-based regimens in the first-line setting for metastatic renal cell carcinoma (mRCC). Currently, there is limited real-world data for clinical outcomes and toxicity in mRCC patients treated with first-line ICI-based regimens. Methods: We performed a retrospective review of 49 mRCC patients treated with ICI-based combination regimens in the standard of care setting at the Winship Cancer Institute of Emory University from 2015-2020. We collected baseline data from the electronic medical record including demographic information and disease characteristics. Immune-related adverse events (irAEs) were collected from clinic notes and laboratory values. The primary clinical outcomes measured were overall survival (OS), progression-free survival (PFS), and objective response rate (ORR). Results: The median age was 65 years, and most patients (80%) were males. The majority were White (86%) and had clear cell RCC (83%). Most patients had an Eastern Cooperative Oncology Group (ECOG) performance status score of 0 (43%) or 1 (45%). Approximately one-half (49%) had at least three sites of distant metastatic disease. Most patients (88%) received nivolumab and ipilimumab. More than one-half (53%) of patients experienced an irAE, with 13 (27%) patients having treatment delayed and 18% discontinuing treatment for toxicity. The median OS was not reached, and the median PFS was 8.0 months per a Kaplan-Meier estimation. More than half of patients (53%) had a PFS > 6 months, and 22% had PFS > 1 year. The ORR was 33% for the entire cohort, and 7% of patients had a complete response. Conclusion: We presented real-world efficacy and toxicity data for front-line ICI combination treatment regimens. The ORR and median PFS were lower in our cohort of patients compared to the available data in the clinical trial setting. This was likely because of more advanced disease in this study. Future studies should provide additional data that will allow comparisons between different ICI combination regimens for untreated mRCC.

Sustaining standardized opioid prescribing practices after pediatric tonsillectomy.

INTRODUCTION: Opioid prescribing patterns after pediatric tonsillectomy are highly variable, and opioids may not improve pain control compared to over-the-counter pain relievers. We evaluated whether a standardized, opioid-sparing analgesic protocol effectively reduced opioid prescriptions without compromising patient outcomes. METHODS: A quality improvement project was initiated in July 2019 to standardize analgesic prescribing after hospital-based tonsillectomy with/without adenoidectomy. An electronic order set provided weight-based dosing and defaulted to non-opioid prescriptions (acetaminophen and ibuprofen). Patients ages 0-6 received non-opioid analgesics alone. Patients ages 7-18 received non-opioid analgesics as first-line pain control, and providers could manually add hydrocodone-acetaminophen for breakthrough pain. Opioid prescriptions and quantities were compared for 18 months of cases pre- versus post-standardization. Postoperative returns to the system were reviewed as a balancing measure. RESULTS: From 2018 through 2020, 1817 cases were reviewed. The frequency of opioid prescriptions decreased significantly post-standardization, from 64.9% to 33.5% of cases (P < .001). Opioid prescribing for young children steadily decreased from over 50% to 2.4%. Protocol adherence improved over time; outlier prescriptions were eliminated. Opioid quantities per prescription decreased by 16.3 doses on average (P < .001), and variance decreased significantly post-standardization (P < .001). The incidence of returns to the system did not change (P = .33), including returns for pain or decreased intake (P = .28). CONCLUSION: An age-based and weight-based analgesic protocol reduced post-tonsillectomy opioid prescriptions without a commensurate increase in returns for postoperative complaints. Standardized protocols can facilitate sustained changes in prescribing patterns and limit potentially unnecessary pediatric opioid exposure.

Functional Characterization of Non-Ankyrin Repeat Domains of Orientia tsutsugamushi Ank Effectors Reveals Their Importance for Molecular Pathogenesis.

Orientia tsutsugamushi is a genetically intractable obligate intracellular bacterium, causes scrub typhus, and has one of the largest known armamentariums of ankyrin repeat-containing effectors (Anks). Most have a C-terminal F-box presumed to interact with the SCF ubiquitin ligase complex primarily based on their ability to bind overexpressed Skp1. Whether all F-box-containing Anks bind endogenous SCF components and the F-box residues essential for such interactions has gone unexplored. Many O. tsutsugamushi Ank F-boxes occur as part of a PRANC (pox protein repeats of ankyrin-C-terminal) domain. Roles of the non-F-box portion of the PRANC and intervening sequence region (ISR) that links the ankyrin repeat and F-box/PRANC domains are unknown. The functional relevance of these effectors' non-ankyrin repeat domains was investigated. The F-box was necessary for Flag-tagged versions of most F-box-containing Anks to precipitate endogenous Skp1, Cul1, and/or Rbx1, while the ISR and PRANC were dispensable. Ank toxicity in yeast was predominantly F-box dependent. Interrogations of Ank1, Ank5, and Ank6 established that L1, P2, E4, I9, and D17 of the F-box consensus are key for binding native SCF components and for Ank1 and Ank6 to inhibit NF-κB. The ISR is also essential for Ank1 and Ank6 to impair NF-κB. Ectopically expressed Ank1 and Ank6 lacking the ISR or having a mutagenized F-box incapable of binding SCF components performed as dominant-negative inhibitors to block O. tsutsugamushi NF-κB modulation. This study advances knowledge of O. tsutsugamushi Ank functional domains and offers an approach for validating their roles in infection.