Klebsiella LPS O1-antigen prevents complement-mediated killing by inhibiting C9 polymerization.

The Gram-negative bacterium Klebsiella pneumoniae is an important human pathogen. Its treatment has been complicated by the emergence of multi-drug resistant strains. The human complement system is an important part of our innate immune response that can directly kill Gram-negative bacteria by assembling membrane attack complex (MAC) pores into the bacterial outer membrane. To resist this attack, Gram-negative bacteria can modify their lipopolysaccharide (LPS). Especially the decoration of the LPS outer core with the O-antigen polysaccharide has been linked to increased bacterial survival in serum, but not studied in detail. In this study, we characterized various clinical Klebsiella pneumoniae isolates and show that expression of the LPS O1-antigen correlates with resistance to complement-mediated killing. Mechanistic data reveal that the O1-antigen does not inhibit C3b deposition and C5 conversion. In contrast, we see more efficient formation of C5a, and deposition of C6 and C9 when an O-antigen is present. Further downstream analyses revealed that the O1-antigen prevents correct insertion and polymerization of the final MAC component C9 into the bacterial membrane. Altogether, we show that the LPS O1-antigen is a key determining factor for complement resistance by K. pneumoniae and provide insights into the molecular basis of O1-mediated MAC evasion.

Neuropsychiatric Symptoms in Rhombencephalosynapsis: A Clinical Report.

Rhombencephalosynapsis (RES) is a hindbrain malformation characterized by a missing cerebellar vermis with apposition or fusion of the cerebellar hemispheres. The present clinical case report provides a comprehensive, longitudinal overview of cognitive and affective manifestations in a 22-year-old patient with RES. The patient shows clinical signs of emotional reactivity and dysregulation, impulsivity, and impairments in executive functioning since early childhood. These features fit the constellation of neuropsychiatric symptoms observed in patients with congenital and acquired abnormalities of the posterior vermis. It is proposed that patients with RES may show affective and cognitive difficulties which increase their vulnerability to psychological stress and risk of developing mental health issues.

Tunability of topological edge states in germanene at room temperature.

Germanene is a two-dimensional topological insulator with a large topological band gap. For its use in low-energy electronics, such as topological field effect transistors and interconnects, it is essential that its topological edge states remain intact at room temperature. In this study, we examine these properties in germanene using scanning tunneling microscopy and spectroscopy at 300 K and compare the results with data obtained at 77 K. Our findings show that the edge states persist at room temperature, although thermal effects cause smearing of the bulk band gap. Additionally, we demonstrate that, even at room temperature, applying an external perpendicular electric field switches the topological states of germanene off. These findings indicate that germanene's topological properties can be maintained and controlled at room temperature, making it a promising material for low-energy electronic applications.

Estimation of Gestational Age-Specific Reference Intervals for Coagulation Assays in a Neonatal Intensive Care Unit Using Real-World Data.

BACKGROUND: Interpretation of coagulation testing in neonates currently relies on reference intervals (RIs) defined from older patient cohorts. Direct RI studies are difficult, but indirect estimation may allow us to infer normative neonatal distributions from routinely collected clinical data. METHODS: We analyzed first-in-life coagulation testing results from all patients admitted to a level IV neonatal intensive care unit between 1/1/2018-1/1/2024. Results obtained after transfusion of any blood product were excluded. Indirect RIs were estimated across gestational age groups using refineR and compared to currently reported intervals for patients less than one year of age. RESULTS: Prothrombin times (PT) and international normalized ratios (INR) were available for 1,128 neonates, while activated partial thromboplastin times (aPTT) were available for 790 neonates. The indirect RI was 10-25s in preterm, 10-22s in term, and 10-24s in all neonates for PT, 0.7-2.1 in preterm, 0.8-1.8 in term, and 0.8-1.9 in all neonates for INR, and 25-68s in preterm, 25-58s in term, and 25-62s in all neonates for aPTT. Compared to our current intervals, the indirect RIs would flag 58% fewer PT, 43% fewer INR, and 17% fewer aPTT results as abnormal. CONCLUSIONS: Indirectly estimated RIs in neonates admitted to intensive care show substantial divergence from current, first-year-of-life RIs, leading to an abundance of abnormal flags. The associations between these flags and provider behavior, transfusion practice, or clinical outcomes is an area of future exploration.

Parent and patient knowledge and attitudes about cancer predisposition syndrome genetic testing in pediatric oncology: Understanding sociodemographic and parent-child differences.

BACKGROUND: Cancer predisposition syndromes (CPS) impact about 10% of patients with pediatric cancer. Genetic testing (CPS-GT) has multiple benefits, but few studies have described parent and child knowledge and attitudes regarding CPS-GT decision-making. This study examined parent and patient CPS-GT decision-making knowledge and attitudes. PROCEDURE: English- or Spanish-speaking parents of children with pediatric cancer and patients with pediatric cancer ages 15-18 within 12 months of diagnosis or relapse were eligible to participate. Seventy-five parents and 19 parent-patient dyads (N = 94 parents, 77.7% female, 43.6% Latino/a/Hispanic; 19 patients, 31.6% female) completed surveys measuring CPS-GT-related beliefs. Independent samples t-tests compared parent responses across sociodemographic characteristics and parent-patient responses within dyads. RESULTS: Spanish-speaking parents were significantly more likely than English-speaking parents to believe that CPS-GT not being helpful (p < .001) and possibly causing personal distress (p = .002) were important considerations for deciding whether to obtain CPS-GT. Parents with less than four-year university education, income less than $75,000, or Medicaid (vs. private insurance) were significantly more likely to endorse that CPS-GT not being helpful was an important consideration for deciding whether to obtain CPS-GT (p < .001). Parents felt more strongly than patients that they understood what CPS-GT was (p = .01) and that parents should decide whether patients under 18 should receive CPS-GT (p = .002). CONCLUSIONS: Spanish-speaking parents and parents with lower socioeconomic statuses were more strongly influenced by the potential disadvantages of CPS-GT in CPS-GT decision-making. Parents felt more strongly than patients that parents should make CPS-GT decisions. Future studies should investigate mechanisms behind these differences and how to best support CPS-GT knowledge and decision-making.

Strategies to reduce bladder tumor recurrences following surgery for upper tract urothelial carcinoma.

The incidence of upper tract urothelial carcinoma (UTUC) has been on the rise and the malignancy is more commonly managed surgically as higher proportions of in situ disease are being detected. One challenge facing urologists is the high rate of post-treatment intravesical recurrence (IVR) of UTUC (23 - 50%). Genomic research indicated that cells of recurrent bladder lesions are most often clonally derived from the primary UTUC and are likely to seed into the bladder after tumor manipulation. This calls for effective strategies to prevent the spread of UTUC. The methods we discuss here are the use of a ureteral access sheath during diagnostic ureteroscopy, application and timing of intravesical chemoprophylaxis, early ureteral ligation distal to UTUC, and formal bladder cuff excision. Urologic surgeons should aim to achieve a reduced rate of IVR when applying these techniques.

Far-red light photoacclimation in a desert Chroococcidiopsis strain with a reduced FaRLiP gene cluster and expression of its chlorophyll f synthase in space-resistant isolates.

Introduction: Some cyanobacteria can use far-red light (FRL) to drive oxygenic photosynthesis, a phenomenon known as Far-Red Light Photoacclimation (FaRLiP). It can expand photosynthetically active radiation beyond the visible light (VL) range. Therefore, it holds promise for biotechnological applications and may prove useful for the future human exploration of outer space. Typically, FaRLiP relies on a cluster of ~20 genes, encoding paralogs of the standard photosynthetic machinery. One of them, a highly divergent D1 gene known as chlF (or psbA4), is the synthase responsible for the formation of the FRL-absorbing chlorophyll f (Chl f) that is essential for FaRLiP. The minimum gene set required for this phenotype is unclear. The desert cyanobacterium Chroococcidiopsis sp. CCMEE 010 is unusual in being capable of FaRLiP with a reduced gene cluster (15 genes), and it lacks most of the genes encoding FR-Photosystem I. Methods: Here we investigated whether the reduced gene cluster of Chroococcidiopsis sp. CCMEE 010 is transcriptionally regulated by FRL and characterized the spectral changes that occur during the FaRLiP response of Chroococcidiopsis sp. CCMEE 010. In addition, the heterologous expression of the Chl f synthase from CCMEE 010 was attempted in three closely related desert strains of Chroococcidiopsis. Results: All 15 genes of the FaRLiP cluster were preferentially expressed under FRL, accompanied by a progressive red-shift of the photosynthetic absorption spectrum. The Chl f synthase from CCMEE 010 was successfully expressed in two desert strains of Chroococcidiopsis and transformants could be selected in both VL and FRL. Discussion: In Chroococcidiopsis sp. CCME 010, all the far-red genes of the unusually reduced FaRLiP cluster, are transcriptionally regulated by FRL and two closely related desert strains heterologously expressing the chlF010 gene could grow in FRL. Since the transformation hosts had been reported to survive outer space conditions, such an achievement lays the foundation toward novel cyanobacteria-based technologies to support human space exploration.

Early detection of ovarian cancer using cell-free DNA fragmentomes and protein biomarkers.

Ovarian cancer is a leading cause of death for women worldwide in part due to ineffective screening methods. In this study, we used whole-genome cell-free DNA (cfDNA) fragmentome and protein biomarker (CA-125 and HE4) analyses to evaluate 591 women with ovarian cancer, benign adnexal masses, or without ovarian lesions. Using a machine learning model with the combined features, we detected ovarian cancer with specificity >99% and sensitivity of 72%, 69%, 87%, and 100% for stages I-IV, respectively. At the same specificity, CA-125 alone detected 34%, 62%, 63%, and 100% of ovarian cancers for stages I-IV. Our approach differentiated benign masses from ovarian cancers with high accuracy (AUC=0.88, 95% CI=0.83-0.92). These results were validated in an independent population. These findings show that integrated cfDNA fragmentome and protein analyses detect ovarian cancers with high performance, enabling a new accessible approach for noninvasive ovarian cancer screening and diagnostic evaluation.

Structure-guided engineering of type I-F CASTs for targeted gene insertion in human cells.

Conventional genome editing tools rely on DNA double-strand breaks (DSBs) and host recombination proteins to achieve large insertions, resulting in a heterogeneous mixture of undesirable editing outcomes. We recently leveraged a type I-F CRISPR-associated transposase (CAST) from the Pseudoalteromonas Tn 7016 transposon ( Pse CAST) for DSB-free, RNA-guided DNA integration in human cells, taking advantage of its programmability and large payload capacity. Pse CAST is the only characterized CAST system that has achieved human genomic DNA insertions, but multiple lines of evidence suggest that DNA binding may be a critical bottleneck that limits high-efficiency activity. Here we report structural determinants of target DNA recognition by the Pse CAST QCascade complex using single-particle cryogenic electron microscopy (cryoEM), which revealed novel subtype-specific interactions and RNA-DNA heteroduplex features. By combining our structural data with target DNA library screens and rationally engineered protein mutations, we uncovered CAST variants that exhibit increased integration efficiency and modified PAM stringency. Structure predictions of key interfaces in the transpososome holoenzyme also revealed opportunities for the design of hybrid CASTs, which we leveraged to build chimeric systems that combine high-activity DNA binding and DNA integration modules. Collectively, our work provides unique structural insights into type I-F CAST systems while showcasing multiple diverse strategies to investigate and engineer new RNA-guided transposase architectures for human genome editing applications.

Preventability of injury-related morbidity & mortality at four hospitals in Cameroon: A systematic approach to trauma quality improvement.

BACKGROUND: Adverse events reviews are a fundamental component of trauma quality improvement (QI) that facilitate the correction of systemic issues in care. Although injury-related mortality in Cameroon is substantial, to our knowledge, opportunities for QI have not been formally assessed. Thus, a formal review of adverse events in Cameroonian trauma patients was implemented as a first step toward identifying targets for systems modification. METHODS: A QI committee composed of multidisciplinary experts at four hospitals in Cameroon was formed to review adverse events including deaths among trauma patients from 2019 to 2021. Events were discussed at newly established morbidity and mortality conferences and committee meetings to identify contributing factors and overall preventability. RESULTS: During 50 meetings, 95 adverse events were reviewed, including 58 deaths (61%). Other adverse events were delays in diagnosis/treatment (22%) and surgical site infections (17%). Overall, 34 deaths (59%) were classified as preventable, 21% potentially preventable, and 21% not preventable. Over half (52%) of the 46 preventable or potentially preventable deaths occurred in the emergency department (ED); while brain injury (57%), respiratory failure (41%), and hemorrhage (39%) were the most frequent physiologic factors associated with mortality. Contributory factors identified include lack of a structured approach to patient management, absence of continuous training for personnel, and locally adapted protocols. CONCLUSIONS: Basic improvements in evaluation and management of life-threatening issues in the ED can significantly reduce the high rate of preventable trauma-related deaths across Cameroon. Formal trauma QI methods can be utilized in low-resource environments to determine mortality root causes and identify intervention targets.