Targeting soluble amyloid-beta oligomers with a novel nanobody.

The classical amyloid cascade hypothesis postulates that the aggregation of amyloid plaques and the accumulation of intracellular hyperphosphorylated Tau tangles, together, lead to profound neuronal death. However, emerging research has demonstrated that soluble amyloid-ß oligomers (SAßOs) accumulate early, prior to amyloid plaque formation. SAßOs induce memory impairment and disrupt cognitive function independent of amyloid-ß plaques, and even in the absence of plaque formation. This work describes the development and characterization of a novel anti-SAßO (E3) nanobody generated from an alpaca immunized with SAßO. In-vitro assays and in-vivo studies using 5XFAD mice indicate that the fluorescein (FAM)-labeled E3 nanobody recognizes both SAßOs and amyloid-ß plaques. The E3 nanobody traverses across the blood-brain barrier and binds to amyloid species in the brain of 5XFAD mice. Imaging of mouse brains reveals that SAßO and amyloid-ß plaques are not only different in size, shape, and morphology, but also have a distinct spatial distribution in the brain. SAßOs are associated with neurons, while amyloid plaques reside in the extracellular matrix. The results of this study demonstrate that the SAßO nanobody can serve as a diagnostic agent with potential theragnostic applications in Alzheimer's disease.

Digital droplet PCR analysis of organoids generated from mouse mammary tumors demonstrates proof-of-concept capture of tumor heterogeneity.

Breast cancer metastases exhibit many different genetic alterations, including copy number amplifications (CNA). CNA are genetic alterations that are increasingly becoming relevant to breast oncology clinical practice. Here we identify CNA in metastatic breast tumor samples using publicly available datasets and characterize their expression and function using a metastatic mouse model of breast cancer. Our findings demonstrate that our organoid generation can be implemented to study clinically relevant features that reflect the genetic heterogeneity of individual tumors.

The Fisher Subunit Anatomical Approach for Cleft Lip Revisions: An Evolution and Case Series.

ABSTRACT: Many techniques exist to reapproximate a cleft lip but can leave unsatisfactory results with nonanatomic scars and a short upper lip, creating a need for revision. Many revisions focus on adjacent tissue transfers and realignment of landmarks, but in the senior authors' experience, recreating the defect and utilizing the Fisher repair for revision have led to aesthetically pleasing results and less noticeable scars. A database was collected that included all cleft lip revisions performed at a large, comprehensive children's hospital from October 2018 to July 2021. Inclusion criteria included any cleft patient with a cleft lip revision performed by two craniofacial surgeons. Data collected included sex, characteristics of the cleft lip, age at initial and index repair, type of initial repair, previous revisions, type of revision with any additional tissue rearrangement, and any nose repair. Sixty-five patients were included in the study for analysis. The type of initial repair was known in sixty-four cases (98%), and fifty-four were Millard repairs (83%). Twenty-two patients (33%) had a previous revision prior to their index revision. Sixty patients (92%) underwent the Fisher repair technique for their index revision and forty-six patients (70%) underwent nasal revision. In follow-up, all patients demonstrated an improvement in lip aesthetics. This study demonstrates a large subset of patients that have undergone cleft lip revision using the Fisher technique. In the senior surgeons' experience, the Fisher repair technique in the setting of cleft lip revision is an ideal way to address the shortcomings of historical repair techniques.

Synthesis and Evaluation of a Novel PET Radioligand for Imaging Glutaminyl Cyclase Activity as a Biomarker for Detecting Alzheimer's Disease.

Several new lines of research have demonstrated that a significant number of amyloid-ß peptides found in Alzheimer's disease (AD) are truncated and undergo post-translational modification by glutaminyl cyclase (QC) at the N-terminal. Notably, QC's products of Abeta-pE3 and Abeta-pE11 have been active targets for investigational drug development. This work describes the design, synthesis, characterization, and in vivo validation of a novel PET radioligand, [18F]PB0822, for targeted imaging of QC. We report herein a simplified and robust chemistry for the synthesis of the standard compound, [19F]PB0822, and the corresponding [18F]PB0822 radioligand. The PET probe was developed with 99.9% radiochemical purity, a molar activity of 965 Ci.mmol-1, and an IC50 of 56.3 nM, comparable to those of the parent PQ912 inhibitor (62.5 nM). Noninvasive PET imaging showed that the probe is distributed in the brain 5 min after intravenous injection. Further, in vivo PET imaging with [18F]PB0822 revealed that AD 5XFAD mice harbor significantly higher QC activity than WT counterparts. The data also suggested that QC activity is found across different brain regions of the tested animals.

Targeting Acute Myeloid Leukemia Stem Cells Through Perturbation of Mitochondrial Calcium.

Acute myeloid leukemia stem cells (LSCs) are uniquely reliant on oxidative phosphorylation (OXPHOS) for survival. Moreover, maintenance of OXPHOS is dependent on BCL-2, creating a therapeutic opportunity to target LSCs using the BCL-2 inhibitor venetoclax. While venetoclax-based regimens have shown promising clinical activity, the emergence of drug resistance is prevalent. Thus, in the present study, we investigated how mitochondrial properties may influence venetoclax responsiveness. Our data show that utilization of mitochondrial calcium is fundamentally different between drug-responsive and non-responsive LSCs. By comparison, venetoclax-resistant LSCs demonstrate a more active metabolic (i.e. OXPHOS) status with relatively high levels of calcium. Consequently, we tested genetic and pharmacological approaches to target the mitochondrial calcium uniporter, MCU. We demonstrate that inhibition of calcium uptake reduces OXPHOS and leads to eradication of venetoclax-resistant LSCs. These findings demonstrate a central role for calcium signaling in LSCs and provide an avenue for clinical management of venetoclax resistance.

Three-Dimensional Printing of Ultrasoft Silicone with a Functional Stiffness Gradient.

A methodology for three-dimensionally printing ultrasoft silicone with a functional stiffness gradient is presented. Ultraviolet-cure silicone was deposited via two independently controlled extruders into a thixotropic, gel-like, silicone oil-based support matrix. Each extruder contained a different liquid silicone formulation. The extrusion rates were independently varied during printing such that the combined selectively deposited material contained different ratios of the two silicones, resulting in localized control of material stiffness. Tests to validate the process are reported, including tensile testing of homogeneous cubic specimens to quantify the range of material stiffness that could be printed, indentation testing of cuboid specimens to characterize printed stiffness gradients, and vibratory testing of synthetic multilayer vocal fold (VF) models to demonstrate that the method may be applied to the fabrication of biomechanical models for voice production research. The cubic specimens exhibited linear stress-strain data with tensile elasticity modulus values between 1.11 and 27.1 kPa, more than a factor of 20 in stiffness variation. The cuboid specimens exhibited material variations that were visually recognizable and quantifiable via indentation testing. The VF models withstood rigorous phonatory flow-induced vibration and exhibited vibratory characteristics comparable to those of previous models. Overall, while process refinements are needed, the results of these tests demonstrate the ability to print ultrasoft silicone with stiffness gradients.

TARGETING SOLUBLE AMYLOID-BETA OLIGOMERS WITH A NOVEL NANOBODY.

The classical amyloid cascade hypothesis postulates that the aggregation of amyloid plaques and the accumulation of intracellular hyperphosphorylated Tau tangles, together, lead to profound neuronal death. However, emerging research has demonstrated that soluble amyloid-ß oligomers (SAßOs) accumulate early, prior to amyloid plaque formation. SAßOs induce memory impairment and disrupt cognitive function independent of amyloid-ß plaques, and even in the absence of plaque formation. This work describes the development and characterization of a novel anti-SAßO (E3) nanobody generated from an alpaca immunized with SAßO. In-vitro assays and in-vivo studies using 5XFAD mice indicate that the fluorescein (FAM)-labeled E3 nanobody recognizes both SAßOs and amyloid-ß plaques. The E3 nanobody traverses across the blood-brain barrier and binds to amyloid species in the brain of 5XFAD mice. Imaging of mouse brains reveals that SAßO and amyloid-ß plaques are not only different in size, shape, and morphology, but also have a distinct spatial distribution in the brain. SAßOs are associated with neurons, while amyloid plaques reside in the extracellular matrix. The results of this study demonstrate that the SAßO nanobody can serve as a diagnostic agent with potential theragnostic applications in Alzheimer's disease.

Long-term outcomes after recurrent acute thoracic aortic dissection: Insights from the International Registry of Aortic Dissection.

OBJECTIVE: With an aging population and advancements in imaging, recurrence of thoracic aortic dissection is becoming more common. METHODS: All patients enrolled in the International Registry of Aortic Dissection from 1996 to 2023 with type A and type B acute aortic dissection were identified. Among them, initial dissection and recurrent dissection were discerned. The study period was categorized into 3 eras: historic era, 1996 to 2005; middle era, 2006 to 2015; most recent era, 2016 to 2023. Propensity score matching was applied between initial dissection and recurrent dissection. Outcome of interests included long-term survival and cumulative incidence of major aortic events defined by the composite of reintervention, aortic rupture, and new dissection. RESULTS: The proportion of recurrent dissection increased from 5.9% in the historic era to 8.0% in the most recent era in the entire dissection cohort. In patients with type A dissection, propensity score matching between initial dissection and recurrent dissection yielded 326 matched pairs. Kaplan-Meier curves showed similar long-term survival between the 2 groups. However, the cumulative incidence of major aortic events was significantly higher in the recurrent dissection group (40.3% ± 6.2% vs 17.8% ± 5.1% at 4 years in the initial dissection group, P = .02). For type B dissection, 316 matched pairs were observed after propensity score matching. Long-term survival and the incidence of major aortic events were equivalent between the 2 groups. CONCLUSIONS: The case volume of recurrent dissection or the ability to detect recurrent dissection has increased over time. Acute type A recurrent dissection was associated with a higher risk of major aortic events than initial dissection. Further judicious follow-up may be crucial after type A recurrent dissection.

Breaking barriers in Candida spp. detection with Electronic Noses and artificial intelligence.

The timely and accurate diagnosis of candidemia, a severe bloodstream infection caused by Candida spp., remains challenging in clinical practice. Blood culture, the current gold standard technique, suffers from lengthy turnaround times and limited sensitivity. To address these limitations, we propose a novel approach utilizing an Electronic Nose (E-nose) combined with Time Series-based classification techniques to analyze and identify Candida spp. rapidly, using culture species of C. albicans, C.kodamaea ohmeri, C. glabrara, C. haemulonii, C. parapsilosis and C. krusei as control samples. This innovative method not only enhances diagnostic accuracy and reduces decision time for healthcare professionals in selecting appropriate treatments but also offers the potential for expanded usage and cost reduction due to the E-nose's low production costs. Our proof-of-concept experimental results, carried out with culture samples, demonstrate promising outcomes, with the Inception Time classifier achieving an impressive average accuracy of 97.46% during the test phase. This paper presents a groundbreaking advancement in the field, empowering medical practitioners with an efficient and reliable tool for early and precise identification of candidemia, ultimately leading to improved patient outcomes.

Plastic Surgery: Beware the Candle Burning at Both Ends.

Background: Plastic surgery dates back to 800 BC, where forehead flaps were used to reconstruct noses in India. Today, it is one of the most romanticized fields in medicine. Due to the influence of social media, there has never been a larger spotlight. Ironically, this spotlight brings a narrowed perception of the scope of plastic surgery. This study aimed to assess the scope through the eyes of the average American to identify gaps in knowledge to better represent the field. Methods: A series of questions were developed under survey methodologists and administered by Qualtrics. Responses were gathered, and data were analyzed to assess the public's knowledge of plastic surgery's scope. Results: Two thousand five hundred responses were obtained, balanced across demographics similar to that of the United States. The US population has a poor understanding of the scope of plastic surgery and how to obtain board certification. Conclusions: This survey demonstrates a gap in awareness of plastic surgery as a field and the scope outside aesthetic procedures dramatized by the media. There remains no clear understanding of the qualifications of plastic surgeons or the provider makeup of the field of cosmetic surgery. Subspecialties proved to be overlooked, and knowledge of board certification was sparse. Further effort is needed to educate both the public and patients of the scope of plastic surgery, so that they might seek and gain access to appropriate treatment in the most efficient manner to optimize outcomes regarding the form and function of the body.

Targeting Acute Myeloid Leukemia Stem Cells Through Perturbation of Mitochondrial Calcium.

We previously reported that acute myeloid leukemia stem cells (LSCs) are uniquely reliant on oxidative phosphorylation (OXPHOS) for survival. Moreover, maintenance of OXPHOS is dependent on BCL2, creating a therapeutic opportunity to target LSCs using the BCL2 inhibitor drug venetoclax. While venetoclax-based regimens have indeed shown promising clinical activity, the emergence of drug resistance is prevalent. Thus, in the present study, we investigated how mitochondrial properties may influence mechanisms that dictate venetoclax responsiveness. Our data show that utilization of mitochondrial calcium is fundamentally different between drug responsive and non-responsive LSCs. By comparison, venetoclax-resistant LSCs demonstrate a more active metabolic (i.e., OXPHOS) status with relatively high steady-state levels of calcium. Consequently, we tested genetic and pharmacological approaches to target the mitochondrial calcium uniporter, MCU. We demonstrate that inhibition of calcium uptake sharply reduces OXPHOS and leads to eradication of venetoclax-resistant LSCs. These findings demonstrate a central role for calcium signaling in the biology of LSCs and provide a therapeutic avenue for clinical management of venetoclax resistance.

Complete genome sequences of microbacterium phages Tedro and BAjuniper.

We purified two novel bacteriophages from soil collected in Sioux County, Iowa: BAjuniper and Tedro. These bacteriophages were isolated from the host, Microbacterium foliorum. BAjuniper was assigned to cluster EB, and Tedro was assigned to cluster EF. Both phages display genomes typical of other phages in their clusters.

Diagnostic performance of circulating biomarkers for non-alcoholic steatohepatitis.

There are no approved diagnostic biomarkers for at-risk non-alcoholic steatohepatitis (NASH), defined by the presence of NASH, high histological activity and fibrosis stage ≥2, which is associated with higher incidence of liver-related events and mortality. FNIH-NIMBLE is a multi-stakeholder project to support regulatory approval of NASH-related biomarkers. The diagnostic performance of five blood-based panels was evaluated in an observational (NASH CRN DB2) cohort (n = 1,073) with full spectrum of non-alcoholic fatty liver disease (NAFLD). The panels were intended to diagnose at-risk NASH (NIS4), presence of NASH (OWLiver) or fibrosis stages >2, >3 or 4 (enhanced liver fibrosis (ELF) test, PROC3 and FibroMeter VCTE). The prespecified performance metric was an area under the receiver operating characteristic curve (AUROC) ≥0.7 and superiority over alanine aminotransferase for disease activity and the FIB-4 test for fibrosis severity. Multiple biomarkers met these metrics. NIS4 had an AUROC of 0.81 (95% confidence interval: 0.78-0.84) for at-risk NASH. The AUROCs of the ELF test, PROC3 and FibroMeterVCTE for clinically significant fibrosis (≥stage 2), advanced fibrosis (≥stage 3) or cirrhosis (stage 4), respectively, were all ≥0.8. ELF and FibroMeter VCTE outperformed FIB-4 for all fibrosis endpoints. These data represent a milestone toward qualification of several biomarker panels for at-risk NASH and also fibrosis severity in individuals with NAFLD.

Tauopathy severely disrupts homeostatic set-points in emergent neural dynamics but not in the activity of individual neurons.

The homeostatic regulation of neuronal activity is essential for robust computation; key set-points, such as firing rate, are actively stabilized to compensate for perturbations. From this perspective, the disruption of brain function central to neurodegenerative disease should reflect impairments of computationally essential set-points. Despite connecting neurodegeneration to functional outcomes, the impact of disease on set-points in neuronal activity is unknown. Here we present a comprehensive, theory-driven investigation of the effects of tau-mediated neurodegeneration on homeostatic set-points in neuronal activity. In a mouse model of tauopathy, we examine 27,000 hours of hippocampal recordings during free behavior throughout disease progression. Contrary to our initial hypothesis that tauopathy would impact set-points in spike rate and variance, we found that cell-level set-points are resilient to even the latest stages of disease. Instead, we find that tauopathy disrupts neuronal activity at the network-level, which we quantify using both pairwise measures of neuron interactions as well as measurement of the network's nearness to criticality, an ideal computational regime that is known to be a homeostatic set-point. We find that shifts in network criticality 1) track with symptoms, 2) predict underlying anatomical and molecular pathology, 3) occur in a sleep/wake dependent manner, and 4) can be used to reliably classify an animal's genotype. Our data suggest that the critical set-point is intact, but that homeostatic machinery is progressively incapable of stabilizing hippocampal networks, particularly during waking. This work illustrates how neurodegenerative processes can impact the computational capacity of neurobiological systems, and suggest an important connection between molecular pathology, circuit function, and animal behavior.

A Novel Type of Monocytic Leukemia Stem Cell Revealed by the Clinical Use of Venetoclax-Based Therapy.

The BCL2 inhibitor venetoclax has recently emerged as an important component of acute myeloid leukemia (AML) therapy. Notably, use of this agent has revealed a previously unrecognized form of pathogenesis characterized by monocytic disease progression. We demonstrate that this form of disease arises from a fundamentally different type of leukemia stem cell (LSC), which we designate as monocytic LSC (m-LSC), that is developmentally and clinically distinct from the more well-described primitive LSC (p-LSC). The m-LSC is distinguished by a unique immunophenotype (CD34-, CD4+, CD11b-, CD14-, CD36-), unique transcriptional state, reliance on purine metabolism, and selective sensitivity to cladribine. Critically, in some instances, m-LSC and p-LSC subtypes can co-reside in the same patient with AML and simultaneously contribute to overall tumor biology. Thus, our findings demonstrate that LSC heterogeneity has direct clinical significance and highlight the need to distinguish and target m-LSCs as a means to improve clinical outcomes with venetoclax-based regimens. SIGNIFICANCE: These studies identify and characterize a new type of human acute myeloid LSC that is responsible for monocytic disease progression in patients with AML treated with venetoclax-based regimens. Our studies describe the phenotype, molecular properties, and drug sensitivities of this unique LSC subclass. This article is featured in Selected Articles from This Issue, p. 1949.

Efficacy and Profitability of Fungicides for Managing Frogeye Leaf Spot on Soybean in the United States: A 10-Year Quantitative Summary.

Frogeye leaf spot (FLS), caused by Cercospora sojina, is an economically important disease of soybean in the United States. Data from 66 uniform fungicide trials (UFTs) conducted from 2012 to 2021 across eight states (Alabama, Arkansas, Illinois, Iowa, Kentucky, Louisiana, Mississippi, and Tennessee) were gathered and analyzed to determine the efficacy and profitability of the following fungicides applied at the beginning pod developmental stage (R3): azoxystrobin + difenoconazole (AZOX + DIFE), difenoconazole + pydiflumetofen (DIFE + PYDI), pyraclostrobin (PYRA), pyraclostrobin + fluxapyroxad + propiconazole (PYRA + FLUX + PROP), tetraconazole (TTRA), thiophanate-methyl (TMET), thiophanate-methyl + tebuconazole (TMET + TEBU), and trifloxystrobin + prothioconazole (TFLX + PROT). A network meta-analytic model was fitted to the log of the means of FLS severity data and to the nontransformed mean yield for each treatment, including the nontreated. The percent reduction in disease severity (%) and the yield response (kg/ha) relative to the nontreated was the lowest for PYRA (11%; 136 kg/ha) and the greatest for DIFE + PYDI (57%; 441 kg/ha). A significant decline in efficacy over time was detected for PYRA (18 percentage points [p.p.]), TTRA (27 p.p.), AZOX + DIFE (18 p.p.), and TMET + TEBU (19 p.p.) by using year as a continuous covariate in the model. Finally, probabilities of breaking even were the greatest (>65%) for the most effective fungicide DIFE + PYDI and the lowest (<55%) for PYRA. Results of this meta-analysis may be useful to support decisions when planning fungicide programs.

Venetoclax is safe and tolerable as post-transplant maintenance therapy for AML patients at high risk for relapse.

Relapse is the most common cause of mortality in acute myeloid leukemia (AML) patients after allogeneic stem cell transplant (SCT). Post-SCT maintenance strategies that prevent relapse are desirable but must be well tolerated and convenient to administer. We hypothesized single agent venetoclax (ven) may be an effective maintenance therapy among high relapse risk patients. Between February 2019 and December 2021, we administered post-SCT ven maintenance to 49 AML patients at high-risk for relapse as a prospectively defined off-label practice at our institution. Ven was planned to be administered until 1-year post-SCT. While temporary interruptions were common (67.3% of all patients), of those with >1 year follow up, 22/25 (88%) completed the full year of planned therapy. Cytopenias (40.8%) and gastrointestinal adverse events (34.7%) were the most common toxicities. At 1-year post-SCT, overall survival (OS) and relapse-free survival (RFS) were 70% and 67% respectively. Our experience demonstrates single agent ven is a safe, tolerable, and feasible maintenance therapy that may improve RFS and OS in high relapse risk post-SCT patients.

Meta-Analytic Modeling of the Severity-Yield Relationships in Soybean Frogeye Leaf Spot Epidemics.

Frogeye leaf spot (FLS), caused by Cercospora sojina, is an important foliar disease affecting soybean in the United States. A meta-analytic approach including 39 fungicide trials conducted from 2012 to 2021 across eight states (Alabama, Arkansas, Illinois, Iowa, Kentucky, Louisiana, Mississippi, Tennessee) was used to assess the relationship between FLS severity and soybean yield. Correlation and regression analyses were performed separately to determine Fisher's transformation of correlation coefficients (Zr), intercept (ß0) and slope (ß1). Disease pressure (low severity, ≤34.5; high severity, >34.5%) and yield class (low, ≤3,352; high, >3,352 kg/ha) were included as categorical moderators. Pearson's [Formula: see text], obtained from back-transforming the [Formula: see text]r estimated by an overall random-effects model, showed a significant negative linear relationship between FLS severity and yield ([Formula: see text] = -0.60). The [Formula: see text]r was affected by disease pressure (P = 0.0003) but not by yield class (P = 0.8141). A random-coefficient model estimated a slope of -19 kg/ha for each percent severity for a mean attainable yield of 3,719.9 kg/ha. Based on the overall mean (95% CI) of the intercept and slope estimated by the random-coefficients model, the estimated overall relative damage coefficient was 0.51% (0.36 to 0.69), indicating that a percent increase in FLS severity reduced yield by 0.51%. The best model included yield class as a covariate, and population-average intercepts differed significantly between low (3,455.1 kg/ha) and high (3,842.7 kg/ha) yield classes. This highlights the potential impact of FLS on soybean yield if not managed and may help in disease management decisions.

Appropriateness of cardiovascular computed tomography and magnetic resonance imaging in patients with conotruncal defects.

BACKGROUND: To promote the rational use of cardiovascular imaging in patients with congenital heart disease, the American College of Cardiology developed Appropriate Use Criteria (AUC), but its clinical application and pre-release benchmarks have not been evaluated. We aimed to evaluate the appropriateness of indications for cardiovascular magnetic resonance (CMR) and cardiovascular computed tomography (CCT) in patients with conotruncal defects and to identify factors associated with maybe or rarely appropriate (M/R) indications. METHODS: Twelve centers each contributed a median of 147 studies performed prior to AUC publication (01/2020) on patients with conotruncal defects. To incorporate patient characteristics and center-level effects, a hierarchical generalized linear mixed model was used. RESULTS: Of the 1753 studies (80% CMR, and 20% CCT), 16% were rated M/R. Center M/R ranged from 4 to 39%. Infants accounted for 8.4% of studies. In multivariable analyses, patient- and study-level factors associated with M/R rating included: age <1 year (OR 1.90 [1.15-3.13]), truncus arteriosus (vs. tetralogy of Fallot, OR 2.55 [1.5-4.35]), and CCT (vs. CMR, OR 2.67 [1.87-3.83]). None of the provider- or center-level factors reached statistical significance in the multivariable model. CONCLUSIONS: Most CMRs and CCTs ordered for the follow-up care of patients with conotruncal defects were rated appropriate. However, there was significant center-level variation in appropriateness ratings. Younger age, CCT, and truncus arteriosus were independently associated with higher odds of M/R rating. These findings could inform future quality improvement initiatives and further exploration of factors resulting in center-level variation.