Modularity and diversity of target selectors in Tn7 transposons.

To spread, transposons must integrate into target sites without disruption of essential genes while avoiding host defense systems. Tn7-like transposons employ multiple mechanisms for target-site selection, including protein-guided targeting and, in CRISPR-associated transposons (CASTs), RNA-guided targeting. Combining phylogenomic and structural analyses, we conducted a broad survey of target selectors, revealing diverse mechanisms used by Tn7 to recognize target sites, including previously uncharacterized target-selector proteins found in newly discovered transposable elements (TEs). We experimentally characterized a CAST I-D system and a Tn6022-like transposon that uses TnsF, which contains an inactivated tyrosine recombinase domain, to target the comM gene. Additionally, we identified a non-Tn7 transposon, Tsy, encoding a homolog of TnsF with an active tyrosine recombinase domain, which we show also inserts into comM. Our findings show that Tn7 transposons employ modular architecture and co-opt target selectors from various sources to optimize target selection and drive transposon spread.

Disease-specific tau filaments assemble via polymorphic intermediates.

Intermediate species in the assembly of amyloid filaments are believed to play a central role in neurodegenerative diseases and may constitute important targets for therapeutic intervention1,2. However, structural information about intermediate species has been scarce and the molecular mechanisms by which amyloids assemble remain largely unknown. Here we use time-resolved cryogenic electron microscopy to study the in vitro assembly of recombinant truncated tau (amino acid residues 297-391) into paired helical filaments of Alzheimer's disease or into filaments of chronic traumatic encephalopathy3. We report the formation of a shared first intermediate amyloid filament, with an ordered core comprising residues 302-316. Nuclear magnetic resonance indicates that the same residues adopt rigid, ß-strand-like conformations in monomeric tau. At later time points, the first intermediate amyloid disappears and we observe many different intermediate amyloid filaments, with structures that depend on the reaction conditions. At the end of both assembly reactions, most intermediate amyloids disappear and filaments with the same ordered cores as those from human brains remain. Our results provide structural insights into the processes of primary and secondary nucleation of amyloid assembly, with implications for the design of new therapies.

Highly Parallel Profiling of Cas9 Variant Specificity.

Determining the off-target cleavage profile of programmable nucleases is an important consideration for any genome editing experiment, and a number of Cas9 variants have been reported that improve specificity. We describe here tagmentation-based tag integration site sequencing (TTISS), an efficient, scalable method for analyzing double-strand breaks (DSBs) that we apply in parallel to eight Cas9 variants across 59 targets. Additionally, we generated thousands of other Cas9 variants and screened for variants with enhanced specificity and activity, identifying LZ3 Cas9, a high specificity variant with a unique +1 insertion profile. This comprehensive comparison reveals a general trade-off between Cas9 activity and specificity and provides information about the frequency of generation of +1 insertions, which has implications for correcting frameshift mutations.

Lecanemab in Early Alzheimer's Disease.

BACKGROUND: The accumulation of soluble and insoluble aggregated amyloid-beta (Aß) may initiate or potentiate pathologic processes in Alzheimer's disease. Lecanemab, a humanized IgG1 monoclonal antibody that binds with high affinity to Aß soluble protofibrils, is being tested in persons with early Alzheimer's disease. METHODS: We conducted an 18-month, multicenter, double-blind, phase 3 trial involving persons 50 to 90 years of age with early Alzheimer's disease (mild cognitive impairment or mild dementia due to Alzheimer's disease) with evidence of amyloid on positron-emission tomography (PET) or by cerebrospinal fluid testing. Participants were randomly assigned in a 1:1 ratio to receive intravenous lecanemab (10 mg per kilogram of body weight every 2 weeks) or placebo. The primary end point was the change from baseline at 18 months in the score on the Clinical Dementia Rating-Sum of Boxes (CDR-SB; range, 0 to 18, with higher scores indicating greater impairment). Key secondary end points were the change in amyloid burden on PET, the score on the 14-item cognitive subscale of the Alzheimer's Disease Assessment Scale (ADAS-cog14; range, 0 to 90; higher scores indicate greater impairment), the Alzheimer's Disease Composite Score (ADCOMS; range, 0 to 1.97; higher scores indicate greater impairment), and the score on the Alzheimer's Disease Cooperative Study-Activities of Daily Living Scale for Mild Cognitive Impairment (ADCS-MCI-ADL; range, 0 to 53; lower scores indicate greater impairment). RESULTS: A total of 1795 participants were enrolled, with 898 assigned to receive lecanemab and 897 to receive placebo. The mean CDR-SB score at baseline was approximately 3.2 in both groups. The adjusted least-squares mean change from baseline at 18 months was 1.21 with lecanemab and 1.66 with placebo (difference, -0.45; 95% confidence interval [CI], -0.67 to -0.23; P<0.001). In a substudy involving 698 participants, there were greater reductions in brain amyloid burden with lecanemab than with placebo (difference, -59.1 centiloids; 95% CI, -62.6 to -55.6). Other mean differences between the two groups in the change from baseline favoring lecanemab were as follows: for the ADAS-cog14 score, -1.44 (95% CI, -2.27 to -0.61; P<0.001); for the ADCOMS, -0.050 (95% CI, -0.074 to -0.027; P<0.001); and for the ADCS-MCI-ADL score, 2.0 (95% CI, 1.2 to 2.8; P<0.001). Lecanemab resulted in infusion-related reactions in 26.4% of the participants and amyloid-related imaging abnormalities with edema or effusions in 12.6%. CONCLUSIONS: Lecanemab reduced markers of amyloid in early Alzheimer's disease and resulted in moderately less decline on measures of cognition and function than placebo at 18 months but was associated with adverse events. Longer trials are warranted to determine the efficacy and safety of lecanemab in early Alzheimer's disease. (Funded by Eisai and Biogen; Clarity AD ClinicalTrials.gov number, NCT03887455.).

Lipid droplets are intracellular mechanical stressors that impair hepatocyte function.

Matrix stiffening and external mechanical stress have been linked to disease and cancer development in multiple tissues, including the liver, where cirrhosis (which increases stiffness markedly) is the major risk factor for hepatocellular carcinoma. Patients with nonalcoholic fatty liver disease and lipid droplet-filled hepatocytes, however, can develop cancer in noncirrhotic, relatively soft tissue. Here, by treating primary human hepatocytes with the monounsaturated fatty acid oleate, we show that lipid droplets are intracellular mechanical stressors with similar effects to tissue stiffening, including nuclear deformation, chromatin condensation, and impaired hepatocyte function. Mathematical modeling of lipid droplets as inclusions that have only mechanical interactions with other cellular components generated results consistent with our experiments. These data show that lipid droplets are intracellular sources of mechanical stress and suggest that nuclear membrane tension integrates cell responses to combined internal and external stresses.

HSP90ß prevents aging-related cataract formation through regulation of the charged multivesicular body protein (CHMP4B) and p53.

Cataract is a leading ocular disease causing global blindness. The mechanism of cataractogenesis has not been well defined. Here, we demonstrate that the heat shock protein 90ß (HSP90ß) plays a fundamental role in suppressing cataractogenesis. HSP90ß is the most dominant HSP in normal lens, and its constitutive high level of expression is largely derived from regulation by Sp1 family transcription factors. More importantly, HSP90ß is significantly down-regulated in human cataract patients and in aging mouse lenses, whereas HSP90ß silencing in zebrafish causes cataractogenesis, which can only be rescued by itself but not other HSP90 genes. Mechanistically, HSP90ß can directly interact with CHMP4B, a newly-found client protein involved in control of cytokinesis. HSP90ß silencing causes upregulation of CHMP4B and another client protein, the tumor suppressor p53. CHMP4B upregulation or overexpression induces excessive division of lens epithelial cells without proper differentiation. As a result, these cells were triggered to undergo apoptosis due to activation of the p53/Bak-Bim pathway, leading to cataractogenesis and microphthalmia. Silence of both HSP90ß and CHMP4B restored normal phenotype of zebrafish eye. Together, our results reveal that HSP90ß is a critical inhibitor of cataractogenesis through negative regulation of CHMP4B and the p53-Bak/Bim pathway.

Short-term response of primary human meniscus cells to simulated microgravity.

BACKGROUND: Mechanical unloading of the knee articular cartilage results in cartilage matrix atrophy, signifying the osteoarthritic-inductive potential of mechanical unloading. In contrast, mechanical loading stimulates cartilage matrix production. However, little is known about the response of meniscal fibrocartilage, a major mechanical load-bearing tissue of the knee joint, and its functional matrix-forming fibrochondrocytes to mechanical unloading events. METHODS: In this study, primary meniscus fibrochondrocytes isolated from the inner avascular region of human menisci from both male and female donors were seeded into porous collagen scaffolds to generate 3D meniscus models. These models were subjected to both normal gravity and mechanical unloading via simulated microgravity (SMG) for 7 days, with samples collected at various time points during the culture. RESULTS: RNA sequencing unveiled significant transcriptome changes during the 7-day SMG culture, including the notable upregulation of key osteoarthritis markers such as COL10A1, MMP13, and SPP1, along with pathways related to inflammation and calcification. Crucially, sex-specific variations in transcriptional responses were observed. Meniscus models derived from female donors exhibited heightened cell proliferation activities, with the JUN protein involved in several potentially osteoarthritis-related signaling pathways. In contrast, meniscus models from male donors primarily regulated extracellular matrix components and matrix remodeling enzymes. CONCLUSION: These findings advance our understanding of sex disparities in knee osteoarthritis by developing a novel in vitro model using cell-seeded meniscus constructs and simulated microgravity, revealing significant sex-specific molecular mechanisms and therapeutic targets.

Smartphone-based optical sectioning (SOS) microscopy with a telecentric design for fluorescence imaging.

We propose a smartphone-based optical sectioning (SOS) microscope based on the HiLo technique, with a single smartphone replacing a high-cost illumination source and a camera sensor. We built our SOS with off-the-shelf optical, mechanical cage systems with 3D-printed adapters to seamlessly integrate the smartphone with the SOS main body. The liquid light guide can be integrated with the adapter, guiding the smartphone's LED light to the digital mirror device (DMD) with neglectable loss. We used an electrically tuneable lens (ETL) instead of a mechanical translation stage to realise low-cost axial scanning. The ETL was conjugated to the objective lens's back pupil plane (BPP) to construct a telecentric design by a 4f configuration to maintain the lateral magnification for different axial positions. SOS has a 571.5 µm telecentric scanning range and an 11.7 µm axial resolution. The broadband smartphone LED torch can effectively excite fluorescent polystyrene (PS) beads. We successfully used SOS for high-contrast fluorescent PS beads imaging with different wavelengths and optical sectioning imaging of multilayer fluorescent PS beads. To our knowledge, the proposed SOS is the first smartphone-based HiLo optical sectioning microscopy (£1965), which can save around £7035 compared with a traditional HiLo system (£9000). It is a powerful tool for biomedical research in resource-limited areas.

Effects of Age, Sex, and Extracellular Matrix Integrity on Aortic Dilatation and Rupture in a Mouse Model of Marfan Syndrome.

BACKGROUND: Transmural failure of the aorta is responsible for substantial morbidity and mortality; it occurs when mechanical stress exceeds strength. The aortic root and ascending aorta are susceptible to dissection and rupture in Marfan syndrome, a connective tissue disorder characterized by a progressive reduction in elastic fiber integrity. Whereas competent elastic fibers endow the aorta with compliance and resilience, cross-linked collagen fibers confer stiffness and strength. We hypothesized that postnatal reductions in matrix cross-linking increase aortopathy when turnover rates are high. METHODS: We combined ex vivo biaxial mechanical testing with multimodality histological examinations to quantify expected age- and sex-dependent structural vulnerability of the ascending aorta in Fbn1C1041G/+ Marfan versus wild-type mice without and with 4-week exposures to ß-aminopropionitrile, an inhibitor of lysyl oxidase-mediated cross-linking of newly synthesized elastic and collagen fibers. RESULTS: We found a strong ß-aminopropionitrile-associated sexual dimorphism in aortic dilatation in Marfan mice and aortic rupture in wild-type mice, with dilatation correlating with compromised elastic fiber integrity and rupture correlating with compromised collagen fibril organization. A lower incidence of rupture of ß-aminopropionitrile-exposed Marfan aortas associated with increased lysyl oxidase, suggesting a compensatory remodeling of collagen that slows disease progression in the otherwise compromised Fbn1C1041G/+ aorta. CONCLUSIONS: Collagen fiber structure and function in the Marfan aorta are augmented, in part, by increased lysyl oxidase in female and especially male mice, which improves structural integrity, particularly via fibrils in the adventitia. Preserving or promoting collagen cross-linking may represent a therapeutic target for an otherwise vulnerable aorta.

FN (Fibronectin)-Integrin α5 Signaling Promotes Thoracic Aortic Aneurysm in a Mouse Model of Marfan Syndrome.

BACKGROUND: Marfan syndrome, caused by mutations in the gene for fibrillin-1, leads to thoracic aortic aneurysms (TAAs). Phenotypic modulation of vascular smooth muscle cells (SMCs) and ECM (extracellular matrix) remodeling are characteristic of both nonsyndromic and Marfan aneurysms. The ECM protein FN (fibronectin) is elevated in the tunica media of TAAs and amplifies inflammatory signaling in endothelial and SMCs through its main receptor, integrin α5ß1. We investigated the role of integrin α5-specific signals in Marfan mice in which the cytoplasmic domain of integrin α5 was replaced with that of integrin α2 (denoted α5/2 chimera). METHODS: We crossed α5/2 chimeric mice with Fbn1mgR/mgR mice (mgR model of Marfan syndrome) to evaluate the survival rate and pathogenesis of TAAs among wild-type, α5/2, mgR, and α5/2 mgR mice. Further biochemical and microscopic analysis of porcine and mouse aortic SMCs investigated molecular mechanisms by which FN affects SMCs and subsequent development of TAAs. RESULTS: FN was elevated in the thoracic aortas from Marfan patients, in nonsyndromic aneurysms, and in mgR mice. The α5/2 mutation greatly prolonged survival of Marfan mice, with improved elastic fiber integrity, mechanical properties, SMC density, and SMC contractile gene expression. Furthermore, plating of wild-type SMCs on FN decreased contractile gene expression and activated inflammatory pathways whereas α5/2 SMCs were resistant. These effects correlated with increased NF-kB activation in cultured SMCs and mgR aortas, which was alleviated by the α5/2 mutation or NF-kB inhibition. CONCLUSIONS: FN-integrin α5 signaling is a significant driver of TAA in the mgR mouse model. This pathway thus warrants further investigation as a therapeutic target.

Adjunctive cariprazine for major depressive disorder: a systematic review and meta-analysis.

Converging evidence has suggested that treatment augmentation with a second-generation atypical antipsychotic (SGA) may improve treatment outcomes in major depressive disorder (MDD) patients after an incomplete response to a first-line antidepressant. Cariprazine is a recently approved SGA for MDD augmentation. Herein, we evaluate both continuous (ie, change in depressive symptom severity scores over time) and categorical (ie, remission and response rates) outcomes. Following a full-text review, four randomized controlled trials (RCTs) were included in our meta-analysis, while five studies were included for a qualitative review. Risk ratios (RRs) were calculated for all included randomized controlled studies to determine the relative response and remission rates of cariprazine compared to placebo augmentation. The RR for all-cause dropout was also determined as a proxy for overall acceptability. Two studies found a statistically significant treatment response using cariprazine augmentation. One study observed depressive symptom remission for cariprazine compared to placebo. Our random-effects model revealed moderate antidepressant effects of cariprazine, with a standardized mean difference (SMD) in Montgomery-Åsberg Depression Rating Scale (MADRS) scores of -1.79 (95% CI): -2.89, -0.69). Our pooled response RR and remission RR were calculated as 1.21 (95% CI: 1.05, 1.39, P=0.008) and 0.99 (95% CI: 0.84, 1.17, P=0.91), respectively. The RR for response was statistically significant (P<0.05). However, the RR for remission was not statistically significant. The findings from our meta-analysis include a variable magnitude of effects. Evidence suggests cariprazine may be an effective treatment for MDD; however, further results are needed to clarify this relation.

High-performance reconstruction method combining total variation with a video denoiser for compressed ultrafast imaging.

Compressed ultrafast photography (CUP) is a novel two-dimensional (2D) imaging technique to capture ultrafast dynamic scenes. Effective image reconstruction is essential in CUP systems. However, existing reconstruction algorithms mostly rely on image priors and complex parameter spaces. Therefore, in general, they are time-consuming and result in poor imaging quality, which limits their practical applications. In this paper, we propose a novel reconstruction algorithm, to the best of our knowledge, named plug-in-plug-fast deep video denoising net-total variation (PnP-TV-FastDVDnet), which exploits an image's spatial features and correlation features in the temporal dimension. Therefore, it offers higher-quality images than those in previously reported methods. First, we built a forward mathematical model of the CUP, and the closed-form solution of the three suboptimization problems was derived according to plug-in and plug-out frames. Secondly, we used an advanced video denoising algorithm based on a neural network named FastDVDnet to solve the denoising problem. The peak signal-to-noise ratio (PSNR) and structural similarity index measure (SSIM) are improved on actual CUP data compared with traditional algorithms. On benchmark and real CUP datasets, the proposed method shows the comparable visual results while reducing the running time by 96% over state-of-the-art algorithms.

The effects of an IV fluid bolus on mitral annular velocity and the assessment of diastolic function: a prospective non-randomized study.

BACKGROUND: Abnormal diastolic function is an independent predictor of adverse postoperative outcomes. Mitral annular tissue Doppler velocity (e') is a key parameter for assessing diastolic function. The purpose of this study was to confirm that an acute increase in preload did not significantly impact the intraoperative measurement of e' and secondarily evaluate the impact of this acute intravascular volume increase on the clinical assessment of diastolic function using a previously described simplified algorithm. METHODS: This was a prospective, non-randomized study in adult patients undergoing elective cardiac surgeries requiring transesophageal echocardiographic monitoring, arterial pressure and Swan-Ganz catheter placements as part of the surgical procedure. Following baseline echocardiographic and hemodynamic measurements, 500 ml of crystalloid solution was infused over 10 min. Hemodynamic and echocardiographic measurements were repeated 5 min after fluid administration. RESULTS: Complete data sets were available from 84 of the 100 patients who were enrolled in this study. There was no significant change in the values of e'. The average baseline was 7.8 ± 2.0 cm/s (95%CI: 7.4, 8.2) and 8.1 ± 2.4 (95%CI: 7.6, 8.6) following the fluid bolus (p = 0.10). All hemodynamic variables associated with increased intravascular volume (central venous pressure, pulmonary arterial pressures and stroke volume variation) changed significantly. The overall distribution of diastolic function grades did not change following fluid administration (p = 0.69). However, there were many individual patient differences. When using this simplified algorithm, functional grading changed in 35 patients. Thirty of these 35 changes were only a single grade shift. 22 patients had worse functional grading after fluid administration while 13 had improved grading. Nine patients with normal diastolic function at baseline demonstrated diastolic dysfunction after fluid administration while 6 patients with baseline dysfunction normalized following the fluid bolus. CONCLUSION: We confirmed that e' is a robust measurement that is reproducible in the intraoperative setting despite variable vascular volume loading conditions, however, the clinical assessment of diastolic function was still altered in 42% of the patients following an intravenous fluid bolus.

Living with multimorbidity: A qualitative exploration of shared experiences of patients, family caregivers, and healthcare professionals in managing symptoms in the United States.

AIMS: To elicit experiences of patients, family caregivers, and healthcare professionals in intermediate care units (IMCUs) in an academic medical centre in Baltimore, MD related to the challenges and intricacies of multimorbidity management to inform development of a multimorbidity symptom management toolkit. DESIGN: Experience-based co-design. METHODS: Between July and October 2021, patients aged 55 years and older with multimorbidity admitted to IMCUs at an academic medical centre in Baltimore, Maryland, USA were recruited and interviewed in person. Interdisciplinary healthcare professionals working in the IMCU were interviewed virtually. Participants were asked questions about their role in recognizing and treating symptoms, factors affecting the quality of life, symptom burden and trajectory over time, and strategies that have and have not worked for managing symptoms. An inductive thematic analysis approach was used for analysis. RESULTS: Twenty-three interviews were conducted: 9 patients, 2 family caregivers, and 12 healthcare professionals. Patients' mean age was 67.5 (±6.5) years, over half (n = 5) were Black or Hispanic, and the average number of comorbidities was 3.67. Five major themes that affect symptom management emerged: (1) the patient-provider relationship; (2) open and honest communication; (3) accessibility of resources during hospitalization and at discharge; (4) caregiver support, training, and education; and (5) care coordination and follow-up care. CONCLUSION: Patients, caregivers, and healthcare professionals often have similar goals but different priorities for multimorbidity management. It is imperative to identify shared priorities and target holistic interventions that consider patient and caregiver experiences to improve outcomes. IMPLICATIONS FOR THE PROFESSION AND/OR PATIENT CARE AND IMPACT: This paper addresses the paucity of research related to the shared experience of disease trajectory and symptom management for people living with multimorbidity. We found that patients, caregivers, and healthcare professionals often have similar goals but different care and communication priorities. Understanding differing priorities will help better design interventions to support symptom management so people with multimorbidity can have the best possible quality of life. REPORTING METHOD: We have adhered to the Consolidated Criteria for Reporting Qualitative Studies (COREQ) guidelines in our reporting. PATIENT OR PUBLIC CONTRIBUTION: This study has been designed and implemented with patient and public involvement throughout the process, including community advisory board engagement in the project proposal phase and interview guide development, and member checking in the data collection and analysis phases. The method we chose, experience-based co-design, emphasizes the importance of engaging members of a community to act as experts in their own life challenges. In the coming phases of the study, the public will be involved in developing and testing a new intervention, informed by these qualitative interviews and co-design events, to support symptom management for people with multimorbidity.

Patterns of cognitive domain abnormalities enhance discrimination of dementia risk prediction: The ARIC study.

INTRODUCTION: The contribution of neuropsychological assessments to risk assessment for incident dementia is underappreciated. METHODS: We analyzed neuropsychological testing results in dementia-free participants in the Atherosclerosis Risk in Communities (ARIC) study. We examined associations of index domain-specific neuropsychological test performance with incident dementia using cumulative incidence curves and Cox proportional hazards models. RESULTS: Among 5296 initially dementia-free participants (mean [standard deviation] age of 75.8 [5.1] years; 60.1% women, 22.2% Black) over a median follow-up of 7.9 years, the covariate-adjusted hazard ratio varied substantially depending on the pattern of domain-specific performance and age, in an orderly manner from single domain language abnormalities (lowest risk) to single domain executive or memory abnormalities, to multidomain abnormalities including memory (highest risk). DISCUSSION: By identifying normatively defined cognitive abnormalities by domains based on neuropsychological test performance, there is a conceptually orderly and age-sensitive spectrum of risk for incident dementia that provides valuable information about the likelihood of progression. HIGHLIGHTS: Domain-specific cognitive profiles carry enhanced prognostic value compared to mild cognitive impairment. Single-domain non-amnestic cognitive abnormalities have the most favorable prognosis. Multidomain amnestic abnormalities have the greatest risk for incident dementia. Patterns of domain-specific risks are similar by sex and race.

Nephrotoxicity of Vancomycin in Combination With Beta-Lactam Agents: Ceftolozane-Tazobactam vs Piperacillin-Tazobactam.

BACKGROUND: Vancomycin (VAN)-associated acute kidney injury (AKI) is increased when VAN is combined with certain beta-lactams (BLs) such as piperacillin-tazobactam (TZP) but has not been evaluated with ceftolozane-tazobactam (C/T). Our aim was to investigate the AKI incidence of VAN in combination with C/T (VAN/C/T) compared with VAN in combination to TZP (VAN-TZP). METHODS: We conducted a multicenter, observational, comparative study across the United States. The primary analysis was a composite outcome of AKI and risk, injury, failure, loss, end stage renal disease; Acute Kidney Injury Network; or VAN-induced nephrotoxicity according to the consensus guidelines. Multivariable logistic regression analysis was conducted to adjust for confounding variables and stratified Kaplan-Meir analysis to assess the time to nephrotoxicity between the 2 groups. RESULTS: We included VAN/C/T (n = 90) and VAN-TZP (n = 284) at an enrollment ratio of 3:1. The primary outcome occurred in 12.2% vs 25.0% in the VAN-C/T and VAN-TZP groups, respectively (P = .011). After adjusting for confounding variables, VAN-TZP was associated with increased odds of AKI compared with VAN-C/T; with an adjusted odds ratio of 3.308 (95% confidence interval, 1.560-6.993). Results of the stratified Kaplan-Meir analysis with log-rank time-to-nephrotoxicity analysis indicate that time to AKI was significantly shorter among patients who received VAN-TZP (P = .004). Cox proportional hazards analysis demonstrated that TZP was consistent with the primary analysis (P = .001). CONCLUSIONS: Collectively, our results suggest that the AKI is not likely to be related to tazobactam but rather to piperacillin, which is a component in VAN-TZP but not in VAN-C/T.

A fetal wound healing program after intrauterine bile duct injury may contribute to biliary atresia.

BACKGROUND & AIMS: Biliary atresia (BA) is an obstructive cholangiopathy that initially affects the extrahepatic bile ducts (EHBDs) of neonates. The etiology is uncertain, but evidence points to a prenatal cause. Fetal tissues have increased levels of hyaluronic acid (HA), which plays an integral role in fetal wound healing. The objective of this study was to determine whether a program of fetal wound healing is part of the response to fetal EHBD injury. METHODS: Mouse, rat, sheep, and human EHBD samples were studied at different developmental time points. Models included a fetal sheep model of prenatal hypoxia, human BA EHBD remnants and liver samples taken at the time of the Kasai procedure, EHBDs isolated from neonatal rats and mice, and spheroids and other models generated from primary neonatal mouse cholangiocytes. RESULTS: A wide layer of high molecular weight HA encircling the lumen was characteristic of the normal perinatal but not adult EHBD. This layer, which was surrounded by collagen, expanded in injured ducts in parallel with extensive peribiliary gland hyperplasia, increased mucus production and elevated serum bilirubin levels. BA EHBD remnants similarly showed increased HA centered around ductular structures compared with age-appropriate controls. High molecular weight HA typical of the fetal/neonatal ducts caused increased cholangiocyte spheroid growth, whereas low molecular weight HA induced abnormal epithelial morphology; low molecular weight HA caused matrix swelling in a bile duct-on-a-chip device. CONCLUSION: The fetal/neonatal EHBD, including in human EHBD remnants from Kasai surgeries, demonstrated an injury response with prolonged high levels of HA typical of fetal wound healing. The expanded peri-luminal HA layer may swell and lead to elevated bilirubin levels and obstruction of the EHBD. IMPACT AND IMPLICATIONS: Biliary atresia is a pediatric cholangiopathy associated with high morbidity and mortality rates; although multiple etiologies have been proposed, the fetal response to bile duct damage is largely unknown. This study explores the fetal pathogenesis after extrahepatic bile duct damage, thereby opening a completely new avenue to study therapeutic targets in the context of biliary atresia.

The BET PROTAC inhibitor dBET6 protects against retinal degeneration and inhibits the cGAS-STING in response to light damage.

BACKGROUND: Chronic inflammation significantly contributes to photoreceptor death in blinding retinal diseases such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP). Bromodomain and extraterminal domain (BET) proteins are epigenetic readers that act as key proinflammatory factors. We recently found the first-generation BET inhibitor JQ1 alleviated sodium iodate-induced retinal degeneration by suppressing cGAS-STING innate immunity. Here, we investigated the effects and mechanism of dBET6, a proteolysis­targeting chimera (PROTAC) small molecule that selectively degrades BET by the ubiquitin‒proteasome system, in light-induced retinal degeneration. METHODS: Mice were exposed to bright light to induce retinal degeneration, and the activation of cGAS-STING was determined by RNA-sequencing and molecular biology. Retinal function, morphology, photoreceptor viability and retinal inflammation were examined in the presence and absence of dBET6 treatment. RESULTS: Intraperitoneal injection of dBET6 led to the rapid degradation of BET protein in the retina without detectable toxicity. dBET6 improved retinal responsiveness and visual acuity after light damage (LD). dBET6 also repressed LD-induced retinal macrophages/microglia activation, Müller cell gliosis, photoreceptor death and retinal degeneration. Analysis of single-cell RNA-sequencing results revealed cGAS-STING components were expressed in retinal microglia. LD led to dramatic activation of the cGAS-STING pathway, whereas dBET6 suppressed LD-induced STING expression in reactive macrophages/microglia and the related inflammatory response. CONCLUSIONS: This study indicates targeted degradation of BET by dBET6 exerts neuroprotective effects by inhibiting cGAS-STING in reactive retinal macrophages/microglia, and is expected to become a new strategy for treatment of retinal degeneration.

Light damage induces inflammatory factors in mouse retina and vitreous humor.

Purpose: Increased inflammatory factor levels have been reported in the vitreous humor (VH) of diabetic retinopathy and neovascular age-related macular degeneration, ocular diseases generally associated with the formation of new retinal blood vessels and leakage. However, the levels of inflammatory mediators are less known in retinal degeneration without neovascularization. Human retinitis pigmentosa (RP) and animal models of light-induced retinal degeneration (LIRD) share several features, such as photoreceptor death and retinal inflammation. Here, we aimed to determine the levels of inflammatory factors in the VH of the LIRD mouse model. Methods: LIRD was induced by exposing BALB/c mice to white light (15,000 lx, 2 h), and the mice were recovered for 2 days before analysis (n = 50 mice). We assessed retinal morphology using optical coherence tomography and hematoxylin and eosin staining; retinal cell viability was determined using terminal deoxynucleotidyl transferase dUTP nick-end labeling, and retinal responses were measured based on electroretinogram signals. Total retinal RNAs were extracted and subjected to RNA sequencing analysis. VH samples from control (n = 4) and LIRD mice (n = 9) were assayed in triplicate for a panel of four inflammatory mediators using the Simple Plex Cartridge on an Ella System. Results: Retinal degeneration, photoreceptor death, infiltration of microglia/macrophages into the photoreceptor layer, and loss of a- and b-waves were obviously detected after LIRD. RNA sequencing revealed that light damage (LD) led to the significant upregulation of inflammatory factors in mouse retinas. In the VH, LD increased the total protein concentration. Dramatic induction of CCL2 (~3000 fold) and IL6 (~10 fold) was detected in VH in response to LD. Increased but not significant levels of TNFα and IL1ß were also detected in light-exposed VH. Conclusions: Given that the LIRD model mimics RP pathogenesis in some aspects, these results suggest a causative link between retinal degeneration and VH inflammation in RP progression, and the increased CCL2 level in VH may reflect similar elevated CCL2 expression in the degenerative retina.

Non-fitting FLIM-FRET facilitates analysis of protein interactions in live zebrafish embryos.

Molecular interactions are key to all cellular processes, and particularly interesting to investigate in the context of gene regulation. Protein-protein interactions are challenging to examine in vivo as they are dynamic, and require spatially and temporally resolved studies to interrogate them. Foerster Resonance Energy Transfer (FRET) is a highly sensitive imaging method, which can interrogate molecular interactions. FRET can be detected by Fluorescence Lifetime Imaging Microscopy (FLIM-FRET), which is more robust to concentration variations and photobleaching than intensity-based FRET but typically needs long acquisition times to achieve high photon counts. New variants of non-fitting lifetime-based FRET perform well in samples with lower signal and require less intensive instrument calibration and analysis, making these methods ideal for probing protein-protein interactions in more complex live 3D samples. Here we show that a non-fitting FLIM-FRET variant, based on the Average Arrival Time of photons per pixel (AAT- FRET), is a sensitive and simple way to detect and measure protein-protein interactions in live early stage zebrafish embryos.