Apolipoprotein A1 Protects Against Necrotic Core Development in Atherosclerotic Plaques: PDZK1-Dependent High-Density Lipoprotein Suppression of Necroptosis in Macrophages.

BACKGROUND: Atherosclerosis is a chronic disease affecting artery wall and a major contributor to cardiovascular diseases. Large necrotic cores increase risk of plaque rupture leading to thrombus formation. Necrotic cores are rich in debris from dead macrophages. Programmed necrosis (necroptosis) contributes to necrotic core formation. HDL (high-density lipoprotein) exerts direct atheroprotective effects on different cells within atherosclerotic plaques. Some of these depend on the SR-B1 (scavenger receptor class B type I) and the adapter protein PDZK1 (postsynaptic density protein/Drosophila disc-large protein/Zonula occludens protein containing 1). However, a role for HDL in protecting against necroptosis and necrotic core formation in atherosclerosis is not completely understood. METHODS: Low-density lipoprotein receptor-deficient mice engineered to express different amounts of ApoA1 (apolipoprotein A1), or to lack PDZK1 were fed a high fat diet for 10 weeks. Atherosclerotic plaque areas, necrotic cores, and key necroptosis mediators, RIPK3 (receptor interacting protein kinase 3), and MLKL (mixed lineage kinase domain-like protein) were characterized. Cultured macrophages were treated with HDL to determine its effects, as well as the roles of SR-B1, PDZK1, and the PI3K (phosphoinositide 3-kinase) signaling pathway on necroptotic cell death. RESULTS: Genetic overexpression reduced, and ApoA1 knockout increased necrotic core formation and RIPK3 and MLKL within atherosclerotic plaques. Macrophages were protected against necroptosis by HDL and this protection required SR-B1, PDZK1, and PI3K/Akt pathway. PDZK1 knockout increased atherosclerosis in LDLRKO mice, increasing necrotic cores and phospho-MLKL; both of which were reversed by restoring PDZK1 in BM-derived cells. CONCLUSIONS: Our findings demonstrate that HDL in vitro and ApoA1, in vivo, protect against necroptosis in macrophages and necrotic core formation in atherosclerosis, suggesting a pathway that could be a target for the treatment of atherosclerosis.

Digital filtering of ghost signal in phase measuring deflectometry.

We introduce a method of geometric screen modification to remove ghost reflections commonly observed in deflectometry optical testing. The proposed method modifies the optical layout and illumination source area to bypass the generation of reflected rays from the undesired surface. The layout flexibility of deflectometry allows us to design specific system layouts that avoid the generation of interrupting secondary rays. The proposed method is supported by optical raytrace simulations, and experimental results are demonstrated with convex and concave lens case studies. Finally, the limitations of the digital masking method are discussed.

Modeling and characterization of OASIS inflatable primary antenna by dual modality metrology.

OASIS (Orbiting Astronomical Satellite for Investigating Stellar Systems) is a space-based observatory with a 14 m diameter inflatable primary antenna that will perform high spectral resolution observations at terahertz frequencies. The large inflatable aperture, non-traditional surface configuration, and the double layered membrane structure afford unique challenges to the modeling and testing of the primary antenna. A 1-meter prototype of the primary antenna (A1) was built to validate our technical approach. A laser radar coordinate measuring system was adopted to measure the shape of A1. In addition, deflectometry was performed to monitor the stability of A1 during the radar measurement. Test cases pertaining to specific operational conditions expected for the 14 m OASIS primary were explored. The measured data were then compared to the Fichter model and Finite-element Analyzer for Inflatable Membranes (FAIM).

Non-planar illumination deflectometry for axicon metrology.

We introduce an on-axis deflectometry test configuration for axicon metrology. Axicons are challenging to measure due to their characteristically steep, convex geometry. However, if an axicon is coaxially aligned with a camera and a surrounding cylindrical illumination source, high-resolution surface measurements can be obtained via the principle of deflectometry. Emitted from the temporally modulated source, light deflects at the conical surface and into the entrance pupil of a camera, illuminating the full axicon aperture except the ø 0.5-mm rounded tip. Deflectometry measurements of a 100° and 140° axicon show holistic cone angle agreement within 0.035° against touch probe data and up to 7.93 root µm mean square difference from a best-fit cone. We discuss the non-planar illumination architecture, sensitivity, and experimental results of arbitrary apex angle axicons.

Benefit and toxicity of programmed death-1 blockade vary by ethnicity in patients with advanced melanoma: an international multicentre observational study.

BACKGROUND: Programmed cell death receptor-1 (PD-1) monotherapy is a standard treatment for advanced cutaneous melanoma, but its efficacy and toxicity are defined in white populations and remain poorly characterized in other ethnic groups, such as East Asian, Hispanic and African. OBJECTIVES: To determine the efficacy and toxicity of PD-1 monotherapy in different ethnic groups. METHODS: Clinical data for patients with unresectable or advanced melanoma treated with anti-PD-1 monotherapy between 2009 and 2019 were collected retrospectively from five independent institutions in the USA, Australia and China. Tumour response, survival and immune-related adverse events (irAEs) were compared by ethnicity (white vs. East Asian/Hispanic/African) across different melanoma subtypes: nonacral cutaneous (NAC)/unknown primary (UP) and acral/mucosal/uveal. RESULTS: In total, 1135 patients were included. White patients had significantly higher objective response rate (ORR) [54%, 95% confidence interval (CI) 50-57% vs. 20%, 95% CI 13-28%; adjusted P < 0·001] and longer progression-free survival (14·2 months, 95% CI 10·7-20·3 vs. 5·4 months, 95% CI 4·5-7·0; adjusted P < 0·001) than East Asian, Hispanic and African patients in the NAC and UP subtypes. White ethnicity remained independently associated with a higher ORR (odds ratio 4·10, 95% CI 2·48-6·81; adjusted P < 0·001) and longer PFS (hazard ratio 0·58, 95% CI 0·46-0·74; adjusted P < 0·001) in multivariate analyses after adjustment for age, sex, primary anatomical location, metastasis stage, baseline lactate dehydrogenase level, mutational status and prior systemic treatment. White and East Asian/Hispanic/African patients shared similar ORR and progression-free survival in acral/mucosal/uveal melanomas. Similar melanoma-subtype-specific ethnic discrepancies were observed in complete response rate and overall survival. White patients had higher rates of gastrointestinal irAEs but lower rates of endocrine, liver and other rare types of irAEs. These differences in irAEs by ethnicity were not attributable to varying melanoma subtypes. CONCLUSIONS: Ethnic discrepancy in clinical benefit is specific to melanoma subtype, and East Asian, Hispanic and African patients with NAC and UP melanomas have poorer clinical benefits than previously recognized. The ethnic discrepancy in toxicity observed across different melanoma subtypes warrants an ethnicity-based irAE surveillance strategy. More research is needed to elucidate the molecular and immunological determinants of these differences. What is already known about this topic? There is a great difference in response to immunotherapy between different subtypes of melanoma (cutaneous, mucosal, acral and uveal) in patients with advanced disease. What does this study add? Our data show for the first time that there are differences between different ethnic groups in terms of both response and toxicity to immunotherapy beyond the well-appreciated discrepancies due to melanoma subtype.

Gene therapy for cystic fibrosis: new tools for precision medicine.

The discovery of the Cystic fibrosis (CF) gene in 1989 has paved the way for incredible progress in treating the disease such that the mean survival age of individuals living with CF is now ~58 years in Canada. Recent developments in gene targeting tools and new cell and animal models have re-ignited the search for a permanent genetic cure for all CF. In this review, we highlight some of the more recent gene therapy approaches as well as new models that will provide insight into personalized therapies for CF.

Computational vector fiducial for deflectometry system alignment.

One of deflectometry's cardinal strengths is its ability to measure highly dynamically sloped optics without needing physical null references. Accurate surface measurements using deflectometry, however, require precise calibration processes. In this Letter, we introduce an alignment technique using a computational fiducial to align a deflectometry system without additional hardware equipment (i.e., algorithmic innovation). Using the ray tracing program, we build relationships between the plane of the screen and detector and algorithmically generate a fiducial pattern for the deflectometry configuration. Since the fiducial pattern is based on ideal system geometry, misalignment of the unit under test with its target position causes a discrepancy between the actual image on the camera detector and the ideal fiducial image. We leverage G and C vector polynomials to quantify misalignment and estimate the alignment status through a reverse optimization method. Simulation and experimental results demonstrate that the proposed algorithm can align the 195mm×80mm of a rectangular aperture freeform optic within 10 µm of peak-to-valley accuracy. The computational fiducial-based alignment algorithm is simple to apply and can be an essential procedure for conventional methods of deflectometry system alignment.

Cutaneous adverse events caused by immune checkpoint inhibitors.

IMPORTANCE: Immune checkpoint inhibitors (ICIs) have emerged as active therapies for a variety of cancers. Cutaneous toxicities are common immune-related adverse events and patients will often be referred to dermatologists for evaluation. OBSERVATIONS: Cutaneous adverse events to ICIs can have a variety of clinical presentations. Among the more common are eczematous, morbilliform, and lichenoid dermatoses, as well as vitiligo and pruritus. Less common adverse events include psoriasiform dermatoses, bullous disorders, and severe cutaneous adverse reactions, including Stevens-Johnson syndrome, toxic epidermal necrolysis, and drug reaction with eosinophilia and systemic symptoms. Because of the immunologic mechanism of ICIs, there are also a variety of rheumatologic adverse reactions with cutaneous manifestations, such as scleroderma, dermatomyositis, cutaneous lupus erythematosus, and various vasculitides. These cutaneous reactions often respond to topical or systemic steroids, although specific toxicities may have alternative treatments available. CONCLUSIONS AND RELEVANCE: As they become more widely prescribed, dermatologists will see an increasing number of patients with cutaneous adverse events caused by ICI therapies. Accurately diagnosing and treating these toxicities is paramount to achieving the most favorable outcomes for patients.

How are economic evaluations carried out for primary caries prevention strategies?

Aims To summarise evidence on economic evaluations (EEs) of primary caries prevention in preschool children aged 2-5 years and to evaluate the reporting quality of full EE studies using a quality assessment tool.Methods Systematic literature search from several databases. The reporting quality was assessed using the Consolidated Health Economic Evaluation Reporting Standards (CHEERS) checklist.Results In total, 808 studies were identified and 39 were included in review. Complex multi-component interventions were the most common followed by water fluoridation. Cost analysis and cost-effectiveness were the most common EEs. Parameters that were not reported well were: characterising uncertainty, study perspective, sensitivity analysis and discount rate.Conclusions The number of EEs has increased but there is inconsistency in how EEs are conducted and reported.

Health care utilization and steroid-refractory toxicities from immune checkpoint inhibitors.

BACKGROUND: Anti-programmed death protein 1 (anti-PD-1) agents have transformed the treatment of advanced melanoma and other cancers, but the rates of steroid-refractory toxicities and health care utilization are not well described. This study assessed these endpoints in patients with melanoma treated with anti-PD-1 with or without ipilimumab. METHODS: This study retrospectively evaluated 344 patients with metastatic melanoma treated with anti-PD-1 or a combination of ipilimumab and nivolumab at Vanderbilt University Medical Center from 2009 to 2018. The incidence, types, grades, management, and outcomes of immune-related adverse events (irAEs) and hospitalizations for irAEs and disease progression were assessed. RESULTS: Patients on combination therapy were more likely to develop irAEs than those on monotherapy (72% vs 37%; P < .001) and were more likely to require systemic steroids (61% vs 20%; P < .001), steroid dose re-escalation (23% vs 6%; P < .001), and second-line immunosuppressive use (17% vs 2%; P < .001) and to suffer high-dose steroid-refractory toxicities (23% vs 3%; P < .001). Combination-treated patients were more likely to have any hospitalization (32% vs 7%; P < .001) or multiple hospitalizations for irAEs (11% vs 3%; P = .001) and had a longer average time of hospitalization (mean, 1.92 vs 0.62 days; P = .002). Among 176 hospitalizations related to disease progression in patients who died during evaluable follow-up, 69% occurred within the 90 days before death. Early hospitalizations for disease-related reasons portended a very poor prognosis (median time from admission to death, 58 days). CONCLUSIONS: Patients treated with a combination of ipilimumab and nivolumab had higher rates of hospitalization and steroid-refractory toxicities than those treated with anti-PD-1 monotherapy. Disease-associated hospitalizations were similar between the 2 groups, portended a poor prognosis, and mostly occurred in the last months of life.

How can children be involved in developing oral health education interventions?

This review examined the oral health interventions that have been developed for elementary school age children and the involvement of children in these intervention studies. Eight randomised controlled trials involving 3,232 children were analysed using deductive content analysis by two authors. Child involvement was categorised using the Typology of Youth Participation and Empowerment TYPE model. In all eight studies, the interventions were designed by the researchers and adult-led without involvement of children. Further studies with participatory research methodology are recommended to better understand the role of involvement of children in oral health education.Data sources The data search was carried out in April 2018 using PubMed, CINAHL, Embase and Scopus databases. The search focused on elementary age school children (6-12 years) who were involved in oral health education intervention studies. Exclusion criteria were studies involving children with mental or physical impairment, undergoing hospital or orthodontic treatment, preventative treatments or solely targeted towards parents/caregivers or teachers.Study selection A systematic review method of randomised controlled trials (RCTs) was selected. Titles and abstracts were included or excluded before full text analysis for eligibility was carried out. Studies were assessed by two authors with a third author to be consulted in cases of disagreement.Data extraction and synthesis Study sample, intervention duration and content, selection, use of educational methods including use of a theoretical framework and outcomes were extracted from the data. Child involvement was categorised using the Typology of Youth Participation and Empowerment (TYPE) pyramid (Wong et al., 2010).1 The quality of studies was assessed using the CONSORT checklist and the Cochrane risk of bias tool. The methodological quality of the studies was relatively low with a score of 14-22 out of 36. Three studies were rated as being fair quality in terms of risk of bias. The remaining five were rated as either unclear or high risk of bias.Results Eight RCTs were selected for review. Sixty-five articles were assessed for eligibility and 57 articles were excluded due to non-RCT design, excluded/unreported ages and full text unavailability. Nine different methods of oral health education were identified: lecture; printed material; demonstration; toothbrushing diary; game; video; workshop; discussion; and oral hygiene training. None of the reported studies demonstrated a rationale for selecting their educational method. Four reports described a theoretical framework for development their intervention: social learning theory (Parcel and Baranowski, 1981) was used by Haleem et al. (2012); Health Belief Model (Becker, 1974) by Yekaninejad et al. (2012), Wolf's health learning capacity (2009) by Freeman et al. (2016) and Ajzen's theory of planned behaviour (1991) by Simpriano and Mialhe (2017). However, the use of these theories was limited and no attempts to introduce children's perspectives into the theoretical framework was identified.Five outcomes to measure effectiveness were found: clinical oral health status; oral health-related behaviour; oral health knowledge; attitudes towards oral health; and oral health related quality of life. Clinical oral health status was the most commonly used outcome (seven of the eight studies). Positive outcomes were found in all eight studies. None of the reports considered the potential for enhancing intervention effects by involving children more actively. The children's role in the interventions was mostly the Vessel type of participation from the TYPE pyramid model. Partially symbolic participation was detected in two studies - Haleem et al. (2012) trained children to conduct oral health education in the role of peer educators and Simpriano and Mialhe (2017) trained children to create their own plans for daily toothbrushing and how to overcome situations preventing that task. There were no reports of consultation or collaboration with children for their perspectives before or after the interventions. Interventions appear to have been created by the researchers alone.Conclusions In all of the included studies, children were only involved during the intervention implementation phase. The interventions were all adult designed and adult led. Lack of detail in the reports meant that reported positive outcomes could not be clearly attributed to the activities carried out by the children. Further studies with participatory research methodology are recommended to better understand the potential role of children in oral health education and research.

Severe Epididymo-Orchitis and Encephalitis Complicating Anti-PD-1 Therapy.

BACKGROUND: Immune checkpoint inhibitors such as pembrolizumab and nivolumab have emerged as active treatment options for patients with many cancers, including metastatic melanoma, but can also cause symptomatic or life-threatening immune-related adverse events, including encephalitis. Epididymitis and orchitis are rare complications of these therapies. CASE PRESENTATION: We describe herein a patient with metastatic melanoma who developed epididymo-orchitis followed by encephalitis while receiving pembrolizumab. The patient developed testicular pain and fever after his third dose of pembrolizumab; ultrasound evaluation demonstrated bilateral epididymo-orchitis. He then developed headaches, fever, and altered mental status over the next week and was admitted to the hospital. Lumbar puncture revealed inflammatory changes consistent with meningoencephalitis; he did not improve with broad-spectrum antibiotics, and an extensive workup for infectious etiologies, including cerebrospinal fluid testing using a clinical metagenomic next-generation sequencing assay, was negative. He received high-dose steroids for suspected autoimmune encephalitis, and both his orchitis and meningoencephalitis improved rapidly after one dose. He fully recovered after a 5-week taper of oral steroids. DISCUSSION: Here, we report a case of epididymo-orchitis complicating immune checkpoint inhibitor therapy. This patient subsequently developed severe encephalitis but rapidly improved with steroids. Clinicians should be aware of rare complications of these agents. KEY POINTS: Epididymo-orchitis is a rare and potentially life-threatening complication of anti-programmed death protein 1 (anti-PD-1) therapy.For patients on anti-PD-1 therapy who develop either epididymo-orchitis or epididymitis without clear infectious cause, immune-related adverse events should be considered in the differential diagnosis.If severe, epididymo-orchitis related to anti-PD-1 therapy may be treated with high-dose corticosteroids.

High contrast thermal deflectometry using long-wave infrared time modulated integrating cavity source.

We introduce a scalable temporally modulated long-wave infrared source design. The design makes use of an array of resistive blackbody heating elements which radiate into a custom aluminum integrating cavity. The output of the box is a rectangular slit, built to match the traditional tungsten ribbon profile for an infrared deflectometry source. Temporal modulation allows for signal isolation and improved resilience to background fluctuations in an infrared deflectometry source. Infrared deflectometry measurements using the new source design and a traditional tungsten ribbon, both with similar radiant flux, were compared for a ground glass surface, an aluminum blank, and an aluminum blank under thermal load (150 °C). Signal-to-noise ratio was ∼4 times higher for the new design and demonstrated improved source temporal stability and geometry. Further, the new design successfully measured the previously untestable hot aluminum flat. The new design improves infrared deflectometry and allows for high contrast thermal deflectometry measurements of optics under thermal load.

Incidence of acute kidney injury among pediatric hematology/oncology patients receiving vancomycin in combination with piperacillin/tazobactam or cefepime.

There is mounting evidence that combination of antibiotic therapy with vancomycin and piperacillin/tazobactam (pip/tazo) is associated with acute kidney injury (AKI). To determine whether vancomycin plus pip/tazo is associated with higher rates of AKI compared to vancomycin plus cefepime among pediatric hematology/oncology (heme/onc) patients, we examined 121 heme/onc patients receiving at least two consecutive days of therapy with vancomycin and either pip/tazo or cefepime. Rate of AKI was higher in the pip/tazo than the cefepime group (4/27 [14.8%] vs 2/94 [2.1%], P = 0.022).

Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabet.

The natural four-letter genetic alphabet, comprised of just two base pairs (dA-dT and dG-dC), is conserved throughout all life, and its expansion by the development of a third, unnatural base pair has emerged as a central goal of chemical and synthetic biology. We recently developed a class of candidate unnatural base pairs, exemplified by the pair formed between d5SICS and dNaM. Here, we examine the PCR amplification of DNA containing one or more d5SICS-dNaM pairs in a wide variety of sequence contexts. Under standard conditions, we show that this DNA may be amplified with high efficiency and greater than 99.9% fidelity. To more rigorously explore potential sequence effects, we used deep sequencing to characterize a library of templates containing the unnatural base pair as a function of amplification. We found that the unnatural base pair is efficiently replicated with high fidelity in virtually all sequence contexts. The results show that, for PCR and PCR-based applications, d5SICS-dNaM is functionally equivalent to a natural base pair, and when combined with dA-dT and dG-dC, it provides a fully functional six-letter genetic alphabet.

The motivation and actions of Australians concerning brain health and dementia risk reduction.

ISSUE ADDRESSED: Alzheimer's disease and dementia are recognised as critical public health priorities. This study investigated intentions and behaviours concerning brain health and dementia risk reduction among Australians. METHODS: A cross-sectional survey of 1000 persons aged 20-75 years measured knowledge, beliefs, intentions and behaviours concerning brain health and dementia. The demographic, experiential and cognitive factors associated with intentions and actions were examined. RESULTS: Around half of respondents were motivated to improve brain health. Behaviours most often reported were mental activity (19%), physical activity (9.6%) and dietary action (6.5%). Actions were most likely among women (OR 1.59, 95% CI 1.19-2.14), those aged 60 years and over (OR 3.07, 95% CI 2.01-2.58), with university education (OR 1.67, 95% CI 1.08-2.58) or with prior contact with a person with dementia (OR 1.99, 95% CI 1.12-3.56). Both intentions and actions were associated with moderate to high knowledge, and beliefs and confidence that favoured dementia risk reduction. CONCLUSIONS: A lower proportion of Australians reported taking action to improve brain health than who expressed intentions in this regard. Strategies are needed to improve knowledge about the range of behaviours that contribute to dementia risk reduction and to increase confidence that this outcome is personally achievable. SO WHAT? The burden of disease due to Alzheimer's disease and dementia is growing dramatically. It is essential to promote awareness that dementia is not an inevitable result of ageing and to increase understanding that action can be taken throughout the life course to promote brain health.

Public knowledge and beliefs about dementia risk reduction: a national survey of Australians.

BACKGROUND: With the dramatically increasing contribution of Alzheimer's Disease and other forms of dementia to the global burden of disease, countries are being urged to address this as a public health priority. This study investigated whether Australian adults recognise this as an important health issue, and hold beliefs and knowledge that are consistent with recommendations concerning dementia risk reduction. This research was undertaken to guide national brain health awareness and education strategies. METHODS: A cross-sectional telephone survey was undertaken of 1,003 Australians aged 20-75 years. This measured the importance placed on dementia, beliefs and confidence related to risk reduction, knowledge of risk reduction methods, and the perceived age-relevance of these. In analysis the data were stratified by sex, age, educational attainment, household income, language preference and previous exposure to dementia. Multivariable logistic regression was undertaken to identify variables independently associated with beliefs and knowledge. RESULTS: People aged 60 years and over identified dementia as very important (17.2%) more often than those aged 40-59 years (5.1%) or 20-39 years (2.1%). While 41.5% of respondents believed the risk of dementia could be reduced, 26.9% were very confident that they could achieve this. Mental activity (57.1%) was identified as beneficial much more often than physical activity (31.3%), healthy eating (23.3%) and other cardiovascular health behaviours. Women, people of English-speaking origin, and those having contact with a person with dementia, showed better knowledge of several health behaviours. CONCLUSIONS: Growing attention is being given to population risk reduction to combat the dramatic increase in the burden of disease due to dementia. In Australia many people do not yet hold beliefs and knowledge that support this, which highlights the need for concerted awareness raising that dementia is not an inevitable aspect of ageing, and for education about the role of vascular health in dementia risk reduction.

Pediatric Dermatology for People of Color.

When evaluating pediatric patients of color, it is essential to consider the unique diagnostic and treatment factors that apply to this population. Certain dermatologic conditions are more common in these patients, including postinflammatory hyperpigmentation, pityriasis alba, progressive macular hypomelanosis, tinea capitis, traction alopecia, keloids, hypertrophic scars, pseudofolliculitis barbae, acne keloidalis nuchae, and hidradenitis suppurativa. Furthermore, conditions such as vitiligo are more noticeable in people of color. This can lead to a significantly diminished quality of life, so these conditions should be quickly recognized and treated. Notably, inflammation can be difficult to recognize on the skin of people of color, which can lead to the underestimation of severity as well as inappropriate treatment. Treatment recommendations can also differ based on lifestyle or cultural norms, such as the use of tinted sunscreens and the consideration of hair care practices. Pediatricians should be aware of these conditions and treatment considerations to best treat pediatric patients of color. [Pediatr Ann. 2024;53(4):e146-e151.].

Early human fetal lung atlas reveals the temporal dynamics of epithelial cell plasticity.

Studying human fetal lungs can inform how developmental defects and disease states alter the function of the lungs. Here, we sequenced >150,000 single cells from 19 healthy human pseudoglandular fetal lung tissues ranging between gestational weeks 10-19. We capture dynamic developmental trajectories from progenitor cells that express abundant levels of the cystic fibrosis conductance transmembrane regulator (CFTR). These cells give rise to multiple specialized epithelial cell types. Combined with spatial transcriptomics, we show temporal regulation of key signalling pathways that may drive the temporal and spatial emergence of specialized epithelial cells including ciliated and pulmonary neuroendocrine cells. Finally, we show that human pluripotent stem cell-derived fetal lung models contain CFTR-expressing progenitor cells that capture similar lineage developmental trajectories as identified in the native tissue. Overall, this study provides a comprehensive single-cell atlas of the developing human lung, outlining the temporal and spatial complexities of cell lineage development and benchmarks fetal lung cultures from human pluripotent stem cell differentiations to similar developmental window.

Expanding the scope of replicable unnatural DNA: stepwise optimization of a predominantly hydrophobic base pair.

As part of an ongoing effort to expand the genetic alphabet for in vitro and eventually in vivo applications, we have synthesized a wide variety of predominantly hydrophobic unnatural base pairs exemplified by d5SICS-dMMO2 and d5SICS-dNaM. When incorporated into DNA, the latter is replicated and transcribed with greater efficiency and fidelity than the former; however, previous optimization efforts identified the para and methoxy-distal meta positions of dMMO2 as particularly promising for further optimization. Here, we report the stepwise optimization of dMMO2 via the synthesis and evaluation of 18 novel para-derivatized analogs of dMMO2, followed by further derivatization and evaluation of the most promising analogs with meta substituents. Subject to size constraints, we find that para substituents can optimize replication via both steric and electronic effects and that meta methoxy groups are unfavorable, while fluoro substituents can be beneficial or deleterious depending on the para substituent. In addition, we find that improvements in the efficiency of unnatural triphosphate insertion translate most directly into higher fidelity replication. Importantly, we identify multiple, unique base pair derivatives that when incorporated into DNA are well replicated. The most promising, d5SICS-dFEMO, is replicated under some conditions with greater efficiency and fidelity than d5SICS-dNaM. These results clearly demonstrate the generality of hydrophobic forces for the control of base pairing within DNA, provide a wealth of new SAR data, and importantly identify multiple new candidates for eventual in vivo evaluation.