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Item Cell behaviors underlying Myxococcus xanthus aggregate dispersal(American Society for Microbiology, 2023) Murphy, Patrick; Comstock, Jessica; Khan, Trosporsha; Zhang, Jiangguo; Welch, Roy; Igoshin, Oleg A.; Center for Theoretical Physical BiologyThe soil bacterium Myxococcus xanthus is a model organism with a set of diverse behaviors. These behaviors include the starvation-induced multicellular development program, in which cells move collectively to assemble multicellular aggregates. After initial aggregates have formed, some will disperse, with smaller aggregates having a higher chance of dispersal. Initial aggregation is driven by two changes in cell behavior: cells slow down inside of aggregates and bias their motion by reversing direction less frequently when moving toward aggregates. However, the cell behaviors that drive dispersal are unknown. Here, we use fluorescent microscopy to quantify changes in cell behavior after initial aggregates have formed. We observe that after initial aggregate formation, cells adjust the bias in reversal timings by initiating reversals more rapidly when approaching unstable aggregates. Using agent-based modeling, we then show dispersal is predominantly generated by this change in bias, which is strong enough to overcome slowdown inside aggregates. Notably, the change in reversal bias is correlated with the nearest aggregate size, connecting cellular activity to previously observed correlations between aggregate size and fate. To determine if this connection is consistent across strains, we analyze a second M. xanthus strain with reduced levels of dispersal. We find that far fewer cells near smaller aggregates modified their bias. This implies that aggregate dispersal is under genetic control, providing a foundation for further investigations into the role it plays in the life cycle of M. xanthus.Item Nanobomb optical coherence elastography in multilayered phantoms(Optica Publishing Group, 2023) Hatami, Maryam; Nevozhay, Dmitry; Singh, Manmohan; Schill, Alexander; Boerner, Paul; Aglyamov, Salavat; Sokolov, Konstantin; Larin, Kirill V.Many tissues are composed of layered structures, and a better understanding of the changes in the layered tissue biomechanics can enable advanced guidance and monitoring of therapy. The advent of elastography using longitudinally propagating shear waves (LSWs) has created the prospect of a high-resolution assessment of depth-dependent tissue elasticity. Laser activation of liquid-to-gas phase transition of dye-loaded perfluorocarbon (PFC) nanodroplets (a.k.a., nanobombs) can produce highly localized LSWs. This study aims to leverage the potential of photoactivation of nanobombs to incudce LSWs with very high-frequency content in wave-based optical coherence elastography (OCE) to estimate the elasticity gradient with high resolution. In this work, we used multilayered tissue-mimicking phantoms to demonstrate that highly localized nanobomb (NB)-induced LSWs can discriminate depth-wise tissue elasticity gradients. The results show that the NB-induced LSWs rapidly change speed when transitioning between layers with different mechanical properties, resulting in an elasticity resolution of ∼65 µm. These results show promise for characterizing the elasticity of multilayer tissue with a fine resolution.Item Real-time, deep-learning aided lensless microscope(Optica Publishing Group, 2023) Wu, Jimin; Boominathan, Vivek; Veeraraghavan, Ashok; Robinson, Jacob T.Traditional miniaturized fluorescence microscopes are critical tools for modern biology. Invariably, they struggle to simultaneously image with a high spatial resolution and a large field of view (FOV). Lensless microscopes offer a solution to this limitation. However, real-time visualization of samples is not possible with lensless imaging, as image reconstruction can take minutes to complete. This poses a challenge for usability, as real-time visualization is a crucial feature that assists users in identifying and locating the imaging target. The issue is particularly pronounced in lensless microscopes that operate at close imaging distances. Imaging at close distances requires shift-varying deconvolution to account for the variation of the point spread function (PSF) across the FOV. Here, we present a lensless microscope that achieves real-time image reconstruction by eliminating the use of an iterative reconstruction algorithm. The neural network-based reconstruction method we show here, achieves more than 10000 times increase in reconstruction speed compared to iterative reconstruction. The increased reconstruction speed allows us to visualize the results of our lensless microscope at more than 25 frames per second (fps), while achieving better than 7 µm resolution over a FOV of 10 mm2. This ability to reconstruct and visualize samples in real-time empowers a more user-friendly interaction with lensless microscopes. The users are able to use these microscopes much like they currently do with conventional microscopes.Item Cervical cancer prevention program in Nepal: a ‘training of trainers’ approach(International Society of Global Health, 2023) Batman, Samantha; Piya, Madan; Chapagain, Sandhya; Lama, Poonam; Maharjan, Pabitra; Aryal, Binod; Neupane, Maya; Pariyar, Shashwat; Phoolcharoen, Natacha; Eaton, Vanessa; Sarchet, Vanessa; Kremzier, Megan; Carns, Jenny; Richards-Kortum, Rebecca; Baker, Ellen; Varon, Melissa Lopez; Salcedo, Mila Pontremoli; Milan, Jessica; Schmeler, Kathleen; Pariyar, JitendraBackground: Cervical cancer remains the leading cause of cancer-related death among Nepalese women. To this effect, Cancer Care Nepal established an international collaboration to implement a 'training of trainers' (TOT) program to expand the reach of cervical cancer prevention techniques. Methods: The Nepal cervical cancer prevention program began with an in-person TOT session in Kathmandu in November 2019. Due to the COVID-19 pandemic, two additional TOT courses were held in October and November 2021 with virtual support, didactic lectures from international faculty, and a hands-on component by Nepalese faculty. The Nepalese providers underwent training in these courses and then held further training in five collaborating centers across Nepal. Participants completed pre- and post-course knowledge assessments. The trainings were supplemented by the creation of a new Project ECHO® (Extension for Community Healthcare Outcomes) telementoring hub at Cancer Care Nepal. A capstone refresher course was held in November 2022. Results: 42 participants attended the initial TOT course in 2019. The two follow-up TOT courses held in October/November 2021 were two days long and included providers from five participating regions in Nepal. The courses included virtual didactic sessions followed by hands-on stations led by the Nepalese faculty who had participated in the 2019 TOT course. The stations included: visual inspection with acetic acid (VIA), colposcopy, thermal ablation, and loop electrosurgical excision procedure (LEEP). There were 41 participants in the October/November TOT courses. The trainers who received the TOT education then conducted local courses of similar content in each of the five regions for 152 local providers. Participants had improved mean knowledge scores after the training (0.70, 95% CI=0.67-0.72) in comparison to prior to training (0.50, 95% CI=0.47-0.53), p\<0.001. The program concluded with a capstone course in November 2022 attended by 26 participants. To date, 11 Project ECHO sessions have been held, with an average of 20 participants per session. Conclusions: Nepal's cervical cancer prevention program has increased the number of providers trained in cervical cancer prevention techniques. By increasing provider capacity, individuals will have increased access to cervical cancer screening and treatment of pre-invasive disease, hopefully decreasing the burden of cervical cancer in Nepal.Item Optical imaging technologies for in vivo cancer detection in low-resource settings(Elsevier, 2023) Hou, Huayu; Mitbander, Ruchika; Tang, Yubo; Azimuddin, Ahad; Carns, Jennifer; Schwarz, Richard A.; Richards-Kortum, Rebecca R.Cancer continues to affect underserved populations disproportionately. Novel optical imaging technologies, which can provide rapid, non-invasive, and accurate cancer detection at the point of care, have great potential to improve global cancer care. This article reviews the recent technical innovations and clinical translation of low-cost optical imaging technologies, highlighting the advances in both hardware and software, especially the integration of artificial intelligence, to improve in vivo cancer detection in low-resource settings. Additionally, this article provides an overview of existing challenges and future perspectives of adapting optical imaging technologies into clinical practice, which can potentially contribute to novel insights and programs that effectively improve cancer detection in low-resource settings.Item Cadaveric aortic aneurysm creation: A life-like model for training endovascular repair(Elsevier, 2023) Osztrogonacz, Peter; Benfor, Bright; Haddad, Paul; Barnes, Rebecca; Chinnadurai, Ponraj; Dang, Vy; Hess, John Paul; Corr, Stuart J.; Rahimi, MahamThe recent decline in RAAA incidence and the fast paced scenario with associated challenges regarding training calls for initiative for a better training environment to maximize learning. This led us to the creation of a pulsatile human cadaveric RAAA model. Fresh frozen cadaver was used to create RAAA with BioTissue in hybrid suite with ability to perform CBCTA for sizing. As a proof of concept, the model was used to perform REVAR with proximal CODA balloon control. The model proved to be feasible and we believe it is a better environment to train and gain adequate proficiency in RAAA management.Item Exosomes modified with anti-MEK1 siRNA lead to an effective silencing of triple negative breast cancer cells(Elsevier, 2023) Ferreira, Débora; Santos-Pereira, Cátia; Costa, Marta; Afonso, Julieta; Yang, Sujuan; Hensel, Janine; McAndrews, Kathleen M.; Longatto-Filho, Adhemar; Fernandes, Rui; Melo, Joana B.; Baltazar, Fátima; Moreira, João N.; Kalluri, Raghu; Rodrigues, Ligia R.Triple negative breast cancer (TNBC) is a highly heterogenous disease not sensitive to endocrine or HER2 therapy and standardized treatment regimens are still missing. Therefore, development of novel TNBC treatment approaches is of utmost relevance. Herein, the potential of MAPK/ERK downregulation by RNAi-based therapeutics in a panel of mesenchymal stem-like TNBC cell lines was uncovered. Our data revealed that suppression of one of the central nodes of this signaling pathway, MEK1, affects proliferation, migration, and invasion of TNBC cells, that may be explained by the reversion of the epithelial-mesenchymal transition phenotype, which is facilitated by the MMP-2/MMP-9 downregulation. Moreover, an exosome-based system was successfully generated for the siRNA loading (iExoMEK1). Our data suggested absence of modification of the physical properties and general integrity of the iExoMEK1 comparatively to the unmodified counterparts. Such exosome-mediated downregulation of MEK1 led to a tumor regression accompanied by a decrease of angiogenesis using the chick chorioallantoic-membrane model. Our results highlight the potential of the targeting of MAPK/ERK cascade as a promising therapeutic approach against TNBC.Item Cooperative assembly confers regulatory specificity and long-term genetic circuit stability(Elsevier, 2023) Bragdon, Meghan D. J.; Patel, Nikit; Chuang, James; Levien, Ethan; Bashor, Caleb J.; Khalil, Ahmad S.A ubiquitous feature of eukaryotic transcriptional regulation is cooperative self-assembly between transcription factors (TFs) and DNA cis-regulatory motifs. It is thought that this strategy enables specific regulatory connections to be formed in gene networks between otherwise weakly interacting, low-specificity molecular components. Here, using synthetic gene circuits constructed in yeast, we find that high regulatory specificity can emerge from cooperative, multivalent interactions among artificial zinc-finger-based TFs. We show that circuits “wired” using the strategy of cooperative TF assembly are effectively insulated from aberrant misregulation of the host cell genome. As we demonstrate in experiments and mathematical models, this mechanism is sufficient to rescue circuit-driven fitness defects, resulting in genetic and functional stability of circuits in long-term continuous culture. Our naturally inspired approach offers a simple, generalizable means for building high-fidelity, evolutionarily robust gene circuits that can be scaled to a wide range of host organisms and applications.Item Assessment of spinal cord injury using ultrasound elastography in a rabbit model in vivo(Springer Nature, 2023) Tang, Songyuan; Weiner, Bradley; Taraballi, Francesca; Haase, Candice; Stetco, Eliana; Mehta, Shail Maharshi; Shajudeen, Peer; Hogan, Matthew; De Rosa, Enrica; Horner, Philip J.; Grande-Allen, K. Jane; Shi, Zhaoyue; Karmonik, Christof; Tasciotti, Ennio; Righetti, RaffaellaThe effect of the mechanical micro-environment on spinal cord injury (SCI) and treatment effectiveness remains unclear. Currently, there are limited imaging methods that can directly assess the localized mechanical behavior of spinal cords in vivo. In this study, we apply new ultrasound elastography (USE) techniques to assess SCI in vivo at the site of the injury and at the time of one week post injury, in a rabbit animal model. Eleven rabbits underwent laminectomy procedures. Among them, spinal cords of five rabbits were injured during the procedure. The other six rabbits were used as control. Two neurological statuses were achieved: non-paralysis and paralysis. Ultrasound data were collected one week post-surgery and processed to compute strain ratios. Histologic analysis, mechanical testing, magnetic resonance imaging (MRI), computerized tomography and MRI diffusion tensor imaging (DTI) were performed to validate USE results. Strain ratios computed via USE were found to be significantly different in paralyzed versus non-paralyzed rabbits. The myelomalacia histologic score and spinal cord Young’s modulus evaluated in selected animals were in good qualitative agreement with USE assessment. It is feasible to use USE to assess changes in the spinal cord of the presented animal model. In the future, with more experimental data available, USE may provide new quantitative tools for improving SCI diagnosis and prognosis.Item A novel tailed primer nucleic acid test for detection of HPV 16, 18 and 45 DNA at the point of care(Springer Nature, 2023) Chang, Megan M.; Ma, Ariel; Novak, Emilie Newsham; Barra, Maria; Kundrod, Kathryn A.; Montealegre, Jane Richards; Scheurer, Michael E.; Castle, Philip E.; Schmeler, Kathleen; Richards-Kortum, RebeccaCervical cancer is a leading cause of death for women in low-resource settings despite being preventable through human papillomavirus (HPV) vaccination, early detection, and treatment of precancerous lesions. The World Health Organization recommends high-risk HPV (hrHPV) as the preferred cervical cancer screening strategy, which is difficult to implement in low-resource settings due to high costs, reliance on centralized laboratory infrastructure, and long sample-to-answer times. To help meet the need for rapid, low-cost, and decentralized cervical cancer screening, we developed tailed primer isothermal amplification and lateral flow detection assays for HPV16, HPV18, and HPV45 DNA. We translated these assays into a self-contained cartridge to achieve multiplexed detection of three hrHPV genotypes in a disposable cartridge. The developed test achieves clinically relevant limits of detection of 50–500 copies per reaction with extracted genomic DNA from HPV-positive cells. Finally, we performed sample-to-answer testing with direct lysates of HPV-negative and HPV-positive cell lines and demonstrated consistent detection of HPV16, HPV18, and HPV45 with 5000–50,000 cells/mL in < 35 min. With additional optimization to improve cartridge reliability, incorporation of additional hrHPV types, and validation with clinical samples, the assay could serve as a point-of-care HPV DNA test that improves access to cervical cancer screening in low-resource settings.Item A portable regulatory RNA array design enables tunable and complex regulation across diverse bacteria(Springer Nature, 2023) Liu, Baiyang; Samaniego, Christian Cuba; Bennett, Matthew R.; Franco, Elisa; Chappell, JamesA lack of composable and tunable gene regulators has hindered efforts to engineer non-model bacteria and consortia. Toward addressing this, we explore the broad-host potential of small transcription activating RNA (STAR) and propose a design strategy to achieve tunable gene control. First, we demonstrate that STARs optimized for E. coli function across different Gram-negative species and can actuate using phage RNA polymerase, suggesting that RNA systems acting at the level of transcription are portable. Second, we explore an RNA design strategy that uses arrays of tandem and transcriptionally fused RNA regulators to precisely alter regulator concentration from 1 to 8 copies. This provides a simple means to predictably tune output gain across species and does not require access to large regulatory part libraries. Finally, we show RNA arrays can be used to achieve tunable cascading and multiplexing circuits across species, analogous to the motifs used in artificial neural networks.Item Development of photoreactive demineralized bone matrix 3D printing colloidal inks for bone tissue engineering(Oxford University Press, 2023) Hogan, Katie J; Öztatlı, Hayriye; Perez, Marissa R; Si, Sophia; Umurhan, Reyhan; Jui, Elysa; Wang, Ziwen; Jiang, Emily Y; Han, Sa R; Diba, Mani; Jane Grande-Allen, K; Garipcan, Bora; Mikos, Antonios GDemineralized bone matrix (DBM) has been widely used clinically for dental, craniofacial and skeletal bone repair, as an osteoinductive and osteoconductive material. 3D printing (3DP) enables the creation of bone tissue engineering scaffolds with complex geometries and porosity. Photoreactive methacryloylated gelatin nanoparticles (GNP-MAs) 3DP inks have been developed, which display gel-like behavior for high print fidelity and are capable of post-printing photocrosslinking for control of scaffold swelling and degradation. Here, novel DBM nanoparticles (DBM-NPs, ∼400 nm) were fabricated and characterized prior to incorporation in 3DP inks. The objectives of this study were to determine how these DBM-NPs would influence the printability of composite colloidal 3DP inks, assess the impact of ultraviolet (UV) crosslinking on 3DP scaffold swelling and degradation and evaluate the osteogenic potential of DBM-NP-containing composite colloidal scaffolds. The addition of methacryloylated DBM-NPs (DBM-NP-MAs) to composite colloidal inks (100:0, 95:5 and 75:25 GNP-MA:DBM-NP-MA) did not significantly impact the rheological properties associated with printability, such as viscosity and shear recovery or photocrosslinking. UV crosslinking with a UV dosage of 3 J/cm2 directly impacted the rate of 3DP scaffold swelling for all GNP-MA:DBM-NP-MA ratios with an ∼40% greater increase in scaffold area and pore area in uncrosslinked versus photocrosslinked scaffolds over 21 days in phosphate-buffered saline (PBS). Likewise, degradation (hydrolytic and enzymatic) over 21 days for all DBM-NP-MA content groups was significantly decreased, ∼45% less in PBS and collagenase-containing PBS, in UV-crosslinked versus uncrosslinked groups. The incorporation of DBM-NP-MAs into scaffolds decreased mass loss compared to GNP-MA-only scaffolds during collagenase degradation. An in vitro osteogenic study with bone marrow-derived mesenchymal stem cells demonstrated osteoconductive properties of 3DP scaffolds for the DBM-NP-MA contents examined. The creation of photoreactive DBM-NP-MAs and their application in 3DP provide a platform for the development of ECM-derived colloidal materials and tailored control of biochemical cue presentation with broad tissue engineering applications.Item Visible and short-wave infrared fiber-based snapshot imaging spectrometer with a custom high-throughput relay system(Optica Publishing Group, 2023) Lu, Jiawei; Zheng, Desheng; Stoian, Razvan-Ionut; Flynn, Christopher; Alexander, David; Tkaczyk, Tomasz S.This paper presents the design and fabrication of a fiber-based snapshot imaging spectrometer working in both visible (490 nm-732 nm) and short-wave infrared (1090 nm - 1310 nm) ranges. To maximize the light collection efficiency, a custom relay system with 0.25 NA and 20 mm field of view (FOV) was designed and integrated. The bench setup showed that the custom relay system could fully resolve 10 µm fiber cores over the entire FOV among visible and short-wave infrared ranges. The numerical aperture (NA) match provided a 2.07X fold throughout improvement in the visible range and about 10X fold in the SWIR range compared to the previous generations, enabling imaging with a fast frame rate and under low illumination conditions. The presented imaging spectrometer generated spectral datacubes with 35000 spatial samplings and 23 spectral channels. Spectral urban imaging results obtained by the spectrometer in both visible and SWIR ranges are presented. Finally, we collected spectral images of apple bruising to show potential applications in the food quality industry.Item Fabrication of waveguide directional couplers using 2-photon lithography(Optica Publishing Group, 2023) Flynn, Christopher; Cao, Haimu; Applegate, Brian E.; Tkaczyk, Tomasz S.Advances in 2-photon lithography have enabled in-lab production of sub-micron resolution and millimeter scale 3D optical components. The potential complex geometries are well suited to rapid prototyping and production of waveguide structures, interconnects, and waveguide directional couplers, furthering future development and miniaturization of waveguide-based imaging technologies. System alignment is inherent to the 2-photon process, obviating the need for manual assembly and allowing precise micron scale waveguide geometries not possible in traditional fused fiber coupler fabrication. Here we present the use of 2-photon lithography for direct printing of multi-mode waveguide couplers with air cladding and single mode waveguide couplers with uncured liquid photoresin cladding. Experimental results show reproducible coupling which can be modified by selected design parameters.Item Revolutionizing bone regeneration: advanced biomaterials for healing compromised bone defects(Frontiers Media S.A., 2023) Awad, Kamal; Ahuja, Neelam; Yacoub, Ahmed S.; Brotto, Leticia; Young, Simon; Mikos, Antonios; Aswath, Pranesh; Varanasi, VenuIn this review, we explore the application of novel biomaterial-based therapies specifically targeted towards craniofacial bone defects. The repair and regeneration of critical sized bone defects in the craniofacial region requires the use of bioactive materials to stabilize and expedite the healing process. However, the existing clinical approaches face challenges in effectively treating complex craniofacial bone defects, including issues such as oxidative stress, inflammation, and soft tissue loss. Given that a significant portion of individuals affected by traumatic bone defects in the craniofacial area belong to the aging population, there is an urgent need for innovative biomaterials to address the declining rate of new bone formation associated with age-related changes in the skeletal system. This article emphasizes the importance of semiconductor industry-derived materials as a potential solution to combat oxidative stress and address the challenges associated with aging bone. Furthermore, we discuss various material and autologous treatment approaches, as well as in vitro and in vivo models used to investigate new therapeutic strategies in the context of craniofacial bone repair. By focusing on these aspects, we aim to shed light on the potential of advanced biomaterials to overcome the limitations of current treatments and pave the way for more effective and efficient therapeutic interventions for craniofacial bone defects.Item Protein target highlights in CASP15: Analysis of models by structure providers(Wiley, 2023) Alexander, Leila T.; Durairaj, Janani; Kryshtafovych, Andriy; Abriata, Luciano A.; Bayo, Yusupha; Bhabha, Gira; Breyton, Cécile; Caulton, Simon G.; Chen, James; Degroux, Séraphine; Ekiert, Damian C.; Erlandsen, Benedikte S.; Freddolino, Peter L.; Gilzer, Dominic; Greening, Chris; Grimes, Jonathan M.; Grinter, Rhys; Gurusaran, Manickam; Hartmann, Marcus D.; Hitchman, Charlie J.; Keown, Jeremy R.; Kropp, Ashleigh; Kursula, Petri; Lovering, Andrew L.; Lemaitre, Bruno; Lia, Andrea; Liu, Shiheng; Logotheti, Maria; Lu, Shuze; Markússon, Sigurbjörn; Miller, Mitchell D.; Minasov, George; Niemann, Hartmut H.; Opazo, Felipe; Phillips Jr, George N.; Davies, Owen R.; Rommelaere, Samuel; Rosas-Lemus, Monica; Roversi, Pietro; Satchell, Karla; Smith, Nathan; Wilson, Mark A.; Wu, Kuan-Lin; Xia, Xian; Xiao, Han; Zhang, Wenhua; Zhou, Z. Hong; Fidelis, Krzysztof; Topf, Maya; Moult, John; Schwede, TorstenWe present an in-depth analysis of selected CASP15 targets, focusing on their biological and functional significance. The authors of the structures identify and discuss key protein features and evaluate how effectively these aspects were captured in the submitted predictions. While the overall ability to predict three-dimensional protein structures continues to impress, reproducing uncommon features not previously observed in experimental structures is still a challenge. Furthermore, instances with conformational flexibility and large multimeric complexes highlight the need for novel scoring strategies to better emphasize biologically relevant structural regions. Looking ahead, closer integration of computational and experimental techniques will play a key role in determining the next challenges to be unraveled in the field of structural molecular biology.Item Hierarchically Engineered Artificial Lamellar Bone with High Strength and Toughness(Wiley, 2023) Zhao, Yonggang; Zheng, Jingchuan; Xiong, Yang; Wang, Hetong; Yang, Shuhui; Sun, Xiaodan; Zhao, Lingyun; Mikos, Antonios G.; Wang, XiumeiComplex hierarchical architectures are ubiquitous in natural hard tissues, which comprise an elaborate assembly of hard and soft phases spanning from the nanoscale to the macroscale. The elegant architectures grant unique performance in terms of strength and toughness, but the biomimetic fabrication of synthetic materials with highly consistent structural and mechanical characteristics with natural counterparts remains a great challenge. Here, a centimeter-size artificial lamellar bone is successfully fabricated for the first time via a well-orchestrated “multiscale cascade regulation” strategy combining multiple techniques of molecular self-assembly, electrospinning, and pressure-driven fusion from molecular to macroscopic levels. The bulk artificial lamellar bone that is composed of hierarchically assembled mineralized collagen fibrils with a waiver of any synthetic polymer highly resembles the chemical composition, multiscale structural organization, and rotated plywood-like structure of natural lamellae, thus achieving a good combination of lightweight and high-stiffness (Ey ≈ 15.2 GPa), -strength (σf ≈ 118.4 MPa), and -toughness (KJC ≈ 9.3 MPa m1/2). This multiscale cascade regulation strategy can break through the limitations of a single technique and enable the construction of elaborate composite materials with multiscale step-by-step regulations of hierarchically structural organizations for unique mechanical properties.Item Fabrication of a multifaceted mapping mirror using two-photon polymerization for a snapshot image mapping spectrometer(Optica Publishing Group, 2023) Lu, Jiawei; Ng, Xue Wen; Piston, David; Tkaczyk, Tomasz S.A design and fabrication technique for making high-precision and large-format multifaceted mapping mirrors is presented. The method is based on two-photon polymerization, which allows more flexibility in the mapping mirror design. The mirror fabricated in this paper consists of 36 2D tilted square pixels, instead of the continuous facet design used in diamond cutting. The paper presents a detailed discussion of the fabrication parameters and optimization process, with particular emphasis on the optimization of stitching defects by compensating for the overall tilt angle and reducing the printing field of view. The fabricated mirrors were coated with a thin layer of aluminum (93 nm) using sputter coating to enhance the reflection rate over the target wave range. The mapping mirror was characterized using a white light interferometer and a scanning electron microscope, which demonstrates its optical quality surface (with a surface roughness of 12 nm) and high-precision tilt angles (with an average of 2.03% deviation). Finally, the incorporation of one of the 3D printed mapping mirrors into an image mapping spectrometer prototype allowed for the acquisition of high-quality images of the USAF resolution target and bovine pulmonary artery endothelial cells stained with three fluorescent dyes, demonstrating the potential of this technology for practical applications.Item Identification of unique α4 chain structure and conserved antiangiogenic activity of α3NC1 type IV collagen in zebrafish(Wiley, 2023) LeBleu, Valerie S.; Dai, Jianli; Tsutakawa, Susan; MacDonald, Brian A.; Alge, Joseph L.; Sund, Malin; Xie, Liang; Sugimoto, Hikaru; Tainer, John; Zon, Leonard I.; Kalluri, RaghuBackground Type IV collagen is an abundant component of basement membranes in all multicellular species and is essential for the extracellular scaffold supporting tissue architecture and function. Lower organisms typically have two type IV collagen genes, encoding α1 and α2 chains, in contrast with the six genes in humans, encoding α1–α6 chains. The α chains assemble into trimeric protomers, the building blocks of the type IV collagen network. The detailed evolutionary conservation of type IV collagen network remains to be studied. Results We report on the molecular evolution of type IV collagen genes. The zebrafish α4 non-collagenous (NC1) domain, in contrast with its human ortholog, contains an additional cysteine residue and lacks the M93 and K211 residues involved in sulfilimine bond formation between adjacent protomers. This may alter α4 chain interactions with other α chains, as supported by temporal and anatomic expression patterns of collagen IV chains during the zebrafish development. Despite the divergence between zebrafish and human α3 NC1 domain (endogenous angiogenesis inhibitor, Tumstatin), the zebrafish α3 NC1 domain exhibits conserved antiangiogenic activity in human endothelial cells. Conclusions Our work supports type IV collagen is largely conserved between zebrafish and humans, with a possible difference involving the α4 chain.Item CAGE sequencing reveals CFTR-dependent dysregulation of type I IFN signaling in activated cystic fibrosis macrophages(AAAS, 2023) Gillan, Jonathan L.; Chokshi, Mithil; Hardisty, Gareth R.; Clohisey Hendry, Sara; Prasca-Chamorro, Daniel; Robinson, Nicola J.; Lasota, Benjamin; Clark, Richard; Murphy, Lee; Whyte, Moira K. B.; Baillie, J. Kenneth; Davidson, Donald J.; Bao, Gang; Gray, Robert D.An intense, nonresolving airway inflammatory response leads to destructive lung disease in cystic fibrosis (CF). Dysregulation of macrophage immune function may be a key facet governing the progression of CF lung disease, but the underlying mechanisms are not fully understood. We used 5′ end centered transcriptome sequencing to profile P. aeruginosa LPS-activated human CF macrophages, showing that CF and non-CF macrophages deploy substantially distinct transcriptional programs at baseline and following activation. This includes a significantly blunted type I IFN signaling response in activated patient cells relative to healthy controls that was reversible upon in vitro treatment with CFTR modulators in patient cells and by CRISPR-Cas9 gene editing to correct the F508del mutation in patient-derived iPSC macrophages. These findings illustrate a previously unidentified immune defect in human CF macrophages that is CFTR dependent and reversible with CFTR modulators, thus providing new avenues in the search for effective anti-inflammatory interventions in CF.