Pub Date : 2024-09-13DOI: 10.1101/2024.09.07.611840
EMINE SILA OZDEMIR, Jessica Tolley, Florian Goncalves, Michelle Gomes, Eli Wagnell, Bruce Branchaud, Srivathsan Ranganathan
The variable heavy chain fragments derived from camelid antibodies, called VHHs or nanobodies, have recently shown promise as high-affinity reagents. They offer higher stability compared to conventional antibodies and fragments thereof. Furthermore, their smaller size (~15-20 kDa) allows better targeting of molecules localized inside the cell and in crowded environments, like tissues and protein aggregates. Despite these advantages, nanobody clones screened using phage display can suffer from poor soluble expression, which we hypothesized, is due to the presence of hydrophobic hotspots on their surface. In this work, we propose a novel computationally guided workflow for screening and production of nanobody binders for optimized expression. After an initial round of phage display screens against our target (K-Ras), we modeled the lead candidates to generate Spatial Aggregation Propensity (SAP) maps to highlight the hydrophobic hotspots with single amino acid resolution, which were subsequently used to guide mutagenesis of the binders for soluble expression. We followed two approaches to perform point hydrophilic mutations: i) performing point hydrophilic mutations in the hydrophobic hotspots; ii) combining point mutation resulting from a round of random mutagenesis that show favorable SAP scores. Both approaches led a remarkable increase in soluble expression which allowed production and characterization of their binding to their target (K-Ras) on soluble ELISA, and biolayer interferometry. We observed that the latter approach resulted in clones with stronger binding affinity compared to the former approach. Our results emphasize the need to perform a round of random mutagenesis to identify point mutations, which can then be used in an in-silico guided pipeline to identify the right combination of mutations for high soluble expression.
从驼科动物抗体中提取的可变重链片段（称为 VHHs 或纳米抗体）最近显示出作为高亲和力试剂的前景。与传统抗体及其片段相比，它们具有更高的稳定性。此外，它们的尺寸较小（约 15-20 kDa），可以更好地靶向细胞内和拥挤环境中的分子，如组织和蛋白质聚集体。尽管有这些优点，但利用噬菌体展示筛选出的纳米抗体克隆可能会出现可溶性表达不佳的问题，我们推测这是由于纳米抗体表面存在疏水热点。在这项工作中，我们提出了一种新颖的计算指导工作流程，用于筛选和生产纳米抗体粘合剂，以优化表达。在针对我们的目标（K-Ras）进行了第一轮噬菌体展示筛选后，我们对主要候选者进行建模，生成空间聚集倾向（SAP）图，以单氨基酸分辨率突出疏水热点，随后用于指导诱变结合体的可溶性表达。我们采用两种方法进行点亲水突变：i) 在疏水热点进行点亲水突变；ii) 将一轮随机诱变产生的点突变与显示出有利 SAP 分数的点突变相结合。这两种方法都显著提高了可溶性表达量，从而可以通过可溶性酶联免疫吸附试验（ELISA）和生物层干涉测量（biolayer interferometry）来生产和鉴定它们与靶标（K-Ras）的结合。我们观察到，与前一种方法相比，后一种方法产生的克隆具有更强的结合亲和力。我们的研究结果强调了进行一轮随机诱变以确定点突变的必要性，这些点突变随后可用于一个以硅为指导的流水线，以确定突变的正确组合，从而实现高可溶性表达。
{"title":"A computationally guided approach to improve expression of VHH binders","authors":"EMINE SILA OZDEMIR, Jessica Tolley, Florian Goncalves, Michelle Gomes, Eli Wagnell, Bruce Branchaud, Srivathsan Ranganathan","doi":"10.1101/2024.09.07.611840","DOIUrl":"https://doi.org/10.1101/2024.09.07.611840","url":null,"abstract":"The variable heavy chain fragments derived from camelid antibodies, called VHHs or nanobodies, have recently shown promise as high-affinity reagents. They offer higher stability compared to conventional antibodies and fragments thereof. Furthermore, their smaller size (~15-20 kDa) allows better targeting of molecules localized inside the cell and in crowded environments, like tissues and protein aggregates. Despite these advantages, nanobody clones screened using phage display can suffer from poor soluble expression, which we hypothesized, is due to the presence of hydrophobic hotspots on their surface. In this work, we propose a novel computationally guided workflow for screening and production of nanobody binders for optimized expression. After an initial round of phage display screens against our target (K-Ras), we modeled the lead candidates to generate Spatial Aggregation Propensity (SAP) maps to highlight the hydrophobic hotspots with single amino acid resolution, which were subsequently used to guide mutagenesis of the binders for soluble expression. We followed two approaches to perform point hydrophilic mutations: i) performing point hydrophilic mutations in the hydrophobic hotspots; ii) combining point mutation resulting from a round of random mutagenesis that show favorable SAP scores. Both approaches led a remarkable increase in soluble expression which allowed production and characterization of their binding to their target (K-Ras) on soluble ELISA, and biolayer interferometry. We observed that the latter approach resulted in clones with stronger binding affinity compared to the former approach. Our results emphasize the need to perform a round of random mutagenesis to identify point mutations, which can then be used in an in-silico guided pipeline to identify the right combination of mutations for high soluble expression.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142216567","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-13DOI: 10.1101/2024.09.09.612079
Kendell Pawelec, Jeremy M. L. Hix, Arianna Troia, Matti Kiupel, Erik Shapiro
Successful tissue engineering requires biomedical devices that initially stabilize wounds, then degrade as tissue is regenerated. However, the material degradation rates reported in literature are often conflicting. Incorporation of in situ monitoring functionality into implanted devices would allow real time assessment of degradation and potential failure. This necessitates introduction of contrast agent as most biomedical devices are composed of polymeric materials with no inherent contrast in medical imaging modalities. In the present study, computed tomography (CT)-visible radiopaque composites were created by adding 5-20wt% tantalum oxide (TaOx) nanoparticles into polymers with distinct degradation profiles: polycaprolactone (PCL), poly(lactide-co-glycolide) (PLGA) 85:15 and PLGA 50:50, representing slow, medium and fast degrading materials respectively. Radiopaque phantoms, mimicking porous tissue engineering devices, were implanted into mice intramuscularly or intraperitoneally, and monitored via CT over 20 weeks. Changes in phantom volume, including collapse and swelling, were visualized over time. Phantom degradation profile was determined by polymer matrix, regardless of nanoparticle addition and foreign body response was dictated by the implant site. In addition, degradation kinetics were significantly affected in mid-degrading materials, transitioning from linear degradation intramuscularly to exponential degradation intraperitoneally, due to differences in inflammatory responses and fluid flow. Nanoparticle excretion from degraded phantoms lagged behind polymer, and future studies will modulate nanoparticle clearance. Utilizing in situ monitoring, this study seeks to unify literature and facilitate better tissue engineering devices, by highlighting the relative effect of composition and implant site on important materials properties.
{"title":"Material Composition and Implantation Site Affect in vivo Device Degradation Rate","authors":"Kendell Pawelec, Jeremy M. L. Hix, Arianna Troia, Matti Kiupel, Erik Shapiro","doi":"10.1101/2024.09.09.612079","DOIUrl":"https://doi.org/10.1101/2024.09.09.612079","url":null,"abstract":"Successful tissue engineering requires biomedical devices that initially stabilize wounds, then degrade as tissue is regenerated. However, the material degradation rates reported in literature are often conflicting. Incorporation of in situ monitoring functionality into implanted devices would allow real time assessment of degradation and potential failure. This necessitates introduction of contrast agent as most biomedical devices are composed of polymeric materials with no inherent contrast in medical imaging modalities. In the present study, computed tomography (CT)-visible radiopaque composites were created by adding 5-20wt% tantalum oxide (TaOx) nanoparticles into polymers with distinct degradation profiles: polycaprolactone (PCL), poly(lactide-co-glycolide) (PLGA) 85:15 and PLGA 50:50, representing slow, medium and fast degrading materials respectively. Radiopaque phantoms, mimicking porous tissue engineering devices, were implanted into mice intramuscularly or intraperitoneally, and monitored via CT over 20 weeks. Changes in phantom volume, including collapse and swelling, were visualized over time. Phantom degradation profile was determined by polymer matrix, regardless of nanoparticle addition and foreign body response was dictated by the implant site. In addition, degradation kinetics were significantly affected in mid-degrading materials, transitioning from linear degradation intramuscularly to exponential degradation intraperitoneally, due to differences in inflammatory responses and fluid flow. Nanoparticle excretion from degraded phantoms lagged behind polymer, and future studies will modulate nanoparticle clearance. Utilizing in situ monitoring, this study seeks to unify literature and facilitate better tissue engineering devices, by highlighting the relative effect of composition and implant site on important materials properties.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142256036","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-13DOI: 10.1101/2024.09.09.612090
Xiaokai Wang, Fatimah Alkaabi, Ashley Cornett, Minkyu Choi, Ulrich M. Scheven, Madeleine R. Di Natale, John B. Furness, Zhongming Liu
Introduction: Gastrointestinal (GI) magnetic resonance imaging (MRI) can simultaneously capture gastric peristalsis, emptying, and intestinal filling and transit. Performing GI MRI with animals requires anesthesia, which complicates physiology and confounds interpretation and translation from animals to humans. This study aims to enable MRI in conscious rats, and for the first time, characterize GI motor functions in awake versus anesthetized conditions. Methods: We acclimated rats to remain awake, still, and minimally stressed during MRI. We scanned 14 Sprague-Dawley rats in both awake and anesthetized conditions after voluntarily consuming a contrast-enhanced test meal. Results: Awake rats remained physiologically stable during MRI, showed gastric emptying of 23.7+/-1.4% after 48 minutes, and exhibited strong peristaltic contractions propagating through the antrum with a velocity of 0.72+/-0.04 mm/s, a relative amplitude of 40.7+/-2.3 %, and a frequency of 5.1+/-0.1 cycles per minute. In the anesthetized condition, gastric emptying was about half of that in the awake condition, likely due to the effect of anesthesia in halving the amplitudes of peristaltic contractions rather than their frequency (not significantly changed) or velocity. In awake rats, the intestine filled more quickly and propulsive contractions were more occlusive. Conclusion: We demonstrated the effective acquisition and analysis of GI MRI in awake rats. Awake rats show faster gastric emptying, stronger gastric contraction with a faster propagation speed, and more effective intestinal filling and transit, compared to anesthetized rats. Our protocol is expected to benefit future preclinical studies of GI physiology and pathophysiology.
{"title":"Magnetic Resonance Imaging of Gastric Motility in Conscious Rats","authors":"Xiaokai Wang, Fatimah Alkaabi, Ashley Cornett, Minkyu Choi, Ulrich M. Scheven, Madeleine R. Di Natale, John B. Furness, Zhongming Liu","doi":"10.1101/2024.09.09.612090","DOIUrl":"https://doi.org/10.1101/2024.09.09.612090","url":null,"abstract":"Introduction: Gastrointestinal (GI) magnetic resonance imaging (MRI) can simultaneously capture gastric peristalsis, emptying, and intestinal filling and transit. Performing GI MRI with animals requires anesthesia, which complicates physiology and confounds interpretation and translation from animals to humans. This study aims to enable MRI in conscious rats, and for the first time, characterize GI motor functions in awake versus anesthetized conditions. Methods: We acclimated rats to remain awake, still, and minimally stressed during MRI. We scanned 14 Sprague-Dawley rats in both awake and anesthetized conditions after voluntarily consuming a contrast-enhanced test meal. Results: Awake rats remained physiologically stable during MRI, showed gastric emptying of 23.7+/-1.4% after 48 minutes, and exhibited strong peristaltic contractions propagating through the antrum with a velocity of 0.72+/-0.04 mm/s, a relative amplitude of 40.7+/-2.3 %, and a frequency of 5.1+/-0.1 cycles per minute. In the anesthetized condition, gastric emptying was about half of that in the awake condition, likely due to the effect of anesthesia in halving the amplitudes of peristaltic contractions rather than their frequency (not significantly changed) or velocity. In awake rats, the intestine filled more quickly and propulsive contractions were more occlusive. Conclusion: We demonstrated the effective acquisition and analysis of GI MRI in awake rats. Awake rats show faster gastric emptying, stronger gastric contraction with a faster propagation speed, and more effective intestinal filling and transit, compared to anesthetized rats. Our protocol is expected to benefit future preclinical studies of GI physiology and pathophysiology.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142256038","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-13DOI: 10.1101/2024.09.09.612117
Jose G Munguia-Lopez, Sangeeth Pillai, Yuli Zhang, Amatzia Gantz, Dimitria B. Camasao, Showan N. Nazhat, Joseph M. Kinsella, Simon D. Tran
Xerostomia (dry mouth) is frequently experienced by patients treated with radiotherapy for head and neck cancers or with Sjögren's syndrome, with no permanent cure existing for this debilitating condition. To this end, in vitro platforms are needed to test therapies directed at salivary (fluid-secreting) cells. However, since these are highly differentiated secretory cells, the maintenance of their differentiated state while expanding in numbers is challenging. In this study, the efficiency of three reversible thermo-ionically crosslinked gels: 1) alginate-gelatin (AG), 2) collagen-containing AG (AGC), and 3) hyaluronic acid-containing AG (AGHA), to recapitulate a native-like environment for human salivary gland (SG) cell expansion and 3D spheroid formation was compared. Although all gels were of mechanical properties comparable to human SG tissue (~11 kPa) and promoted the formation of 3D spheroids, AGHA gels produced larger (>100 cells/spheroid), viable (>93%), proliferative, and well-organized 3D SG spheroids while spatially and temporally maintaining the high expression of key SG proteins (aquaporin-5, NKCC1, ZO-1, α-amylase) for 14 days in culture. Moreover, the spheroids responded to agonist-induced stimulation by increasing α-amylase secretory granules. Here, we propose alternative low-cost, reproducible, and reversible AG-based 3D hydrogels that allow the facile and rapid retrieval of intact, highly viable 3D-SG spheroids for downstream applications.
{"title":"Expansion of functional human salivary acinar cell spheroids with reversible thermo-ionically crosslinked 3D hydrogels","authors":"Jose G Munguia-Lopez, Sangeeth Pillai, Yuli Zhang, Amatzia Gantz, Dimitria B. Camasao, Showan N. Nazhat, Joseph M. Kinsella, Simon D. Tran","doi":"10.1101/2024.09.09.612117","DOIUrl":"https://doi.org/10.1101/2024.09.09.612117","url":null,"abstract":"Xerostomia (dry mouth) is frequently experienced by patients treated with radiotherapy for head and neck cancers or with Sjögren's syndrome, with no permanent cure existing for this debilitating condition. To this end, in vitro platforms are needed to test therapies directed at salivary (fluid-secreting) cells. However, since these are highly differentiated secretory cells, the maintenance of their differentiated state while expanding in numbers is challenging. In this study, the efficiency of three reversible thermo-ionically crosslinked gels: 1) alginate-gelatin (AG), 2) collagen-containing AG (AGC), and 3) hyaluronic acid-containing AG (AGHA), to recapitulate a native-like environment for human salivary gland (SG) cell expansion and 3D spheroid formation was compared. Although all gels were of mechanical properties comparable to human SG tissue (~11 kPa) and promoted the formation of 3D spheroids, AGHA gels produced larger (>100 cells/spheroid), viable (>93%), proliferative, and well-organized 3D SG spheroids while spatially and temporally maintaining the high expression of key SG proteins (aquaporin-5, NKCC1, ZO-1, α-amylase) for 14 days in culture. Moreover, the spheroids responded to agonist-induced stimulation by increasing α-amylase secretory granules. Here, we propose alternative low-cost, reproducible, and reversible AG-based 3D hydrogels that allow the facile and rapid retrieval of intact, highly viable 3D-SG spheroids for downstream applications.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142227035","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-13DOI: 10.1101/2024.09.09.612161
Nicolas Acosta, Ruyi Gong, Yuanzhe Su, Jane Frederick, Karla Medina, Wing Shun Li, Kiana Mohammadian, Luay Almassalha, Vadim Backman
Super-resolution microscopy has revolutionized our ability to visualize structures below the diffraction limit of conventional optical microscopy and is particularly useful for investigating complex biological targets like chromatin. Chromatin exhibits a hierarchical organization with structural compartments and domains at different length scales, from nanometers to micrometers. Single molecule localization microscopy (SMLM) methods, such as STORM, are essential for studying chromatin at the supra-nucleosome level due to their ability to target epigenetic marks that determine chromatin organization. Multi-label imaging of chromatin is necessary to unpack its structural complexity. However, these efforts are challenged by the high-density nuclear environment, which can affect antibody binding affinities, diffusivity and non-specific interactions. Optimizing buffer conditions, fluorophore stability, and antibody specificity is crucial for achieving effective antibody conjugates. Here, we demonstrate a sequential immunolabeling protocol that reliably enables three-label studies within the dense nuclear environment. This protocol couples multiplexed localization datasets with a robust analysis algorithm, which utilizes localizations from one target as seed points for distance, density and multi-label joint affinity measurements to explore complex organization of all three targets. Applying this multi-plexed algorithm to analyze distance and joint density reveals that heterochromatin and euchromatin are not-distinct territories, but that localization of transcription and euchromatin couple with the periphery of heterochromatic clusters. This work is a crucial step in molecular imaging of the dense nuclear environment as multi-label capacity enables for investigation of complex multi-component systems like chromatin with enhanced accuracy.
{"title":"Three-color single-molecule localization microscopy in chromatin","authors":"Nicolas Acosta, Ruyi Gong, Yuanzhe Su, Jane Frederick, Karla Medina, Wing Shun Li, Kiana Mohammadian, Luay Almassalha, Vadim Backman","doi":"10.1101/2024.09.09.612161","DOIUrl":"https://doi.org/10.1101/2024.09.09.612161","url":null,"abstract":"Super-resolution microscopy has revolutionized our ability to visualize structures below the diffraction limit of conventional optical microscopy and is particularly useful for investigating complex biological targets like chromatin. Chromatin exhibits a hierarchical organization with structural compartments and domains at different length scales, from nanometers to micrometers. Single molecule localization microscopy (SMLM) methods, such as STORM, are essential for studying chromatin at the supra-nucleosome level due to their ability to target epigenetic marks that determine chromatin organization. Multi-label imaging of chromatin is necessary to unpack its structural complexity. However, these efforts are challenged by the high-density nuclear environment, which can affect antibody binding affinities, diffusivity and non-specific interactions. Optimizing buffer conditions, fluorophore stability, and antibody specificity is crucial for achieving effective antibody conjugates. Here, we demonstrate a sequential immunolabeling protocol that reliably enables three-label studies within the dense nuclear environment. This protocol couples multiplexed localization datasets with a robust analysis algorithm, which utilizes localizations from one target as seed points for distance, density and multi-label joint affinity measurements to explore complex organization of all three targets. Applying this multi-plexed algorithm to analyze distance and joint density reveals that heterochromatin and euchromatin are not-distinct territories, but that localization of transcription and euchromatin couple with the periphery of heterochromatic clusters. This work is a crucial step in molecular imaging of the dense nuclear environment as multi-label capacity enables for investigation of complex multi-component systems like chromatin with enhanced accuracy.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142216565","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-12DOI: 10.1101/2024.09.06.611741
Thomas C Turner, Frank S Pittman, Hongmanlin Zhang, Lauren A Hymel, Tianyi Zheng, Monica Behara, Shannon E Anderson, Julia Andraca Harrer, Kaitlyn A Link, Mashoor Al Ahammed, Kristal Maner-Smith, Xueyun Liu, Xuanzhi Yin, Hong Seo Lim, Matthew Spite, Peng Qiu, Andres J Garcia, Luke J. Mortensen, Young C Jang, Nick J Willett, Edward A Botchwey
Severe tissue loss resulting from extremity trauma, such as volumetric muscle loss (VML), poses significant clinical challenges for both general and military populations. VML disrupts the endogenous tissue repair mechanisms, resulting in acute and unresolved chronic inflammation and immune cell presence, impaired muscle healing, scar tissue formation, persistent pain, and permanent functional deficits. The aberrant healing response is preceded by acute inflammation and immune cell infiltration which does not resolve. We analyzed the biosynthesis of inflammatory and specialized pro-resolving lipid mediators (SPMs) after VML injury in two different models; muscle with critical-sized defects had a decreased capacity to biosynthesize SPMs, leading to dysregulated and persistent inflammation. We developed a modular poly(ethylene glycol)-maleimide hydrogel platform to locally release a stable isomer of Resolvin D1 (AT-RvD1) and promote endogenous pathways of inflammation resolution in the two muscle models. The local delivery of AT-RvD1 enhanced muscle regeneration, improved muscle function, and reduced pain sensitivity after VML by promoting molecular and cellular resolution of inflammation. These findings provide new insights into the pathogenesis of VML and establish a pro-resolving hydrogel therapeutic as a promising strategy for promoting functional muscle regeneration after traumatic injury.
{"title":"Improving Functional Muscle Regeneration in Volumetric Muscle Loss Injuries by Shifting the Balance of Inflammatory and Pro-Resolving Lipid Mediators","authors":"Thomas C Turner, Frank S Pittman, Hongmanlin Zhang, Lauren A Hymel, Tianyi Zheng, Monica Behara, Shannon E Anderson, Julia Andraca Harrer, Kaitlyn A Link, Mashoor Al Ahammed, Kristal Maner-Smith, Xueyun Liu, Xuanzhi Yin, Hong Seo Lim, Matthew Spite, Peng Qiu, Andres J Garcia, Luke J. Mortensen, Young C Jang, Nick J Willett, Edward A Botchwey","doi":"10.1101/2024.09.06.611741","DOIUrl":"https://doi.org/10.1101/2024.09.06.611741","url":null,"abstract":"Severe tissue loss resulting from extremity trauma, such as volumetric muscle loss (VML), poses significant clinical challenges for both general and military populations. VML disrupts the endogenous tissue repair mechanisms, resulting in acute and unresolved chronic inflammation and immune cell presence, impaired muscle healing, scar tissue formation, persistent pain, and permanent functional deficits. The aberrant healing response is preceded by acute inflammation and immune cell infiltration which does not resolve. We analyzed the biosynthesis of inflammatory and specialized pro-resolving lipid mediators (SPMs) after VML injury in two different models; muscle with critical-sized defects had a decreased capacity to biosynthesize SPMs, leading to dysregulated and persistent inflammation. We developed a modular poly(ethylene glycol)-maleimide hydrogel platform to locally release a stable isomer of Resolvin D1 (AT-RvD1) and promote endogenous pathways of inflammation resolution in the two muscle models. The local delivery of AT-RvD1 enhanced muscle regeneration, improved muscle function, and reduced pain sensitivity after VML by promoting molecular and cellular resolution of inflammation. These findings provide new insights into the pathogenesis of VML and establish a pro-resolving hydrogel therapeutic as a promising strategy for promoting functional muscle regeneration after traumatic injury.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142216566","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Chimeric antigen receptor (CAR)-modified macrophages (CAR-Ms) are a promising approach for the treatment of solid tumors due to its high infiltration and immune-regulation activity. Prostate cancer is a typical solid tumor associated with highly immunosuppressive microenvironment. To date, the potential application of CAR-M cell therapy in prostate cancer has been infrequently explored. The prostate-specific membrane antigen (PSMA) functions as a specific biomarker for prostate cancer. In this study, we assessed the antitumor efficacy of PSMA-targeted CAR-Ms in preclinical models. CAR-Ms were engineered to express a PSMA-specific single-chain variable fragment (scFv) and co-stimulatory domains. In vitro data demonstrated specific cytotoxicity of CAR-Ms against PSMA-expressing prostate cancer cells, which was further supported by transcriptome analysis demonstrating the pro-inflammatory phenotypes of CAR-Ms. In vivo studies using xenograft mouse models confirmed significant tumor regression after administration of PSMA-targeted CAR-Ms compared to controls. Histopathological analysis showed infiltration of CAR-Ms into tumor tissues without off-target toxicity. These results highlight the strong antitumor activity and safety of PSMA-targeted CAR-Ms, supporting their potential as a new immunotherapy for prostate cancer.
{"title":"PSMA-specific CAR-engineered macrophages for therapy of prostate cancer","authors":"Yangli Xu, Duoli Xie, Chunhao Cao, Yue Ju, Xinxin Chen, Lili Guan, Xuelong Li, Luo Zhang, Chao Liang, Xiushan Yin","doi":"10.1101/2024.09.07.611792","DOIUrl":"https://doi.org/10.1101/2024.09.07.611792","url":null,"abstract":"Chimeric antigen receptor (CAR)-modified macrophages (CAR-Ms) are a promising approach for the treatment of solid tumors due to its high infiltration and immune-regulation activity. Prostate cancer is a typical solid tumor associated with highly immunosuppressive microenvironment. To date, the potential application of CAR-M cell therapy in prostate cancer has been infrequently explored. The prostate-specific membrane antigen (PSMA) functions as a specific biomarker for prostate cancer. In this study, we assessed the antitumor efficacy of PSMA-targeted CAR-Ms in preclinical models. CAR-Ms were engineered to express a PSMA-specific single-chain variable fragment (scFv) and co-stimulatory domains. In vitro data demonstrated specific cytotoxicity of CAR-Ms against PSMA-expressing prostate cancer cells, which was further supported by transcriptome analysis demonstrating the pro-inflammatory phenotypes of CAR-Ms. In vivo studies using xenograft mouse models confirmed significant tumor regression after administration of PSMA-targeted CAR-Ms compared to controls. Histopathological analysis showed infiltration of CAR-Ms into tumor tissues without off-target toxicity. These results highlight the strong antitumor activity and safety of PSMA-targeted CAR-Ms, supporting their potential as a new immunotherapy for prostate cancer.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142216587","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-11DOI: 10.1101/2024.09.05.611365
Carly A Lockard, Bruce M Damon, Hacene Serrai
Zero echo time (ZTE) sequences capture signal from tissues with extremely short T2* and are useful for qualitative and quantitative imaging of musculoskeletal tissues' ultrashort-T2* components. One such sequence is Pointwise Encoding Time Reduction with Radial Acquisition (PETRA). While this sequence has shown promising results, it has undergone only limited testing at 7 tesla (T). The purpose of this work was to evaluate PETRA at 7T in its standard form and with sequence modifications to allow extended echo times for the purpose of performing ultrashort-T2* mapping. We acquired PETRA images of MnCl2 and collagen phantoms and of the knee in eight participants (5 for optimization and 3 for ultrashort-T2* mapping assessment; 5 male/3 female, 39 +/- 11 years old). Images were acquired using a 1-transmit/28-receive-channel knee coil. Artifacts, signal, signal-to-noise ratio (SNR), ultrashort-T2*, the corresponding curve fit quality, and repeatability were assessed. SNR was high in knee tissues at TE = 0.07 msec compared to a conventional-TE sequence (Dual-Echo Steady State with TE = 2.55 msec), with values ranging between 68 to 337 across the assessed tissues for PETRA versus 16 to 30 for the same tissue regions of interest in the conventional-TE series. Acquisition of series for ultrashort-T2* maps was feasible at 1.50 mm isotropic acquisition resolution and TE less than or equal to 0.58 msec. Strong linear correlations were observed between relaxation times and MnCl2 concentration, and between signal and collagen concentration. Ultrashort-T2* signal decay curve fit R2 and repeatability were high for phantom and knee ultrashort-T2* <1 msec. PETRA imaging with minimal artifacts, high SNR, and scan time < 11 minutes was achieved at 7T at high (0.34 mm isotropic) resolution at TE = 0.07 msec and lower resolution (1.52 mm isotropic) at echo times less than or equal to 0.58 msec. Ultrashort-T2* mapping provided acceptable curve-fitting results for substances with sub-millisecond T2*.
{"title":"Ultrashort-T2* mapping at 7 tesla using an optimized pointwise encoding time reduction with radial acquisition (PETRA) sequence at standard and extended echo times","authors":"Carly A Lockard, Bruce M Damon, Hacene Serrai","doi":"10.1101/2024.09.05.611365","DOIUrl":"https://doi.org/10.1101/2024.09.05.611365","url":null,"abstract":"Zero echo time (ZTE) sequences capture signal from tissues with extremely short T2* and are useful for qualitative and quantitative imaging of musculoskeletal tissues' ultrashort-T2* components. One such sequence is Pointwise Encoding Time Reduction with Radial Acquisition (PETRA). While this sequence has shown promising results, it has undergone only limited testing at 7 tesla (T). The purpose of this work was to evaluate PETRA at 7T in its standard form and with sequence modifications to allow extended echo times for the purpose of performing ultrashort-T2* mapping. We acquired PETRA images of MnCl2 and collagen phantoms and of the knee in eight participants (5 for optimization and 3 for ultrashort-T2* mapping assessment; 5 male/3 female, 39 +/- 11 years old). Images were acquired using a 1-transmit/28-receive-channel knee coil. Artifacts, signal, signal-to-noise ratio (SNR), ultrashort-T2*, the corresponding curve fit quality, and repeatability were assessed. SNR was high in knee tissues at TE = 0.07 msec compared to a conventional-TE sequence (Dual-Echo Steady State with TE = 2.55 msec), with values ranging between 68 to 337 across the assessed tissues for PETRA versus 16 to 30 for the same tissue regions of interest in the conventional-TE series. Acquisition of series for ultrashort-T2* maps was feasible at 1.50 mm isotropic acquisition resolution and TE less than or equal to 0.58 msec. Strong linear correlations were observed between relaxation times and MnCl2 concentration, and between signal and collagen concentration. Ultrashort-T2* signal decay curve fit R2 and repeatability were high for phantom and knee ultrashort-T2* <1 msec. PETRA imaging with minimal artifacts, high SNR, and scan time < 11 minutes was achieved at 7T at high (0.34 mm isotropic) resolution at TE = 0.07 msec and lower resolution (1.52 mm isotropic) at echo times less than or equal to 0.58 msec. Ultrashort-T2* mapping provided acceptable curve-fitting results for substances with sub-millisecond T2*.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142216593","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-11DOI: 10.1101/2024.09.06.611597
Judith Fuentes, Rafael Mestre, Maria Guix, Ibtissam Ghailan, Noela Ruiz-Gonzalez, Tania Patino, Samuel Sanchez
Advances in 3D bioprinting have opened new possibilities in the development of bioengineered muscle models that mimic the structure and functionality of native tissues. The combination of skeletal muscle tissue and artificial elements promotes diverse innovative solutions of interest in both the biomedical field and the development of biohybrid actuators. However, current bioengineering approaches do not fully recreate the complex fascicle-like hierarchical organization of skeletal muscle, impacting on the muscle maturation process due to a lack of oxygen and nutrients supply in the scaffold inner regions. Here we explored co-axial 3D bioprinting as a strategy towards overcoming this challenge, creating individual/non-fused filaments with controlled thickness that present a fascicle-like organization. Compared to conventional 3D-bioprinting, where cell-laden bioink is disposed by a single syringe, our Pluronic-assisted co-axial 3D-bioprinting system (PACA-3D) creates a physical confinement of the bioink during the extrusion process, effectively obtaining thin and independent printed fibers with controlled shape. Fabrication of skeletal muscle-based actuators with PACA-3D resulted in improved cell differentiation, obtaining stronger bioactuators with increased force output when compared to bioactuators fabricated by conventional 3D bioprinting. The versatility of our technology has been demonstrated using different biomaterials, showing its potential to develop more complex biohybrid tissue-based architectures with improved functionality.
{"title":"Bioengineering Fascicle-like Skeletal Muscle Bioactuators via Pluronic-Assisted Co-axial 3D Bioprinting","authors":"Judith Fuentes, Rafael Mestre, Maria Guix, Ibtissam Ghailan, Noela Ruiz-Gonzalez, Tania Patino, Samuel Sanchez","doi":"10.1101/2024.09.06.611597","DOIUrl":"https://doi.org/10.1101/2024.09.06.611597","url":null,"abstract":"Advances in 3D bioprinting have opened new possibilities in the development of bioengineered muscle models that mimic the structure and functionality of native tissues. The combination of skeletal muscle tissue and artificial elements promotes diverse innovative solutions of interest in both the biomedical field and the development of biohybrid actuators. However, current bioengineering approaches do not fully recreate the complex fascicle-like hierarchical organization of skeletal muscle, impacting on the muscle maturation process due to a lack of oxygen and nutrients supply in the scaffold inner regions. Here we explored co-axial 3D bioprinting as a strategy towards overcoming this challenge, creating individual/non-fused filaments with controlled thickness that present a fascicle-like organization. Compared to conventional 3D-bioprinting, where cell-laden bioink is disposed by a single syringe, our Pluronic-assisted co-axial 3D-bioprinting system (PACA-3D) creates a physical confinement of the bioink during the extrusion process, effectively obtaining thin and independent printed fibers with controlled shape. Fabrication of skeletal muscle-based actuators with PACA-3D resulted in improved cell differentiation, obtaining stronger bioactuators with increased force output when compared to bioactuators fabricated by conventional 3D bioprinting. The versatility of our technology has been demonstrated using different biomaterials, showing its potential to develop more complex biohybrid tissue-based architectures with improved functionality.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142227036","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-11DOI: 10.1101/2024.09.06.611636
Pouria Fattahi, Mousa Younesi, Won Dong Lee, Keumrai Whang, Taewook Kang, Joshua D Rabinowitz, Lauren M Aleksunes, Dan Dongeun Huh
Exposure of pregnant women to toxic metals is an environmental health issue associated with various pregnancy complications. Efforts to advance our biological understanding of this problem and mitigate its adverse effects, however, have been challenged by ethical concerns of human subject research during pregnancy. Here, we present an alternative approach that leverages the design flexibility, controllability, and scalability of bioengineered human reproductive tissues to enable experimental simulation and in-depth investigation of placental exposure to environmental metals in maternal circulation. Central to this method is an in vitro analog of the maternal-fetal interface and its dynamic tissue-specific environment constructed using primary human placental cells grown in a microengineered device. Using cadmium as a representative toxicant, we demonstrate the proof-of-concept of emulating the human placental barrier subjected to the flow of cadmium-containing maternal blood to show how this model can be used to examine adverse biological responses and impaired tissue function on both the maternal and fetal sides. Moreover, we present a mechanistic study of maternal-to-fetal cadmium transport in this system to reveal that efflux membrane transporters expressed by trophoblasts may play an important protective role against cadmium-induced toxicity. Finally, we describe metabolomic analysis of our microphysiological system to demonstrate the feasibility of discovering metabolic biomarkers that may potentially be useful for detection and monitoring of cadmium-induced placental dysfunction.
{"title":"A bioengineered model of human placental exposure to environmental metals during pregnancy","authors":"Pouria Fattahi, Mousa Younesi, Won Dong Lee, Keumrai Whang, Taewook Kang, Joshua D Rabinowitz, Lauren M Aleksunes, Dan Dongeun Huh","doi":"10.1101/2024.09.06.611636","DOIUrl":"https://doi.org/10.1101/2024.09.06.611636","url":null,"abstract":"Exposure of pregnant women to toxic metals is an environmental health issue associated with various pregnancy complications. Efforts to advance our biological understanding of this problem and mitigate its adverse effects, however, have been challenged by ethical concerns of human subject research during pregnancy. Here, we present an alternative approach that leverages the design flexibility, controllability, and scalability of bioengineered human reproductive tissues to enable experimental simulation and in-depth investigation of placental exposure to environmental metals in maternal circulation. Central to this method is an in vitro analog of the maternal-fetal interface and its dynamic tissue-specific environment constructed using primary human placental cells grown in a microengineered device. Using cadmium as a representative toxicant, we demonstrate the proof-of-concept of emulating the human placental barrier subjected to the flow of cadmium-containing maternal blood to show how this model can be used to examine adverse biological responses and impaired tissue function on both the maternal and fetal sides. Moreover, we present a mechanistic study of maternal-to-fetal cadmium transport in this system to reveal that efflux membrane transporters expressed by trophoblasts may play an important protective role against cadmium-induced toxicity. Finally, we describe metabolomic analysis of our microphysiological system to demonstrate the feasibility of discovering metabolic biomarkers that may potentially be useful for detection and monitoring of cadmium-induced placental dysfunction.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142216590","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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