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Microbial biosensors hold great promise for engineering high-performance, field-deployable and affordable detection devices for medical and environmental applications. This review explores recent advances in the field, highlighting new sensing strategies and modalities for whole-cell biosensors as well as the remarkable expansion of microbial cell-free systems. We also discuss improvements in robustness that have enhanced the ability of biosensors to withstand the challenging conditions found in biological samples. However, limitations remain in expanding the detection repertoire, particularly for proteins. We anticipate that the AI-powered revolution in protein design will streamline the engineering of custom-made sensing modules and unlock the full potential of microbial biosensors., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

Cell-free transcription-translation (TXTL) systems expressing genes from linear dsDNA enable the rapid prototyping of genetic devices while avoiding cloning steps. However, repetitive inclusion of a reporter gene is an incompressible cost and sometimes accounts for most of the synthesized DNA length. Here we present reporter systems based on split-GFP systems that reassemble into functional fluorescent proteins and can be used to monitor gene expression in E. coli TXTL. The 135 bp GFP10-11 fragment produces a fluorescent signal comparable to its full-length GFP counterpart when reassembling with its complementary protein synthesized from the 535 bp fragment expressed in TXTL. We show that split reporters can be used to characterize promoter libraries, with data qualitatively comparable to full-length GFP and matching in vivo expression measurements. We also use split reporters as small fusion tags to measure the TXTL protein and peptide production yield. Finally, we generalize our concept by providing a luminescent split reporter based on split-nanoluciferase. The ∼80% gene sequence length reduction afforded by split reporters lowers synthesis costs and liberates space for testing larger devices while producing a reliable output. In the peptide production context, the small size of split reporters compared with full-length GFP is less likely to bias peptide solubility assays. We anticipate that split reporters will facilitate rapid and cost-efficient genetic device prototyping, protein production, and interaction assays.

CRISPR-based DNA editing technologies enable rapid and accessible genome engineering of eukaryotic cells. However, the delivery of genetically encoded CRISPR components remains challenging and sustained Cas9 expression correlates with higher off-target activities, which can be reduced via Cas9-protein delivery. Here we demonstrate that baculovirus, alongside its DNA cargo, can be used to package and deliver proteins to human cells. Using protein-loaded baculovirus (pBV), we demonstrate delivery of Cas9 or base editors proteins, leading to efficient genome and base editing in human cells. By implementing a reversible, chemically inducible heterodimerization system, we show that protein cargoes can selectively and more efficiently be loaded into pBVs (spBVs). Using spBVs we achieved high levels of multiplexed genome editing in a panel of human cell lines. Importantly, spBVs maintain high editing efficiencies in absence of detectable off-targets events. Finally, by exploiting Cas9 protein and template DNA co-delivery, we demonstrate up to 5% site-specific targeted integration of a 1.8 kb heterologous DNA payload using a single spBV in a panel of human cell lines. In summary, we demonstrate that spBVs represent a versatile, efficient and potentially safer alternative for CRISPR applications requiring co-delivery of DNA and protein cargoes., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Adenovirus-derived nanoparticles (ADDomer) comprise 60 copies of adenovirus penton base protein (PBP). ADDomer is thermostable, rendering the storage, transport, and deployment of ADDomer-based therapeutics independent of a cold chain. To expand the scope of ADDomers for new applications, we engineered ADDobodies, representing PBP crown domain, genetically separated from PBP multimerization domain. We inserted heterologous sequences into hyper-variable loops, resulting in monomeric, thermostable ADDobodies expressed at high yields in Escherichia coli. The X-ray structure of an ADDobody prototype validated our design. ADDobodies can be used in ribosome display experiments to select a specific binder against a target, with an enrichment factor of ∼10 4 -fold per round. ADDobodies can be re-converted into ADDomers by genetically reconnecting the selected ADDobody with the PBP multimerization domain from a different species, giving rise to a multivalent nanoparticle, called Chimera, confirmed by a 2.2 Å electron cryo-microscopy structure. Chimera comprises 60 binding sites, resulting in ultra-high, picomolar avidity to the target., Competing Interests: Declaration of interests C.S. and I.B. report shareholding in Halo Therapeutics Ltd. unrelated to this Correspondence. I.B. reports shareholding in Geneva Biotech SARL, unrelated to this Correspondence. I.B. and F.G. report shareholding in Imophoron Ltd., related to this Correspondence. Patents and patent applications have been filed related to ADDomer vaccines and therapeutics (WO2017167988A and EP22191583.8). ADDomer and ADDobody are registered trademarks of Imophoron Ltd., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Due to COVID-19, pandemic preparedness emerges as a key imperative, necessitating new approaches to accelerate development of reagents against infectious pathogens., Methods: Here, we developed an integrated approach combining synthetic, computational and structural methods with in vitro antibody selection and in vivo immunization to design, produce and validate nature-inspired nanoparticle-based reagents against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)., Results: Our approach resulted in two innovations: (i) a thermostable nasal vaccine called ADDoCoV, displaying multiple copies of a SARS-CoV-2 receptor binding motif derived epitope and (ii) a multivalent nanoparticle superbinder, called Gigabody, against SARS-CoV-2 including immune-evasive variants of concern (VOCs). In vitro generated neutralizing nanobodies and electron cryo-microscopy established authenticity and accessibility of epitopes displayed by ADDoCoV. Gigabody comprising multimerized nanobodies prevented SARS-CoV-2 virion attachment with picomolar EC 50 . Vaccinating mice resulted in antibodies cross-reacting with VOCs including Delta and Omicron., Conclusion: Our study elucidates Adenovirus-derived dodecamer (ADDomer)-based nanoparticles for use in active and passive immunization and provides a blueprint for crafting reagents to combat respiratory viral infections., Competing Interests: C.S., K.G. and I.B. report shareholding in Halo Therapeutics Ltd unrelated to this Correspondence. I.B. reports shareholding in Geneva Biotech SARL, unrelated to this correspondence. F.G., J.H. and I.B. report shareholding in Imophoron Ltd, related to this Correspondence. Patents and patent applications have been filed related to ADDomer vaccines and therapeutics (WO2017167988A, EP22191583.8). The other authors do not declare competing interests. ADDomer is a registered trademark of Imophoron Ltd., (© The Author(s) 2023. Published by Oxford University Press on behalf of Antibody Therapeutics. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objective: Evaluate patellofemoral cartilage health, as assessed by quantitative magnetic resonance imaging (qMRI) T 2 relaxation times, 24-months after ACL reconstruction (ACLR) and determine if they were associated with patellofemoral contact forces and knee mechanics during gait 3 months after surgery., Design: Thirty individuals completed motion analysis during overground walking at a self-selected speed 3 months after ACLR. An EMG-driven neuromusculoskeletal model was used to determine muscle forces, which were then used in a previously described model to estimate patellofemoral contact forces. Biomechanical variables of interest included peak patellofemoral contact force, peak knee flexion angle and moment, and walking speed. These same participants underwent a sagittal bilateral T 2 mapping qMRI scan 24-months after surgery. T 2 relaxation times were estimated for both patellar and trochlear cartilage. Paired t-tests were used to compare T 2 relaxation times between limbs while Pearson correlations and linear regressions were utilized to assess the association between the biomechanical variables of interest and T 2 relaxation times., Results: Prolonged involved limb trochlear T 2 relaxation times (vs uninvolved) were present 24-months after surgery, indicating worse cartilage health. No differences were detected in patellar cartilage. Significant negative associations were present within the involved limb for all the biomechanical variables of interest 3 months after ACLR and trochlear T 2 relaxation times at 24-months. No associations were found in patellar cartilage or within the uninvolved limb., Conclusions: Altered involved limb trochlear cartilage health is present 24-months after ACLR and may be related to patellofemoral loading and other walking gait mechanics 3 months after surgery., (Copyright © 2022 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

CRISPR-based precise gene-editing requires simultaneous delivery of multiple components into living cells, rapidly exceeding the cargo capacity of traditional viral vector systems. This challenge represents a major roadblock to genome engineering applications. Here we exploit the unmatched heterologous DNA cargo capacity of baculovirus to resolve this bottleneck in human cells. By encoding Cas9, sgRNA and Donor DNAs on a single, rapidly assembled baculoviral vector, we achieve with up to 30% efficacy whole-exon replacement in the intronic β-actin (ACTB) locus, including site-specific docking of very large DNA payloads. We use our approach to rescue wild-type podocin expression in steroid-resistant nephrotic syndrome (SRNS) patient derived podocytes. We demonstrate single baculovirus vectored delivery of single and multiplexed prime-editing toolkits, achieving up to 100% cleavage-free DNA search-and-replace interventions without detectable indels. Taken together, we provide a versatile delivery platform for single base to multi-gene level genome interventions, addressing the currently unmet need for a powerful delivery system accommodating current and future CRISPR technologies without the burden of limited cargo capacity., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Survivors of the novel coronavirus (COVID-19) experience significant morbidity with reduced physical function and impairments in activities of daily living. The use of in-hospital rehabilitation therapy may reduce long-term impairments., Objective: To determine the frequency of therapy referral and treatment amongst hospitalized COVID-19 patients, assess for disparities in referral and receipt of therapy, and identify potentially modifiable factors contributing to disparities in therapy allocation., Design, Setting and Participants: Retrospective cohort study using data collected from the University of Colorado Health Data Compass data warehouse assessing therapy referral rates and estimated delivery based on available administrative billing., Measurements: Multivariable logistic regression was used to determine the association between sex and/or underrepresented minority race with therapy referral or delivery., Results: Amongst 6239 COVID-19-related hospitalization, a therapy referral was present in 3952 patients (51.9%). Hispanic ethnicity was independently associated with lower odds of receipt of therapy referral (adjusted OR [aOR]: 0.78, 95% confidence interval [CI]: 0.67-0.93, p = .001). Advanced age (aOR: 1.53, 95% CI: 1.46-1.62, p < .001), greater COVID illness severity (aOR for intensive care unit admission: 1.63, 95% CI: 1.37-1.94, p < .01) and hospital stay (aOR: 1.14, 95% CI: 1.12-1.15, p < .01) were positively associated with referral., Conclusions and Relevance: In a cohort of patients hospitalized for COVID-19 across a multicenter healthcare system, we found that referral rates and delivery of physical therapy and/or occupational therapy sessions were significantly reduced for patients of Hispanic identity compared with patients of non-Hispanic, Caucasian identity after adjustment for potential confounding by available demographic and illness severity variables., (© 2022 Society of Hospital Medicine.)

Background: Psychological readiness to return to sport has emerged as an important factor associated with outcomes after anterior cruciate ligament reconstruction (ACLR). Psychological factors are potentially modifiable during the course of rehabilitation, and improving them may lead to better outcomes., Purpose: To determine whether athletes with a positive psychological response after participation in a neuromuscular training and second injury prevention program had better self-reported function and activity outcomes compared with athletes who did not have a meaningful change., Study Design: Cohort study; Level of evidence, 3., Methods: After ACLR and the completion of formal rehabilitation, 66 level I/II athletes completed the following self-reported measures at enrollment (pretraining): the Anterior Cruciate Ligament-Return to Sport after Injury (ACL-RSI) scale, the International Knee Documentation Committee (IKDC) subjective knee form, and the 5 subscales of the Knee injury and Osteoarthritis Outcome Score (KOOS). Participants completed these measures after 10 sessions of agility, plyometric, and progressive strength training and at 1 and 2 years after ACLR. Participants who displayed an increase in the ACL-RSI score from pretraining to posttraining that exceeded the minimal clinically important difference (≥10 points) were defined as having a positive psychological response (responders) to training, and those who did not were defined as nonresponders. A mixed-model analysis of variance was used to determine if group differences in IKDC and KOOS scores existed over the 4 time points (pretraining, posttraining, and the 1- and 2-year follow-ups)., Results: The responders reported better self-reported function compared with the nonresponders, regardless of time, on the IKDC form ( P = .001), KOOS-Sport and Recreation ( P = .014), KOOS-Pain ( P = .007), and KOOS-Symptoms ( P = .002) but not on the KOOS-Quality of Life ( P = .078). Overall, 77% of responders and 67% of nonresponders returned to their previous level of sport by 1 year after ACLR ( P = .358), and 82% of responders and 78% of nonresponders returned to their previous level of sport by 2 years after ACLR ( P = .668)., Conclusion: Ultimately, 59% of the athletes in this study displayed a meaningful improvement in their psychological outlook over the course of the training program. Responders demonstrated persistently better self-reported function at posttraining and at 1 and 2 years after ACLR, but there were no between-group differences in return-to-sport rates.

Gait biomechanics after anterior cruciate ligament (ACL) injury are associated with functional outcomes and the development of posttraumatic knee osteoarthritis. However, biomechanical outcomes between patients treated nonoperatively compared with operatively are not well understood. The primary purpose of this study was to compare knee joint contact forces, angles, and moments during loading response of gait between individuals treated with operative compared with nonoperative management at 5 years after ACL injury. Forty athletes treated operatively and 17 athletes treated nonoperatively completed gait analysis at 5 years after ACL reconstruction or completion of nonoperative rehabilitation. Medial compartment joint contact forces were estimated using a previously validated, patient-specific electromyography-driven musculoskeletal model. Knee joint contact forces, angles, and moments were compared between the operative and nonoperative group using mixed model 2 × 2 analyses of variance. Peak medial compartment contact forces were larger in the involved limb of the nonoperative group (Op: 2.37 ± 0.47 BW, Non-Op: 3.03 ± 0.53 BW; effect size: 1.36). Peak external knee adduction moment was also larger in the involved limb of the nonoperative group (Op: 0.25 ± 0.08 Nm/kg·m, Non-Op: 0.32 ± 0.09 Nm/kg·m; effect size: 0.89). No differences in radiographic tibiofemoral osteoarthritis were present between the operative and nonoperative groups. Overall, participants treated nonoperatively walked with greater measures of medial compartment joint loading than those treated operatively, while sagittal plane group differences were not present. Statement of clinical relevance: The differences in medial knee joint loading at 5 years after operative and nonoperative management of ACL injury may have implications on the development of posttraumatic knee osteoarthritis., (© 2020 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), represents a global crisis. Key to SARS-CoV-2 therapeutic development is unraveling the mechanisms that drive high infectivity, broad tissue tropism, and severe pathology. Our 2.85-angstrom cryo-electron microscopy structure of SARS-CoV-2 spike (S) glycoprotein reveals that the receptor binding domains tightly bind the essential free fatty acid linoleic acid (LA) in three composite binding pockets. A similar pocket also appears to be present in the highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). LA binding stabilizes a locked S conformation, resulting in reduced angiotensin-converting enzyme 2 (ACE2) interaction in vitro . In human cells, LA supplementation synergizes with the COVID-19 drug remdesivir, suppressing SARS-CoV-2 replication. Our structure directly links LA and S, setting the stage for intervention strategies that target LA binding by SARS-CoV-2., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

DNA delivery is at the forefront of current research efforts in gene therapy and synthetic biology. Viral vectors have traditionally dominated the field; however, nonviral delivery systems are increasingly gaining traction. Baculoviruses are arthropod-specific viruses that can be easily engineered and repurposed to accommodate and deliver large sequences of exogenous DNA into mammalian cells, tissues, or ultimately organisms. These synthetic virus-derived nanosystems (SVNs) are safe, readily customized, and can be manufactured at scale. By implementing clustered regularly interspaced palindromic repeats (CRISPR) associated protein (CRISPR/Cas) modalities into this system, we developed SVNs capable of inserting complex DNAs into genomes, at base pair precision. We anticipate a major role for SVNs as an attractive alternative to viral vectors in accelerating genome engineering and gene therapy applications in the future.

The mechanism of knee osteoarthritis development after anterior cruciate ligament injuries is poorly understood. The objective of this study was to evaluate knee gait variables, muscle co-contraction indices and knee joint loading in young subjects with anterior cruciate ligament deficiency (ACLD, n = 36), versus control subjects (n = 12). A validated, electromyography-informed model was used to estimate joint loading. For the involved limb of ACLD subjects versus control, muscle co-contraction indices were higher for the medial (p = 0.018, effect size = 0.93) and lateral (p = 0.028, effect size = 0.83) agonist-antagonist muscle pairs. Despite higher muscle co-contraction, medial compartment contact force was lower for the involved limb, compared to both the uninvolved limb (mean difference = 0.39 body weight, p = 0.009, effect size = 0.70) as well as the control limb (mean difference = 0.57 body weight, p = 0.007, effect size = 1.14). Similar observations were made for total contact force. For involved versus uninvolved limb, the ACLD group demonstrated lower vertical ground reaction force (mean difference = 0.08 body weight, p = 0.010, effect size = 0.70) and knee flexion moment (mean difference = 1.32% body weight * height, p = 0.003, effect size = 0.76), during weight acceptance. These results indicate that high muscle co-contraction does not always result in high knee joint loading, which is thought to be associated with knee osteoarthritis. Long-term follow-up is required to evaluate how gait alterations progress in non-osteoarthritic versus osteoarthritic subjects. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res., (© 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

The objective of the study was to evaluate differences in gait mechanics 5 years after unilateral anterior cruciate ligament reconstruction surgery, for non-osteoarthritic (n = 24) versus osteoarthritic (n = 9) subjects. For the involved knee, the osteoarthritic group demonstrated significantly lower peak knee flexion angles (non-osteoarthritic = 24.3 ± 4.6°, osteoarthritic = 19.1 ± 2.9°, p = 0.01) and peak knee flexion moments (non-osteoarthritic = 5.3 ± 1.2% Body Weight × Height, osteoarthritic = 4.4 ± 1.2% Body Weight × Height, p = 0.05). Differences in peak knee adduction moment approached significance, with a higher magnitude for the osteoarthritic group (non-osteoarthritic = 2.4 ± 0.8% Body Weight × Height, osteoarthritic = 2.9 ± 0.5% Body Weight × Height, p = 0.09). Peak medial compartment joint load was evaluated using electromyography-informed neuromusculoskeletal modeling. Peak medial compartment joint load in the involved knee for the two groups was not different (non-osteoarthritic = 2.4 ± 0.4 Body Weight, osteoarthritic = 2.3 ± 0.6 Body Weight). The results suggest that subjects with dissimilar peak knee moments can have similar peak medial compartment joint load magnitudes. There was no evidence of inter-limb asymmetry for either group. Given the presence of inter-group differences (non-osteoarthritic vs. osteoarthritic) for the involved knee, but an absence of inter-limb asymmetry in either group, it may be necessary to evaluate how symmetry is achieved, over time, and to differentiate between good versus bad inter-limb symmetry, when evaluating knee gait parameters. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:625-633, 2017., (© 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)