Foldy: An open-source web application for interactive protein structure analysis.

Foldy is a cloud-based application that allows non-computational biologists to easily utilize advanced AI-based structural biology tools, including AlphaFold and DiffDock. With many deployment options, it can be employed by individuals, labs, universities, and companies in the cloud without requiring hardware resources, but it can also be configured to utilize locally available computers. Foldy enables scientists to predict the structure of proteins and complexes up to 6000 amino acids with AlphaFold, visualize Pfam annotations, and dock ligands with AutoDock Vina and DiffDock. In our manuscript, we detail Foldy's interface design, deployment strategies, and optimization for various user scenarios. We demonstrate its application through case studies including rational enzyme design and analyzing proteins with domains of unknown function. Furthermore, we compare Foldy's interface and management capabilities with other open and closed source tools in the field, illustrating its practicality in managing complex data and computation tasks. Our manuscript underlines the benefits of Foldy as a day-to-day tool for life science researchers, and shows how Foldy can make modern tools more accessible and efficient.

Characterization and Diversification of AraC/XylS Family Regulators Guided by Transposon Sequencing.

In this study, we explored the development of engineered inducible systems. Publicly available data from previous transposon sequencing assays were used to identify regulators of metabolism in Pseudomonas putida KT2440. For AraC family regulators (AFRs) represented in these data, we posited AFR/promoter/inducer groupings. Twelve promoters were characterized for a response to their proposed inducers in P. putida, and the resultant data were used to create and test nine two-plasmid sensor systems in Escherichia coli. Several of these were further developed into a palette of single-plasmid inducible systems. From these experiments, we observed an unreported inducer response from a previously characterized AFR, demonstrated that the addition of a P. putida transporter improved the sensor dynamics of an AFR in E. coli, and identified an uncharacterized AFR with a novel potential inducer specificity. Finally, targeted mutations in an AFR, informed by structural predictions, enabled the further diversification of these inducible plasmids.

Automated Platform for the Plasmid Construction Process.

There is a growing need for applications capable of handling large synthesis biology experiments. At the core of synthetic biology is the process of cloning and manipulating DNA as plasmids. Here, we report the development of an application named DNAda capable of writing automation instructions for any given DNA construct design generated by the J5 DNA assembly program. We also describe the automation pipeline and several useful features. The pipeline is particularly useful for the construction of combinatorial DNA assemblies. Furthermore, we demonstrate the platform by constructing a library of polyketide synthase parts, which includes 120 plasmids ranging in size from 7 to 14 kb from 4 to 7 DNA fragments.

REC protein family expansion by the emergence of a new signaling pathway.

IMPORTANCE: We explore when and why large classes of proteins expand into new sequence space. We used an unsupervised machine learning approach to observe the sequence landscape of REC domains of bacterial response regulator proteins. We find that within-gene recombination can switch effector domains and, consequently, change the regulatory context of the duplicated protein.

Module-Based Polyketide Synthase Engineering for de Novo Polyketide Biosynthesis.

Polyketide retrobiosynthesis, where the biosynthetic pathway of a given polyketide can be reversibly engineered due to the colinearity of the polyketide synthase (PKS) structure and function, has the potential to produce millions of organic molecules. Mixing and matching modules from natural PKSs is one of the routes to produce many of these molecules. Evolutionary analysis of PKSs suggests that traditionally used module boundaries may not lead to the most productive hybrid PKSs and that new boundaries around and within the ketosynthase domain may be more active when constructing hybrid PKSs. As this is still a nascent area of research, the generality of these design principles based on existing engineering efforts remains inconclusive. Recent advances in structural modeling and synthetic biology present an opportunity to accelerate PKS engineering by re-evaluating insights gained from previous engineering efforts with cutting edge tools.

Maximizing Heterologous Expression of Engineered Type I Polyketide Synthases: Investigating Codon Optimization Strategies.

Type I polyketide synthases (T1PKSs) hold enormous potential as a rational production platform for the biosynthesis of specialty chemicals. However, despite great progress in this field, the heterologous expression of PKSs remains a major challenge. One of the first measures to improve heterologous gene expression can be codon optimization. Although controversial, choosing the wrong codon optimization strategy can have detrimental effects on the protein and product levels. In this study, we analyzed 11 different codon variants of an engineered T1PKS and investigated in a systematic approach their influence on heterologous expression in Corynebacterium glutamicum, Escherichia coli, and Pseudomonas putida. Our best performing codon variants exhibited a minimum 50-fold increase in PKS protein levels, which also enabled the production of an unnatural polyketide in each of these hosts. Furthermore, we developed a free online tool (https://basebuddy.lbl.gov) that offers transparent and highly customizable codon optimization with up-to-date codon usage tables. In this work, we not only highlight the significance of codon optimization but also establish the groundwork for the high-throughput assembly and characterization of PKS pathways in alternative hosts.

Biosensor Guided Polyketide Synthases Engineering for Optimization of Domain Exchange Boundaries.

Type I modular polyketide synthases (PKSs) are multi-domain enzymes functioning like assembly lines. Many engineering attempts have been made for the last three decades to replace, delete and insert new functional domains into PKSs to produce novel molecules. However, inserting heterologous domains often destabilize PKSs, causing loss of activity and protein misfolding. To address this challenge, here we develop a fluorescence-based solubility biosensor that can quickly identify engineered PKSs variants with minimal structural disruptions. Using this biosensor, we screen a library of acyltransferase (AT)-exchanged PKS hybrids with randomly assigned domain boundaries, and we identify variants that maintain wild type production levels. We then probe each position in the AT linker region to determine how domain boundaries influence structural integrity and identify a set of optimized domain boundaries. Overall, we have successfully developed an experimentally validated, high-throughput method for making hybrid PKSs that produce novel molecules.

Expanding Extender Substrate Selection for Unnatural Polyketide Biosynthesis by Acyltransferase Domain Exchange within a Modular Polyketide Synthase.

Modular polyketide synthases (PKSs) are polymerases that employ α-carboxyacyl-CoAs as extender substrates. This enzyme family contains several catalytic modules, where each module is responsible for a single round of polyketide chain extension. Although PKS modules typically use malonyl-CoA or methylmalonyl-CoA for chain elongation, many other malonyl-CoA analogues are used to diversify polyketide structures in nature. Previously, we developed a method to alter an extension substrate of a given module by exchanging an acyltransferase (AT) domain while maintaining protein folding. Here, we report in vitro polyketide biosynthesis by 13 PKSs (the wild-type PKS and 12 AT-exchanged PKSs with unusual ATs) and 14 extender substrates. Our ∼200 in vitro reactions resulted in 13 structurally different polyketides, including several polyketides that have not been reported. In some cases, AT-exchanged PKSs produced target polyketides by >100-fold compared to the wild-type PKS. These data also indicate that most unusual AT domains do not incorporate malonyl-CoA and methylmalonyl-CoA but incorporate various rare extender substrates that are equal to in size or slightly larger than natural substrates. We developed a computational workflow to predict the approximate AT substrate range based on active site volumes to support the selection of ATs. These results greatly enhance our understanding of rare AT domains and demonstrate the benefit of using the proposed PKS engineering strategy to produce novel chemicals in vitro.

ClusterCAD 2.0: an updated computational platform for chimeric type I polyketide synthase and nonribosomal peptide synthetase design.

Megasynthase enzymes such as type I modular polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) play a central role in microbial chemical warfare because they can evolve rapidly by shuffling parts (catalytic domains) to produce novel chemicals. If we can understand the design rules to reshuffle these parts, PKSs and NRPSs will provide a systematic and modular way to synthesize millions of molecules including pharmaceuticals, biomaterials, and biofuels. However, PKS and NRPS engineering remains difficult due to a limited understanding of the determinants of PKS and NRPS fold and function. We developed ClusterCAD to streamline and simplify the process of designing and testing engineered PKS variants. Here, we present the highly improved ClusterCAD 2.0 release, available at https://clustercad.jbei.org. ClusterCAD 2.0 boasts support for PKS-NRPS hybrid and NRPS clusters in addition to PKS clusters; a vastly enlarged database of curated PKS, PKS-NRPS hybrid, and NRPS clusters; a diverse set of chemical 'starters' and loading modules; the new Domain Architecture Cluster Search Tool; and an offline Jupyter Notebook workspace, among other improvements. Together these features massively expand the chemical space that can be accessed by enzymes engineered with ClusterCAD.

The pterional approach for the surgical resection of an orbital cholesteatoma: a case report.

BACKGROUND: Cholesteatomas are benign tumors mainly composed of cholesterol crystals that rarely arise within the orbit. However, orbital cholesteatomas require a complete surgical resection due to their recidivating potential. Transcranial approaches offering a broad surgical exposure of the orbital cavity have been scarcely used for the management of these tumors. Here, we provide evidence of the benefits of the pterional craniotomy for the surgical resection of orbital tumors by sharing our experience in the surgical management of a cholesteatoma of the superotemporal orbital wall. CASE PRESENTATION: A 45-year-old Hispanic man with a 2-year history of progressive proptosis of the left eye attended to our center complaining of diplopia and migraine. At his arrival, physical examination revealed ptosis, palpebral edema, and exophthalmos of the left eye, as well as the abolishment of the ipsilateral photomotor and consensual responses. Fundoscopy showed mild optic atrophy, whereas a T2-weighted magnetic resonance imaging (MRI) of the head showed a hyperintense mass arising at the superotemporal wall of the orbit that was displacing the eyeball. The tumor was resected using a pterional craniotomy without postoperative complications. The histopathological analysis of the tumor revealed a cholesteatoma. The patient recovered the functionality of the left eye with no visual sensitive deficits nor tumor recurrence 1 year after the surgery. CONCLUSIONS: Our results support the use of the pterional craniotomy as a safe procedure for the surgical resection of cholesteatomas arising at the superotemporal walls of the orbit, with low postoperative morbidity.

Beneficial effects of a multidomain cognitive rehabilitation program for traumatic brain injury-associated diffuse axonal injury: a case report.

BACKGROUND: Neuropsychological rehabilitation is a crucial component of medical care for patients with diffuse axonal injury (DAI). However, current cognitive intervention programs directed to favor the training of specific domains individually have shown controversial results. Here, we evaluated the effectiveness of a neuropsychological rehabilitation program directed to favor training of attention, memory, visuospatial abilities, and executive functioning together in a patient with severe traumatic brain injury (TBI)-associated DAI. CASE PRESENTATION: A 26-year-old Hispanic woman with a recent history of a severe TBI attended our center complaining of memory problems, dysarthria, and difficulty in planning. A comprehensive cognitive assessment revealed dysfunction in sustained, selective, and divided attention, alterations in memory, planning, and organization of executive behavior, as well as impairment of visuospatial cognitive functions. The patient underwent a 24-week neuropsychological rehabilitation program directed to favor attention, memory, visuospatial abilities, and executive functioning together. After the cognitive intervention, we observed a better patient's performance in tasks requiring sustained, selective, and divided attention, improvement of encoding and retrieval memory problems, use of spatial relationships, planning, and organization of behavior skills. We also observed generalization effects on other domains, such as learning, mental flexibility, inhibition functions, and language. CONCLUSIONS: In conclusion, our results suggest that neuropsychological rehabilitation programs favoring multiple domains together are useful in reestablishing cognitive deficits in patients with severe DAI.

Neurological Aspects of SARS-CoV-2 Infection: Mechanisms and Manifestations.

The human infection of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a public health emergency of international concern that has caused more than 16.8 million new cases and 662,000 deaths as of July 30, 2020. Although coronavirus disease 2019 (COVID-19), which is associated with this virus, mainly affects the lungs, recent evidence from clinical and pathological studies indicates that this pathogen has a broad infective ability to spread to extrapulmonary tissues, causing multiorgan failure in severely ill patients. In this regard, there is increasing preoccupation with the neuroinvasive potential of SARS-CoV-2 due to the observation of neurological manifestations in COVID-19 patients. This concern is also supported by the neurotropism previously documented in other human coronaviruses, including the 2002-2003 SARS-CoV-1 outbreak. Hence, in the current review article, we aimed to summarize the spectrum of neurological findings associated with COVID-19, which include signs of peripheral neuropathy, myopathy, olfactory dysfunction, meningoencephalitis, Guillain-Barré syndrome, and neuropsychiatric disorders. Furthermore, we analyze the mechanisms underlying such neurological sequela and discuss possible therapeutics for patients with neurological findings associated with COVID-19. Finally, we describe the host- and pathogen-specific factors that determine the tissue tropism of SARS-CoV-2 and possible routes employed by the virus to invade the nervous system from a pathophysiological and molecular perspective. In this manner, the current manuscript contributes to increasing the current understanding of the neurological aspects of COVID-19 and the impact of the current pandemic on the neurology field.

Yucatán carnivorans shed light on the Great American Biotic Interchange.

The Great American Biotic Interchange is considered to be a punctuated process, primarily occurring during four major pulses that began approximately 2.5 Ma. Central America and southeastern Mexico have a poor fossil record of this dynamic faunal history due to tropical climates. Exploration of submerged caves in the Yucatán, particularly the natural trap Hoyo Negro, is exposing a rich and remarkably well-preserved late Pleistocene fauna. Radiometric dates on megafauna range from approximately 38 400-12 850 cal BP, and extinct species include the ursid Arctotherium wingei and canid Protocyon troglodytes. Both genera were previously thought to be indigenous to and confined to South America and appear to represent an instance of large placental mammals, descended from North American progenitors, migrating back north across the Panama Isthmus. This discovery expands the distribution of these carnivorans greater than 2000 km outside South America. Their presence along with a diverse sloth assemblage suggests a more complex history of these organisms in Middle America. We suggest that landscape and ecological changes caused by latest Pleistocene glaciation supported an interchange pulse that included A. wingei, P. troglodytes and Homo sapiens.