CasPEDIA Database: a functional classification system for class 2 CRISPR-Cas enzymes.

CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic and biochemical diversity of these proteins in the public domain poses a barrier for researchers seeking to leverage their activities. We present CasPEDIA (http://caspedia.org), the Cas Protein Effector Database of Information and Assessment, a curated encyclopedia that integrates enzymatic classification for hundreds of different Cas enzymes across 27 phylogenetic groups spanning the Cas9, Cas12 and Cas13 families, as well as evolutionarily related IscB and TnpB proteins. All enzymes in CasPEDIA were annotated with a standard workflow based on their primary nuclease activity, target requirements and guide-RNA design constraints. Our functional classification scheme, CasID, is described alongside current phylogenetic classification, allowing users to search related orthologs by enzymatic function and sequence similarity. CasPEDIA is a comprehensive data portal that summarizes and contextualizes enzymatic properties of widely used Cas enzymes, equipping users with valuable resources to foster biotechnological development. CasPEDIA complements phylogenetic Cas nomenclature and enables researchers to leverage the multi-faceted nucleic-acid targeting rules of diverse Class 2 Cas enzymes.

"Candidatus Nealsonbacteria" Are Likely Biomass Recycling Ectosymbionts of Methanogenic Archaea in a Stable Benzene-Degrading Enrichment Culture.

The Candidate Phyla Radiation (CPR), also referred to as superphylum Patescibacteria, is a very large group of bacteria with no pure culture representatives discovered by 16S rRNA sequencing or genome-resolved metagenomic analyses of environmental samples. Within the CPR, candidate phylum Parcubacteria, previously referred to as OD1, is prevalent in anoxic sediments and groundwater. Previously, we had identified a specific member of the Parcubacteria (referred to as DGGOD1a) as an important member of a methanogenic benzene-degrading consortium. Phylogenetic analyses herein place DGGOD1a within the clade "Candidatus Nealsonbacteria." Because of its persistence over many years, we hypothesized that "Ca. Nealsonbacteria" DGGOD1a must play an important role in sustaining anaerobic benzene metabolism in the consortium. To try to identify its growth substrate, we amended the culture with a variety of defined compounds (pyruvate, acetate, hydrogen, DNA, and phospholipid), as well as crude culture lysate and three subfractions thereof. We observed the greatest (10-fold) increase in the absolute abundance of "Ca. Nealsonbacteria" DGGOD1a only when the consortium was amended with crude cell lysate. These results implicate "Ca. Nealsonbacteria" in biomass recycling. Fluorescence in situ hybridization and cryogenic transmission electron microscope images revealed that "Ca. Nealsonbacteria" DGGOD1a cells were attached to larger archaeal Methanothrix cells. This apparent epibiont lifestyle was supported by metabolic predictions from a manually curated complete genome. This is one of the first examples of bacterial-archaeal episymbiosis and may be a feature of other "Ca. Nealsonbacteria" found in anoxic environments. IMPORTANCE An anaerobic microbial enrichment culture was used to study members of candidate phyla that are difficult to grow in the lab. We were able to visualize tiny "Candidatus Nealsonbacteria" cells attached to a large Methanothrix cell, revealing a novel episymbiosis.

Organic radical emitters: nature of doublet excitons in emissive layers.

Organic radical emitters have received significant attention as a new route to efficient organic light-emitting diodes (OLEDs). The electronic structure of radical emitters allows bypassing the triplet harvesting issue in current OLED devices. However, the nature of doublet excited states remains elusive due to the complex nature of emissive layers. To date, the computational efforts have treated radical carrying materials as isolated entities in the gas phase. However, OLED materials are applied as thin solid films where intermolecular interactions significantly impact optoelectronic properties of the devices. Here, we combine molecular dynamics simulations and quantum chemical calculations to evaluate the effect of emitter-host interactions on the performance of radical-based emissive layers. Results demonstrate that intermolecular interactions remarkably modulate the electronic properties of the radicals in the thin solid films. The doublet excitons of isolated emitters demonstrate a hybrid character of charge-transfer (CT) and local-excitation (LE), while the emitter-host clusters present a significant CT character. Further, the impact of static and dynamic disorders on the hole-electron recombination is studied. Although the host-emitter interactions simultaneously decrease radiative rates and increase non-radiative rates, the latter rates are 100 times smaller than the former rates, allowing internal quantum efficiency to reach 100% for the doublet-based emission process. The results of this study highlight the significant impact of host-emitter interactions on radiative and non-radiative recombination processes and offer guidelines to tune these interactions for advancing radical-based OLEDs.

Unusual biology across a group comprising more than 15% of domain Bacteria.

A prominent feature of the bacterial domain is a radiation of major lineages that are defined as candidate phyla because they lack isolated representatives. Bacteria from these phyla occur in diverse environments and are thought to mediate carbon and hydrogen cycles. Genomic analyses of a few representatives suggested that metabolic limitations have prevented their cultivation. Here we reconstructed 8 complete and 789 draft genomes from bacteria representing >35 phyla and documented features that consistently distinguish these organisms from other bacteria. We infer that this group, which may comprise >15% of the bacterial domain, has shared evolutionary history, and describe it as the candidate phyla radiation (CPR). All CPR genomes are small and most lack numerous biosynthetic pathways. Owing to divergent 16S ribosomal RNA (rRNA) gene sequences, 50-100% of organisms sampled from specific phyla would evade detection in typical cultivation-independent surveys. CPR organisms often have self-splicing introns and proteins encoded within their rRNA genes, a feature rarely reported in bacteria. Furthermore, they have unusual ribosome compositions. All are missing a ribosomal protein often absent in symbionts, and specific lineages are missing ribosomal proteins and biogenesis factors considered universal in bacteria. This implies different ribosome structures and biogenesis mechanisms, and underlines unusual biology across a large part of the bacterial domain.

Identical bacterial populations colonize premature infant gut, skin, and oral microbiomes and exhibit different in situ growth rates.

The initial microbiome impacts the health and future development of premature infants. Methodological limitations have led to gaps in our understanding of the habitat range and subpopulation complexity of founding strains, as well as how different body sites support microbial growth. Here, we used metagenomics to reconstruct genomes of strains that colonized the skin, mouth, and gut of two hospitalized premature infants during the first month of life. Seven bacterial populations, considered to be identical given whole-genome average nucleotide identity of >99.9%, colonized multiple body sites, yet none were shared between infants. Gut-associated Citrobacter koseri genomes harbored 47 polymorphic sites that we used to define 10 subpopulations, one of which appeared in the gut after 1 wk but did not spread to other body sites. Differential genome coverage was used to measure bacterial population replication rates in situ. In all cases where the same bacterial population was detected in multiple body sites, replication rates were faster in mouth and skin compared to the gut. The ability of identical strains to colonize multiple body sites underscores the habit flexibility of initial colonists, whereas differences in microbial replication rates between body sites suggest differences in host control and/or resource availability. Population genomic analyses revealed microdiversity within bacterial populations, implying initial inoculation by multiple individual cells with distinct genotypes. Overall, however, the overlap of strains across body sites implies that the premature infant microbiome can exhibit very low microbial diversity.

Structure of bone morphogenetic protein 9 procomplex.

Bone morphogenetic proteins (BMPs) belong to the TGF-ß family, whose 33 members regulate multiple aspects of morphogenesis. TGF-ß family members are secreted as procomplexes containing a small growth factor dimer associated with two larger prodomains. As isolated procomplexes, some members are latent, whereas most are active; what determines these differences is unknown. Here, studies on pro-BMP structures and binding to receptors lead to insights into mechanisms that regulate latency in the TGF-ß family and into the functions of their highly divergent prodomains. The observed open-armed, nonlatent conformation of pro-BMP9 and pro-BMP7 contrasts with the cross-armed, latent conformation of pro-TGF-ß1. Despite markedly different arm orientations in pro-BMP and pro-TGF-ß, the arm domain of the prodomain can similarly associate with the growth factor, whereas prodomain elements N- and C-terminal to the arm associate differently with the growth factor and may compete with one another to regulate latency and stepwise displacement by type I and II receptors. Sequence conservation suggests that pro-BMP9 can adopt both cross-armed and open-armed conformations. We propose that interactors in the matrix stabilize a cross-armed pro-BMP conformation and regulate transition between cross-armed, latent and open-armed, nonlatent pro-BMP conformations.

Genomic resolution of a cold subsurface aquifer community provides metabolic insights for novel microbes adapted to high CO2 concentrations.

As in many deep underground environments, the microbial communities in subsurface high-CO2 ecosystems remain relatively unexplored. Recent investigations based on single-gene assays revealed a remarkable variety of organisms from little studied phyla in Crystal Geyser (Utah, USA), a site where deeply sourced CO2 -saturated fluids are erupted at the surface. To provide genomic resolution of the metabolisms of these organisms, we used a novel metagenomic approach to recover 227 high-quality genomes from 150 microbial species affiliated with 46 different phylum-level lineages. Bacteria from two novel phylum-level lineages have the capacity for CO2 fixation. Analyses of carbon fixation pathways in all studied organisms revealed that the Wood-Ljungdahl pathway and the Calvin-Benson-Bassham Cycle occurred with the highest frequency, whereas the reverse TCA cycle was little used. We infer that this, and selection for form II RuBisCOs, are adaptions to high CO2 -concentrations. However, many autotrophs can also grow mixotrophically, a strategy that confers metabolic versatility. The assignment of 156 hydrogenases to 90 different organisms suggests that H2 is an important inter-species energy currency even under gaseous CO2 -saturation. Overall, metabolic analyses at the organism level provided insight into the biochemical cycles that support subsurface life under the extreme condition of CO2 saturation.

Genome-Resolved Meta-Omics Ties Microbial Dynamics to Process Performance in Biotechnology for Thiocyanate Degradation.

Remediation of industrial wastewater is important for preventing environmental contamination and enabling water reuse. Biological treatment for one industrial contaminant, thiocyanate (SCN-), relies upon microbial hydrolysis, but this process is sensitive to high loadings. To examine the activity and stability of a microbial community over increasing SCN- loadings, we established and operated a continuous-flow bioreactor fed increasing loadings of SCN-. A second reactor was fed ammonium sulfate to mimic breakdown products of SCN-. Biomass was sampled from both reactors for metagenomics and metaproteomics, yielding a set of genomes for 144 bacteria and one rotifer that constituted the abundant community in both reactors. We analyzed the metabolic potential and temporal dynamics of these organisms across the increasing loadings. In the SCN- reactor, Thiobacillus strains capable of SCN- degradation were highly abundant, whereas the ammonium sulfate reactor contained nitrifiers and heterotrophs capable of nitrate reduction. Key organisms in the SCN- reactor expressed proteins involved in SCN- degradation, sulfur oxidation, carbon fixation, and nitrogen removal. Lower performance at higher loadings was linked to changes in microbial community composition. This work provides an example of how meta-omics can increase our understanding of industrial wastewater treatment and inform iterative process design and development.

Critical biogeochemical functions in the subsurface are associated with bacteria from new phyla and little studied lineages.

Nitrogen, sulfur and carbon fluxes in the terrestrial subsurface are determined by the intersecting activities of microbial community members, yet the organisms responsible are largely unknown. Metagenomic methods can identify organisms and functions, but genome recovery is often precluded by data complexity. To address this limitation, we developed subsampling assembly methods to re-construct high-quality draft genomes from complex samples. We applied these methods to evaluate the interlinked roles of the most abundant organisms in biogeochemical cycling in the aquifer sediment. Community proteomics confirmed these activities. The eight most abundant organisms belong to novel lineages, and two represent phyla with no previously sequenced genome. Four organisms are predicted to fix carbon via the Calvin-Benson-Bassham, Wood-Ljungdahl or 3-hydroxyproprionate/4-hydroxybutarate pathways. The profiled organisms are involved in the network of denitrification, dissimilatory nitrate reduction to ammonia, ammonia oxidation and sulfate reduction/oxidation, and require substrates supplied by other community members. An ammonium-oxidizing Thaumarchaeote is the most abundant community member, despite low ammonium concentrations in the groundwater. This organism likely benefits from two other relatively abundant organisms capable of producing ammonium from nitrate, which is abundant in the groundwater. Overall, dominant members of the microbial community are interconnected through exchange of geochemical resources.

Through-space (19)F-(19)F spin-spin coupling in ortho-fluoro Z-azobenzene.

We report through-space (TS) (19)F-(19)F coupling for ortho-fluoro-substituted Z-azobenzenes. The magnitude of the TS-coupling constant ((TS) JFF ) ranged from 2.2-5.9 Hz. Using empirical formulas reported in the literature, these coupling constants correspond to non-bonded F-F distances (dFF) of 3.0-3.5 Å. These non-bonded distances are significantly smaller than those determined by X-ray crystallography or density functional theory, which argues that simple models of (19)F-(19)F TS spin-spin coupling solely based dFF are not applicable. (1)H, (13)C and (19)F data are reported for both the E and Z isomers of ten fluorinated azobenzenes. Density functional theory [B3YLP/6-311++G(d,p)] was used to calculate (19) F chemical shifts, and the calculated values deviated 0.3-10.0 ppm compared with experimental values.

Comparison of environmental and isolate Sulfobacillus genomes reveals diverse carbon, sulfur, nitrogen, and hydrogen metabolisms.

BACKGROUND: Bacteria of the genus Sulfobacillus are found worldwide as members of microbial communities that accelerate sulfide mineral dissolution in acid mine drainage environments (AMD), acid-rock drainage environments (ARD), as well as in industrial bioleaching operations. Despite their frequent identification in these environments, their role in biogeochemical cycling is poorly understood. RESULTS: Here we report draft genomes of five species of the Sulfobacillus genus (AMDSBA1-5) reconstructed by cultivation-independent sequencing of biofilms sampled from the Richmond Mine (Iron Mountain, CA). Three of these species (AMDSBA2, AMDSBA3, and AMDSBA4) have no cultured representatives while AMDSBA1 is a strain of S. benefaciens, and AMDSBA5 a strain of S. thermosulfidooxidans. We analyzed the diversity of energy conservation and central carbon metabolisms for these genomes and previously published Sulfobacillus genomes. Pathways of sulfur oxidation vary considerably across the genus, including the number and type of subunits of putative heterodisulfide reductase complexes likely involved in sulfur oxidation. The number and type of nickel-iron hydrogenase proteins varied across the genus, as does the presence of different central carbon pathways. Only the AMDSBA3 genome encodes a dissimilatory nitrate reducatase and only the AMDSBA5 and S. thermosulfidooxidans genomes encode assimilatory nitrate reductases. Within the genus, AMDSBA4 is unusual in that its electron transport chain includes a cytochrome bc type complex, a unique cytochrome c oxidase, and two distinct succinate dehydrogenase complexes. CONCLUSIONS: Overall, the results significantly expand our understanding of carbon, sulfur, nitrogen, and hydrogen metabolism within the Sulfobacillus genus.

Feasibility and Favorable Responses Following Investigational CAR T-Cell Therapy for Relapsed and Refractory Infant ALL.

Infants with B-cell acute lymphoblastic leukemia (B-ALL) continue to have significantly worse outcomes compared to older children with B-ALL, and those with relapsed or refractory (R/R) infant ALL have especially dismal outcomes with conventional treatment. CD19-targeting chimeric antigen receptor (CAR) T-cell therapy has demonstrated remarkable success in the treatment of R/R childhood B-ALL, though the majority of reports have been in non-infant patients. Barriers to the successful implementation of CAR T-cell therapy in infant B-ALL include challenges related to apheresis, product manufacturing and disease-specific considerations such as lineage switch. We describe our experience utilizing two experimental CD19-CAR T-cell products, SCRI-CAR19 or SCRI-CAR19x22, for 19 patients with R/R infant B-ALL enrolled on three clinical trials. CAR T-cell products were successfully manufactured in 18/19 (94.7%) patients, with a median age of 22.5 months at enrollment (range, 14.5-40.1 months). Sixteen of 17 (94.1%) treated patients achieved a complete remission without detectable minimal residual disease. The 1-year leukemia free survival was 75% and 1-year overall survival was 76.5%, with a median follow up time of 35.8 months (range, 1.7-83.6 months). Cytokine release syndrome (CRS) occurred in 14/17 (82.4%) patients, with only 1 patient experiencing Grade 3 CRS. Neurotoxicity occurred in 2/17 (11.8%) patients with all events ≤ Grade 2. With the successful early clinical experience of CAR T-cell therapy in this population, more systematic evaluation specific to infant ALL is warranted.

Interactions between specific phytoplankton and bacteria affect lake bacterial community succession.

Time-series observations and a phytoplankton manipulation experiment were combined to test the hypothesis that phytoplankton succession effects changes in bacterial community composition. Three humic lakes were sampled weekly May-August and correlations between relative abundances of specific phytoplankton and bacterial operational taxonomic units (OTUs) in each time series were determined. To experimentally characterize the influence of phytoplankton, bacteria from each lake were incubated with phytoplankton from one of the three lakes or no phytoplankton. Following incubation, variation in bacterial community composition explained by phytoplankton treatment increased 65%, while the variation explained by bacterial source decreased 64%. Free-living bacteria explained, on average, over 60% of the difference between phytoplankton and corresponding no-phytoplankton control treatments. Fourteen out of the 101 bacterial OTUs that exhibited positively correlated patterns of abundance with specific algal populations in time-series observations were enriched in mesocosms following incubation with phytoplankton, and one out of 59 negatively correlated bacterial OTUs was depleted in phytoplankton treatments. Bacterial genera enriched in mesocosms containing specific phytoplankton assemblages included Limnohabitans (clade betI-A), Bdellovibrio and Mitsuaria. These results suggest that effects of phytoplankton on certain bacterial populations, including bacteria tracking seasonal changes in algal-derived organic matter, result in correlations between algal and bacterial community dynamics.

Novel CRISPR-Associated Gene-Editing Systems Discovered in Metagenomic Samples Enable Efficient and Specific Genome Engineering.

Development of medicines using gene editing has been hampered by enzymological and immunological impediments. We described previously the discovery and characterization of improved, novel gene-editing systems from metagenomic data. In this study, we substantially advance this work with three such gene-editing systems, demonstrating their utility for cell therapy development. All three systems are capable of reproducible, high-frequency gene editing in primary immune cells. In human T cells, disruption of the T cell receptor (TCR) alpha-chain was induced in >95% of cells, both paralogs of the TCR beta-chain in >90% of cells, and >90% knockout of ß2-microglobulin, TIGIT, FAS, and PDCD1. Simultaneous double knockout of TRAC and TRBC was obtained at a frequency equal to that of the single edits. Gene editing with our systems had minimal effect on T cell viability. Furthermore, we integrate a chimeric antigen receptor (CAR) construct into TRAC (up to ∼60% of T cells), and demonstrate CAR expression and cytotoxicity. We next applied our novel gene-editing tools to natural killer (NK) cells, B cells, hematopoietic stem cells, and induced pluripotent stem cells, generating similarly efficient cell-engineering outcomes including the creation of active CAR-NK cells. Interrogation of our gene-editing systems' specificity reveals a profile comparable with or better than Cas9. Finally, our nucleases lack preexisting humoral and T cell-based immunity, consistent with their sourcing from nonhuman pathogens. In all, we show these new gene-editing systems have the activity, specificity, and translatability necessary for use in cell therapy development.

Novel and Engineered Type II CRISPR Systems from Uncultivated Microbes with Broad Genome Editing Capability.

Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 nucleases have been extensively used in biotechnology and therapeutics. However, many applications are not possible owing to the size, targetability, and potential off-target effects associated with currently known systems. In this study, we identified thousands of CRISPR type II effectors by mining an extensive, genome-resolved metagenomics database encompassing hundreds of thousands of microbial genomes. We developed a high-throughput pipeline that enabled us to predict tracrRNA sequences, to design single guide RNAs, and to demonstrate nuclease activity in vitro for 41 newly described subgroups. Active systems represent an extensive diversity of protein sequences and guide RNA structures and require diverse protospacer adjacent motifs (PAMs) that collectively expand the known targeting capability of current systems. Several nucleases showed activity levels comparable to or significantly higher than SpCas9, despite being smaller in size. In addition, top systems exhibited low levels of off-target editing in mammalian cells, and PAM-interacting domain engineered chimeras further expanded their targetability. These newly discovered nucleases are attractive enzymes for translation into many applications, including therapeutics.

Compact Cas9d and HEARO enzymes for genome editing discovered from uncultivated microbes.

Programmable, RNA-guided nucleases are diverse enzymes that have been repurposed for biotechnological applications. However, to further expand the therapeutic application of these tools there is a need for targetable systems that are small enough to be delivered efficiently. Here, we mined an extensive genome-resolved metagenomics database and identified families of uncharacterized RNA-guided, compact nucleases (between 450 and 1,050 aa). We report that Cas9d, a new CRISPR type II subtype, contains Zinc-finger motifs and high arginine content, features that we also found in nucleases related to HEARO effectors. These enzymes exhibit diverse biochemical characteristics and are broadly targetable. We show that natural Cas9d enzymes are capable of genome editing in mammalian cells with >90% efficiency, and further engineered nickase variants into the smallest base editors active in E. coli and human cells. Their small size, broad targeting potential, and translatability suggest that Cas9d and HEARO systems will enable a variety of genome editing applications.

Divergent activities of osteogenic BMP2, and tenogenic BMP12 and BMP13 independent of receptor binding affinities.

Ectopic expression of recombinant human bone morphogenetic protein 2 (rhBMP2) induces osteogenesis, while ectopic expression of rhBMP12 and rhBMP13 induces the formation of tendon-like tissue. Despite their different in vivo activities, all three ligands bound to the type I bone morphogenic protein receptors (BMPRs), activin receptor-like kinase (ALK)-3 and ALK6, and to the type II BMPRs, activin receptor type-2A, activin receptor type-2B, and BMPR2, with similar affinities. Treatment of C3H10T1/2 cells with rhBMP2 activated SMAD signaling and induced expression of osteoblast markers including osteocalcin mRNA (Ocn). In contrast, treatment with rhBMP12 or rhBMP13 resulted in a dose-dependent induction of a tendon-specific gene (Thbs4) expression with no detectable activation of SMAD 1, 5, and 8. Differential regulation of Thbs4 and Ocn has potential utility as an in vitro biomarker for induction of tenogenic signaling. Such an assay also permits the ability to distinguish between the activities of different BMPs and may prove useful in studies on the molecular mechanisms of BMP tenogenic activity.

Whole-genome sequencing and phenotypic analysis of Bacillus subtilis mutants following evolution under conditions of relaxed selection for sporulation.

Little is known about how genetic variation at the nucleotide level contributes to competitive fitness within species. During a 6,000-generation study of Bacillus subtilis evolved under relaxed selection for sporulation, a new strain, designated WN716, emerged with significantly different colony and cell morphologies; loss of sporulation, competence, acetoin production, and motility; multiple auxotrophies; and increased competitive fitness (H. Maughan and W. L. Nicholson, Appl. Environ. Microbiol. 77:4105-4118, 2011). The genome of WN716 was analyzed by OpGen optical mapping, whole-genome 454 pyrosequencing, and the CLC Genomics Workbench. No large chromosomal rearrangements were found; however, 34 single-nucleotide polymorphisms (SNPs) and +1 frameshifts were identified in WN716 that resulted in amino acid changes in coding sequences of annotated genes, and 11 SNPs were located in intergenic regions. Several classes of genes were affected, including biosynthetic pathways, sporulation, competence, and DNA repair. In several cases, attempts were made to link observed phenotypes of WN716 with the discovered mutations, with various degrees of success. For example, a +1 frameshift was identified at codon 13 of sigW, the product of which (SigW) controls a regulon of genes involved in resistance to bacteriocins and membrane-damaging antibiotics. Consistent with this finding, WN716 exhibited sensitivity to fosfomycin and to a bacteriocin produced by B. subtilis subsp. spizizenii and exhibited downregulation of SigW-dependent genes on a transcriptional microarray, consistent with WN716 carrying a knockout of sigW. The results suggest that propagation of B. subtilis for less than 2,000 generations in a nutrient-rich environment where sporulation is suppressed led to rapid initiation of genomic erosion.

A BMP/Activin A Chimera Induces Posterolateral Spine Fusion in Nonhuman Primates at Lower Concentrations Than BMP-2.

BACKGROUND: Supraphysiologic bone morphogenetic protein (BMP)-2 concentrations are required to induce spinal fusion. In this study, a BMP-2/BMP-6/activin A chimera (BV-265), optimized for BMP receptor binding, delivered in a recombinant human collagen:CDHA [calcium-deficient hydroxyapatite] porous composite matrix (CM) or bovine collagen:CDHA granule porous composite matrix (PCM), engineered for optimal BV-265 retention and guided tissue repair, was compared with BMP-2 delivered in a bovine absorbable collagen sponge (ACS) wrapped around a MASTERGRAFT Matrix (MM) ceramic-collagen rod (ACS:MM) in a nonhuman primate noninstrumented posterolateral fusion (PLF) model. METHODS: In vivo retention of 125I-labeled-BV-265/CM or PCM was compared with 125I-labeled-BMP-2/ACS or BMP-2/buffer in a rat muscle pouch model using scintigraphy. Noninstrumented PLF was performed by implanting CM, BV-265/CM, BV-265/PCM, or BMP-2/ACS:MM across L3-L4 and L5-L6 or L3-L4-L5 decorticated transverse processes in 26 monkeys. Computed tomography (CT) images were acquired at 0, 4, 8, 12, and 24 weeks after surgery, where applicable. Manual palpation, µCT (microcomputed tomography) or nCT (nanocomputed tomography), and histological analysis were performed following euthanasia. RESULTS: Retention of 125I-labeled-BV-265/CM was greater than BV-265/PCM, followed by BMP-2/ACS and BMP-2/buffer. The CM, 0.43 mg/cm3 BMP-2/ACS:MM, and 0.05 mg/cm3 BV-265/CM failed to generate PLFs. The 0.15-mg/cm3 BV-265/CM or 0.075-mg/cm3 BV-265/PCM combinations were partially effective. The 0.25-mg/cm3 BV-265/CM and 0.15 and 0.3-mg/cm3 BV-265/PCM combinations generated successful 2-level PLFs at 12 and 24 weeks. CONCLUSIONS: BV-265/CM or PCM can induce fusion in a challenging nonhuman primate noninstrumented PLF model at substantially lower concentrations than BMP-2/ACS:MM. CLINICAL RELEVANCE: BV-265/CM and PCM represent potential alternatives to induce PLF in humans at substantially lower concentrations than BMP-2/ACS:MM.

Design of Organic Electronic Materials With a Goal-Directed Generative Model Powered by Deep Neural Networks and High-Throughput Molecular Simulations.

In recent years, generative machine learning approaches have attracted significant attention as an enabling approach for designing novel molecular materials with minimal design bias and thereby realizing more directed design for a specific materials property space. Further, data-driven approaches have emerged as a new tool to accelerate the development of novel organic electronic materials for organic light-emitting diode (OLED) applications. We demonstrate and validate a goal-directed generative machine learning framework based on a recurrent neural network (RNN) deep reinforcement learning approach for the design of hole transporting OLED materials. These large-scale molecular simulations also demonstrate a rapid, cost-effective method to identify new materials in OLEDs while also enabling expansion into many other verticals such as catalyst design, aerospace, life science, and petrochemicals.