RCSB Protein Data Bank: powerful new tools for exploring 3D structures of biological macromolecules for basic and applied research and education in fundamental biology, biomedicine, biotechnology, bioengineering and energy sciences.

The Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB), the US data center for the global PDB archive and a founding member of the Worldwide Protein Data Bank partnership, serves tens of thousands of data depositors in the Americas and Oceania and makes 3D macromolecular structure data available at no charge and without restrictions to millions of RCSB.org users around the world, including >660 000 educators, students and members of the curious public using PDB101.RCSB.org. PDB data depositors include structural biologists using macromolecular crystallography, nuclear magnetic resonance spectroscopy, 3D electron microscopy and micro-electron diffraction. PDB data consumers accessing our web portals include researchers, educators and students studying fundamental biology, biomedicine, biotechnology, bioengineering and energy sciences. During the past 2 years, the research-focused RCSB PDB web portal (RCSB.org) has undergone a complete redesign, enabling improved searching with full Boolean operator logic and more facile access to PDB data integrated with >40 external biodata resources. New features and resources are described in detail using examples that showcase recently released structures of SARS-CoV-2 proteins and host cell proteins relevant to understanding and addressing the COVID-19 global pandemic.

Determining the pharmacologic window of bisphosphonates that mitigates severe injury-induced osteoporosis and muscle calcification, while preserving fracture repair.

Following severe injury, biomineralization is disrupted and limited therapeutic options exist to correct these pathologic changes. This study utilized a clinically relevant murine model of polytrauma including a severe injury with concomitant musculoskeletal injuries to identify when bisphosphonate administration can prevent the paradoxical decrease of biomineralization in bone and increased biomineralization in soft tissues, yet not interfere with musculoskeletal repair. INTRODUCTION: Systemic and intrinsic mechanisms in bone and soft tissues help promote biomineralization to the skeleton, while preventing it in soft tissues. However, severe injury can disrupt this homeostatic biomineralization tropism, leading to adverse patient outcomes due to a paradoxical decrease of biomineralization in bone and increased biomineralization in soft tissues. There remains a need for therapeutics that restore the natural tropism of biomineralization in severely injured patients. Bisphosphonates can elicit potent effects on biomineralization, though with variable impact on musculoskeletal repair. Thus, a critical clinical question remains as to the optimal time to initiate bisphosphonate therapy in patients following a polytrauma, in which bone and muscle are injured in combination with a severe injury, such as a burn. METHODS: To test the hypothesis that the dichotomous effects of bisphosphonates are dependent upon the time of administration relative to the ongoing biomineralization in reparative bone and soft tissues, this study utilized murine models of isolated injury or polytrauma with a severe injury, in conjunction with sensitive, longitudinal measure of musculoskeletal repair. RESULTS: This study demonstrated that if administered at the time of injury, bisphosphonates prevented severe injury-induced bone loss and soft tissue calcification, but did not interfere with bone repair or remodeling. However, if administered between 7 and 21 days post-injury, bisphosphonates temporally and spatially localized to sites of active biomineralization, leading to impaired fracture callus remodeling and permanence of soft tissue calcification. CONCLUSION: There is a specific pharmacologic window following polytrauma that bisphosphonates can prevent the consequences of dysregulated biomineralization, yet not impair musculoskeletal regeneration.

Bedside estimates of dead space using end-tidal CO2 are independently associated with mortality in ARDS.

PURPOSE: In acute respiratory distress syndrome (ARDS), dead space fraction has been independently associated with mortality. We hypothesized that early measurement of the difference between arterial and end-tidal CO2 (arterial-ET difference), a surrogate for dead space fraction, would predict mortality in mechanically ventilated patients with ARDS. METHODS: We performed two separate exploratory analyses. We first used publicly available databases from the ALTA, EDEN, and OMEGA ARDS Network trials (N = 124) as a derivation cohort to test our hypothesis. We then performed a separate retrospective analysis of patients with ARDS using University of Chicago patients (N = 302) as a validation cohort. RESULTS: The ARDS Network derivation cohort demonstrated arterial-ET difference, vasopressor requirement, age, and APACHE III to be associated with mortality by univariable analysis. By multivariable analysis, only the arterial-ET difference remained significant (P = 0.047). In a separate analysis, the modified Enghoff equation ((PaCO2-PETCO2)/PaCO2) was used in place of the arterial-ET difference and did not alter the results. The University of Chicago cohort found arterial-ET difference, age, ventilator mode, vasopressor requirement, and APACHE II to be associated with mortality in a univariate analysis. By multivariable analysis, the arterial-ET difference continued to be predictive of mortality (P = 0.031). In the validation cohort, substitution of the arterial-ET difference for the modified Enghoff equation showed similar results. CONCLUSION: Arterial to end-tidal CO2 (ETCO2) difference is an independent predictor of mortality in patients with ARDS.

BrainAGE and regional volumetric analysis of a Buddhist monk: a longitudinal MRI case study.

Yongey Mingyur Rinpoche (YMR) is a Tibetan Buddhist monk, and renowned meditation practitioner and teacher who has spent an extraordinary number of hours of his life meditating. The brain-aging profile of this expert meditator in comparison to a control population was examined using a machine learning framework, which estimates "brain-age" from brain imaging. YMR's brain-aging rate appeared slower than that of controls suggesting early maturation and delayed aging. At 41 years, his brain resembled that of a 33-year-old. Specific regional changes did not differentiate YMR from controls, suggesting that the brain-aging differences may arise from coordinated changes spread throughout the gray matter.

The RCSB protein data bank: integrative view of protein, gene and 3D structural information.

The Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB, http://rcsb.org), the US data center for the global PDB archive, makes PDB data freely available to all users, from structural biologists to computational biologists and beyond. New tools and resources have been added to the RCSB PDB web portal in support of a 'Structural View of Biology.' Recent developments have improved the User experience, including the high-speed NGL Viewer that provides 3D molecular visualization in any web browser, improved support for data file download and enhanced organization of website pages for query, reporting and individual structure exploration. Structure validation information is now visible for all archival entries. PDB data have been integrated with external biological resources, including chromosomal position within the human genome; protein modifications; and metabolic pathways. PDB-101 educational materials have been reorganized into a searchable website and expanded to include new features such as the Geis Digital Archive.

The RCSB Protein Data Bank: views of structural biology for basic and applied research and education.

The RCSB Protein Data Bank (RCSB PDB, http://www.rcsb.org) provides access to 3D structures of biological macromolecules and is one of the leading resources in biology and biomedicine worldwide. Our efforts over the past 2 years focused on enabling a deeper understanding of structural biology and providing new structural views of biology that support both basic and applied research and education. Herein, we describe recently introduced data annotations including integration with external biological resources, such as gene and drug databases, new visualization tools and improved support for the mobile web. We also describe access to data files, web services and open access software components to enable software developers to more effectively mine the PDB archive and related annotations. Our efforts are aimed at expanding the role of 3D structure in understanding biology and medicine.

Prevalence and management of recurrent respiratory papillomatosis (RRP) in the UK: cross-sectional study.

OBJECTIVES: To estimate the number of patients with recurrent respiratory papillomatosis currently managed in secondary and tertiary health care in the UK and the frequency of its treatment with radiofrequency cold ablation (Coblation™ ). DESIGN: Cross-sectional survey of ENT consultants in the UK with validation using Hospital Episode Statistics (HES) inpatient data. SETTING: Online survey. PARTICIPANTS: ENT consultants in the UK. MAIN OUTCOME MEASURES: Number of recurrent respiratory papillomatosis patients currently managed in acute care in the UK and frequency of use of Coblation. RESULTS: A total of 283 ENT consultants from 128 UK NHS healthcare trusts and health boards completed the online survey. Responses were received from 86% of surveyed organisations, and an estimated 45% of all ENT consultants in UK. The estimated number of recurrent respiratory papillomatosis patients from the cross-sectional survey was 918 (at August 2015) which included 730 patients in England. The number of recurrent respiratory papillomatosis patients in England estimated from Hospital Episode Statistics (2014/15 financial year) was up to 741. A total of 42 Coblation procedures conducted in the UK were identified from the cross-sectional survey; 36 were conducted in England compared with 34 identified from Hospital Episode Statistics. CONCLUSIONS: The numbers of recurrent respiratory papillomatosis patients and Coblation procedures identified in England from a cross-sectional survey and Hospital Episode Statistics were in broad agreement. Our study estimated 1.42 recurrent respiratory papillomatosis patients per 100 000 in the general UK population. We also estimated that Coblation procedures accounted for 3% of interventional treatments conducted in the UK recurrent respiratory papillomatosis population.

Tyrosine-Coordinated P-Cluster in G. diazotrophicus Nitrogenase: Evidence for the Importance of O-Based Ligands in Conformationally Gated Electron Transfer.

The P-cluster is a unique iron-sulfur center that likely functions as a dynamic electron (e(-)) relay site between the Fe-protein and the catalytic FeMo-cofactor in nitrogenase. The P-cluster has been shown to undergo large conformational changes upon 2-e(-) oxidation which entail the coordination of two of the Fe centers to a Ser side chain and a backbone amide N, respectively. Yet, how and if this 2-e(-) oxidized state (P(OX)) is involved in catalysis by nitrogenase is not well established. Here, we present the crystal structures of reduced and oxidized MoFe-protein (MoFeP) from Gluconacetobacter diazotrophicus (Gd), which natively possesses an Ala residue in the position of the Ser ligand to the P-cluster. While reduced Gd-MoFeP is structurally identical to previously characterized counterparts around the FeMo-cofactor, oxidized Gd-MoFeP features an unusual Tyr coordination to its P-cluster along with ligation by a backbone amide nitrogen. EPR analysis of the oxidized Gd-MoFeP P-cluster confirmed that it is a 2-e(-) oxidized, integer-spin species. Importantly, we have found that the sequence positions corresponding to the Ser and Tyr ligands are almost completely covariant among Group I nitrogenases. These findings strongly support the possibility that the P(OX) state is functionally relevant in nitrogenase catalysis and that a hard, O-based anionic ligand serves to stabilize this state in a switchable fashion.

RCSB PDB Mobile: iOS and Android mobile apps to provide data access and visualization to the RCSB Protein Data Bank.

SUMMARY: The Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) resource provides tools for query, analysis and visualization of the 3D structures in the PDB archive. As the mobile Web is starting to surpass desktop and laptop usage, scientists and educators are beginning to integrate mobile devices into their research and teaching. In response, we have developed the RCSB PDB Mobile app for the iOS and Android mobile platforms to enable fast and convenient access to RCSB PDB data and services. Using the app, users from the general public to expert researchers can quickly search and visualize biomolecules, and add personal annotations via the RCSB PDB's integrated MyPDB service. AVAILABILITY AND IMPLEMENTATION: RCSB PDB Mobile is freely available from the Apple App Store and Google Play (http://www.rcsb.org).

What Factors Contribute to Intimate Partner Violence Against Women in Urban, Conflict-Affected Settings? Qualitative Findings from Abidjan, Côte d'Ivoire.

Rapid urbanization is a key driver of the unique set of health risks facing urban populations. One of the most critical health hazards facing urban women is intimate partner violence (IPV). In post-conflict urban areas, women may face an even greater risk of IPV. Yet, few studies have examined the IPV experiences of urban-dwelling, conflict-affected women, including those who have been internally displaced. This study qualitatively examined the social and structural characteristics of the urban environment that contributed to the IPV experiences of women residing in post-conflict Abidjan, Côte d'Ivoire. Ten focus groups were conducted with men and women, both internally displaced (IDPs) and non-displaced. Lack of support networks, changing gender roles, and tensions between traditional gender norms and those of the "modern" city were reported as key contributors to IPV. Urban poverty and with it unemployment, food insecurity, and housing instability also played a role. Finally, IDPs faced heightened vulnerability to IPV as a result of displacement and discrimination. The relationship between economic strains and IPV are similar to other conflict-affected settings, but Abidjan's urban environment presented other unique characteristics contributing to IPV. Understanding these factors is crucial to designing appropriate services for women and for implementing IPV reduction interventions in urban areas. Strengthening formal and informal mechanisms for help-seeking, utilizing multi-modal interventions that address economic stress and challenge inequitable gender norms, as well as tailoring programs specifically for IDPs, are some considerations for IPV program planning focused on conflict-affected women in urban areas.

Evidence for Functionally Relevant Encounter Complexes in Nitrogenase Catalysis.

Nitrogenase is the only enzyme that can convert atmospheric dinitrogen (N2) into biologically usable ammonia (NH3). To achieve this multielectron redox process, the nitrogenase component proteins, MoFe-protein (MoFeP) and Fe-protein (FeP), repeatedly associate and dissociate in an ATP-dependent manner, where one electron is transferred from FeP to MoFeP per association. Here, we provide experimental evidence that encounter complexes between FeP and MoFeP play a functional role in nitrogenase catalysis. The encounter complexes are stabilized by electrostatic interactions involving a positively charged patch on the ß-subunit of MoFeP. Three single mutations (ßAsn399Glu, ßLys400Glu, and ßArg401Glu) in this patch were generated in Azotobacter vinelandii MoFeP. All of the resulting variants displayed decreases in specific catalytic activity, with the ßK400E mutation showing the largest effect. As simulated by the Thorneley-Lowe kinetic scheme, this single mutation lowered the rate constant for FeP-MoFeP association 5-fold. We also found that the ßK400E mutation did not affect the coupling of ATP hydrolysis with electron transfer (ET) between FeP and MoFeP. These data suggest a mechanism where FeP initially forms encounter complexes on the MoFeP ß-subunit surface en route to the ATP-activated, ET-competent complex over the αß-interface.

The RCSB Protein Data Bank: new resources for research and education.

The Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) develops tools and resources that provide a structural view of biology for research and education. The RCSB PDB web site (http://www.rcsb.org) uses the curated 3D macromolecular data contained in the PDB archive to offer unique methods to access, report and visualize data. Recent activities have focused on improving methods for simple and complex searches of PDB data, creating specialized access to chemical component data and providing domain-based structural alignments. New educational resources are offered at the PDB-101 educational view of the main web site such as Author Profiles that display a researcher's PDB entries in a timeline. To promote different kinds of access to the RCSB PDB, Web Services have been expanded, and an RCSB PDB Mobile application for the iPhone/iPad has been released. These improvements enable new opportunities for analyzing and understanding structure data.

Mutational hotspot in the human biotinidase gene causes profound biotinidase deficiency.

Biotinidase deficiency is an autosomal recessive inherited disorder that is characterized by neurological and cutaneous symptoms. Biotinidase-deficient children cannot recycle endogenous biotin, an essential water-soluble B vitamin. Biotin is covalently attached to epsilon-amino groups of lysyl residues of four carboxylases. These carboxylases are subsequently degraded to biocytin (biotin-epsilon-lysine). Biotinidase cleaves biocytin to biotin and lysine, thereby completing the biotin cycle. The symptoms of biotinidase deficiency can be resolved or prevented by treatment with biotin. Therefore, it is important that biotinidase deficiency is diagnosed early so that permanent neurological damage can be prevented. Many states and countries currently perform newborn screening for biotinidase deficiency. We have recently isolated and characterized the cDNA for normal human biotinidase and localized the gene to chromosome 3p25 (ref. 9). We have now identified the first mutation that causes profound biotinidase deficiency. It occurs in a distinct region of the gene that encodes the putative signal peptide. Fifty percent of symptomatic children studied have a 7-bp deletion coupled with a 3-bp insertion in at least one of their alleles of the biotinidase gene. This mutation appears to be a common cause of biotinidase deficiency in symptomatic children.

PFAS in municipal landfill leachate: Occurrence, transformation, and sources.

To understand sources and processes affecting per- and polyfluoroalkyl substances (PFAS), 32 PFAS were measured in landfill leachate from 17 landfills across Washington State in both pre-and post-total oxidizable precursor (TOP) assay samples, using an analytical method that was the precursor to EPA Draft Method 1633. As in other studies, 5:3FTCA was the dominant PFAS in the leachate, suggesting that carpets, textiles, and food packaging were the main sources of PFAS. Total PFAS concentrations (Σ32PFAS) ranged from 61 to 172,976 ng/L and 580-36,122 ng/L in pre-TOP and post-TOP samples, respectively, suggesting that little or no uncharacterized precursors remained in landfill leachate. Furthermore, due to chain-shortening reactions, the TOP assay often resulted in a loss of overall PFAS mass. Positive matrix factorization (PMF) analysis of the combined pre- and post-TOP samples produced five factors that represent sources and processes. Factor 1 consisted primarily of 5:3FTCA (intermediate of 6:2 fluorotelomer degradation and characteristic of landfill leachate), while factor 2 was dominated by PFBS (degradant of C-4 sulfonamide chemistry) and, to a lesser extent, by several PFCAs and 5:3FTCA. Factor 3 consisted primarily of both short-chain PFCAs (end-products of 6:2 fluorotelomer degradation) and PFHxS (derived from C-6 sulfonamide chemistry), while the main component of factor 4 was PFOS (dominant in many environmental media but minor in landfill leachate, perhaps reflecting a production shift from longer to shorter chain PFAS). Factor 5, highly loaded with PFCAs, was dominant in post-TOP samples and therefore represented the oxidation of precursors. Overall, PMF analysis suggests that the TOP assay approximates some redox processes which occur in landfills, including chain-shortening reactions which yield biodegradable products.

Allogeneic Blood or Marrow Transplantation with Post-Transplantation Cyclophosphamide for Peripheral T Cell Lymphoma: The Importance of Graft Source.

The use of post-transplantation cyclophosphamide (PTCy) for graft-versus host-disease (GVHD) prophylaxis has revolutionized allogeneic blood or marrow transplantation (alloBMT), but there is limited published experience in peripheral T cell lymphoma (PTCL). We sought to assess outcomes in patients with PTCL who underwent alloBMT with PTCy. We reviewed the charts of all adult patients age ≥18 years who underwent alloBMT with nonmyeloablative conditioning and PTCy-based GVHD prophylaxis at the Sidney Kimmel Comprehensive Cancer Center between January 2004 and December 2020. Sixty-five patients were identified. The median age was 59 years (range, 24 to 75 years). Lymphoma histology included PTCL not otherwise specified (n = 24), anaplastic lymphoma kinase-negative anaplastic large cell lymphoma (n = 14), angioimmunoblastic T cell lymphoma (n = 7), enteropathy-associated T cell lymphoma (n = 6), hepatosplenic T cell lymphoma (n = 4), and others (n = 10). Eleven patients were in first complete remission (17%); the remaining patients were in first partial remission or underwent salvage therapy to at least PR prior to transplantation. Forty-eight patients underwent alloBMT from a haploidentical related donor (74%), 10 from a fully matched donor (15%), and 7 from a mismatched unrelated donor (11%). All patients received fludarabine, cyclophosphamide, and total body irradiation (TBI). The graft source was bone marrow (BM) in 46 patients (71%) and peripheral blood (PB) in 19 patients (29%); all patients in the BM cohort received 200 cGy TBI, and most patients in the PB cohort (15 of 19) received 400 cGy TBI. GVHD prophylaxis comprised PTCy, mycophenolate mofetil, and a calcineurin inhibitor or sirolimus. With a median follow-up of 2.8 years (range, 290 days to 14.2 years), the 2-year progression-free survival (PFS) for the entire cohort was 49% (95% confidence interval [CI], 38% to 64%), and the 2-year overall survival (OS) was 55% (95% CI, 44% to 69%). Outcomes were significantly improved in those receiving PB compared to those receiving BM, including a 2-year PFS of 79% (95% CI 63% to 100%) versus 39% (95% CI, 27% to 56%), 2-year OS of 84% (95% CI, 69% to 100%) versus 46% (95% CI, 33% to 63%), and 1-year cumulative incidence of relapse of 5% (95% CI, 0 to 16%) versus 33% (95% CI, 19% to 46%), with no difference in GVHD and nonrelapse mortality. AlloBMT with PTCy is safe and well-tolerated in patients with PTCL. Our data suggest that increasing the TBI dose to 400 cGy and using PB allografts may offer improved disease control and better survival outcomes, though additional studies are needed to confirm these findings.

Phantom rivers filter birds and bats by acoustic niche.

Natural sensory environments, despite strong potential for structuring systems, have been neglected in ecological theory. Here, we test the hypothesis that intense natural acoustic environments shape animal distributions and behavior by broadcasting whitewater river noise in montane riparian zones for two summers. Additionally, we use spectrally-altered river noise to explicitly test the effects of masking as a mechanism driving patterns. Using data from abundance and activity surveys across 60 locations, over two full breeding seasons, we find that both birds and bats avoid areas with high sound levels, while birds avoid frequencies that overlap with birdsong, and bats avoid higher frequencies more generally. We place 720 clay caterpillars in willows, and find that intense sound levels decrease foraging behavior in birds. For bats, we deploy foraging tests across 144 nights, consisting of robotic insect-wing mimics, and speakers broadcasting bat prey sounds, and find that bats appear to switch hunting strategies from passive listening to aerial hawking as sound levels increase. Natural acoustic environments are an underappreciated niche axis, a conclusion that serves to escalate the urgency of mitigating human-created noise.

NK cells associate with ALS in a sex- and age-dependent manner.

NK cells are innate immune cells implicated in ALS; whether NK cells impact ALS in a sex- and age-specific manner was investigated. Herein, NK cells were depleted in male and female SOD1G93A ALS mice, survival and neuroinflammation were assessed, and data were stratified by sex. NK cell depletion extended survival in female but not male ALS mice with sex-specific effects on spinal cord microglia. In humans, NK cell numbers, NK cell subpopulations, and NK cell surface markers were examined in prospectively blood collected from subjects with ALS and control subjects; longitudinal changes in these metrics were correlated to revised ALS functional rating scale (ALSFRS-R) slope and stratified by sex and age. Expression of NK cell trafficking and cytotoxicity markers was elevated in subjects with ALS, and changes in CXCR3+ NK cells and 7 trafficking and cytotoxicity markers (CD11a, CD11b, CD38, CX3CR1, NKG2D, NKp30, NKp46) correlated with disease progression. Age affected the associations between ALSFRS-R and markers NKG2D and NKp46, whereas sex impacted the NKp30 association. Collectively, these findings suggest that NK cells contribute to ALS progression in a sex- and age-specific manner and demonstrate that age and sex are critical variables when designing and assessing ALS immunotherapy.

Allogeneic Blood or Marrow Transplantation with Nonmyeloablative Conditioning and High-Dose Cyclophosphamide-Based Graft-versus-Host Disease Prophylaxis for Secondary Central Nervous System Lymphoma.

Secondary central nervous system (CNS) lymphoma is a rare and often fatal complication of non-Hodgkin lymphoma (NHL). Treatment options include radiation therapy, high-dose systemic chemotherapy, intrathecal chemotherapy, and high-dose chemotherapy with autologous stem cell rescue, but outcomes remain poor. Allogeneic blood or marrow transplantation (alloBMT) is widely used in patients with relapsed/refractory systemic NHL. We sought to understand whether a graft-versus-lymphoma effect could maintain remission in CNS disease. We reviewed outcomes in 20 consecutive patients with secondary CNS lymphoma who underwent alloBMT with nonmyeloablative conditioning using fludarabine, cyclophosphamide, and 200 cGy total body irradiation. For graft-versus-host disease prophylaxis, all patients received post-transplantation cyclophosphamide, mycophenolate mofetil, and a calcineurin inhibitor. With a median follow up of 4.1 years, the median overall survival for the entire cohort was not reached. Median progression-free survival was 3.8 years (95% confidence interval [CI], 5.3 months to not reached). The cumulative incidence of relapse was 25% (95% CI, 5% to 45%), and nonrelapse mortality was 30% (95% CI, 5% to 54%) at 4 years. Of the 5 patients who relapsed, 2 were CNS only, 1 was systemic only, and 2 were combined CNS/systemic. The use of alloBMT in CNS lymphoma merits further investigation.

EEG alpha activity reflects attentional demands, and beta activity reflects emotional and cognitive processes.

Two experiments were designed to examine the effects of attentional demands on the electroencephalogram during cognitive and emotional tasks. We found an interaction of task with hemisphere as well as more overall parietal alpha for tasks not requiring attention to the environment, such as mental arithmetic, than for those requiring such attention. Differential hemispheric activation for beta was found most strongly in the temporal areas for emotionally positive or negative tasks and in the parietal areas for cognitive tasks.

Diphtheria toxin subunit active in vitro.

Exposure of diphtheria toxin to dithiothreitol (and similar thiols) resulted in a subunit which was active in catalyzing the adenosine diphosphateribosylation of mammalian aminoacyl-transferase II in the presence of nicotinamide adenine dinucleotide. At the same time there was a marked increase in total ADP-ribosylation activity. A molecule which was apparently identical to the derived subunit in size and activity was detected in partially purified preparations of toxin.