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BACKGROUND: Disparities in survival and clinical outcomes between African American and White patients with breast cancer (BC) are well documented, but African American patients have not been well represented in randomized clinical trials of CDK4/6 inhibitors. Real-world studies can provide evidence for effective treatment strategies for underreported patient populations. PATIENTS AND METHODS: This retrospective analysis of African American patients with HR+/HER2- metastatic breast cancer (mBC) from the Flatiron Health longitudinal database evaluated treatments for patients with BC in routine clinical practice in the US. Patients initiated first-line therapy with palbociclib plus an aromatase inhibitor (AI) or AI alone between February 2015 and March 2020. Outcomes assessed included overall survival (OS) and real-world progression-free survival (rwPFS) until September 2020. RESULTS: Of 270 eligible patients, 127 (median age 64 years) were treated with palbociclib + AI, and 143 (median age 68 years) were treated with an AI. Median follow-up was 24.0 months for palbociclib + AI and 18.2 months for AI-treated patients. Median OS was not reached (NR; 95% CI, 38.2-NR) in the palbociclib + AI group versus 28.2 months (95% CI, 19.2-52.8) in the AI group (adjusted HR, 0.56; 95% CI, 0.36-0.89; P = .013). Median rwPFS was 18.0 months (95% CI, 12.4-26.7) in the palbociclib + AI group and 10.5 months (95% CI, 7.0-13.4) in the AI group (adjusted HR, 0.74; 95% CI, 0.47-1.17; P = .199). CONCLUSION: This comparative analysis of palbociclib + AI versus AI alone indicates that palbociclib combined with endocrine therapy in the first line is associated with improved effectiveness for African American patients with HR+/HER2- mBC in real-world settings. TRIAL NUMBER: NCT05361655.

Environmental epidemiology has proven critical to study various associations between environmental exposures and adverse human health effects. However, there is a perception that it often does not sufficiently inform quantitative risk assessment. To help address this concern, in 2017, the Health and Environmental Sciences Institute initiated a project engaging the epidemiology, exposure science, and risk assessment communities with tripartite representation from government agencies, industry, and academia, in a dialogue on the use of environmental epidemiology for quantitative risk assessment and public health decision making. As part of this project, four meetings attended by experts in epidemiology, exposure science, toxicology, statistics, and risk assessment, as well as one additional meeting engaging funding agencies, were organized to explore incentives and barriers to realizing the full potential of epidemiological data in quantitative risk assessment. A set of questions was shared with workshop participants prior to the meetings, and two case studies were used to support the discussion. Five key ideas emerged from these meetings as areas of desired improvement to ensure that human data can more consistently become an integral part of quantitative risk assessment: 1) reducing confirmation and publication bias, 2) increasing communication with funding agencies to raise awareness of research needs, 3) developing alternative funding channels targeted to support quantitative risk assessment, 4) making data available for reuse and analysis, and 5) developing cross-disciplinary and cross-sectoral interactions, collaborations, and training. We explored and integrated these themes into a roadmap illustrating the need for a multi-stakeholder effort to ensure that epidemiological data can fully contribute to the quantitative evaluation of human health risks, and to build confidence in a reliable decision-making process that leverages the totality of scientific evidence.

BackgroundDepression and anxiety are leading causes of disability worldwide. Though effective treatments exist, depression and anxiety remain undertreated. Blended care psychotherapy, combining the scalability of online interventions with the personalization and engagement of a live therapist, is a promising approach for increasing access to evidence-based care.ObjectivesTo evaluate the effectiveness and individual contribution of two components - i) digital tools and ii) video-based therapist-led sessions - in a blended care CBT-based intervention under real world conditions.MethodsA retrospective cohort design was used to analyze N = 1372 US-based individuals who enrolled in blended care psychotherapy. Of these, at baseline, 761 participants had depression symptoms in the clinical range (based on PHQ-9), and 1254 had anxiety symptoms in the clinical range (based on GAD-7). Participants had access to the program as a mental health benefit offered by their employer. The CBT-based blended care psychotherapy program consisted of regular video sessions with therapists, complemented by digital lessons and digital exercises assigned by the clinician and completed in between sessions. Depression and anxiety levels and clients' treatment engagement were tracked throughout treatment. A 3-level individual growth curve model incorporating time-varying covariates was utilized to examine symptom trajectories of PHQ-9 scores (for those with clinical range of depression at baseline) and GAD-7 scores (for those with clinical range of anxiety at baseline).ResultsOn average, individuals exhibited a significant decline in depression and anxiety symptoms during the initial weeks of treatment (P

Data on real-world effectiveness of cyclin-dependent kinase 4/6 inhibitor combination therapy versus endocrine therapy alone are limited. The Flatiron Health Analytic Database was used to assess overall survival (OS) in patients with hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+/HER2-) metastatic breast cancer (MBC) treated with first-line palbociclib plus an aromatase inhibitor (AI) versus an AI alone in routine US clinical practice. In total, 2888 patients initiated treatment during February 3, 2015-March 31, 2020, with a potential ≥6-month follow-up (cutoff date, September 30, 2020). After stabilized inverse probability treatment weighting, median OS (95% CI) is significantly longer among palbociclib versus AI recipients (49.1 [45.2-57.7] versus 43.2 [37.6-48.0] months; hazard ratio, 0.76 [95% CI, 0.65-0.87]; P

BackgroundBoth the gut microbiota and chronic viral infections have profound effects on host immunity, but interactions between these influences have been only superficially explored. Cytomegalovirus (CMV), for example, infects approximately 80% of people globally and drives significant changes in immune cells. Similarly, certain gut-resident bacteria affect T-cell development in mice and nonhuman primates. It is unknown if changes imposed by CMV on the intestinal microbiome contribute to immunologic effects of the infection.ResultsWe show that rhesus cytomegalovirus (RhCMV) infection is associated with specific differences in gut microbiota composition, including decreased abundance of Firmicutes, and that the extent of microbial change was associated with immunologic changes including the proliferation, differentiation, and cytokine production of CD8+ T cells. Furthermore, RhCMV infection disrupted the relationship between short-chain fatty acid producers and Treg/Th17 balance observed in seronegative animals, showing that some immunologic effects of CMV are due to disruption of previously existing host-microbe relationships.ConclusionsGut microbes have an important influence on health and disease. Diet is known to shape the microbiota, but the influence of concomitant chronic viral infections is unclear. We found that CMV influences gut microbiota composition to an extent that is correlated with immunologic changes in the host. Additionally, pre-existing correlations between immunophenotypes and gut microbes can be subverted by CMV infection. Immunologic effects of CMV infection on the host may therefore be mediated by two different mechanisms involving gut microbiota. Video Abstract.

The contribution of the host immune system to the efficacy of new anti-hepatitis C virus (HCV) drugs is unclear. We undertook a longitudinal prospective study of 33 individuals with chronic HCV treated with combination pegylated IFN-α, ribavirin, and telaprevir/boceprevir. We characterized innate and adaptive immune cells to determine whether kinetics of the host response could predict sustained virologic response (SVR). We show that characteristics of the host immune system present before treatment were correlated with successful therapy. Augmentation of adaptive immune responses during therapy was more impressive among those achieving SVR. Most importantly, active memory T cell proliferation before therapy predicted SVR and was associated with the magnitude of the HCV-specific responses at week 12 after treatment start. After therapy initiation, the most important correlate of success was minimal monocyte activation, as predicted by previous in vitro work. In addition, subjects achieving SVR had increasing expression of the transcription factor T-bet, a driver of Th1 differentiation and cytotoxic effector cell maturation. These results show that host immune features present before treatment initiation predict SVR and eventual development of a higher frequency of functional virus-specific cells in blood. Such host characteristics may also be required for successful vaccine-mediated protection.

The HIV reservoir forming at the earliest stages of infection is likely composed of CCR5+ cells, because these cells are the targets of transmissible virus. Restriction of the CCR5+ reservoir, particularly in the gut, may be needed for subsequent cure attempts. Strategies for killing or depleting CCR5+ cells have been described, but none have been tested in vivo in nonhuman primates, and the extent of achievable depletion from tissues is not known. In this study we investigate the efficacy of two novel cytotoxic treatments for targeting and eliminating CCR5+ cells in young rhesus macaques. The first, an immunotoxin consisting of the endogenous CCR5 ligand RANTES fused with Pseudomonas exotoxin (RANTES-PE38), killed CCR5+ lamina propria lymphocytes (LPLs) ex vivo, but had no detectable effect on CCR5+ LPLs in vivo. The second, a primatized bispecific antibody for CCR5 and CD3, depleted all CCR5+ cells from blood and the vast majority of such cells from the colonic mucosa (up to 96% of CD4+CCR5+). Absence of CCR5-expressing cells from blood endured for at least 1 week, while CCR5+ cells in colon were substantially replenished over the same time span. These data open an avenue to investigation of combined early ART treatment and CCR5+ reservoir depletion for cure of HIV-infected infants.

The United Statess Clean Water Act stipulates in section 303(d) that states must identify impaired water bodies for which total maximum daily loads (TMDLs) of pollution inputs into water bodies are developed. Decision-making procedures about how to list, or delist, water bodies as impaired, or not, per Clean Water Act 303(d) differ across states. In states such as California, whether or not a particular monitoring sample suggests that water quality is impaired can be regarded as a binary outcome variable, and Californias current regulatory framework invokes a version of the exact binomial test to consolidate evidence across samples and assess whether the overall water body complies with the Clean Water Act. Here, we contrast the performance of Californias exact binomial test with one potential alternative, the Sequential Probability Ratio Test (SPRT). The SPRT uses a sequential testing framework, testing samples as they become available and evaluating evidence as it emerges, rather than measuring all the samples and calculating a test statistic at the end of the data collection process. Through simulations and theoretical derivations, we demonstrate that the SPRT on average requires fewer samples to be measured to have comparable Type I and Type II error rates as the current fixed-sample binomial test. Policymakers might consider efficient alternatives such as SPRT to current procedure.

To gain insight into the cellular and molecular cues that promote neurovascular co-patterning at the earliest stages of human embryogenesis, we developed a human embryonic stem cell model to mimic the developing epiblast. Contact of ectoderm-derived neural cells with mesoderm-derived vasculature is initiated via the neural crest (NC), not the neural tube (NT). Neurovascular co-patterning then ensues with specification of NC toward an autonomic fate requiring vascular endothelial cell (EC)-secreted nitric oxide (NO) and direct contact with vascular smooth muscle cells (VSMCs) via T-cadherin-mediated homotypic interactions. Once a neurovascular template has been established, NT-derived central neurons then align themselves with the vasculature. Our findings reveal that, in early human development, the autonomic nervous system forms in response to distinct molecular cues from VSMCs and ECs, providing a model for how other developing lineages might coordinate their co-patterning.

Abstract Background Adenosine triphosphate (ATP) is used as an intracellular energy source by all living organisms. It plays a central role in the respiration and metabolism, and is the most important energy supplier in many enzymatic reactions. Its critical role as the energy storage molecule makes it extremely valuable to all cells. Results We report here the detection of extracellular ATP in the cultures of a variety of bacterial species. The levels of the extracellular ATP in bacterial cultures peaked around the end of the log phase and decreased in the stationary phase of growth. Extracellular ATP levels were dependent on the cellular respiration as bacterial mutants lacking cytochrome bo oxidase displayed lower extracellular ATP levels. We have also shown that Escherichia coli (E. coli) and Salmonella actively depleted extracellular ATP and an ATP supplement in culture media enhanced the stationary survival of E. coli and Salmonella. In addition to E. coli and Salmonella the presence of the extracellular ATP was observed in a variety of bacterial species that contain human pathogens such as Acinetobacter, Pseudomonas, Klebsiella and Staphylococcus. Conclusion Our results indicate that extracellular ATP is produced by many bacterial species during growth and extracellular ATP may serve a role in the bacterial physiology.

Sufu (Suppressor of Fused), a two-domain protein, plays a critical role in regulating Hedgehog signaling and is conserved from flies to humans. A few bacterial Sufu-like proteins have previously been identified based on sequence similarity to the N-terminal domain of eukaryotic Sufu proteins, but none have been structurally or biochemically characterized and their function in bacteria is unknown. We have determined the crystal structure of a more distantly related Sufu-like homolog, NGO1391 from Neisseria gonorrhoeae, at 1.4 Å resolution, which provides the first biophysical characterization of a bacterial Sufu-like protein. The structure revealed a striking similarity to the N-terminal domain of human Sufu (r.m.s.d. of 2.6 Å over 93% of the NGO1391 protein), despite an extremely low sequence identity of ∼15%. Subsequent sequence analysis revealed that NGO1391 defines a new subset of smaller, Sufu-like proteins that are present in ∼200 bacterial species and has resulted in expansion of the SUFU (PF05076) family in Pfam.

The crystal structures of BB2672 and SPO0826 were determined to resolutions of 1.7 and 2.1 Å by single-wavelength anomalous dispersion and multiple-wavelength anomalous dispersion, respectively, using the semi-automated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG) as part of the NIGMS Protein Structure Initiative (PSI). These proteins are the first structural representatives of the PF06684 (DUF1185) Pfam family. Structural analysis revealed that both structures adopt a variant of the Bacillus chorismate mutase fold (BCM). The biological unit of both proteins is a hexamer and analysis of homologs indicates that the oligomer interface residues are highly conserved. The conformation of the critical regions for oligomerization appears to be dependent on pH or salt concentration, suggesting that this protein might be subject to environmental regulation. Structural similarities to BCM and genome-context analysis suggest a function in amino-acid synthesis.

The crystal structure of a putative NTPase, YP_001813558.1 from Exiguobacterium sibiricum 255-15 (PF09934, DUF2166) was determined to 1.78 Å resolution. YP_001813558.1 and its homologs (dimeric dUTPases, MazG proteins and HisE-encoded phosphoribosyl ATP pyrophosphohydrolases) form a superfamily of all-α-helical NTP pyrophosphatases. In dimeric dUTPase-like proteins, a central four-helix bundle forms the active site. However, in YP_001813558.1, an unexpected intertwined swapping of two of the helices that compose the conserved helix bundle results in a `linked dimer' that has not previously been observed for this family. Interestingly, despite this novel mode of dimerization, the metal-binding site for divalent cations, such as magnesium, that are essential for NTPase activity is still conserved. Furthermore, the active-site residues that are involved in sugar binding of the NTPs are also conserved when compared with other α-helical NTPases, but those that recognize the nucleotide bases are not conserved, suggesting a different substrate specificity.

Membrane-attack complex/perforin (MACPF) proteins are transmembrane pore-forming proteins that are important in both human immunity and the virulence of pathogens. Bacterial MACPFs are found in diverse bacterial species, including most human gut-associated Bacteroides species. The crystal structure of a bacterial MACPF-domain-containing protein BT_3439 (Bth-MACPF) from B. thetaiotaomicron, a predominant member of the mammalian intestinal microbiota, has been determined. Bth-MACPF contains a membrane-attack complex/perforin (MACPF) domain and two novel C-terminal domains that resemble ribonuclease H and interleukin 8, respectively. The entire protein adopts a flat crescent shape, characteristic of other MACPF proteins, that may be important for oligomerization. This Bth-MACPF structure provides new features and insights not observed in two previous MACPF structures. Genomic context analysis infers that Bth-MACPF may be involved in a novel protein-transport or nutrient-uptake system, suggesting an important role for these MACPF proteins, which were likely to have been inherited from eukaryotes via horizontal gene transfer, in the adaptation of commensal bacteria to the host environment.

Examination of the genomic context for members of the FmdE Pfam family (PF02663), such as the protein encoded by the fmdE gene from the methanogenic archaeon Methanobacterium thermoautotrophicum, indicates that 13 of them are co-transcribed with genes encoding subunits of molybdenum formylmethanofuran dehydrogenase (EC 1.2.99.5), an enzyme that is involved in microbial methane production. Here, the first crystal structures from PF02663 are described, representing two bacterial and one archaeal species: B8FYU2_DESHY from the anaerobic dehalogenating bacterium Desulfitobacterium hafniense DCB-2, Q2LQ23_SYNAS from the syntrophic bacterium Syntrophus aciditrophicus SB and Q9HJ63_THEAC from the thermoacidophilic archaeon Thermoplasma acidophilum. Two of these proteins, Q9HJ63_THEAC and Q2LQ23_SYNAS, contain two domains: an N-terminal thioredoxin-like α+β core domain (NTD) consisting of a five-stranded, mixed β-sheet flanked by several α-helices and a C-terminal zinc-finger domain (CTD). B8FYU2_DESHY, on the other hand, is composed solely of the NTD. The CTD of Q9HJ63_THEAC and Q2LQ23_SYNAS is best characterized as a treble-clef zinc finger. Two significant structural differences between Q9HJ63_THEAC and Q2LQ23_SYNAS involve their metal binding. First, zinc is bound to the putative active site on the NTD of Q9HJ63_THEAC, but is absent from the NTD of Q2LQ23_SYNAS. Second, whereas the structure of the CTD of Q2LQ23_SYNAS shows four Cys side chains within coordination distance of the Zn atom, the structure of Q9HJ63_THEAC is atypical for a treble-cleft zinc finger in that three Cys side chains and an Asp side chain are within coordination distance of the zinc.

Dipeptidyl-peptidase VI from Bacillus sphaericus and YkfC from Bacillus subtilis have both previously been characterized as highly specific γ-D-glutamyl-L-diamino acid endopeptidases. The crystal structure of a YkfC ortholog from Bacillus cereus (BcYkfC) at 1.8 Å resolution revealed that it contains two N-terminal bacterial SH3 (SH3b) domains in addition to the C-terminal catalytic NlpC/P60 domain that is ubiquitous in the very large family of cell-wall-related cysteine peptidases. A bound reaction product (L-Ala-γ-D-Glu) enabled the identification of conserved sequence and structural signatures for recognition of L-Ala and γ-D-Glu and, therefore, provides a clear framework for understanding the substrate specificity observed in dipeptidyl-peptidase VI, YkfC and other NlpC/P60 domains in general. The first SH3b domain plays an important role in defining substrate specificity by contributing to the formation of the active site, such that only murein peptides with a free N-terminal alanine are allowed. A conserved tyrosine in the SH3b domain of the YkfC subfamily is correlated with the presence of a conserved acidic residue in the NlpC/P60 domain and both residues interact with the free amine group of the alanine. This structural feature allows the definition of a subfamily of NlpC/P60 enzymes with the same N-terminal substrate requirements, including a previously characterized cyanobacterial L-alanine-γ-D-glutamate endopeptidase that contains the two key components (an NlpC/P60 domain attached to an SH3b domain) for assembly of a YkfC-like active site.

BT2081 from Bacteroides thetaiotaomicron (GenBank accession code NP_810994.1) is a member of a novel protein family consisting of over 160 members, most of which are found in the different classes of Bacteroidetes. Genome-context analysis lends support to the involvement of this family in carbohydrate metabolism, which plays a key role in B. thetaiotaomicron as a predominant bacterial symbiont in the human distal gut microbiome. The crystal structure of BT2081 at 2.05 Å resolution represents the first structure from this new protein family. BT2081 consists of an N-terminal domain, which adopts a β-sandwich immunoglobulin-like fold, and a larger C-terminal domain with a β-sandwich jelly-roll fold. Structural analyses reveal that both domains are similar to those found in various carbohydrate-active enzymes. The C-terminal β-jelly-roll domain contains a potential carbohydrate-binding site that is highly conserved among BT2081 homologs and is situated in the same location as the carbohydrate-binding sites that are found in structurally similar glycoside hydrolases (GHs). However, in BT2081 this site is partially occluded by surrounding loops, which results in a deep solvent-accessible pocket rather than a shallower solvent-exposed cleft.

Proteins that contain the DUF2874 domain constitute a new Pfam family PF11396. Members of this family have predominantly been identified in microbes found in the human gut and oral cavity. The crystal structure of one member of this family, BVU2987 from Bacteroides vulgatus, has been determined, revealing a β-lactamase inhibitor protein-like structure with a tandem repeat of domains. Sequence analysis and structural comparisons reveal that BVU2987 and other DUF2874 proteins are related to β-lactamase inhibitor protein, PepSY and SmpA_OmlA proteins and hence are likely to function as inhibitory proteins.

KPN03535 (gi|152972051) is a putative lipoprotein of unknown function that is secreted by Klebsiella pneumoniae MGH 78578. The crystal structure reveals that despite a lack of any detectable sequence similarity to known structures, it is a novel variant of the OB-fold and structurally similar to the bacterial Cpx-pathway protein NlpE, single-stranded DNA-binding (SSB) proteins and toxins. K. pneumoniae MGH 78578 forms part of the normal human skin, mouth and gut flora and is an opportunistic pathogen that is linked to about 8% of all hospital-acquired infections in the USA. This structure provides the foundation for further investigations into this divergent member of the OB-fold family.

The crystal structure of the Bacteroides thetaiotaomicron protein BT_3984 was determined to a resolution of 1.7 Å and was the first structure to be determined from the extensive SusD family of polysaccharide-binding proteins. SusD is an essential component of the sus operon that defines the paradigm for glycan utilization in dominant members of the human gut microbiota. Structural analysis of BT_3984 revealed an N-terminal region containing several tetratricopeptide repeats (TPRs), while the signature C-terminal region is less structured and contains extensive loop regions. Sequence and structure analysis of BT_3984 suggests the presence of binding interfaces for other proteins from the polysaccharide-utilization complex.

Proteins with the DUF2063 domain constitute a new Pfam family, PF09836. The crystal structure of a member of this family, NGO1945 from Neisseria gonorrhoeae, has been determined and reveals that the N-terminal DUF2063 domain is likely to be a DNA-binding domain. In conjunction with the rest of the protein, NGO1945 is likely to be involved in transcriptional regulation, which is consistent with genomic neighborhood analysis. Of the 216 currently known proteins that contain a DUF2063 domain, the most significant sequence homologs of NGO1945 (∼40-99% sequence identity) are from various Neisseria and Haemophilus species. As these are important human pathogens, NGO1945 represents an interesting candidate for further exploration via biochemical studies and possible therapeutic intervention.

The crystal structures of SPO0140 and Sbal_2486 were determined using the semiautomated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG) as part of the NIGMS Protein Structure Initiative (PSI). The structures revealed a conserved core with domain duplication and a superficial similarity of the C-terminal domain to pleckstrin homology-like folds. The conservation of the domain interface indicates a potential binding site that is likely to involve a nucleotide-based ligand, with genome-context and gene-fusion analyses additionally supporting a role for this family in signal transduction, possibly during oxidative stress.

BT1062 from Bacteroides thetaiotaomicron is a homolog of Mfa2 (PGN0288 or PG0179), which is a component of the minor fimbriae in Porphyromonas gingivalis. The crystal structure of BT1062 revealed a conserved fold that is widely adopted by fimbrial components.