Methods for Biophysical Characterization of SznF, a Member of the Heme-Oxygenase-Like Diiron Oxidase/Oxygenase Superfamily.

Nonheme diiron enzymes harness the chemical potential of oxygen to catalyze challenging reactions in biology. In their resting state, these enzymes have a diferrous cofactor that is coordinated by histidine and carboxylate ligands. Upon exposure to oxygen, the cofactor oxidizes to its diferric state forming a peroxo- adduct, capable of catalyzing a wide range of oxidative chemistries such as desaturation and heteroatom oxidation. Despite their versatility and prowess, an emerging subset of nonheme diiron enzymes has inherent cofactor instability making them resistant to structural characterization. This feature is widespread among members of the heme-oxygenase-like diiron oxidase/oxygenase (HDO) superfamily. HDOs have a flexible core structure that remodels upon metal binding. Although ~9600 HDOs have been unearthed, few have undergone functional characterization to date. In this chapter, we describe the methods that have been used to characterize the HDO N-oxygenase, SznF. We demonstrate the overexpression and purification of apo-SznF and methodology specifically designed to aid in obtaining an X-ray structure of holo-SznF. We also describe the characterization of the transient SznF-peroxo-Fe(III)2 complex by stopped-flow absorption and Mössbauer spectroscopies. These studies provide the framework for the characterization of new members of the HDO superfamily.

Out of the empirical box: A mixed-methods study of tool innovation among Congolese BaYaka forager and Bondongo fisher-farmer children.

Tool innovation has played a crucial role in human adaptation. Yet, this capacity seems to arise late in development. Before 8 years of age, many children struggle to solve the hook task, a common measure of tool innovation that requires modification of a straight pipe cleaner into a hook to extract a prize. Whether these findings are generalizable beyond postindustrialized Western children remains unclear. In many small-scale subsistence societies, children engage in daily tool use and modification, experiences that theoretically could enhance innovative capabilities. Although two previous studies found no differences in innovative ability between children from Western and small-scale subsistence societies, these did not account for the latter's inexperience with pipe cleaners. Thus, the current study investigated how familiarity with pipe cleaners affected hook task success in 132 Congolese BaYaka foragers (57 girls) and 59 Bondongo fisher-farmers (23 girls) aged 4-12 years. We contextualized these findings within children's interview responses and naturalistic observations of how pipe cleaners were incorporated into daily activities. Counter to our expectation, prior exposure did not improve children's performance during the hook task. Bondongo children innovated significantly more hooks than BaYaka children, possibly because they participate in hook-and-line fishing. Observations and interviews showed that children imagined and innovated novel uses for pipe cleaners outside the experimental context, including headbands, bracelets, and suspenders. We relate our findings to ongoing debates regarding systematic versus unsystematic tool innovation, the importance of prior experience for the ontogeny of tool innovation, and the external validity of experimental paradigms.

Structure and assembly of the diiron cofactor in the heme-oxygenase-like domain of the N-nitrosourea-producing enzyme SznF.

In biosynthesis of the pancreatic cancer drug streptozotocin, the tridomain nonheme-iron oxygenase SznF hydroxylates Nδ and Nω' of Nω-methyl-l-arginine before oxidatively rearranging the triply modified guanidine to the N-methyl-N-nitrosourea pharmacophore. A previously published structure visualized the monoiron cofactor in the enzyme's C-terminal cupin domain, which promotes the final rearrangement, but exhibited disorder and minimal metal occupancy in the site of the proposed diiron cofactor in the N-hydroxylating heme-oxygenase-like (HO-like) central domain. We leveraged our recent observation that the N-oxygenating µ-peroxodiiron(III/III) intermediate can form in the HO-like domain after the apo protein self-assembles its diiron(II/II) cofactor to solve structures of SznF with both of its iron cofactors bound. These structures of a biochemically validated member of the emerging heme-oxygenase-like diiron oxidase and oxygenase (HDO) superfamily with intact diiron cofactor reveal both the large-scale conformational change required to assemble the O2-reactive Fe2(II/II) complex and the structural basis for cofactor instability-a trait shared by the other validated HDOs. During cofactor (dis)assembly, a ligand-harboring core helix dynamically (un)folds. The diiron cofactor also coordinates an unanticipated Glu ligand contributed by an auxiliary helix implicated in substrate binding by docking and molecular dynamics simulations. The additional carboxylate ligand is conserved in another N-oxygenating HDO but not in two HDOs that cleave carbon-hydrogen and carbon-carbon bonds to install olefins. Among ∼9,600 sequences identified bioinformatically as members of the emerging HDO superfamily, ∼25% conserve this additional carboxylate residue and are thus tentatively assigned as N-oxygenases.

Enhancing point-of-care testing through standardized training and redeployment of pharmacy technicians in the community setting.

Objectives: The primary objective of this study was to assess the impact of a standardized training model for technician-supported point-of-care testing (POCT) on the number of health screenings performed across two states in a large community chain pharmacy. Secondary objectives included the assessment of pharmacist and technician perceptions of advanced roles of the pharmacy technician in POCT service delivery. Practice description: Certified pharmacy technicians (CPhTs) across six regional divisions of a large community chain pharmacy in Tennessee and Ohio participated in a standardized training program prior to implementation of technician-supported POCT. Practice innovation: Standardized training consisted of pre-training assessments, online training modules, post-training assessments, followed by in-person skills-based assessments. CPhT participation was limited to technical tasks of POCT (e.g. sample collections, quality assurance). Evaluation methods: The study addressed its primary objective by comparing total number of health screenings for included pharmacies in 2019 as compared to 2020. Descriptive and inferential statistics were used. Perceptions were assessed using an electronic, Likert-type scale questionnaire. Results: Pharmacies with technician-supported POCT showed a 46% increase in the total number of health screenings performed vs. 2019. The survey found that 74% (106/144) of pharmacists and 83% (34/41) of CPhTs agreed or strongly agreed that technician-supported POCT is acceptable for their practice site. Most pharmacy personnel agreed or strongly agreed that the service was appropriate and feasible for their respective practice sites. Conclusion: This study provided supporting evidence that technician-supported POCT may positively impact the number of health screenings conducted in a community pharmacy setting. Standardization of training may allow for expansion of this service across additional states. Furthermore, pharmacy personnel perceptions were overall positive.

Where innovations flourish: an ethnographic and archaeological overview of hunter-gatherer learning contexts.

Research in developmental psychology suggests that children are poor tool innovators. However, such research often overlooks the ways in which children's social and physical environments may lead to cross-cultural variation in their opportunities and proclivity to innovate. In this paper, we examine contemporary hunter-gatherer child and adolescent contributions to tool innovation. We posit that the cultural and subsistence context of many hunter-gatherer societies fosters behavioural flexibility, including innovative capabilities. Using the ethnographic and developmental literature, we suggest that socialisation practices emphasised in hunter-gatherer societies, including learning through autonomous exploration, adult and peer teaching, play and innovation seeking may bolster children's ability to innovate. We also discuss whether similar socialisation practices can be interpreted from the archaeological record. We end by pointing to areas of future study for understanding the role of children and adolescents in the development of tool innovations across cultures in the past and present.

Optional-switch cognitive flexibility in primates: Chimpanzees' (Pan troglodytes) intermediate susceptibility to cognitive set.

Within human problem-solving, the propensity to use a familiar approach, rather than switch to a more efficient alternative is pervasive. This susceptibility to "cognitive set" prevents optimization by biasing response patterns toward known solutions. In a recent study, which used a nonverbal touch screen task, baboons exhibited a striking ability to deviate from their learned strategy to use a more efficient shortcut. Humans, on the other hand, displayed the opposite response pattern and almost exclusively used a less efficient, but familiar, response. In the current study, we sought to further explore variation in susceptibility to cognitive set within the primate lineage by conducting the Learned Strategy-Direct Strategy task with 10 chimpanzees (Pan troglodytes). Using multilevel multinomial modeling, we found that chimpanzees' shortcut use was intermediate to baboons' and humans'. However, unlike either baboons or humans, there was pronounced inter- and intraindividual variability in chimpanzees' shortcut use. Additionally, a subset of chimpanzees employed a unique solution, wherein they switched strategies midtrial. Further, we found that chimpanzees did not exhibit switch costs when switching between the learned strategy and the shortcut, but humans did. We propose that differences in abstract rule encoding may underlie differences in susceptibility to cognitive set on the Learned Strategy-Direct Strategy task within the primate lineage. (PsycINFO Database Record (c) 2020 APA, all rights reserved).

Capuchin and rhesus monkeys but not humans show cognitive flexibility in an optional-switch task.

Learned rules help us accurately solve many problems, but by blindly following a strategy, we sometimes fail to find more efficient alternatives. Previous research found that humans are more susceptible to this "cognitive set" bias than other primates in a nonverbal computer task. We modified the task to test one hypothesis for this difference, that working memory influences the advantage of taking a shortcut. During training, 60 humans, 7 rhesus macaques, and 22 capuchin monkeys learned to select three icons in sequence. They then completed 96 baseline trials, in which only this learned rule could be used, and 96 probe trials, in which they could also immediately select the final icon. Rhesus and capuchin monkeys took this shortcut significantly more often than humans. Humans used the shortcut more in this new, easier task than in previous work, but started using it significantly later than the monkeys. Some participants of each species also used an intermediate strategy; they began the learned rule but switched to the shortcut after selecting the first item in the sequence. We suggest that these species differences arise from differences in rule encoding and in the relative efficiency of exploiting a familiar strategy versus exploring alternatives.

Changes in Frontoparietotemporal Connectivity following Do-As-I-Do Imitation Training in Chimpanzees ( Pan troglodytes).

Human imitation is supported by an underlying "mirror system" principally composed of inferior frontal, inferior parietal, and superior temporal cortical regions. Across primate species, differences in frontoparietotemporal connectivity have been hypothesized to explain phylogenetic variation in imitative abilities. However, if and to what extent these regions are involved in imitation in nonhuman primates is unknown. We hypothesized that "Do As I Do" (DAID) imitation training would enhance white matter integrity within and between frontoparietotemporal regions. To this end, four captive chimpanzees ( Pan troglodytes) were trained to reproduce 23 demonstrated actions, and four age-/sex-matched controls were trained to produce basic husbandry behaviors in response to manual cues. Diffusion tensor images were acquired before and after 600 min of training over an average of 112 days. Bilateral and asymmetrical changes in frontoparietotemporal white matter integrity were compared between DAID trained subjects and controls. We found that imitation trained subjects exhibited leftward shifts in both mean fractional anisotropy and tract strength asymmetry measures in brain regions within the mirror system. This is the first report of training-induced changes in white matter integrity in chimpanzees and suggests that frontoparietotemporal connectivity, particularly in the left hemisphere, may have facilitated the emergence of increasingly complex imitation learning abilities.

Genetic Factors and Orofacial Motor Learning Selectively Influence Variability in Central Sulcus Morphology in Chimpanzees (Pan troglodytes).

Captive chimpanzees (Pan troglodytes) have been shown to learn the use of novel attention-getting (AG) sounds to capture the attention of humans as a means of requesting or drawing their attention to a desired object or food. There are significant individual differences in the use of AG sounds by chimpanzees and, here, we examined whether changes in cortical organization of the central sulcus (CS) were associated with AG sound production. MRI scans were collected from 240 chimpanzees, including 122 that reliably produced AG sounds and 118 that did not. For each subject, the depth of CS was quantified along the superior-inferior plane with specific interest in the inferior portion corresponding to the region of the motor cortex where the mouth and orofacial movements are controlled. Results indicated that CS depth in the inferior, but not superior, portion was significantly greater in chimpanzees that reliably produced AG sounds compared with those who did not. Quantitative genetic analyses indicated that overall CS surface area and depth were significantly heritable, particularly in the superior regions, but less so in the inferior and central portions. Further, heritability in CS depth was altered as a function of acquisition of AG sounds. The collective results suggest that learning to produce AG sounds resulted in region-specific cortical reorganization within the inferior portion of the CS, a finding previously undocumented in chimpanzees or any nonhuman primate.SIGNIFICANCE STATEMENT Recent studies in chimpanzees (Pan troglodytes) have shown that some can learn to produce novel sounds by configuring different orofacial movement patterns and these sounds are used in communicatively relevant contexts. Here, we examined the neuromorphological correlates in the production of these sounds in chimpanzees. We show that chimpanzees that have learned to produce these sounds show significant differences in central sulcus (CS) morphology, particularly in the inferior region. We further show that overall CS morphology and regions within the superior portion are significantly heritable, whereas central and inferior portions of the CS are not. The collective findings suggest chimpanzees exhibit cortical plasticity in regions of the brain that were central to the emergence of speech functions in humans.

Motor skill for tool-use is associated with asymmetries in Broca's area and the motor hand area of the precentral gyrus in chimpanzees (Pan troglodytes).

Among nonhuman primates, chimpanzees are well known for their sophistication and diversity of tool use in both captivity and the wild. The evolution of tool manufacture and use has been proposed as a driving mechanism for the development of increasing brain size, complex cognition and motor skills, as well as the population-level handedness observed in modern humans. Notwithstanding, our understanding of the neurological correlates of tool use in chimpanzees and other primates remains poorly understood. Here, we assessed the hand preference and performance skill of chimpanzees on a tool use task and correlated these data with measures of neuroanatomical asymmetries in the inferior frontal gyrus (IFG) and the pli-de-passage fronto-parietal moyen (PPFM). The IFG is the homolog to Broca's area in the chimpanzee brain and the PPFM is a buried gyrus that connects the pre- and post-central gyri and corresponds to the motor-hand area of the precentral gyrus. We found that chimpanzees that performed the task better with their right compared to left hand showed greater leftward asymmetries in the IFG and PPFM. This association between hand performance and PPFM asymmetry was particularly robust for right-handed individuals. Based on these findings, we propose that the evolution of tool use was associated with increased left hemisphere specialization for motor skill. We further suggest that lateralization in motor planning, rather than hand preference per se, was selected for with increasing tool manufacture and use in Hominid evolution.

Behavioral and brain asymmetries in primates: a preliminary evaluation of two evolutionary hypotheses.

Contrary to many historical views, recent evidence suggests that species-level behavioral and brain asymmetries are evident in nonhuman species. Here, we briefly present evidence of behavioral, perceptual, cognitive, functional, and neuroanatomical asymmetries in nonhuman primates. In addition, we describe two historical accounts of the evolutionary origins of hemispheric specialization and present data from nonhuman primates that address these specific theories. Specifically, we first discuss the evidence that genes play specific roles in determining left-right differences in anatomical and functional asymmetries in primates. We next consider and present data on the hypothesis that hemispheric specialization evolved as a by-product of increasing brain size relative to the surface area of the corpus callosum in different primate species. Last, we discuss some of the challenges in the study of hemispheric specialization in primates and offer some suggestions on how to advance the field.

Baboons (Papio papio), but not humans, break cognitive set in a visuomotor task.

Cognitive set can be both helpful and harmful in problem solving. A large set of similar problems may be solved mechanically by applying a single-solution method. However, efficiency might be sacrificed if a better solution exists and is overlooked. Despite half a century of research on cognitive set, there have been no attempts to investigate whether it occurs in nonhuman species. The current study utilized a nonverbal, computer task to compare cognitive set between 104 humans and 15 baboons (Papio papio). A substantial difference was found between humans' and baboons' abilities to break cognitive set. Consistent with previous studies, the majority of humans were highly impaired by set, yet baboons were almost completely unaffected. Analysis of the human data revealed that children (aged 7-10) were significantly better able to break set than adolescents (11-18) and adults (19-68). Both the evolutionary and developmental implications of these findings are discussed.

Multimodal communication in chimpanzees.

A fundamental characteristic of human language is multimodality. In other words, humans use multiple signaling channels concurrently when communicating with one another. For example, people frequently produce manual gestures while speaking, and the words a person perceives are impacted by visual information. For this study, we hypothesized that similar to the way that humans regularly couple their spoken utterances with gestures and facial expressions, chimpanzees regularly produce vocalizations in conjunction with other communicative signals. To test this hypothesis, data were collected from 101 captive chimpanzees living in mixed-sex social groupings of seven to twelve individuals. A total of 2,869 vocal events were collected. The data indicate that approximately 50% of the vocal events were produced in conjunction with another communicative modality. In addition, approximately 68% were directed to a specific individual, and these directed vocalizations were more likely to include a signal from another communicative modality than were vocalizations that were not directed to a specific individual. These results suggest that, like humans, chimpanzees often pair their vocalizations with signals from other communicative modalities. In addition, chimpanzees appear to use their communicative signals strategically to meet specific socio-communicative ends, providing support for the growing literature that indicates that at least some chimpanzee vocal signaling is intentional.

The association between imitation recognition and socio-communicative competencies in chimpanzees (Pan troglodytes).

Imitation recognition provides a viable platform from which advanced social cognitive skills may develop. Despite evidence that non-human primates are capable of imitation recognition, how this ability is related to social cognitive skills is unknown. In this study, we compared imitation recognition performance, as indicated by the production of testing behaviors, with performance on a series of tasks that assess social and physical cognition in 49 chimpanzees. In the initial analyses, we found that males were more responsive than females to being imitated and engaged in significantly greater behavior repetitions and testing sequences. We also found that subjects who consistently recognized being imitated performed better on social but not physical cognitive tasks, as measured by the Primate Cognitive Test Battery. These findings suggest that the neural constructs underlying imitation recognition are likely associated with or among those underlying more general socio-communicative abilities in chimpanzees. Implications regarding how imitation recognition may facilitate other social cognitive processes, such as mirror self-recognition, are discussed.

Ixabepilone in combination with capecitabine and as monotherapy for treatment of advanced breast cancer refractory to previous chemotherapies.

PURPOSE: To describe the considerations leading to marketing approval of ixabepilone in combination with capecitabine and as monotherapy for the treatment of advanced breast cancer that is refractory to other chemotherapies. EXPERIMENTAL DESIGN: Data from one randomized multicenter trial comparing combination therapy with ixabepilone and capecitabine to capecitabine alone were analyzed for support of the combination therapy indication. For monotherapy, a single-arm trial of ixabepilone was analyzed. Supporting data came from an additional single-arm combination therapy study and two single-arm monotherapy studies. RESULTS: In patients with metastatic or locally advanced breast cancer who had disease progression on or following an anthracycline and a taxane, ixabepilone plus capecitabine showed an improvement in progression-free survival compared with capecitabine alone {median progression-free survival, 5.7 [95% confidence interval (95% CI), 4.8-6.7] versus 4.1 (95% CI, 3.1-4.3) months, stratified log-rank P < 0.0001; hazard ratio, 0.69 (95% CI, 0.58-0.83)}. As monotherapy for patients who had disease progression on or following an anthracycline, a taxane, and capecitabine, ixabepilone as monotherapy showed a 12% objective response rate by independent blinded review and 18% by investigator assessment. The major toxicities from ixabepilone therapy were peripheral neuropathy and myelosuppression, particularly neutropenia. CONCLUSIONS: On October 16, 2007, the Food and Drug Administration approved ixabepilone for injection in combination with capecitabine or as monotherapy for the treatment of patients with advanced breast cancer who have experienced disease progression on previous chemotherapies.

Sprycel for chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia resistant to or intolerant of imatinib mesylate.

PURPOSE: On June 28, 2006, the U.S. Food and Drug Administration approved dasatinib (Sprycel; Bristol-Myers Squibb), a new small-molecule inhibitor of multiple tyrosine kinases, for the treatment of adults with chronic phase, accelerated phase, or myeloid or lymphoid blast phase chronic myeloid leukemia (CML) or Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+) ALL) with resistance or intolerance to prior therapy including imatinib. This summary reviews the database supporting this approval. EXPERIMENTAL DESIGN: Four single-arm multicenter studies supported the efficacy and safety of dasatinib. The primary efficacy end point in chronic phase CML was major cytogenetic response. The primary end point in accelerated phase, myeloid phase, and lymphoid blast phase CML, and Ph(+) ALL was major hematologic response. RESULTS: The four studies combined enrolled 445 patients. In patients with chronic phase CML, the major cytogenetic response rate was 45% with a complete cytogenetic response rate of 33%. Major hematologic response rates in patients with accelerated phase CML, myeloid CML, lymphoid blast CML, and Ph(+) ALL were 59%, 32%, 31%, and 42%, respectively. Median response durations in chronic phase, accelerated phase, and myeloid phase CML had not been reached. The median durations of major hematologic response were 3.7 months in lymphoid blast CML and 4.8 months in Ph(+) ALL. Common toxicities with dasatinib included myelosuppression, bleeding, and fluid retention. CONCLUSIONS: This report describes the Food and Drug Administration review supporting the approval of dasatinib for CML and Ph(+) ALL based on the rates and durability of cytogenetic and hematologic responses.

Vorinostat for treatment of cutaneous manifestations of advanced primary cutaneous T-cell lymphoma.

PURPOSE: To discuss vorinostat approval for treatment of cutaneous manifestations of advanced cutaneous T-cell lymphoma (CTCL). EXPERIMENTAL DESIGN: Data from 1 single-arm, open-label, multicenter pivotal trial and 11 other trials submitted to support the new drug application for vorinostat in the treatment of advanced primary CTCL were reviewed. The pivotal trial assessed responses by changes in overall skin disease score using a severity-weighted assessment tool (SWAT). Vorinostat could be considered active in CTCL if observed response rate was at least 20% and the lower bound of the corresponding 95% confidence interval (95% CI) excluded 5%. Patients reported pruritus relief using a questionnaire and a visual analogue scale. RESULTS: The pivotal trial enrolled 74 patients with stage IB or higher CTCL. Median number of prior treatments was 3, and 61 patients (82%) had stage IIB or higher disease. The objective response rate in the skin disease assessed by change in the overall SWAT score from the baseline was 30% (95% CI, 18.5 to 42.6) in patients with stage IIB or higher disease. Median response duration (end of response defined by 50% increase in SWAT score from the nadir) was 168 days. Median time to tumor progression was 148 days for overall population and 169 days for patients with stage IIB or higher disease. Assessment of pruritus relief was considered unreliable. CONCLUSIONS: Vorinostat showed activity in CTCL, and skin responses were a clinical benefit. Vorinostat was approved for treatment of cutaneous manifestations of CTCL. A nonblinded, single-arm trial did not allow a reliable assessment of pruritus relief.