Introduction to Plenary Session

SR Leigh

Department of Anthropology, University of Colorado, Boulder, CO

Methodologies comprise critical elements of research in biological anthropology. Fortunately, the abundance and richness of methods seems ever-increasing in service of exciting new paradigms, theories, and hypotheses. This talk shares lessons learned about shifting to new problems, deploying unfamiliar or new theories, and the importance of methods in these processes.

Consistently prioritizing scholarly renewal has been a valuable lesson learned. The idea is that a path to success in scientific research and, in fact, just about any scholarly field, is to pursue ways to actively renew one’s scholarship. This means consciously seeking out new, unfamiliar, sometimes difficult or even uncomfortable new problems or topics for investigation. Scholarly renewal becomes a form of “meta-method” that guides new learning, investigations, methods, collaborations, and new research.

Collaboration is central to this sort of approach, especially when a direction of scholarly renewal leads to unfamiliar fields. As our symposium proposal notes, we “benefit from partnership more often than not.” Fortunately, most of us belong to institutions of higher learning, intrinsically spanning wide and divergent fields of knowledge. Moreover, our field engenders curiosity and intrinsically interests potential collaborators. I advocate dedicated efforts to work with colleagues across a broad spectrum. My own research in fields beginning in bioarchaeology, through to comparative primate anatomy and human evolution, to evolutionary microbiomics, provides examples of how this might be accomplished.

In this presentation, I will briefly reflect on field research conducted over the past 30+ years in different parts of the world. I will particularly focus on the challenges involved in collecting and transporting biological samples from the field to the lab for genetic analysis. Overall, this work has revealed how such efforts can be affected by logistical constraints, local politics, sampling methods, shifting bioethical practices, participant recruitment, and other random factors. Suffice it to say that, while collecting biological samples for DNA analysis in the field is generally straightforward from a methodological perspective, the context in which they are obtained and transported from the field can vary widely by the time, location, community and sociopolitical environment in which fieldwork is being conducted.

Gastrointestinal infections with parasitic organisms (e.g., soil-transmitted helminths, waterborne protozoal infections) affect billions of people globally. Soil-transmitted helminths alone are estimated to infect almost a quarter of the world’s population. While these infections can drive important health outcomes related to immune and gut function, chronic disease risk, and even mortality rates, they remain understudied and are often not incorporated into human biology field studies. Because these infections can lead to altered levels of other biomarkers, their inclusion in research focused on immune, digestive, and chronic disease-related biomarkers is crucial. Beginning research that incorporates stool sample collection to test for intestinal pathogens can be challenging due to many unknowns, including type and abundance of species present, level of comfort for participants collecting their own stool samples and researchers processing samples, and access to field laboratory and storage space. These factors are important considerations that should drive site-specific methodological decisions. Here, we discuss lessons learned over more than a decade of stool sample collection for parasitological analyses across several highly variable field sites in Ecuador and the United States. These sometimes hard-learned lessons include: 1) creating collection kits to maximize participant comfort based on level of sanitation and hygiene infrastructure available; 2) setting up a sanitary laboratory space for processing, analyzing, and disposing of samples in varying climates and environments; and 3) choosing the most accurate analysis methods based on expected pathogens encountered, equipment availability, and budgetary constraints. More comparative global studies on parasitological infections will benefit holistic human biology research.

Previously validated psychological and behavioral instruments are often applied to field-based anthropology. These surveys have the benefit of internal and external validity, accuracy, and comparability with previous work. However, most instruments are used and validated only in WEIRD settings, making their applicability to small-scale societies suspect. WEIRD instruments typically rely on many difficult-to-translate Likert-scale items and often reference beliefs and constructs that may not be cross-culturally applicable. Drawing on lessons from 10+ years of fieldwork with Kunene pastoralists of Namibia, we argue that, when the goal is to understand the internal cultural dynamics of a group, validated WEIRD instruments have limited utility and introduce significant problems. Instead, developing locally relevant questions, tasks, and instruments that are embedded in the norms and beliefs of the communities of study is a more expeditious and scientifically valid way do to conduct anthropological research.

The use of point-of-care tests (POCT) has become increasingly common in human biology research. Instruments that deliver immediate results from minimally invasive samples at the location of collection can facilitate rapid data sharing with participants and enhance community outreach efforts. However, concerns around equipment reliability and results interpretation limit POCT usefulness in some contexts. This presentation draws on past experiences incorporating POCT in the Rural Embodiment and Community Health (REACH) study – a community-engaged project assessing how lived experiences impact intestinal health and immune function – to describe unforeseen POCT complications, including equipment malfunctions and navigating IRB concerns surrounding data sharing. Additionally, our lab has previously documented statistically significant differences in test sensitivity between POCT and standard laboratory analysis techniques. For example, enzyme-linked immunosorbent assays detected significantly more positive cases of Helicobacter pylori (a common gastrointestinal bacteria; X2(1) = 42.68, p < .001) and more cases of elevated fecal calprotectin (a measure of intestinal inflammation; X2(4) = 149.89, p < .001) compared to POCT from the same manufacturers. Fully understanding test strengths and limitations can help ensure POCT are used appropriately. Finally, this presentation will consider lessons learned from past community-engaged work, including strategies for incorporating POCT to address community concerns and provide easily interpretable information requested by community members. Challenges in sharing health information and connecting participants with affordable follow-up medical care will also be discussed. Cumulatively, the knowledge gained from these past challenges has shaped the REACH project in profound ways and led to exciting new research foci.

Saliva reflects mucosal immune function at a major portal of pathogen entry. Because collection is noninvasive and inexpensive, salivary measures enable repeated sampling and inclusion of populations where venipuncture is impractical. Functional bacteria-killing assays (BKA) offer an integrative endpoint that captures combined activity of complement, lysozyme, antimicrobial peptides, and natural antibodies. To present a standardized saliva bacteria-killing assay (BKA) suitable for human field and lab studies, and summarize validation tests. The assay uses passive drool saliva, heat inactivation at 42 °C for 8 min, 1:2 dilution in CO₂-independent medium, incubation with E. coli ATCC 8739 for 30 min at 37 °C/95% humidity, and triplicate plating with matched positive controls. To limit end-of-run effects, samples are processed in batches of ≤20. We evaluated effects of heat-inactivation, storage, freeze–thaw cycles, and diurnal timing. Heat-inactivation produced values closely aligned with untreated samples. Percent killing was similar across one to five freeze–thaw cycles. Storage at −80 °C, −20 °C, or 4 °C performed comparably; room-temperature storage reduced killing. Across the day, mean killing was stable with modest within-day curvature, supporting first-morning collections to reduce variation. No effect of salivary flow rate was detected. The protocol yields a practical functional readout of salivary innate activity and is feasible across settings. Key implementation points include clear labeling and plate matching for controls, batch processing, and triplicate counts. The BKA can support comparative, ecological, and health research that requires a noninvasive functional immune measure.

Most researchers measure the prevalence of hot flashes with questionnaires. For example, we administered questionnaires in Paraguay and Slovenia to assess frequency of hot flash experience among women at midlife during the past two weeks: 43% of Paraguayan women reported hot flashes in Asunción compared to 24% of women in the Selška Valley of Slovenia. In Puebla, Mexico, we added biometric measures and learned that polygraph tracings do not always reflect self-report. Hot flashes were commonly experienced on the back of the neck, but these were not measured by the conventional method of placing electrodes on either side of the sternum. In Bangladesh, 64% of participants felt hot flashes on the top of their head versus 46% of participants in Mexico and 27% in the US. In Hilo, Hawaii, we placed electrodes on the back of the neck, as well as on either side of the sternum. Discord with questionnaire results, particularly among Japanese Americans, led us to conclude that differences in self-report were influenced in part by population variation in sensing, labeling, and willingness to talk about hot flashes. Our work in Campeche, Mexico, showed that extreme heat and humidity can compromise self-reported hot flashes. Questionnaires alone are not the best way to compare hot flash experience across populations; however, biometric measures have their own set of challenges. A combination of methods contributes to an appreciation for how differences in hot flash frequencies across populations are associated with variation in physiology, culture, and environment.

The early days of work with degraded/ancient DNA (aDNA) from various historic and archaeological sources recognized the potential for contamination from modern DNA, but how to protect against exogenous DNA took time to be fully implemented. It was, in some ways, the Wild West, of genetic research, rife with “black hat” research that often reported more of the researcher than DNA from the purported source. Early publications on everything from Egyptian mummies to Neanderthals ran afoul of the dreaded contamination. However, the scope of contamination and how to control it was wrangled, particularly with the technologies such as Next Generation Sequencing (NGS). The lessons learned, by and large, have led to degraded DNA being able to be sequenced and utilized in studies of human evolution, migration, and selection in a myriad of important ways. And while there are still great divides in the representation of aDNA from many parts of the world, the ability to trust in the data that is published now goes a long way to aid in our understanding of human biology past, present, and maybe even future. It’ll probably never help us find Bigfoot, though, no matter how many times someone brings me a “sample.”

Gut function is central to human biology. It influences our ability to extract energy from our environments (e.g., the absorption of consumed food). It also influences how our bodies allocate the resources that we have available to competing metabolic tasks (e.g., the translocation of microbes across the gut-blood barrier and resulting immune activation). Despite clear implications for human evolution, variation, and health, gut function has been poorly studied in the field of human biology. We have begun to address this shortcoming in the Human Evolutionary Biology and Health Lab. Over the past few years, we have implemented several minimally invasive approaches for assessing gut function in population research. This includes the application of several established assessment methods that are common in other fields but have rarely/never been adopted in human biology (e.g., urinary lactulose-mannitol testing of intestinal absorption and permeability), methods that are based on newly available point-of-care technology (e.g., breath-based testing of small intestinal bacterial overgrowth), and methods that have been validated using minimally invasive approaches for the first time in our lab (e.g., finger-prick dried blood spot biomarkers of environmental enteric dysfunction). In this talk, I highlight some of these methods and our initial findings on children’s gut function in projects in Ecuador, Kenya, and elsewhere. I also share advice on what works, what doesn’t, and how human biologists might best contribute to this exciting area of research.

Hair is a widely used biological matrix for measuring steroid hormones, with hair cortisol concentration (HCC) most commonly employed as a retrospective indicator of long-term systemic exposure over months (and sometimes even years). HCC offers a useful alternative to biological fluid matrices for estimating cortisol exposure, which is otherwise complicated by the hormone’s circadian and pulsatile secretion.

To date, HCC has been linked to dozens of health outcomes—spanning cardiometabolic, endocrine, mental health, sleep, perinatal, pain, and cognitive domains—suggesting a broad association with health risk.

Nonetheless, key methodological questions remain. These include the physiological meaning and temporal dynamics of HCC (e.g., the assumption that hair grows ~1 cm per month, contributions from secondary cortisol sources, and the lag between acute systemic changes and their appearance in the proximal hair segment) and evidence-informed best practices for laboratory handling (e.g., the effectiveness of washing steps in removing exogenous steroids and other factors that influence extraction efficiency). Such issues ultimately shape observed associations and likely contribute to mixed findings across studies.

In this presentation, I will synthesize findings from the literature and present data from my prior and ongoing projects on hair steroids, including associations between HCC and indices of acute HPA-axis reactivity. I will also discuss how downstream protocol steps affect extraction efficiency and quantification, and address confounding factors unique to hair-based cortisol measures. Finally, I will highlight implications for study design and laboratory protocols to improve the interpretability and reproducibility of HCC and other hair-steroid measures.