Fluid inclusions in evaporite minerals (halite, gypsum, etc.) potentially preserve genetic records of microbial diversity and changing environmental conditions of Earth's hydrosphere for nearly one billion years. Here we describe a robust protocol for surface sterilization and retrieval of DNA from fluid inclusions in halite that, unlike previously published methods, guarantees removal of potentially contaminating surface-bound DNA. The protocol involves microscopic visualization of cell structures, deliberate surface contamination followed by surface sterilization with acid and bleach washes, and DNA extraction using Amicon centrifugal filters. Methods were verified on halite crystals of four different ages from Saline Valley, California (modern, 36 ka, 64 ka, and 150 ka), with retrieval of algal and archaeal DNA, and characterization of the algal community using ITS1 sequences. The protocol we developed opens up new avenues for study of ancient microbial ecosystems in fluid inclusions, understanding microbial evolution across geological time, and investigating the antiquity of life on earth and other parts of the solar system.

Background Archaeological and linguistic evidence suggests the Marianas Islands were settled around 3,600 years before present (ybp) from Island Southeast Asia (ISEA). Around 1,000 ybp latte stone pillars and the first evidence of rice cultivation appear in the Marianas. Both traditions are absent in the rest of prehistoric Oceania. Objective To examine the genetic origins and postsettlement gene flow of Chamorros of the Marianas Islands. Methods To infer the origins of the Chamorros we analyzed ~360 base pairs of the hypervariable-region 1 (HVS1) of mitochondrial DNA from 105 Chamorros from Guam, Rota, and Saipan, and the complete mitochondrial genome of 32 Guamanian Chamorros, and compared them to lineages from ISEA and neighboring Pacific archipelagoes from the database. Results Results reveal that 92% of Chamorros belong to haplogroup E, also found in ISEA but rare in Oceania. The two most numerous E lineages were identical to lineages currently found in Indonesia, while the remaining E lineages differed by only one or two mutations and all were unique to the Marianas. Seven percent of the lineages belonged to a single Chamorro-specific lineage within haplogroup B4, common to ISEA as well as Micronesia and Polynesia. Conclusions These patterns suggest a small founding population had reached and settled the Marianas from ISEA by 4,000 ybp, and developed unique mutations in isolation. A second migration from ISEA may have arrived around 1,000 ybp, introducing the latte pillars, rice agriculture and the homogeneous minority B4 lineage.

Background The immune system of milk protects against infections and guides immune system development. A system-level understanding of milk immune activity is critical for research into infant infectious disease risk and lifelong health. Research aim To describe a protocol to characterize immune activity in human milk via in vitro stimulation for use in population-based (rather than clinical) research. Methods This study proceeded in two phases, each with a cross-sectional design. Human milk specimens were incubated for 24 hr at 37 °C in mammalian cell culture medium with stimuli (e.g., Salmonella enterica) in a CO2-enriched environment. Immune responses to stimuli were characterized as the change in cytokine: [stimulated]/[baseline]. Predictors of cytokine responses were evaluated with generalized linear models. Results Patterns were detectable across mother–child dyads: Interleukin-6 responses to stimuli were generally positively associated with child age and with maternal autoimmune disease. Conclusions Our method allows characterization of pro-inflammatory milk immune activity in vitro in population-based (rather than clinical) research settings. In vitro activity has a system-level interpretation and is likely to be of broad utility in global health research in settings with high infectious disease risk, where understanding the immune system of milk is critical to understanding maternal and child health.

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Seasonal variation in spatial distribution and pathogen prevalence of Borrelia burgdorferi in blacklegged ticks (Ixodes scapularis) influences human population risk of Lyme disease in peri-urban built environments. Parks, gardens, playgrounds, school campuses and neighborhoods represent a significant risk for Lyme disease transmission. From June 2012 through May 2014, ticks were collected using 1 m2 corduroy cloths dragged over low-lying vegetation parallel to walkways with high human foot traffic. DNA was extracted from ticks, purified and presence of B. burgdorferi assessed by polymerase chain reaction amplification. Summer is reported as the time of highest risk for Lyme disease transmission in the United States and our results indicate a higher tick density of 26.0/1000 m2 in summer vs. 0.2/1000 m2 to 10.5/1000 m2 in spring and fall. However, our findings suggest that tick infection rate is proportionally higher during the fall and spring than summer (30.0–54.7% in fall and 36.8–65.6% in spring vs. 20.0–28.2% in summer). Seasonal variation in infected tick density has significant implications for Lyme disease transmission as people are less likely to be aware of ticks in built environments, and unaware of increased infection in ticks in spring and fall. These factors may lead to more tick bites resulting in Lyme infection.