lacZ Reporter System for Use in Borrelia burgdorferi

Regulation of gene expression is critical for the ability of Borrelia burgdorferi to adapt to different environments during its natural infectious cycle. Reporter genes have been used successfully to study gene regulation in multiple organisms. We have introduced a lacZ gene into B. burgdorferi, and we show that B. burgdorferi produces a protein with detectable β-galactosidase activity in both liquid and solid media when lacZ is expressed from a constitutive promoter. Furthermore, when lacZ is expressed from the ospC promoter, β-galactosidase activity is detected only in B. burgdorferi clones that express ospC, and it accurately monitors endogenous gene expression. The addition of lacZ to the repertoire of genetic tools available for use in B. burgdorferi should contribute to a better understanding of how B. burgdorferi gene expression is regulated during the infectious cycle.Borrelia burgdorferi sensu lato, the pathogen that causes Lyme disease (7), alternates between two distinct environments, an arthropod vector and a vertebrate host. As B. burgdorferi moves from one milieu to the other, its ability to adapt and survive requires dramatic changes in gene expression. Many studies have shown that different B. burgdorferi gene products are upregulated or downregulated at specific times during the infectious cycle (19, 31) and in response to host and environmental signals (6, 8a, 15, 24, 25). Although it is clear that B. burgdorferi alters gene expression to adapt to different environments, the genetic tools for studying gene regulation in B. burgdorferi are limited.Within the last 2 decades, the complete genomic sequence of B. burgdorferi strain B31 was published (10, 14) and techniques for basic genetic manipulation of B. burgdorferi became available (5, 11, 13, 27-29, 36). A chloramphenicol acetyltransferase (CAT) gene was the first reporter gene that was fused to B. burgdorferi promoters for analysis of promoter strength (33). The development of luciferase (4) and multiple fluorescent proteins (9, 11, 30) as reporter systems in B. burgdorferi followed. Although these systems have value, there are limitations with each. β-Galactosidase, encoded by lacZ, has been used extensively as a convenient reporter gene in Escherichia coli and is still applicable to a broad range of organisms, both prokaryotic and eukaryotic, but has not yet been used with B. burgdorferi. β-Galactosidase activity can be monitored easily and quickly by simple colorimetric assays in both liquid and solid media, neither of which require expensive or specialized equipment. Additionally, a wide variety of substrates for β-galactosidase allow for different levels of sensitivity in either in vitro or in vivo detection formats (17). Having lacZ available as a genetic tool for B. burgdorferi would enhance investigation of the complex regulatory events that are integral to the spirochete''s infectious cycle. To this end, we developed lacZ as a reporter gene in B. burgdorferi and demonstrated its utility.     

Multiple trophic pathways support fish on floodplains of California's Central Valley

We used compound-specific isotope analysis of carbon isotopes in amino acids (AAs) to determine the biosynthetic source of AAs in fish from major tributaries to California's Sacramento-San Joaquin river delta (i.e., the Sacramento, Cosumnes and Mokelumne rivers). Using samples collected in winter and spring between 2016 and 2019, we confirmed that algae are a critical component of floodplain food webs in California's Central Valley. Results from bulk stable isotope analysis of carbon and nitrogen in producers and consumers were adequate to characterize a general trophic structure and identify potential upstream and downstream migration into our study site by American shad Alosa sapidissima and rainbow trout Oncorhynchus mykiss, respectively. However, owing to overlap and variability in source isotope compositions, our bulk data were unsuitable for conventional bulk isotope mixing models. Our results from compound-specific carbon isotope analysis of AAs clearly indicate that algae are important sources of organic matter to fish of conservation concern, such as Chinook salmon Oncorhynchus tshawytscha in California's Central Valley. However, algae were not the exclusive source of energy to metazoan food webs. We also revealed that other sources of AAs, such as bacteria, fungi and higher plants, contributed to fish as well. While consistent with the well-supported notion that algae are critical to aquatic food webs, our results highlight the possibility that detrital subsidies might intermittently support metazoan food webs.     

The Environment and the Microbial Ecology of Human Skin

Microbial flora of the skin of three human population groups representing different natural environments was examined quantitatively and qualitatively to determine whether environmental differences in temperature and humidity can influence the microbial flora of normal skin. Five anatomical skin sites - hands, back, axillae, groin, and feet - were sampled from 10 subjects working in a high-humidity, high-temperature environment, 10 subjects from a low-temperature, high-humidity environment, and 10 subjects working in a moderate-temperature and low-humidity environment. Bacterial populations were significantly larger from the back, axillae, and feet in individuals from the high-temperature and high-humidity environment as compared to the moderate-temperature, low-humidity environment. High humidity and low temperature had no significant effect on total populations, but this group showed a higher frequency of isolation of fungi, and gram-negative bacteria from the back and feet. Although there was an indication that increase in the environmental humidity could result in an increased frequency of isolation of gram-negative bacteria, there was no evidence that an increase in either temperature or humidity altered the relative proportions of gram-negative bacteria in the predominantly gram-positive microbial flora found on normal skin. It was concluded that, although climatic changes may cause fluctation in microbial populations from certain sites, they are not a major influence on the ecology of the microbial flora of normal skin in the natural environment. The variables introduced by studying individuals in their natural environment and the influence of these on the results are discussed.     

No evidence for postcopulatory inbreeding avoidance in Drosophila melanogaster

Selection to avoid inbreeding is predicted to vary across species due to differences in population structure and reproductive biology. Over the past decade, there have been numerous investigations of postcopulatory inbreeding avoidance, a phenomenon that first requires discrimination of mate (or sperm) relatedness and then requires mechanisms of male ejaculate tailoring and/or cryptic female choice to avoid kin. The number of studies that have found a negative association between male-female genetic relatedness and competitive fertilization success is roughly equal to the number of studies that have not found such a relationship. In the former case, the underlying mechanisms are largely unknown. The present study was undertaken to verify and expand upon a previous report of postcopulatory inbreeding avoidance in D. melanogaster, as well as to resolve underlying mechanisms of inbreeding avoidance using transgenic flies that express a sperm head-specific fluorescent tag. However, siblings did not have a lower fertilization success as compared to unrelated males in either the first (P(1) ) or second (P(2) ) mate role in sperm competition with a standard unrelated competitor male in our study population of D. melanogaster. Analyses of mating latency, copulation duration, egg production rate, and remating interval further revealed no evidence for inbreeding avoidance.     

Mucosal luminal manipulation of T cell geography switches on protective efficacy by otherwise ineffective parenteral genetic immunization

Genetic immunization holds great promise for future vaccination against mucosal infectious diseases. However, parenteral genetic immunization is ineffective in control of mucosal intracellular infections, and the underlying mechanisms have remained unclear. By using a model of parenteral i.m. genetic immunization and pulmonary tuberculosis (TB), we have investigated the mechanisms that determine the failure and success of parenteral genetic immunization. We found that lack of protection from pulmonary Mycobacterium tuberculosis (M.tb) challenge by i.m. immunization with a recombinant adenovirus-vectored tuberculosis vaccine was linked to the absence of M.tb Ag-specific T cells within the airway lumen before M.tb challenge despite potent T cell activation in the systemic compartments. Furthermore, pulmonary mycobacterial challenge failed to recruit CD8 T cells into the airway lumen of i.m. immunized mice. Such defect in T cell recruitment, intra-airway CTL, and immune protection was restored by creating acute inflammation in the airway with inflammatory agonists such as virus. However, the Ag-specific T cells recruited as such were not retained in the airway lumen, resulting in a loss of protection. In comparison, airway exposure to low doses of soluble M.tb Ags not only recruited but retained Ag-specific CD8 T cells in the airway lumen over time that provided robust protection against M.tb challenge. Thus, our study reveals that mucosal protection by parenteral immunization is critically determined by T cell geography, i.e., whether Ag-specific T cells are within or outside of the mucosal lumen and presents a feasible solution to empower parenteral immunization strategies against mucosal infectious diseases.     

Systematic Investigation of the Stability of Food Preferences in Captive Orangutans: Implications for Positive Reinforcement Training

Using preference-assessment tests with humans in conjunction with behavioral modification sessions has been a regular component of almost all operant conditioning programs with mentally challenged humans. This has been very effective in improving the efficiency of behavioral training in these settings and could be similarly effective in zoological and research environments. This study investigated the preferences of 9 captive orangutans for different food items. The study used a pairwise presentation to record each nonhuman animal's preferences for 5 different foods on 6 different occasions over the course of 6 months. Results of a Friedman's 2-way ANOVA indicated that the orangutans showed a clear overall preference for apple. However, there was significant variability among different orangutans in preference ranking for the 5 foods, as shown by a Kendall's tau. In addition, there was variability in preference rankings across time for each orangutan. Because the orangutans' preferences change over time and vary according to individual, regular assessments should identify items to be used as rewards in behavioral husbandry training or as part of feeding enrichment strategies.     

Quantitation of Endogenous C-type Virion Production in Several Murine Cell Lines

Quantitation by enumeration of virion particles, measurement of absorbancy at 260 nm, and densitometry on sodium dodecyl sulfate-polyacrylamide gels has shown that mouse L-M cells yielded 7- to 10-fold less endogenous C-type virions than the parental lines, L or L929. The previously noted stimulation of L-M cell virion production by a concomitant 10% increase in fetal calf serum concentration (D. A. Kindig, R. Karp, and W. H. Kirsten, 1968), was not observed.     

Positive Reinforcement Training Moderates Only High Levels of Abnormal Behavior in Singly Housed Rhesus Macaques

This study evaluated the application of positive reinforcement training (PRT) as an intervention for abnormal behaviors in singly housed laboratory rhesus macaques at 2 large primate facilities. Training involved basic control behaviors and body-part presentation. The study compared baseline behavioral data on 30 adult males and 33 adult females compared with 3 treatment phases presented in counterbalanced order: 6 min per week of PRT, 20 or 40 min per week of PRT, and 6 min per week of unstructured human interaction (HI). Within-subject parametric tests detected no main or interaction effects involving experimental phase. However, among a subset of subjects with levels of abnormal in the top quartile of the range (n = 15), abnormal behavior was reduced from 35% to 25% of samples with PRT but not with HI. These results suggest that short durations of PRT applied as enrichment for this species and in this context may not in itself be sufficient intervention for abnormal behavior because levels remained high. However, it may be appropriate as an adjunct to other interventions and may be best targeted to the most severely affected individuals.