Temperature Effects on DNA Damage during Hibernation.

AbstractDuring multiday torpor, deep-hibernating mammals maintain a hypometabolic state where heart rate and ventilation are reduced to 2%-4% of euthermic rates. It is hypothesized that this ischemia-like condition may cause DNA damage through reactive oxygen species production. The reason for intermittent rewarming (arousal) during hibernation might be to repair the accumulated DNA damage. Because increasing ambient temperatures (Ta's) shortens torpor bout duration, we hypothesize that hibernating at higher Ta's will result in a faster accumulation of genomic DNA damage. To test this, we kept 39 male and female garden dormice at a Ta of either 5°C or 10°C and obtained tissue at 1, 4, and 8 d in torpor to assess DNA damage and recruitment of DNA repair markers in splenocytes. DNA damage in splenocytes measured by comet assay was significantly higher in almost all torpor groups than in summer euthermic groups. Damage accumulates in the first days of torpor at Ta=5°C (between days 1 and 4) but not at Ta=10°C. At the higher Ta, DNA damage is high at 24 h in torpor, indicating either a faster buildup of DNA damage at higher Ta's or an incomplete repair during arousals in dormice. At 5°C, recruitment of the DNA repair protein 53BP1 paralleled the increase in DNA damage over time during torpor. In contrast, after 1 d in torpor at 10°C, DNA damage levels were high, but 53BP1 was not recruited to the nuclear DNA yet. The data suggest a potential mismatch in the DNA damage/repair dynamics during torpor at higher Ta's.

Evaluating the Clinical and Immune Responses to Spotted Fever Rickettsioses in the Guinea Pig-Tick-Rickettsia System.

The guinea pig was the original animal model developed for investigating spotted fever rickettsiosis (SFR). This model system has persisted on account of the guinea pig's conduciveness to tick transmission of SFR agents and ability to recapitulate SFR in humans through clinical signs that include fever, unthriftiness, and in some cases the development of an eschar. The guinea pig is the smallest animal model for SFR that allows the collection of multiple blood and skin samples antemortem for longitudinal studies. This unit provides the basic protocols necessary to establish, maintain, and utilize a guinea pig-tick-Rickettsia model for monitoring the course of infection and immune response to an infection by spotted fever group Rickettsia (SFGR) that can be studied at biosafety level 2 (BSL-2) and arthropod containment level 2 (ACL-2); adaptations must be made for BSL-3 agents. The protocols cover methods for tick feeding and colony development, laboratory infection of ticks, tick transmission of Rickettsia to guinea pigs, and monitoring of the course of infection through clinical signs, rickettsial burden, and immune response. It should be feasible to adapt these methods to study other tick-borne pathogens. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Tick transmission of SFGR to guinea pigs Support Protocol 1: Laboratory infection of ticks by injection Alternate Protocol 1: Needle inoculation of SFGR to guinea pigs Basic Protocol 2: Monitoring the course of guinea pig rickettsial infection: clinical signs Basic Protocol 3: Monitoring the course of guinea pig rickettsial infection: collection of biological specimens Support Protocol 2: Guinea pig anesthesia Basic Protocol 4: Monitoring rickettsial burden in guinea pigs by multiplex qPCR Basic Protocol 5: Monitoring guinea pig immune response to infection: blood leukocytes by flow cytometry Basic Protocol 6: Monitoring immune response to guinea pig rickettsial infection: leukocyte infiltration of skin at the tick bite site by flow cytometry Basic Protocol 7: Monitoring the immune response to guinea pig rickettsial infection: antibody titer by ELISA Support Protocol 4: Coating ELISA Plates Alternate Protocol 2: Monitoring immune response to guinea pig rickettsial infection: antibody titer by immunofluorescence assay.

Multiplex TaqMan® Quantitative PCR Assays for Host-Tick-Pathogen Studies Using the Guinea Pig-Tick-Rickettsia System.

Spotted Fever Rickettsiosis (SFR) is caused by spotted fever group Rickettsia spp. (SFGR), and is associated with symptoms common to other illnesses, making it challenging to diagnose before detecting SFGR-specific antibodies. The guinea pig is a valuable biomedical model for studying Spotted Fever Rickettsiosis (SFR); its immune system is more like the human immune system than that of the murine model, and guinea pigs develop characteristic clinical signs. Thus, we have a compelling interest in developing, expanding, and optimizing tools for use in our guinea pig-Amblyomma-Rickettsia system for understanding host-tick-pathogen interactions. With the design and optimization of the three multiplex TaqMan® qPCR assays described here, we can detect the two SFGR, their respective primary Amblyomma sp. vectors, and the guinea pig model as part of controlled experimental studies using tick-transmission of SFGR to guinea pigs. We developed qPCR assays that reliably detect each specific target down to 10 copies by producing plasmid standards for each assay target, optimizing the individual primer-probe sets, and optimizing the final multiplex reactions in a methodical, stepwise fashion. We anticipate that these assays, currently designed for in vivo studies, will serve as a foundation for optimal SFGR detection in other systems, including fieldwork.

Skin in the Game: An Assay to Monitor Leukocyte Infiltration in Dermal Lesions of a Guinea Pig Model for Tick-Borne Rickettsiosis.

Intact, the skin typically serves as an effective barrier to the external world; however, once pathogens have breached this barrier via a wound, such as a tick bite, the surrounding tissues must recruit immune cells from the blood to neutralize the pathogen. With innate and adaptive immune systems being similar between the guinea pig and human systems, the ability of guinea pigs to show clinical signs of many infectious diseases, and the large size of guinea pigs relative to a murine model, the guinea pig is a valuable model for studying tick-borne and other pathogens that invade the skin. Here, we report a novel assay for assessing guinea pig leukocyte infiltration in the skin. Briefly, we developed an optimized six-color/eight-parameter polychromatic flow cytometric panel that combines enzymatic and mechanical dissociation of skin tissue with fluorescent antibody staining to allow for the immunophenotyping of guinea pig leukocytes that have migrated into the skin, resulting in inflammation. We designed this assay using a guinea pig model for tick-borne rickettsiosis to further investigate host-pathogen interactions in the skin, with preliminary data demonstrating immunophenotyping at skin lesions from infected ticks. We anticipate that future applications will include hypothesis testing to define the primary immune cell infiltrates responding to exposure to virulent, avirulent tick-borne rickettsiae, and tick-borne rickettsiae of unknown virulence. Other relevant applications include skin lesions resulting from other vector-borne pathogens, Staphylococcus aureus infection, and Buruli ulcer caused by Mycobacterium ulcerans.

Beyond the IFA: Revisiting the ELISA as a More Sensitive, Objective, and Quantitative Evaluation of Spotted Fever Group Rickettsia Exposure.

Based on limited serological studies, at least 10% of the US population has been exposed to spotted fever group Rickettsia (SFGR) species. The immunofluorescence antibody assay (IFA) has been the gold standard for the serodiagnosis of rickettsial infections such as spotted fever rickettsiosis (SFR). However, the IFA is semi-quantitative and subjective, requiring a high level of expertise to interpret it correctly. Here, we developed an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Rickettsia parkeri infection in the guinea pig. Our ELISA is an objective, quantitative, and high-throughput assay that shows greater sensitivity and resolution in observed titers than the IFA. We methodically optimized relevant parameters in sequence for optimal signal-to-noise ratio and low coefficient of variation% values. We used a guinea pig model as it is a part of our overall research efforts to understand the immunological and clinical response to SFGR species after tick transmission. Guinea pigs are a useful model to study SFR and show clinical signs of SFR, such as fever and eschars. We anticipate that this assay will be easily adapted to other hosts, including humans and other SFGR species.

An optimized five-color/seven-parameter flow cytometry panel for immunophenotyping guinea pig peripheral blood lymphocytes.

Guinea pigs are an ideal animal model for the study of several infectious diseases, including tuberculosis, legionellosis, brucellosis, and spotted fever rickettsiosis. In comparison to the murine model, clinical signs in guinea pigs are more representative of disease in humans, the guinea pig immune system is more similar to that of the human, and their large size offers logistic advantages for sample collection while following disease progression. Unfortunately, the advantage of using guinea pigs in biomedical research, particularly in understanding the immune response to infectious agents, is limited in large part by the paucity of available reagents and lack of genetically manipulated strains. Here, we expand the utility of guinea pigs in biomedical research by establishing an optimized five-color/seven-parameter polychromatic flow cytometric assay for immunophenotyping lymphocytes. This assay fills a need for immunophenotyping peripheral blood lymphocytes and is an improvement over current published flow cytometry assays for guinea pigs. We anticipate that our approach will be an important starting point for developing new assays to evaluate the cellular immune response to infectious diseases in the guinea pig model. Importantly, we are currently using this assay for evaluating immunity to spotted fever rickettsiosis in a guinea pig-tick-Rickettsia system, where CD8+ T cells are a critical contributor to the immune response. Developing resources to utilize the guinea pig more effectively will enhance our ability to understand infectious diseases where the guinea pig would otherwise be the ideal model.

MEGARes: an antimicrobial resistance database for high throughput sequencing.

Antimicrobial resistance has become an imminent concern for public health. As methods for detection and characterization of antimicrobial resistance move from targeted culture and polymerase chain reaction to high throughput metagenomics, appropriate resources for the analysis of large-scale data are required. Currently, antimicrobial resistance databases are tailored to smaller-scale, functional profiling of genes using highly descriptive annotations. Such characteristics do not facilitate the analysis of large-scale, ecological sequence datasets such as those produced with the use of metagenomics for surveillance. In order to overcome these limitations, we present MEGARes (https://megares.meglab.org), a hand-curated antimicrobial resistance database and annotation structure that provides a foundation for the development of high throughput acyclical classifiers and hierarchical statistical analysis of big data. MEGARes can be browsed as a stand-alone resource through the website or can be easily integrated into sequence analysis pipelines through download. Also via the website, we provide documentation for AmrPlusPlus, a user-friendly Galaxy pipeline for the analysis of high throughput sequencing data that is pre-packaged for use with the MEGARes database.

P50 sensory gating in infants.

Attentional deficits are common in a variety of neuropsychiatric disorders including attention deficit-hyperactivity disorder, autism, bipolar mood disorder, and schizophrenia. There has been increasing interest in the neurodevelopmental components of these attentional deficits; neurodevelopmental meaning that while the deficits become clinically prominent in childhood or adulthood, the deficits are the results of problems in brain development that begin in infancy or even prenatally. Despite this interest, there are few methods for assessing attention very early in infancy. This report focuses on one method, infant auditory P50 sensory gating. Attention has several components. One of the earliest components of attention, termed sensory gating, allows the brain to tune out repetitive, noninformative sensory information. Auditory P50 sensory gating refers to one task designed to measure sensory gating using changes in EEG. When identical auditory stimuli are presented 500 ms apart, the evoked response (change in the EEG associated with the processing of the click) to the second stimulus is generally reduced relative to the response to the first stimulus (i.e. the response is "gated"). When response to the second stimulus is not reduced, this is considered a poor sensory gating, is reflective of impaired cerebral inhibition, and is correlated with attentional deficits. Because the auditory P50 sensory gating task is passive, it is of potential utility in the study of young infants and may provide a window into the developmental time course of attentional deficits in a variety of neuropsychiatric disorders. The goal of this presentation is to describe the methodology for assessing infant auditory P50 sensory gating, a methodology adapted from those used in studies of adult populations.

Effects of pH on the ability of flavonoids to act as Pickering emulsion stabilizers.

The flavonoids tiliroside, rutin and naringin have been investigated as stabilizers of Pickering oil-in-water (O/W) emulsions. The mean droplet size of tetradecane emulsions was considerably smaller at higher pH, especially for rutin. The solubility of flavonoids in the aqueous phase was 4-6 times higher at pH 8 compared to pH 2 for tiliroside and rutin, although all absolute solubilities remained low (<1 mM). This agreed with a slight increase in surface activity of tiliroside and rutin at the O-W interface at pH 8 compared to pH 2. However, improved emulsion stabilization at higher pH is better explained by the significant increase in ζ-potential of the flavonoid particles to more negative values at pH 8, which will improve particle dispersion and increase the charge on the droplets stabilized by them. A buckwheat tea extract, rich in rutin, was also shown to be an effective stabilizer of sunflower O/W emulsions.

Changes in lysosomal hydrolase activity associated with malnutrition in young rats

Total lysosmal hydrolase activities were measured in liver, gastrocnemius muscle and plasma of malnourished and normal rats between 3 and 8 weeks of age. Concurrently, the DNA and protein contents of the livers and muscles were determined. Increased amounts of acid hydrolase activities were found to be associated with subnormal protein: DNA ratios in the tissues of malnourished rats. It was concluded that lysosmal enzymes may be involved in protein catabolism during malnutrition (AU)