Mary Farone

Direct laser trapping for measuring the behavior of transfused erythrocytes in a sickle cell anemia patient.

Using a laser trap, we have studied the properties of erythrocytes from a sickle cell anemia patient (SCA) after receiving an intravenous blood transfusion, and a normal adult individual carrying normal adult hemoglobin. The hemoglobin type and quantitation assessment was carried out by high performance liquid chromatography (HPLC). We conducted an analysis of the size distributions of the cells. By targeting those erythrocytes in the overlapping regions of size distributions, we have investigated their properties when the cells are trapped and released. The efficacy of the transfusion treatment is also studied by comparing the relative changes in deformation and the relaxation-time of the cells in the two samples.

Gardnerella vaginalis triggers NLRP3 inflammasome recruitment in THP-1 monocytes

Gardnerella vaginalis is a Gram-positive bacterium associated with bacterial vaginosis (BV), pelvic inflammatory disease, and preterm birth. BV is the most prevalent vaginal dysbiosis in women of childbearing age characterized by the absence of normal lactobacilli and an overgrowth of G. vaginalis and other bacteria. Although mucosal fluids from BV patients exhibit increases in proinflammatory cytokines and Toll-like receptor 2 and 4 mRNA, G. vaginalis has not been demonstrated to directly induce an inflammatory response. This study tested the hypothesis that G. vaginalis induces an inflammatory response in the human monocyte cell line, THP-1. The objectives of the study were to measure proinflammatory cytokine production, molecular mechanisms by which cytokines are produced, and whether G. vaginalis results in death of the monocytic cells. We found that G. vaginalis induced significant increases in the inflammasome-dependent cytokines IL-1β, IL-18, as well as TNF-α in treated cells. G. vaginalis caused significant cell death by 24h post-treatment compared with untreated controls, but cells remained 66% viable. Caspase-1 cleavage in treated cells confirmed the inflammatory cell death, and NLRP3 knockdown confirmed its involvement through reduction of IL-1β secretion. Using a stably expressing YFP-ASC THP-1 cell model with immunofluorescent staining, YFP-ASC colocalized with NLRP3 in G. vaginalis-treated cells and the addition of a caspase-1 inhibitor wholly ameliorated IL-1β secretion. Our study provides new insight into the role of G. vaginalis in inflammatory conditions in the genital tract.

Antifungal activity of substituted aurones

Novel antifungals are in high demand as there is a growing resistance to antifungals currently in use. In particular, opportunistic fungal infections caused by Candida spp. are on the rise with infections by this genus accounting for the most severe fungal infections following chemotherapy, implantation procedures, and in patients with HIV/AIDS. A series of simple aurone analogs were synthesized and screened for antifungal activity versus Candida spp. Several compounds displayed activity at 100μM, with two having IC50 values below 20μM for three species of Candida. One of the compounds tested here also exhibits anti-biofilm activity for mid-maturation growth.

The potential of in situ hybridization and an immunogold assay to identify Legionella associations with other microorganisms

Based on in vitro studies, bacteria in the genus Legionella are believed to multiply within protozoa such as amoebae in aquatic environments. Current methods used for detection of Legionella species, however, are not designed to show this relationship. Thus the natural intimate association of Legionella with other microorganisms remains to be clearly documented and the extent to which protozoa might be infected with Legionella species remains undefined. In this report we describe methods based on the use of Legionella specific reagents that would prove useful in describing its associations with other microorganisms. An immunogold and in situ hybridization technique have the potential to demonstrate the natural occurrence of Legionella species in free-living amoebae. In preliminary observations, however, bacteria reactive with Legionella specific reagents were often not intimately associated with amoebae. Bacteria occurred as free single cells, as cell aggregates, in proximity to other cells and debris, and only occasionally in close proximity to amoebae. Although some Legionella species replicate within amoebae, these preliminary observations suggest the bacteria may be encountered most frequently as extracellular microorganisms, either free-floating or in association with other structures or microorganisms. The future use of these techniques will aid in the elucidation of any naturally occurring relationships between Legionella species and other microorganisms.

Cis- and Trans-gnetin H from Paeonia suffruticosa suppress inhibitor kappa B kinase phosphorylation in LPS-stimulated human THP-1 cells

ETHNOPHARMACOLOGICAL RELEVANCE The inflammatory response is an important mechanism in host defense; however, overstimulation and chronic inflammation are involved in many important human diseases. Currently, tumor necrosis factor-alpha blockers such as infliximab and adalimumab along with methotrexate are used in cases of severe and chronic disease. However, there are severe side effects and limitations associated with these treatments. Cis- and trans-gnetin H are compounds isolated from the seeds of Paeonia suffruticosa, a medicinal plant used in traditional Chinese medicine for the treatment of many conditions, including inflammatory diseases. In this study, we investigated possible anti-inflammatory mechanisms of cis- and trans-gnetin H against LPS-stimulated human THP-1 cells. MATERIAL AND METHODS PMA-differentiated THP-1 cells were pretreated with increasing concentrations of cis- and trans-gnetin H with or without LPS. Following treatment, cytotoxicity and the TNF-α, IL-1β, and IL-8 response were measured. We also characterized the nuclear translocation of NF-κB subunit p65 (RelA) by immunofluorescence and then investigated NF-κB activation by measuring the phosphorylation of NF-κB mediators, IKK-β, IκB α, and p65 by western blotting. RESULTS We found that cis- and trans-gnetin H significantly inhibited the cytokine response in a concentration-dependent manner without affecting cell viability. Cis- and trans-gnetin H effectively inhibited nuclear translocation of p65 and phosphorylation of IKK-β, IκB α, and p65. While both compounds showed promising anti-inflammatory effects, trans-gnetin H was determined to be more effective in suppressing cytokine responses. CONCLUSION We demonstrated that cis- and trans-gnetin H suppress cytokine response in LPS-stimulated THP-1 cells by preventing activation of key signaling molecules, IKK-β, IκB α, and p65, involved in the NF-κB pathway and suggest the use of cis- and trans-gnetin H in potential therapies for conditions and diseases associated with chronic inflammation.

Bacterial enteropathogens associated with diarrhea in a rural population of Haiti

Purpose Diarrheal disease is one of the leading causes of morbidity in developing countries. To further understand the epidemiology of diarrheal disease among a rural population surrounding Robillard, Haiti, fecal swabs from patients with diarrhea were screened for the presence of enteropathogenic bacteria. Patients and methods Fecal swabs were collected from 34 patients with signs and symptoms of diarrhea and stored in BBL™ Cary-Blair transport medium (Becton, Dickinson and Company, Sparks, MD) until transit to the USA. Swab material was inoculated on to different enrichment and selective agars for incubation. Fermenting and nonfermenting bacteria that grew on the enteric selection media were identified by the BBL™ Crystal™ Enteric/Nonferementing Identification system (Becton, Dickinson and Company). Organisms identified as Escherichia coli were further screened for the presence of virulence factors by polymerase chain reaction (PCR). Results Of 34 patients, no Campylobacter, Shigella, Salmonella, or Vibrio spp. were isolated from swabs transported to the USA for culture. Of 73 E. coli isolates cultured from the swabs, one enteropathogenic strain of E. coli was identified by multiplex PCR. Escherichia fergusonii and Cronobacter sakazakii, both potential gastrointestinal pathogens, were also isolated from patient stools. Conclusion This study was undertaken to determine if bacterial enteropathogens could be detected in the stools of patients suffering from diarrhea or dysentery and, in the absence of sufficient facilities, rectal swabs could be transported to the USA for culture. Although several genera of overt enteropathogens were not detected, one enteropathogenic E. coli and other pathogenic enterobacteriaceae were successfully cultured and identified.

Suppression of LPS-induced NF-κB activity in macrophages by the synthetic aurone, (Z)-2-((5-(hydroxymethyl) furan-2-yl) methylene) benzofuran-3(2H)-one.

&NA; Suppressing cytokine responses has frequently been shown to have promising therapeutic effects for many chronic inflammatory and autoimmune diseases. However, the severe side effects associated with the long‐term use of current treatments, such as allergic reactions and increased risk of stroke, have focused attention towards the targeting of intracellular signaling mechanisms, such as NF‐&kgr;B, that regulate inflammation. We synthesized a series of non‐natural aurone derivatives and investigated their ability to suppress pro‐inflammatory signaling in human monocyte (THP‐1) and murine macrophage‐like (RAW 267.4) cell lines. One of these derivatives, (Z)‐2‐((5‐(hydroxymethyl) furan‐2‐yl) methylene) benzofuran‐3(2H)‐one (aurone 1), was found to inhibit LPS‐induced secretion of the pro‐inflammatory cytokines, tumor‐necrosis factor &agr; (TNF&agr;), interleukin 1&bgr; (IL‐1&bgr;), and IL‐8 by THP‐1 cells. To investigate the mechanism, we probed the effect of aurone 1 on LPS‐induced MAPK and NF‐&kgr;B signaling in both THP‐1 and RAW264.7. While aurone 1 pre‐treatment had no effect on the phosphorylation of ERK, JNK, or p38 MAPK, it strongly suppressed activation of IKK‐&bgr;, as indicated by attenuation of Ser176/180 phosphorylation, resulting in decreased phosphorylation of p65 (ser536) as well as phosphorylation (ser32) and degradation of I&kgr;B&agr;. Consistent with this, aurone 1 significantly reduced LPS‐stimulated nuclear translocation of p65‐containing NF‐&kgr;B transcription factors and expression of an mCherry reporter of TNF&agr; gene transactivation in RAW264.7 cells. Inhibition of TNF&agr; expression at the transcription level was also demonstrated in THP‐1 by qRT‐PCR. In addition to its effects on cytokine expression, aurone 1 pre‐treatment decreased expression of iNOS, a bona fide NF‐&kgr;B target gene and marker of macrophage M1 polarization, resulting in decreased NO production in RAW264.7 cells. Together, these data indicate that aurone 1 may have the potential to function as a pharmacological agent for the treatment of chronic inflammation disorders. Graphical abstract: Figure. No caption available. HighlightsAurone 1 inhibits TNF&agr;, IL‐1&bgr;, and IL‐8 expression in LPS‐stimulated THP‐1 cells.p65 translocation and NF‐&kgr;B‐dependent transcription is blocked by aurone 1 in macrophages.The mechanism involves inhibition of IKK&bgr; phosphorylation and I&kgr;B&agr; degradation.Aurone 1 has no significant effect on LPS‐induced MAPK phosphorylation.Aurone 1 suppresses iNOS expression and NO production in RAW264.7 macrophages.

Application of a laser trap as a viscometer

A laser tweezer (LT) along with advanced imaging techniques has been widely applied to manipulate and study living as well as nonliving microscopic objects. In this study we present yet another novel application of LTs for a precise measurement of the viscosities of fluids in a micro-volume flow. We have demonstrated this novel application by measuring the viscosity of a fetal bovine serum (FBS) using a LT constructed from a single intensity gradient laser trap. By calibrating the LT using dielectric silica micro-beads in a fluid with a known viscosity, specifically water, and by suspending same size of silica beads in the FBS and trapping with the same trap, we have determined the viscosity of the FBS at different temperatures. We have used the relationship between the trapping and Stoke’s drag force for a constant drag speed to determine the viscosity. We have also analyzed the viscosities determined in comparison with corresponding viscosities measured using an Ostwald viscometer.

A Practical Approach for Computing the Active Site of the Ribonucleoside Hydrolase of E. coli Encoded by rihC

We predict the potential active and catalytic sites, the transition state and how it is stabilized, and the mechanism of rihC ribonucleoside hydrolase of E. coli. Our approach is based on well-known primary sequence analysis techniques. A canonically associated extreme value distribution is used to assess the significance of the prediction. Parameters for the extreme value distribution are computed directly from data. Our practical approach is consistent with known results in the literature. We obtain BLOSUM matrices in a way that is intrinsically tied to the data base, and we employ user-friendly techniques that should be applicable to a range of medically significant scenarios.

Stability of pentobarbital in soil

ABSTRACT Intravenous injection of barbiturates, particularly pentobarbital (5-ethyl-5-pentan-2-yl-1,3-diazinane-2,4,5-trione), is a widely used method to euthanize large animals such as horses. However, one concern with this method is the fate of pentobarbital after the disposal of the carcass. As tissues decompose, pentobarbital may leach into the soil and from there migrate to groundwater. A method using methanol extraction, solid phase concentration, and liquid chromatography (LC/MS) has been developed to measure pentobarbital in soils. Recovery of pentobarbital from soil averaged approximately 85% from different soil types including topsoil, potting soil, sand, stall sweepings, and loam. The method was capable of detecting pentobarbital levels of 0.1 ppm. A calibration curve was constructed with a linear range of 1 ppm to 100 ppm. The limit of quantification was 0.5 ppm. The rate of degradation of pentobarbital in sand, topsoil, and potting soil was measured over a 17-week period. At the end of week 17, approximately 17% of the pentobarbital remained in the sand, 19% remained in the topsoil, and 10% remained in the potting soil. While there was a significant decrease in the pentobarbital recovered from the soil, there were still detectable amounts of pentobarbital present in the soil after 17 weeks. To determine the importance of bacterial degradation, the three soil types were autoclaved before addition of pentobarbital. After autoclaving, no degradation of pentobarbital was observed in sand and one topsoil sample, while there was no difference in the degradation of pentobarbital in autoclaved potting soil versus potting soil that had not undergone autoclaving.