Sohrab Naushad Ali

Probiotic Lactobacillus rhamnosus reduces organophosphate pesticide absorption and toxicity to Drosophila melanogaster

ABSTRACT Organophosphate pesticides used in agriculture can pose health risks to humans and wildlife. We hypothesized that dietary supplementation with Lactobacillus, a genus of commensal bacteria, would reduce absorption and toxicity of consumed organophosphate pesticides (parathion and chlorpyrifos [CP]). Several Lactobacillus species were screened for toleration of 100 ppm of CP or parathion in MRS broth based on 24-h growth curves. Certain Lactobacillus strains were unable to reach stationary-phase culture maxima and displayed an abnormal culture morphology in response to pesticide. Further characterization of commonly used, pesticide-tolerant and pesticide-susceptible, probiotic Lactobacillus rhamnosus strain GG (LGG) and L. rhamnosus strain GR-1 (LGR-1), respectively, revealed that both strains could significantly sequester organophosphate pesticides from solution after 24-h coincubations. This effect was independent of metabolic activity, as L. rhamnosus GG did not hydrolyze CP and no difference in organophosphate sequestration was observed between live and heat-killed strains. Furthermore, LGR-1 and LGG reduced the absorption of 100 μM parathion or CP in a Caco-2 Transwell model of the small intestine epithelium. To determine the effect of sequestration on acute toxicity, newly eclosed Drosophila melanogaster flies were exposed to food containing 10 μM CP with or without supplementation with live LGG. Supplementation with LGG simultaneously, but not with administration of CP 3 days prior (prophylactically), mitigated CP-induced mortality. In summary, the results suggest that L. rhamnosus may be useful for reducing toxic organophosphate pesticide exposure via passive binding. These findings could be transferable to clinical and livestock applications due to affordability and practical ability to supplement products with food-grade bacteria. IMPORTANCE The consequences of environmental pesticide pollution due to widespread usage in agriculture and soil leaching are becoming a major societal concern. Although the long-term effects of low-dose pesticide exposure for humans and wildlife remain largely unknown, logic suggests that these chemicals are not aligned with ecosystem health. This observation is most strongly supported by the agricultural losses associated with honeybee population declines, known as colony collapse disorder, in which pesticide usage is a likely trigger. Lactobacilli are bacteria used as beneficial microorganisms in fermented foods and have shown potentials to sequester and degrade environmental toxins. This study demonstrated that commonly used probiotic strains of lactobacilli could sequester, but not metabolize, organophosphate pesticides (parathion and chlorpyrifos). This Lactobacillus-mediated sequestration was associated with decreased intestinal absorption and insect toxicity in appropriate models. These findings hold promise for supplementing human, livestock, or apiary foods with probiotic microorganisms to reduce organophosphate pesticide exposure.

Neonicotinoid-induced pathogen susceptibility is mitigated by Lactobacillus plantarum immune stimulation in a Drosophila melanogaster model

Pesticides are used extensively in food production to maximize crop yields. However, neonicotinoid insecticides exert unintentional toxicity to honey bees (Apis mellifera) that may partially be associated with massive population declines referred to as colony collapse disorder. We hypothesized that imidacloprid (common neonicotinoid; IMI) exposure would make Drosophila melanogaster (an insect model for the honey bee) more susceptible to bacterial pathogens, heat stress, and intestinal dysbiosis. Our results suggested that the immune deficiency (Imd) pathway is necessary for D. melanogaster survival in response to IMI toxicity. IMI exposure induced alterations in the host-microbiota as noted by increased indigenous Acetobacter and Lactobacillus spp. Furthermore, sub-lethal exposure to IMI resulted in decreased D. melanogaster survival when simultaneously exposed to bacterial infection and heat stress (37 °C). This coincided with exacerbated increases in TotA and Dpt (Imd downstream pro-survival and antimicrobial genes, respectively) expression compared to controls. Supplementation of IMI-exposed D. melanogaster with Lactobacillus plantarum ATCC 14917 mitigated survival deficits following Serratia marcescens (bacterial pathogen) septic infection. These findings support the insidious toxicity of neonicotinoid pesticides and potential for probiotic lactobacilli to reduce IMI-induced susceptibility to infection.

Natural History of Residual Fragments After Percutaneous Nephrolithotomy: Evaluation of Factors Related to Clinical Events and Intervention

OBJECTIVE To determine the natural history of residual fragments (RF) after percutaneous nephrolithotomy in long-term patient follow-up and to identify possible predictive factors for future intervention. MATERIALS AND METHODS We assessed all patients from 2006 to 2013 with postoperative computed tomography scan revealing RF, who did not undergo second-look nephroscopy or immediate ancillary procedures, and with at least 12 months of clinical follow-up. We evaluated factors associated with clinical, radiological, and surgical outcomes. Kaplan-Meier curves were used to calculate the proportion of asymptomatic and treatment-free patients during follow-up. RESULTS From 781 percutaneous nephrolithotomies performed, 202 patients underwent postoperative computed tomography scan and 44 patients with residual stones were included in the analysis. Mean follow-up was 57.9 months. A total of 24 patients (54.5%) developed at least 1 clinical outcome, and 32 (72.7%) patients had a surgical intervention. Only 4 patients had radiological evidence of stone passage. Multivariate analysis found that RF >4 mm and struvite or apatite stones were significant predictors for surgical intervention. The 5-year estimated probability to remain intervention free was 29%. CONCLUSION Despite the size of the RF, the vast majority of patients required an intervention during long-term follow-up. We identified that RFs of diameter >4 mm associate with the need for a surgical therapy, but the rate of clinical events was not affected by the size or location of the stones. Struvite or apatite composition stones had an increased risk of intervention during follow-up. Spontaneous passage was an uncommon event in this cohort.

Novel Methods of Determining Urinary Calculi Composition: Petrographic Thin Sectioning of Calculi and Nanoscale Flow Cytometry Urinalysis

Accurate determination of urinary stone composition has significant bearing on understanding pathophysiology, choosing treatment modalities and preventing recurrence. A need exists for improved methods to determine stone composition. Urine of 31 patients with known renal calculi was examined with nanoscale flow cytometry and the calculi collected during surgery subsequently underwent petrographic thin sectioning with polarized and fluorescent microscopy. Fluorescently labeled bisphosphonate probes (Alendronate-fluorescein/Alendronate-Cy5) were developed for nanoscale flow cytometry to enumerate nanocrystals that bound the fluorescent probes. Petrographic sections of stones were also imaged by fluorescent and polarized light microscopy with composition analysis correlated to alendronate +ve nanocrystal counts in corresponding urine samples. Urine samples from patients with Ca2+ and Mg2+ based calculi exhibited the highest alendronate +ve nanocrystal counts, ranging from 100–1000 nm in diameter. This novel urine based assay was in agreement with composition determined by petrographic thin sections with Alendronate probes. In some cases, high alendronate +ve nanocrystal counts indicated a Ca2+ or Mg2+ composition, as confirmed by petrographic analysis, overturning initial spectrophotometric diagnosis of stone composition. The combination of nanoscale flow cytometry and petrographic thin sections offer an alternative means for determining stone composition. Nanoscale flow cytometry of alendronate +ve nanocrystals alone may provide a high-throughput means of evaluating stone burden.

Drosophila melanogaster as a function-based high-throughput screening model for anti-nephrolithiasis agents in kidney stone patients

ABSTRACT Kidney stone disease involves the aggregation of stone-forming salts consequent to solute supersaturation in urine. The development of novel therapeutic agents for this predominantly metabolic and biochemical disorder have been hampered by the lack of a practical pre-clinical model amenable to drug screening. Here, Drosophila melanogaster, an emerging model for kidney stone disease research, was adapted as a high-throughput functional drug screening platform independent of the multifactorial nature of mammalian nephrolithiasis. Through functional screening, the therapeutic potential of a novel compound commonly known as arbutin that specifically binds to oxalate, a key component of kidney calculi, was identified. Through isothermal titration calorimetry, high-performance liquid chromatography and atomic force microscopy, arbutin was determined to interact with calcium and oxalate in both free and bound states, disrupting crystal lattice structure, growth and crystallization. When used to treat patient urine samples, arbutin significantly abrogated calculus formation in vivo and outperformed potassium citrate in low pH urine conditions, owing to its oxalate-centric mode of action. The discovery of this novel antilithogenic compound via D. melanogaster, independent of a mammalian model, brings greater recognition to this platform, for which metabolic features are primary outcomes, underscoring the power of D. melanogaster as a high-throughput drug screening platform in similar disorders. This is the first description of the use of D. melanogaster as the model system for a high-throughput chemical library screen. This article has an associated First Person interview with the first authors of the paper. Summary: Chemical library screens using Drosophila melanogaster as a model of nephrolithiasis can be performed in a high-throughput and efficient manner, leading to candidate drugs with clinical potential in kidney stone patients.

MP67-09 HIGH-THROUGHPUT AND NON-INVASIVE FUNCTIONAL DRUG SCREENING PLATFORM FOR DROSOPHILA MELANOGASTER MODELS OF NEPHROLITHIASIS

INTRODUCTION AND OBJECTIVES: An experimental study in rats was performed to evaluate the potential protective effects of a calcium channel blocking agent (Verapamil) on the ischemia induced oxidative stress related changes during hyperoxaluric phase. METHODS: A total of 32 rats have been included into the study program. Hyperoxaluria was induced by continuous administration of ethyleneglycol (EG) (0.75%). In addition to hyperoxaluria induction applied in Group1 animals in Groups 2 did receive a calcium channel blocking agent (verapamil) and animals in Group 3 consituted the control group. Histologic alterations of the kidneys including crystal formation were evaluated on days 1,7 and 28, respectively. In addition to the scavenger enzymes (catalase (CAT), superoxide dismutase (SOD), total glutathione (GSH)),malonil dialdehid (MDA) levels, (nitric oxide)NO levelsand the total antioxidant capacity (TAOC) of the kidney were also assessed both in the serum as well as the kidney tissue specimens of the animals in all groups. RESULTS: While the evlautaion of the antioxidant enzyme levels did reveal a significant reduction in animals receiving EG only; application of verapamil was found to be protective with limited reduction with a statistical significance when compared with the control group animals (Sidak-Bonferroni method, alpha1⁄41.000%). Moreover, comparison of kidney tissue T-AOC as well as NO levels in a comparative manner between the groups agan showed a statistically significant difefrence between Group 1 and the other group animals where the antioxidant capacity of the kidney seemed to be protected by verapamil application (Sidak-Bonferroni method, alpha1⁄4 1.000%). Lastly, both serum as well as tissue MDA levels were also statistically significant high in animals receiving EG only when compared with Group 2 and control animals during day7 as well as day 28 evaluation. CONCLUSIONS: Our current results indicate that as a potent calcium channel blocking agent Verapamil may limit the extent of oxidative stress related changes in renal tubules following hyperoxaluria induction and preservation of antioxidant capacity of the kidney along with the limitation of crystallization by this agent may allow us to understand the pathophysiology of stone formation as well as the possible use of this agent to limit both stone formation and recurrence.