Quantifying the fitness effects of resistance alleles with and without anthelmintic selection pressure using Caenorhabditis elegans.

Albendazole (a benzimidazole) and ivermectin (a macrocyclic lactone) are the two most commonly co-administered anthelmintic drugs in mass-drug administration programs worldwide. Despite emerging resistance, we do not fully understand the mechanisms of resistance to these drugs nor the consequences of delivering them in combination. Albendazole resistance has primarily been attributed to variation in the drug target, a beta-tubulin gene. Ivermectin targets glutamate-gated chloride channels (GluCls), but it is unknown whether GluCl genes are involved in ivermectin resistance in nature. Using Caenorhabditis elegans, we defined the fitness costs associated with loss of the drug target genes singly or in combinations of the genes that encode GluCl subunits. We quantified the loss-of-function effects on three traits: (i) multi-generational competitive fitness, (ii) fecundity, and (iii) development. In competitive fitness and development assays, we found that a deletion of the beta-tubulin gene ben-1 conferred albendazole resistance, but ivermectin resistance required the loss of two GluCl genes (avr-14 and avr-15). The fecundity assays revealed that loss of ben-1 did not provide any fitness benefit in albendazole conditions and that no GluCl deletion mutants were resistant to ivermectin. Next, we searched for evidence of multi-drug resistance across the three traits. Loss of ben-1 did not confer resistance to ivermectin, nor did loss of any single GluCl subunit or combination confer resistance to albendazole. Finally, we assessed the development of 124 C. elegans wild strains across six benzimidazoles and seven macrocyclic lactones to identify evidence of multi-drug resistance between the two drug classes and found a strong phenotypic correlation within a drug class but not across drug classes. Because each gene affects various aspects of nematode physiology, these results suggest that it is necessary to assess multiple fitness traits to evaluate how each gene contributes to anthelmintic resistance.

House Officer-Driven Reduction in Laboratory Utilization.

OBJECTIVES: To determine whether sharing laboratory charge and personal utilization information with physicians can reduce laboratory test orders and expenditures, thereby decreasing the overutilization of laboratory testing. METHODS: This was a prospective study. By querying our electronic medical records, we calculated the median laboratory charges per patient/per day (PP/PD) and median laboratory tests ordered PP/PD for the resident general internal medicine and hospitalist services. For 10 weeks, we shared this team-based information with physicians with weekly updates. We calculated total laboratory charges for the 10 most common discharge diagnoses to capture laboratory charges for entire episodes of care. RESULTS: During the intervention, the mean number of laboratory tests ordered PP/PD by resident service decreased from 5.56 to 5.17 (-0.389, P <0.001); the mean charge PP/PD decreased from $488 to $461 (-$27, P < 0.001). The hospitalist service decreased the number of laboratory tests ordered PP/PD from 3.54 to 3.36 (-0.18, P = 0.77) and the mean charge PP/PD decreased from $331 to $301 (-$30, P = 0.96). The statistically significant decline in laboratory charges persisted after controlling for the 10 most common discharge diagnoses. Compared with the 3-month period before the study began, physicians in the 10-week intervention period ordered 1464 fewer laboratory tests, resulting in a $188,000 reduction in charges and a 3% to 4% reduction in utilization. CONCLUSIONS: Informing physicians of the charges for laboratory tests and their personal utilization patterns can reduce the number of laboratory tests ordered and laboratory expenditures, especially for physicians in training.

Quantitative tests of albendazole resistance in beta-tubulin mutants.

Benzimidazole (BZ) anthelmintics are among the most important treatments for parasitic nematode infections in the developing world. Widespread BZ resistance in veterinary parasites and emerging resistance in human parasites raise major concerns for the continued use of BZs. Knowledge of the mechanisms of resistance is necessary to make informed treatment decisions and circumvent resistance. Benzimidazole resistance has traditionally been associated with mutations and natural variants in the C. elegans beta-tubulin gene ben-1 and orthologs in parasitic species. However, variants in ben-1 alone do not explain the differences in BZ responses across parasite populations. Here, we examine the roles of five C. elegans beta-tubulin genes (tbb-1, mec-7, tbb-4, ben-1, and tbb-6) to identify the role each gene plays in BZ response. We generated C. elegans strains with a loss of each beta-tubulin gene, as well as strains with a loss of tbb-1, mec-7, tbb-4, or tbb-6 in a genetic background that also lacks ben-1 to test beta-tubulin redundancy in BZ response. We found that only the individual loss of ben-1 conferred a substantial level of BZ resistance, although the loss of tbb-1 was found to confer a small benefit in the presence of albendazole (ABZ). The loss of ben-1 was found to confer an almost complete rescue of animal development in the presence of 30 µM ABZ, likely explaining why no additive effects caused by the loss of a second beta-tubulin were observed. We demonstrate that ben-1 is the only beta-tubulin gene in C. elegans where loss confers substantial BZ resistance.

Quantifying the fitness effects of resistance alleles with and without anthelmintic selection pressure using Caenorhabditis elegans.

Albendazole and ivermectin are the two most commonly co-administered anthelmintic drugs in mass-drug administration programs worldwide. Despite emerging resistance, we do not fully understand the mechanisms of resistance to these drugs nor the consequences of delivering them in combination. Albendazole resistance has primarily been attributed to variation in the drug target, a beta-tubulin gene. Ivermectin targets glutamate-gated chloride channel (GluCl) genes, but it is unknown whether these genes are involved in ivermectin resistance in nature. Using Caenorhabditis elegans, we defined the fitness costs associated with loss of the drug target genes singly or in combinations of the genes that encode GluCl subunits. We quantified the loss-of function effects on three traits: (i) multi-generational competitive fitness, (ii) fecundity, and (iii) development. In competitive fitness and development assays, we found that a deletion of the beta-tubulin gene ben-1 conferred albendazole resistance, but ivermectin resistance required loss of two GluCl genes (avr-14 and avr-15) or loss of three GluCl genes (avr-14, avr-15, and glc-1). The fecundity assays revealed that loss of ben-1 did not provide any fitness benefit in albendazole and that no GluCl deletion mutants were resistant to ivermectin. Next, we searched for evidence of multi-drug resistance across the three traits. Loss of ben-1 did not confer resistance to ivermectin, nor did loss of any single GluCl subunit or combination confer resistance to albendazole. Finally, we assessed the development of 124 C. elegans wild strains across six benzimidazoles and seven macrocyclic lactones to identify evidence of multi-drug resistance between the two drug classes and found a strong phenotypic correlation within a drug class but not across drug classes. Because each gene affects various aspects of nematode physiology, these results suggest that it is necessary to assess multiple fitness traits to evaluate how each gene contributes to anthelmintic resistance.

Quantitative tests of albendazole resistance in Caenorhabditis elegans beta-tubulin mutants.

Benzimidazole (BZ) anthelmintics are among the most important treatments for parasitic nematode infections in the developing world. Widespread BZ resistance in veterinary parasites and emerging resistance in human parasites raise major concerns for the continued use of BZs. Knowledge of the mechanisms of resistance is necessary to make informed treatment decisions and circumvent resistance. Benzimidazole resistance has traditionally been associated with mutations and natural variants in the C. elegans beta-tubulin gene ben-1 and orthologs in parasitic species. However, variants in ben-1 alone do not explain the differences in BZ responses across parasite populations. Here, we examined the roles of five C. elegans beta-tubulin genes (tbb-1, mec-7, tbb-4, ben-1, and tbb-6) in the BZ response as well as to determine if another beta-tubulin acts redundantly with ben-1. We generated C. elegans strains with a loss of each beta-tubulin gene, as well as strains with a loss of tbb-1, mec-7, tbb-4, or tbb-6 in a genetic background that also lacks ben-1. We found that the loss of ben-1 conferred the maximum level of resistance following exposure to a single concentration of albendazole, and the loss of a second beta-tubulin gene did not alter the level of resistance. However, additional traits other than larval development could be affected by the loss of additional beta-tubulins, and the roles of other beta-tubulin genes might be revealed at different albendazole concentrations. Therefore, further work is needed to fully define the possible roles of other beta-tubulin genes in the BZ response.

Cyprocide selectively kills nematodes via cytochrome P450 bioactivation.

Left unchecked, plant-parasitic nematodes have the potential to devastate crops globally. Highly effective but non-selective nematicides are justifiably being phased-out, leaving farmers with limited options for managing nematode infestation. Here, we report our discovery of a 1,3,4-oxadiazole thioether scaffold called Cyprocide that selectively kills nematodes including diverse species of plant-parasitic nematodes. Cyprocide is bioactivated into a lethal reactive electrophilic metabolite by specific nematode cytochrome P450 enzymes. Cyprocide fails to kill organisms beyond nematodes, suggesting that the targeted lethality of this pro-nematicide derives from P450 substrate selectivity. Our findings demonstrate that Cyprocide is a selective nematicidal scaffold with broad-spectrum activity that holds the potential to help safeguard our global food supply.

The turkey ascarid, Ascaridia dissimilis, as a model genetic system.

Parasitic nematodes cause significant effects on humans each year, with the most prevalent being Ascaris lumbricoides. Benzimidazoles (BZ) are the most widely used anthelmintic drug in humans, and although the biology of resistance to this drug class is understood in some species, resistance is poorly characterized in ascarids. Models such as Caenorhabditis elegans were essential in developing our current understanding of BZ resistance, but more closely related model nematodes are needed to understand resistance in ascarids. Here, we propose a new ascarid model species that infects turkeys, Ascaridia dissimilis, to develop a better understanding of BZ resistance.

Comparison of ketanserin and metoprolol in the treatment of essential hypertension.

Ketanserin, a serotonin receptor antagonist (S2), lowered blood pressure in patients with essential hypertension; at three months 72% (13/18) had a successful reduction in pressure. No marked orthostatic changes were noted. Older patients responded better when standing. Compared with metoprolol, ketanserin provided no significant difference in response at three months. With ketanserin, the heart rate was reduced only in the supine position, whereas it was reduced in the supine and standing positions with metoprolol. Response to ketanserin could not be predicted from baseline renin, aldosterone, or cortisol levels in blood, nor were there any changes in these factors or in plasma hydroxyindole levels with therapy. Ketanserin was generally well tolerated. Cholesterol values were significantly reduced with ketanserin, and there were no adverse hematologic or biochemical changes. Ketanserin should have a significant role in managing hypertension.

Synthesis and anxiolytic activity of 6-(substituted-phenyl)-1,2,4-triazolo[4,3-b]pyridazines.

The synthesis of a series of 6-(substituted-phenyl)-1,2,4-triazolo[4,3-b]pyridazines (VIII) is reported. Some of these derivatives show activity in tests predictive of anxiolytic activity [(a) protection against pentylenetetrazole-induced convulsions; (b) thirsty rat conflict procedure]. They also represent a new class of compound which inhibits [3H]diazepam binding. Structure--activity correlations, as well as the ability of structures VIII to inhibit [3H]diazepam binding (in vitro), are discussed.

Short bowel syndrome.

Improvements in parenteral nutrition and supportive therapy have led to a growing population of patients who survive for prolonged periods with short bowel syndrome. Definitive treatment for these patients requires innovative therapy based on a sound knowledge of small intestinal physiology and adaptation. Current understanding of short bowel pathophysiology and of intestinal adaptation are reviewed. Medical and surgical therapeutic options are described, highlighting the promotion of small bowel adaptation and methods to increase the small intestinal mucosal mass.

Reconsidering "good teaching" across the continuum of medical education.

There is no shortage of sustained inquiry into the nature and evaluation of teaching in medical education. For the most part, however, this growing and respectable body of inquiry has uncritically adopted a single model of effective teaching that is assumed to be appropriate across variations in context, learners, and teachers. This article presents five alternative views of "good teaching" and challenges the trend toward any single, dominant view of what constitutes good teaching. Based on 10 years of research, in five different countries, studying hundreds of educators in adult and higher education across a wide range of disciplines, contexts, and cultures, we have evidence of five different perspectives on good teaching: transmission, developmental, apprenticeship, nurturing, and social reform. Each perspective represents a philosophical orientation to knowledge, learning, and the role and responsibility of being an educator. A "snapshot" of each perspective is provided, including an example from continuing medical education (CME), a set of key beliefs, primary responsibilities, typical strategies, and common difficulties. Readers are encouraged to use the five perspectives as a means of identifying, articulating, and revisiting assumptions and beliefs they hold regarding their view of effective teaching. They are also encouraged to resist a "one-size-fits-all" approach to the investigation, improvement, or evaluation of teaching in CME.

Comparison of open and laparoscopic gastrostomy and fundoplication in 120 patients.

The rapid development and incorporation of minimally invasive surgical techniques has abruptly changed adult surgical practices. These minimally invasive procedures are now being successfully applied to pediatric surgical problems. The anticipated benefits of these techniques include less postoperative pain, quicker return of bowel function, shorter hospital stay, and lower hospital costs, with a quicker return to normal activity. This report compares the first 60 infants and children to undergo laparoscopic gastrostomy and/or fundoplication at our institution with the same number of patients that underwent these procedures in the traditional open fashion. The two groups were similar with respect to age, sex, concurrent illness, presenting symptoms, neurological status, and procedures performed. Patients in the laparoscopic group were found to have shorter mean hospital and postoperative stays and tolerated feeding earlier. The mean hospital stay was 13.8 days for the laparoscopic group versus 16.4 days in the open group. The mean postoperative stay was 6.8 days for the laparoscopic group versus 10.7 days for the open group. The mean postoperative day on which feeding was tolerated was 2.3 in the laparoscopic group versus 4.8 in the open group. Postoperative complications were similar between the two groups. These results seem to reflect the less traumatic nature of the laparoscopic procedures as compared with the open procedures. Laparoscopic fundoplication and gastrostomy is an attractive alternative to open fundoplication and gastrostomy in infants and children.

Menopause.

The body of evidence now swings the scale toward the benefit of HRT for women beginning at the menopause. Based on newer studies, the risks for osteoporosis, cardiovascular morbidity, breast carcinoma, symptomatic vasomotor and anatomic changes occurring postmenopausally outweigh the risks of hormone replacement therapy in the end of the 20th century. Women should be instructed in adequate calcium intake, 1000 mg per day premenopausally and 1500 mg per day postmenopausally. Osteoporotic, breast carcinoma, and cardiovascular risks should be investigated at age 35 with appropriate lab screening, including lipoprotein analysis. Screening mammography should begin at age 40, continuing every 5 years until age 50, and yearly between ages 50 and 65. A diet high in calcium, low in cholesterol and fat, and a weight reduction program should be made available as early as possible and continued indefinitely. HRT should be made available beginning at menopause and continued to age 70. Moderate exercise should be encouraged at all ages. The next 5 to 10 years will answer some of the questions about the benefits of long-term HRT postmenopausally, especially with respect to its influence on cardiovascular risk. New progestational agents will probably be developed that will have fewer adverse effects on lipid profiles, while maintaining the protective effect on the endometrium and breast and further influencing the benefits of HRT postmenopausally. Modern medicine certainly cannot ensure living forever. The body of knowledge now available can modify the major causes for morbidity and mortality as the baby boom population reaches their middle age and golden years.

Sentinel node biopsy in breast cancer: results in a large series.

Sentinel lymph node biopsy (SLNB) is an appropriate method for the evaluation of axillary status in cases of early breast cancer. We report our experience in treating cases evaluated using SLNB. We analyzed a total of 1192 cases assessed by means of SLNB from July 1999 to December 2007. SLNB processing was successfully completed in 1154 cases with the use of blue dye or radiolabeled 99mTc-Dextran-500, or both. Of these 1154 patients, 857 were N0(i-) (no regional lymph node metastasis, negative immunohistochemistry, IHC), 96 were N0(i+) (no regional lymph node metastasis histologically, positive IHC, no IHC cluster greater than 0.2 mm) and 201 were N1mi (greater than 0.2 mm, none greater than 2.0 mm). Most of the tumors (70%) were invasive ductal carcinomas and tumors were staged as T1 in 770 patients (65%). A total of 274 patients underwent SLNB and axillary dissections up to April 2003. The inclusion criteria were tumor size equal to or less than 3 cm in diameter, no clinically palpable axillary lymph nodes, no neoadjuvant therapy. In 19 cases, the SLN could not be identified intraoperatively. A false-negative rate of 11% and a negative predictive value of 88.2% were obtained for the 255 assessable patients. The overall concordance between SLNB and axillary lymph node status was 92%. SLNB sensitivity for nodes was 81% and specificity was 100%. The higher sensitivity, specificity, accuracy, and lower false-negative rates of SLNB suggest that this method may be an appropriate alternative to total axillary dissection in early breast cancer patients.