Genotypes of Hepatitis C Virus and Efficacy of Direct-Acting Antiviral Drugs among Chronic Hepatitis C Patients in a Tertiary Care Hospital.

Hepatitis C virus (HCV) chronic infection is a major causative factor for several chronic liver diseases, including liver cirrhosis, liver cell failure, and hepatocellular carcinoma. The HCV has seven major genotypes. Genotype 4 is the most prevalent genotype in the Middle East, including Saudi Arabia, followed by genotype 1. The HCV genotype affects the response to different HCV treatments and the progression of liver disease. Currently, combinations of direct-acting antiviral drugs (DAAs) approved for the treatment of HCV achieve high cure rates with minimal adverse effects. Because real-world data from Saudi Arabia about the efficacy of DAAs are still limited, this study was conducted to assess the effectiveness of DAAs in treating patients with chronic hepatitis C and to identify the variables related to a sustained virologic response (SVR) in a real-world setting in Saudi Arabia. This prospective cohort study included 200 Saudi patients with chronic HCV who were 18 years of age or older and had been treated with DAAs at King Abdul-Aziz Specialized Hospital in Taif, Saudi Arabia, between September 2018 and March 2021. The response to treatment was assessed by whether or not an SVR had been achieved at week 12 post treatment (SVR12). An SVR12 was reached in 97.5% of patients. SVR12 rates were comparable for patients of different ages, between men and women, and between patients with and without cirrhosis. In addition, the SVR12 rates did not differ according to the infecting HCV genotype. In this study, the presence of cirrhosis and the patient's gender were independent predictors of who would not reach an SVR12 (known here as the non-SVR12 group) according to the results of univariate and multivariate binary logistic regression analyses based on the determinants of SVR12. In this population of patients with chronic HCV infection, all DAA regimens achieved very high SVR12 rates. The patients' gender and the presence of cirrhosis were independent factors of a poor response.

Prevalence and Risk Factors for Trichomonas Vaginalis Infection Among Women: a Population-Based Controlled Study in Saudi Arabia.

BACKGROUND: Information on Trichomonas vaginalis (T. vaginalis) infection in Saudi Arabia is scarce. The aim of study was to assess the burden and risk factors of T. vaginalis infection for a cohort of women living in Saudi Arabia. METHODS: Women aged ≥ 18 years who were seeking medical care at the King Faisal Medical Complex Gynecology Clinic in Taif city, Western Saudi Arabia, were enrolled in a non-randomized case-control study between June 2018 and May 2019. Participants were interviewed using a standard questionnaire for a number of sociodemographic and clinical characteristics. Vaginal swabs obtained from each participant were screened for T. vaginalis infection with direct wet mount smear microscopy, the OSOM Trichomonas rapid test 'OSOM Trich' (Genzyme Diagnostics, Cambridge, MA, USA) and a published nested PCR. RESULTS: Over the study period, 155 women were recruited: 79 with symptoms of vaginitis (i.e. cases) and 76 with no symptoms (i.e. controls). The T. vaginalis infection was detected in ~20% (16/79) of cases and ~9% (7/76) of the controls by the nested PCR. Using the PCR test results as a gold standard, the wet mount microscopy's sensitivity, specificity, negative predictive value, and positive predictive value were 69.5%, 100%, 94.9%, and 100%, respectively, whereas the OSOM Trich's were 86.9%, 100%, 97.7%, and 100%, respectively. The main high-risk factors included age between 30 and 39 years (~35%), marriage for 10 - 30 years (~62%), non-education (~41%), urban residence (~29%), and employment (~36%). Highly significant differences were observed concerning infection distribution among cases for the presence of lower abdominal pain (~64%) and abnormal vaginal discharge (38%) as presenting symptoms (χ2 = 20.42; p < 0.001 and χ2 = 5.63; p = 0.017, respectively). CONCLUSIONS: The burden of infection with T. vaginalis is unexpectedly high in the population studied. Regular screening for T. vaginalis infection, particularly in high-risk women, is required.

Effect of COVID-19 Pandemic on Neonatal Sepsis.

BACKGROUND: There is a sudden rise in infectious diseases, with special concern to the most recent SARS-CoV 2 outbreak. A retrospective study was conducted to study the effect of this outbreak on neonatal sepsis as a global issue that poses a challenge for pediatric management and to identify its risk factors, microbial profile, and mortality rate at King Faisal Medical Complex, Taif, KSA, a COVID-19-tertiary care segregation hospital. METHODS: This research included 111 neonates with a culture-proven diagnosis of neonatal sepsis (4 and 62 cases during 2019 and 2020, respectively). RESULTS: During 2019 early onset sepsis (EOS) occurred in 6/49 (12.2%) while in 2020 22/62 (35.5%), and during 2019 late onset sepsis (LOS) occurred in 43/49 (87.7%) while in 2020 40/62 (64.5%). Premature rupture of membrane was the major neonatal risk factor for EOS during 2019 and 2020 with proportions of 4 (66.7%), 20 (90.9%); respectively. As regards LOS, the peripherally inserted central catheters and peripheral lines were the top neonatal risk factors. In the two-year outbreak, the most prevalent causative organism for EOS neonates was Escherichia coli and for LOS neonates it was Klebsiella. There was non-significant change in the mortality rate of neonatal sepsis between 2019 and 2020. However, the mortality rate was higher in EOS 9/22 (40.9%) in 2020 in comparison to 2/6 (33.3%) in 2019. CONCLUSIONS: Neonatal sepsis remains a major health problem causing serious morbidity and mortality, and health care policy makers have to implement EOS preventive measures.

Prevalence and Molecular Characterization of Mycoplasma Species, Pasteurella multocida, and Staphylococcus aureus Isolated from Calves with Respiratory Manifestations.

Bovine respiratory disease (BRD) is a complex syndrome associated with high mortality in young calves and causes severe economic losses in the cattle industry worldwide. The current study investigated the prevalence and molecular characterization of common bacterial pathogens associated with respiratory symptoms in young calves from Sadat City, one of the largest industrial cities in Menoufiya Governorate, Egypt. In between December 2020 and March 2021, 200 mixed-breed young calves of 6-12 months were examined clinically. Of them, sixty (30%) calves showed signs of respiratory manifestations, such as coughing, serous to mucopurulent nasal discharges, fever, and abnormal lung sound. Deep nasal (Nasopharyngeal) swabs were collected from the affected calves for bacteriological investigation. Phenotypic characterization and identification revealed Mycoplasma bovis, Mycoplasma bovigenitalium, Pasteurella multocida, and Staphylococcus aureus in 8.33%, 5%, 5%, and 5% of the tested samples, respectively. The PCR technique using species-specific primer sets successfully amplified the target bacterial DNA in all culture-positive samples, confirming the identity of the isolated bacterial species. Partial gene sequencing of 16S rRNA gene of M. bovigenitalium, P. multocida, and S. aureus, and mb-mp 81 gene of M. bovis revealed high nucleotide similarity and genetic relationship with respective bacterial species reported from Egypt and around the world, suggesting transmission of these bacterial species between animal host species and localities. Our study highlights the four important bacterial strains associated with respiratory disorders in calves and suggests the possible spread of these bacterial pathogens across animal species and different geographic locations. Further studies using WGS and a large number of isolates are required to investigate the realistic lineage of Egyptian isolates and globally.

Exploration of Community Care Awareness about COVID-19 Sterilization.

BACKGROUND: COVID-19 affects millions of people worldwide so WHO declared the COVID-19 pandemic on 11 March 2020. Since the vaccine is in the early trial phase and until it proves its efficacy, the need of finding alternative methods, which can help to curb this pandemic is urgent, so its prevention depends on standard infection control measures. This study's aim is to assess the knowledge, awareness, and practice level of Taif population towards Corona Virus disease - 2019 (COVID-19) sterilization. METHODS: A cross sectional study was conducted on 504 participants by administering a well-structured questionnaire comprising three sections including demographics, knowledge, attitude, and practice among the general population in Taif governorate KSA, over a duration of three months from July until September 2020. The descriptive analysis was carried out for demographics and dependent variables using the statistical program for social sciences. The t-test was used to detect any relationship between knowledge and practice score percentage of the general population response with respect to their gender and level of education. A p-value of < 0.05 was considered statistically significant. RESULTS: A total of 504 respondents willingly participated in the survey, there is a highly significant difference in the knowledge score percentage in respondents aged between 41 - 60 years old in comparison to the age group < 20 - 40 years old also between urban residence in comparison to rural residence, and a highly significant difference in the knowledge and practice score percentage in post graduate respondents in comparison to undergraduate. In addition, there was a significant difference in the practice score percentage in respondents aged between 41 - 60 years old in comparison to age group < 20 - 40, and a highly significant difference was seen in the practice score percentage in respondents living in urban areas in comparison to rural areas. CONCLUSIONS: The suggestion of this study was that knowledge and practice gaps among population, especially in the young age group, had to be covered by holding training programs through workshops or to include courses in the curriculum of ministry of health.

Profiling Inflammatory Cytokines in a Cohort Study of Egyptian Patients with COVID-19 Infection.

BACKGROUND: Coronavirus disease 2019 (COVID-19) pandemic is an international public health emergency with major disruptions and devastating health consequences resulting from the associated cytokine storm syndrome. The aim of our research was to assess the inflammatory biomarkers and risk factors associated with severity of (COVID-19) patients. METHODS: A cross-sectional study was conducted and included 150 Egyptian patients with COVID-19. Patients were classified into mild, moderate, and severe according to the clinical and CT chest findings. Blood samples were collected from patients for laboratory assessment of inflammatory biomarkers. RESULTS: Our results showed significant negative correlation between oxygen saturation percent and serum levels of inflammatory markers. The correlations were statistically significant with IL-6, CRP, ferritin, LDH, and D-dimer which can be used as sensitive biomarkers for assessment of the risk of severity of infection in COVID 19 patients. CONCLUSIONS: The study revealed that the risk factors associated with severity of COVID 19 infection included older age, male gender, presence of underlying chronic disease, and increased levels of inflammatory biomarkers: CRP, LDH, ferritin, IL-6, and D-dimer.

Prevalence, toxin gene profile, genotypes and antibiotic susceptibility of Clostridium difficile in a tertiary care hospital in Taif, Saudi Arabia.

Purpose: Clostridium difficile (C. difficile) is an important causative agent of nosocomial diarrhoea and has become a major worldwide public health concern. The current study was conducted to determine the prevalence of C. difficile infection (CDI) amongst patients with nosocomial diarrhoea in a large tertiary care hospital in Taif, Saudi Arabia, and to define molecular characteristics and antimicrobial sensitivity profiles of C. difficile strains isolated from those patients. Materials and Methods: Stool specimens were collected from 456 patients and were cultured for C. difficile isolation. The isolates were subjected to multiplex polymerase chain reaction (PCR) for detecting genes encoding the toxins (toxin A, toxin B and binary toxin [CDT]), genotyping by PCR ribotyping method and antimicrobial sensitivity testing using E test strips. Results: Seventy-four C. difficile strains were recovered, of which 44 (59.5%) were A+B+CDT-, 14 (18.9%) were A-B+CDT-, 4 (5.4%) were A+B+CDT+ and 12 (16.2%) were A-B-CDT-. Toxigenic strains, and hence CDI, were detected in 13.6% of the patients (62/456). Fourteen different ribotypes were distinguished amongst bacterial isolates, of which ribotypes 002, 001, 017, 014 and 020 were the most prevalent (20.3%, 18.9%, 18.9%, 9.5% and 8.1%, respectively). Four isolates (5.4%) belonged to ribotype 027. All bacterial isolates showed sensitivity to metronidazole, vancomycin and piperacillin-tazobactam. The isolates exhibited resistance to linezolid (2.7%), chloramphenicol (5.4%), rifampicin (13.5%), tetracycline (21.6%), moxifloxacin (48.6%), clindamycin (54%) and imipenem (83.8%). Multiple drug resistance was observed in 56.8% of the isolates. Conclusion: Further larger studies are required for an accurate understanding of CDI epidemiology in Saudi Arabia.

Association of toll-like receptors 2, 4, 9 and 10 genes polymorphisms and Helicobacter pylori-related gastric diseases in Saudi patients.

Purpose: Helicobacter pylori is one of the most prevalent human pathogens worldwide. However, the outcomes of H. pylori infection are markedly variable from asymptomatic mild lesion to malignant transformation. Many factors are suggested to influence these infection outcomes, including host immunity and genetic susceptibility. Toll-like receptors (TLRs) can recognise different microbial components and play an essential role in the mucosal immune response against H. pylori infection. Materials and Methods: The association between the common single nucleotide polymorphisms (SNPs) in the genes of TLR2, 4, 9 and 10 and H. pylori-related gastric diseases were investigated by molecular methods after the confirmation of H. pylori infection. The study included 210 patients in three groups; chronic gastritis (n = 90), peptic ulcer disease (PUD) (n = 75) and gastric carcinoma (n = 45). Results: The results showed a significant association between TLR4 SNPs (rs 4986790 and rs 4986791) and the presence of H. pylori infection, especially in chronic gastritis patient group. Furthermore, TLR9-rs352140 TT genotype was more prevalent among chronic gastritis patient group. TLR10-rs 10004195 TT genotype was found to be less prevalent among H. pylori-related chronic gastritis and PUD and was suspected to have a protective effect. TLR2 SNPs (rs3804099 and rs3804100) showed no significant statistical difference between H. pylori-infected patients and the controls. Conclusion: TLR genes polymorphisms may play a role in H. pylori infection susceptibility and may influence its outcomes; however, the ethnic and other factors may modify this effect.

Dientamoeba fragilis Infection in Patients with Digestive and Non-Digestive Symptoms: A Case-Control Study.

In most developing countries, Dientamoeba fragilis infection is an obscure protozoan infection. We aimed to determine a frequency and clinical importance of D. fragilis infection in Taif, Saudi Arabia. A 1-year case control study included patients with gastrointestinal (cases, n=114) or non-gastrointestinal symptoms (controls, n=90). The fecal samples were examined with the classical parasitological methods for intestinal protozoa, and by real time PCR for D. fragilis. The infection by D. fragilis was detected in 5.8% by PCR and in 4.4% patients by microscopy. The infection was identified more in control group (n=9) than in cases (n=3); a sole infection in 11 patients and mixed with Giardia in 1 patient. The other enteric parasites detected were Blastocystis sp. (8.3%), Giardia sp. (5.3%), Cryptosporidium sp. (2.9%), Entamoeba histolytica (1.4%), Entamoeba coli (0.9%) and Hymenolepis nana (0.4%). Our results tend to reinforce the need to increase awareness of D. fragilis infection in Saudi Arabia.

Cancerous Inhibitor of Protein Phosphatase 2A as a Molecular Marker for Aggressiveness and Survival in Oral Squamous Cell Carcinoma.

Cancerous inhibitor of protein phosphatase 2A (CIP2A) has been identified as one of the most commonly altered proteins in human cancers. It blocks the tumor-suppressive action of protein phosphatase 2A (PP2A) complex and enhances malignancy. Thirty-five patients with squamous cell carcinoma of the oral cavity underwent surgical resection of the tumor. CIP2A was assessed by quantitative real-time PCR in the resected tumor tissues and in their adjacent normal tissues. CIP2A was found to be overexpressed in all oral squamous cell carcinoma (OSCC) specimens in comparison to their surrounding normal tissue. CIP2A overexpression was statistically correlated with poor prognostic feature of the tumor. Thus, a high expression level of CIP2A was associated with shorter survival. In conclusion, CIP2A is upregulated in OSCC, and its overexpression is correlated with aggressiveness of the tumor and poor outcome and survival. It may serve as a prognostic marker of OSCC.

Discovery of a novel DNA binding agent via design and synthesis of new thiazole hybrids and fused 1,2,4-triazines as potential antitumor agents: Computational, spectrometric and in silico studies.

New series of furan-thiazole hybrids (3a-f), thiazolo[2,3-c]-1,2,4-triazines (4a-f), their bioisosteres 1,3,4-thiadiazolo[2,3-c]-1,2,4-triazines (8a-d) and 1,2,4-triazino[4,3-b]-1,2,4-triazines (13a-e) were designed, synthesized and evaluated for their in vitro antitumor activities at the National Cancer Institute (NCI, USA). Among the synthesized compounds, 3d was found to exhibit promising broad spectrum antitumor activity (GI50 MG-MID = 14.22 µM) in a five-dose assay against the full panel NCI-cancer cell lines. 3d displayed higher antitumor activity against most tested cancer cell lines than 5-FU as reference. COMPARE analysis and molecular electrostatic potential computational study revealed that 3d probably exerts its antitumor properties through DNA binding similar to Clomesone. Further DNA binding studies using fluorescent terbium (Tb+3) probe revealed increased fluroresence of DNA-3d-Tb+3 mixture due to damage of the double-stranded DNA. Also, UV-vis absorption study was conducted which showed hyperchromic shift in DNA absorption confirming 3d-induced DNA damage. The assessed potency of 3d-induced DNA damage of calf thymus DNA showed a concentration as low as 2.04 ng/mL for a detectable DNA damage. Moreover, in silico calculation of physicochemical properties and druglikeness were in compliance to Lipinski's rule.

Molecular diagnosis of Helicobacter pylori antibiotic resistance in the Taif region, Saudi Arabia.

Success in eradication of Helicobacter pylori is declining globally because H. pylori has developed resistance against most of the antibiotics proposed for eradication regimens, mainly through point mutations. The present study included 200 patients with dyspepsia attending Taif Hospital. Gastric biopsies were obtained during gastroscopy and subjected to rapid urease testing. Molecular methods were used to confirm diagnoses of H. pylori infection and to identify resistance gene variants of four antibiotics; namely, clarithromycin, metronidazole, fluoroquinolones and tetracycline (23S rRNA, gyrA, rdxA and 16S rRNA respectively). Of all investigated patients, Molecular diagnoses were made in 143 of all investigated patients; thus, the prevalence was .5%. The overall rate of resistance to clarithromycin among the H. pylori-positive patients was high (39.9%) and the rate of resistance significantly greater (48.2%) among the secondary resistance group, secondary resistance being defined as resistance as a result of previous exposure to the relevant antibiotic. The rate of resistance to fluoroquinolones was considered moderate; the difference in rate of resistance between the primary and secondary resistance groups (8.4% and 9.5%, respectively) was not significant Also, there was a low prevalence of both primary and the secondary tetracycline resistance in the study cohort. In contrast, the prevalence of metronidazole resistance was considered high with no significant difference between the two resistance groups. H. pylori showed an increased prevalence of resistance to all four of the commonly used therapeutic agents. Thus, eradication therapy should be based on the regional results of susceptibility testing. Moreover, treatment tailored according to individually determined H. pylori susceptibility may be a reasonable future goal.

Diagnosis, Treatment and Clinical Features of Cutaneous Leishmaniasis in Saudi Arabia.

Cutaneous leishmaniasis (CL) has been one of the most common parasitic diseases in Saudi Arabia. This study exhibits the clinical features, diagnosis, cytokine profile and treatment of CL patients in Al-Taif province. Ninety CL suspects at a tertiary care general hospital were enrolled in one-year study. Patients were interviewed, clinically-examined, and subjected to laboratory tests: skin scraping smear microscopy, OligoC-TesT commercial PCR (Coris BioConcept) and kinetoplast DNA (kDNA) PCR for Leishmania diagnosis. Interferon-gamma (RayBio; Human IFN-γ) and nitric oxide (NO) levels in patients' sera were evaluated before treatment with sodium stibogluconate (pentostam) with 20-day intramuscular drug regimen. Positive rates of microscopy, commercial PCR and kDNA PCR were 74.4%, 95.5% and 100%, respectively. Patients came to hospital mostly in winter (45.0%). CL was frequently exhibited in Saudi patients (78.8%), male gender (70.7%), age <20 years (50.0%), rural-dwellers (75.5%) and patients with travel history (86.6%). Lesion was mostly single ulcer (93.3%), occurred in the face (67.7%). Upon pentostam treatment, 85.1% of ulcers showed rapid healing signs. Levels of IFN-γ and NO were significantly higher in the healing than the non-healing cases (P<0.001). The kDNA PCR proved more sensitive than microscopy and OligoC-TesT commercial PCR. Our results open perspectives for IFN-γ use as a biomarker predicting treatment response.

High Frequency of Enteric Protozoan, Viral, and Bacterial Potential Pathogens in Community-Acquired Acute Diarrheal Episodes: Evidence Based on Results of Luminex Gastrointestinal Pathogen Panel Assay.

Infectious diarrhea is endemic in most developing countries. We aimed to investigate the protozoan, viral, and bacterial causes of acute diarrhea in Taif, Saudi Arabia. A cross-sectional prospective 1-year study was conducted on 163 diarrheal patients of various ages. Stool samples were collected, 1 per patient, and tested for 3 protozoa, 3 viruses, and 9 bacteria with the Luminex Gastrointestinal Pathogen Panel. Overall, 53.4% (87/163) of samples were positives (20.8% protozoa, 19.6% viruses, 2.8% bacteria, and 9.8% mixed). Rotavirus (19.6%), Giardia duodenalis (16.5%), and Cryptosporidium spp. (8.5%) were the mostly detected pathogens. Adenovirus 40/41 (4.2%), Salmonella (3%), Shiga toxin-producing Escherichia coli (3%), and Entamoeba histolytica (2.4%) were also detected. Norovirus GI/II, Vibrio cholerae, Yersinia enterocolitica, and Clostridium difficile toxin A/B were not detected in any patients. All pathogens were involved in coinfections except E. histolytica. Giardia (5.5%) and rotavirus (3%) were the most commonly detected in co-infections. Enterotoxigenic E. coli (2.4%), Campylobacter spp. (2.4%), E. coli 0157 (1.8%), and Shigella spp. (1.2%) were detected in patients only as co-infections. Infections were more in children 0-4 years, less in adults <40 years, and least >40 years, with statistically significant differences in risk across age groups observed with rotavirus (P<0.001), Giardia (P=0.006), and Cryptosporidium (P=0.036) infections. Lastly, infections were not significantly more in the spring. This report demonstrates the high burden of various enteropathogens in the setting. Further studies are needed to define the impact of these findings on the clinical course of the disease.

Synthesis, biological evaluation and molecular docking studies of some pyrimidine derivatives.

Some novel pyrimidine-5-carbonitrile derivatives bearing various substituent have been synthesized. The structures of target compounds were confirmed by elemental analysis and spectral data. Some selected members of the newly synthesized compounds were investigated for their cytotoxic potency against certain human tumor cell lines. Five representative active anticancer compounds 6a, 6c, 6d, 17a and 18a were subjected to docking using MOE program on the 3D structure of two enzymes, namely; thymidylate synthase and dihydrofolate reductase. The antimicrobial activities of the synthesized compounds were tested against Staphylococcus aureus, Pseudomonas aeruginosa, Shigella flexneri and Candida albicans. Compounds 2c, 7a and 9c showed broad spectrum antimicrobial activity.

Molecular identification of coproantigens of Cryptosporidium parvum compared to conventional staining techniques.

To compare the immunologic techniques to the conventional staining methods (mainly modified Ziehl Neelsen = MZN), 93 children (65 immunocompromised and 28 immunocompetent) potentially at risk of Cryptosporidium parvum were studied. Using MZN, a prevalence of 10.7% in diarrhoeic children was found. ELISA coproantigen and detection of 23 KDa band of immunoblotting by serum IgG were sensitive and specific. They gave 85.7% sensitivity, 100% specificity, 100% diagnostic accuracy, 100% positive predictive value and 98.9% negative predicttive value. ELISA detection of serum IgG gave 85.7% sensitivity, 97.7% specificity, 96.8% diagnostic accuracy, 75% positive predictive value and 98.9% negative predictive value. So, 23KDa band determined by ELISA is a valuable sensitive and specific mean of diagnosing cryptosporidiosis as this antigen is a consistent target of humoral immune response.

Structure activity studies of ring E analogues of methyllycaconitine. Part 2: Synthesis of antagonists to the alpha3beta4* nicotinic acetylcholine receptors through modifications to the ester.

The development of novel agents for the differentiation of neuronal nicotinic acetylcholine receptors (nAChRs) is important for the treatment of a variety of pathological conditions. We have prepared and evaluated a number of simpler analogues of the norditerpeniod alkaloid methyllycaconitine (MLA) in an effort to understand molecular determinants of nAChR*small molecule interactions. We have previously reported the synthesis and evaluation of a series of ring E analogues of MLA. We report here the optimization of the alpha3beta4* functional activity of this series of compounds through modification of the ester.

Structure-activity studies with ring E analogues of methyllycaconitine on bovine adrenal alpha3beta4* nicotinic receptors.

The development of new agents that selectively interact with subtypes of neuronal nicotinic receptors (nAChRs) is of primary importance for the study of physiological processes and pathophysiological conditions involving these receptors. Our laboratory has evidence that simple ring E analogues of methyllycaconitine (MLA) act as antagonists to bovine adrenal alpha3beta4* nAChRs. The following studies were designed to characterize the concentration-response effects of several ring E analogues of MLA in order to assess structural requirements involved with their inhibitory activity on bovine adrenal alpha3beta4* nAChRs. Ring E analogues with various substitutions on the ring E nitrogen were tested for their ability to inhibit nicotinic stimulated adrenal catecholamine release and [3H]epibatidine binding to a bovine adrenal membrane preparation. Several N-alkyl derivatives inhibited secretion with IC50 values in the low micromolar range. The N-phenpropyl analogue was the most potent of the analogues tested (IC50, 11 microM) on adrenal secretion. Competition binding studies suggest a noncompetitive interaction of the analogues with bovine adrenal nAChRs. These studies identify several structural features of ring E analogues of MLA which significantly affect their inhibitory activity on bovine adrenal alpha3beta4* nAChRs.