Boswellic acids, as novel inhibitor targeting peptidoglycan biosynthetic enzyme UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) in Escherichia coli.

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is an essential cytosolic enzyme in the biosynthesis of peptidoglycan. It becomes a potential bacterial target for screening promising antibacterial compounds as it is associated with the early phases of peptidoglycan production. MurA enzyme is conserved and necessary for bacterial viability with no mammalian homolog, which is a well-proven therapeutic research target. The present study reports the natural compounds from Boswellia serrata targeting the MurA enzyme. The identified inhibitors against MurA Escherichia coli (E. coli): ß-boswellic acid (IC50 33.65 µM), Acetyl-ß-boswellic acid (IC50 30.17 µM), and Acetyl-11-keto-ß-boswellic acid (IC50 37.67 µM). Inhibitors showed a fourfold decrease in IC50 values on pre-incubation with substrate-UDP-N-acetyl-glucosamine (UDP-GlcNAc). Mode-of-inhibition studies revealed their uncompetitive nature with both the substrates. Although these boswellic acids have been explored for their pharmacological potential, this is the first study reporting these compounds' E. coli MurA inhibiting potential.

Screening of compound library identifies novel inhibitors against the MurA enzyme of Escherichia coli.

Bacterial cell has always been an attractive target for anti-infective drug discovery. MurA (UDP-N-acetylglucosamine enolpyruvyl transferase) enzyme of Escherichia coli (E.coli) is crucial for peptidoglycan biosynthetic pathway, as it is involved in the early stages of bacterial cell wall biosynthesis. In the present study we aim to identify novel chemical structures targeting the MurA enzyme. For screening purpose, we used in silico approach (pharmacophore based strategy) for 52,026 library compounds (Chembridge, Chemdiv and in house synthetics) which resulted in identification of 50 compounds. These compounds were screened in vitro against MurA enzyme and release of inorganic phosphate (Pi) was estimated. Two compounds (IN00152 and IN00156) were found to inhibit MurA enzyme > 70% in primary screening and IC50 of 14.03 to 32.30 µM respectively. These two hits were further evaluated for their mode of inhibition studies and whole-cell activity where we observed 2-4 folds increase in activity in presence of Permeabilizer EDTA (Ethylenediaminetetraacetic acid). Combination studies were also performed with known antibiotics in presence of EDTA. Hits are reported for the first time against this target and our report also support the use of OM permeabilizer in combination with antibacterial compounds to address the permeability and efficacy issue. These lead hits can be further optimized for drug discovery. KEY POINTS: • Emerging Gram negative resistant strains is a matter of concern. • Need for new screening strategies to cope with drying up antibiotics pipeline. • Outer membrane permeabilizers could be useful to improve potency of molecules to reach its target.

Progress Toward Polio Eradication - Worldwide, January 2016-March 2018.

In 1988, when an estimated 350,000 cases of poliomyelitis occurred in 125 countries, the World Health Assembly resolved to eradicate polio globally. Transmission of wild poliovirus (WPV) continues uninterrupted in only three countries (Afghanistan, Nigeria, and Pakistan) (1), and among the three serotypes, WPV type 1 (WPV1) remains the only confirmed circulating type. This report describes global progress toward polio eradication during January 2016-March 2018, and updates previous reports (2). In 2017, 22 WPV1 cases were reported, a 41% decrease from the 37 WPV1 cases reported in 2016. As of April 24, 2018, eight WPV1 cases have been reported (seven in Afghanistan and one in Pakistan), compared with five cases during the same period in 2017. In Pakistan, continuing WPV1 transmission has been confirmed in multiple areas in 2018 by isolation from wastewater samples. In Nigeria, ongoing endemic WPV1 transmission was confirmed in 2016 (3); although WPV was not detected in 2017 or in 2018 to date, limitations in access for vaccination and surveillance in insurgent-held areas in northeastern Nigeria might permit continued undetected poliovirus transmission. Substantial progress toward polio eradication has continued in recent years; however, interruption of WPV transmission will require overcoming remaining challenges to reaching and vaccinating every missed child. Until poliovirus eradication is achieved, all countries must remain vigilant by maintaining high population immunity and sensitive poliovirus surveillance.

Assessment of an Innovative Voluntary Substance Abuse Treatment Program Designed to Replace Compulsory Drug Detention Centers in Malaysia.

BACKGROUND: As part of an ongoing initiative by the Malaysian government to implement alternative approaches to involuntary detention of people who use drugs, the National Anti-Drug Agency has created new voluntary drug treatment programs known as Cure and Care (C&C) Centers that provide free access to addiction treatment services, including methadone maintenance therapy, integrated with social and health services. OBJECTIVES: We evaluated early treatment outcomes and client satisfaction among patients accessing C&C treatment and ancillary services at Malaysia's second C&C Center located in Kota Bharu, Kelantan. METHODS: In June-July 2012, a cross-sectional convenience survey of 96 C&C inpatients and outpatients who entered treatment >30 days previously was conducted to assess drug use, criminal justice experience, medical co-morbidities, motivation for seeking treatment, and attitudes towards the C&C. Drug use was compared for the 30-day-period before C&C entry and the 30-day-period before the interview. RESULTS: Self-reported drug use levels decreased significantly among both inpatient and outpatient clients after enrolling in C&C treatment. Higher levels of past drug use, lower levels of social support, and more severe mental health issues were reported by participants who were previously imprisoned. Self-reported satisfaction with C&C treatment services was high. Conclusions/Importance: Preliminary evidence of reduced drug use and high levels of client satisfaction among C&C clients provide support for Malaysia's ongoing transition from compulsory drug detention centers (CDDCs) to these voluntary drug treatment centers. If C&C centers are successful, Malaysia plans to gradually transition away from CDDCs entirely.

Pretreatment drug use characteristics and experiences among patients in a voluntary substance abuse treatment center in Malaysia: A mixed-methods approach.

BACKGROUND: Drug use in Malaysia remains a significant public health and social problem despite implementation of harsh punitive drug policies such as forcibly placing suspected drug users into compulsory drug detention centers (CDDCs). Following criticism over human right violations in CDDCs, Malaysia has begun to transition towards voluntary drug treatment centers known as Cure & Care (C&C) centers. To best serve the needs of regional C&C centers, data on drug use are essential among patients accessing treatment. Using a mixed-methods approach, the authors examined pretreatment drug use characteristics and experiences with addiction treatment among C&C patients in Kelantan-a religiously conservative state in northeast Malaysia with high prevalence of drug use but where limited data are available on drug use patterns. METHODS: A mixed-methods study utilizing surveys (n = 96) and semistructured interviews (n = 20) was conducted among a convenience sample of inpatients and outpatients at the Pengkalan Chepa C&C Center in Kelantan. RESULTS: Survey results showed that 89.6% of participants met screening criteria for moderate to severe addiction severity. Nearly 90% reported lifetime illicit amphetamine (syabu, meth, ice, and pil kuda) use, followed by alcohol (60.4%) and opioids (52.1%). Qualitative results pointed to the powerful influence of peer networks in drug initiation and relapse, and the positive effect of the C&C center on drug rehabilitation. CONCLUSIONS: The drug use profile of the Kelantan C&C center enrollees shows extensive pretreatment amphetamine use, polysubstance use, and injection drug use, including high-risk behaviors such as sharing needles, syringes, and containers. Evidence points to the need for integration of social support-oriented practices and behavioral interventions into the rehabilitation of drug users in this region.

Discovery of new Mycobacterium tuberculosis proteasome inhibitors using a knowledge-based computational screening approach.

Mycobacterium tuberculosis bacteria cause deadly infections in patients [Corrected]. The rise of multidrug resistance associated with tuberculosis further makes the situation worse in treating the disease. M. tuberculosis proteasome is necessary for the pathogenesis of the bacterium validated as an anti-tubercular target, thus making it an attractive enzyme for designing Mtb inhibitors. In this study, a computational screening approach was applied to identify new proteasome inhibitor candidates from a library of 50,000 compounds. This chemical library was procured from the ChemBridge (20,000 compounds) and the ChemDiv (30,000 compounds) databases. After a detailed analysis of the computational screening results, 50 in silico hits were retrieved and tested in vitro finding 15 compounds with IC50 values ranging from 35.32 to 64.15 µM on lysate. A structural analysis of these hits revealed that 14 of these compounds probably have non-covalent mode of binding to the target and have not reported for anti-tubercular or anti-proteasome activity. The binding interactions of all the 14 protein-inhibitor complexes were analyzed using molecular docking studies. Further, molecular dynamics simulations of the protein in complex with the two most promising hits were carried out so as to identify the key interactions and validate the structural stability.

Initial Safety and Feasibility Results From a Phase 1, Diagnose-and-Treat Trial of Neoadjuvant Intratumoral Cisplatin for Stage IV NSCLC.

Neoadjuvant intratumoral cisplatin has the potential to generate substantial cytotoxicity and immune priming within the tumor environment, while minimizing systemic, off-target, adverse events. We initiated a phase 1A, 3+3 dose-ranging study of neoadjuvant, intratumoral cisplatin, delivered through endobronchial ultrasound bronchoscopy, in the same procedure as the initial diagnosis. There were no dose-limiting toxicity identified at the 20mg level.

Identification of novel MurA inhibitors using in silico approach, their validation and elucidation of mode of inhibition.

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is an important enzyme involved in the first cytosolic step of bacterial cell wall synthesis. In this study a combination of ligand based and structure based in silico virtual screening methods were utilised for screening of more than 50,000 drug-like compounds from CSIR-IIIM in-house compound library in order to identify potent inhibitors of MurA. The identified hits were validated in vitro under various incubation conditions using Malachite green phosphate assay, and two potent hits viz 3772-9534 and D396-0012 were identified. Among these hits, compound 3772-9534 showed significant changes in the activity values in different assay conditions. The MD simulation study of 3772-9534 suggested a novel binding site in MurA enzyme, independent of the two-substrate binding sites. Binding of inhibitors at the allosteric site induces conformational changes in the enzyme, which leads to inhibition of enzymatic activity. Overall, the study offers new insight for targeting MurA, which may promote the discovery of novel MurA allosteric site inhibitors.

Visualizing intratumoral injections in lung tumors by endobronchial ultrasound.

Real-time endobronchial ultrasound images are crucial for the accurate placement of the needle in peribronchial lung tumors and lymph nodes for diagnostic sampling. Beyond its role as a diagnostic tool, ultrasound-guided bronchoscopy can also aid the delivery of anti-cancer agents intratumorally, enabling diagnosis, staging, and treatment to occur within the same anesthesia, reducing the patient's burden. However, determining drug retention and distribution in situ remains challenging, albeit pivotal in assessing the success or failure of the therapeutic intervention. We hypothesized that ultrasound images acquired by the bronchoscope during the injection can provide qualitative and quantitative real-time information about drug transport. As a proof-of-concept, we retrospectively analyzed 13 videos of intratumoral cisplatin injections in advanced non-small cell lung cancers. We identified the injection and performed quantitative analysis through image processing and segmentation algorithms and mathematical models in 5 of them. We were able to infer the unlikeliness of a laminar flow through interstitial pores in favor of the emergence of tissue fractures. These data imply that the structural integrity of the tumor is a critical determinant of the ultimate distribution of an intratumorally delivered agent.

Endobronchial ultrasound-guided transbronchial needle injection of cisplatin results in dynamic changes in the tumor immune microenvironment.

RATIONALE: Direct intratumoral delivery of cisplatin via endobronchial ultrasound guided-transbronchial needle injections (EBUS-TBNI) is a novel approach for salvage treatment of advanced stage non-small cell lung cancer (NSCLC). The goal of this study was to evaluate changes in the tumor immune microenvironment during the course of EBUS-TBNI cisplatin therapy. METHODS: Under an IRB approved protocol, patients with recurrence after radiation therapy who were not receiving other cytotoxic therapy, were prospectively enrolled, and underwent weekly treatments with EBUS-TBNI with additional biopsies obtained for research. Needle aspiration was performed prior to cisplatin delivery at each procedure. Samples were evaluated by flow cytometry for the presence of immune cell types. RESULTS: Three of the six patients responded to the therapy based on RECIST criteria. Compared to the pre-treatment baseline, intratumoral neutrophils increased in 5 of the 6 patients (p = 0.041), with an average increase of 27.1%, but was not associated with response. A lower pre-treatment CD8+/CD4+ ratio at baseline was associated with response (P = 0.01). Responders demonstrated a lower final proportion of PD-1+ CD8+ T cells compared to non-responders (8.6% vs. 62.3%, respectively, P<0.001. Lower doses of intratumoral cisplatin were associated with subsequent increases in CD8+ T cells within the tumor microenvironment (P = 0.008). CONCLUSIONS: EBUS-TBNI cisplatin resulted in significant alterations in the tumor immune microenvironment. Further studies are needed to determine if the changes seen here generalize to larger cohorts.

Burden and Predictors of Non-Alcoholic Fatty Liver Disease in a Retrospective Cohort of Patients With Crohn's Disease.

Background: Non-alcoholic fatty liver disease (NAFLD) is an emerging extraintestinal manifestation (EIM) of Crohn's disease (CD). We aimed to investigate the prevalence and comorbid predictors of NAFLD in patients with CD. Methods: We conducted a nationwide retrospective cohort study to determine the prevalence, characteristics, comorbidities, and hospitalization outcomes associated with NAFLD in patients with CD. Comparison between groups was performed by Mann-Whitney test for continuous variables and Chi-square test for categorical variables. We performed a binary logistic regression analysis for predictors of NAFLD among patients with CD. Results: We extracted 215,049 index hospital discharges with CD; 2.4% had NAFLD. CD patients, with NAFLD, had increased length of stay (4 days; interquartile range (IQR): 2 - 6 vs. 3; IQR: 2 - 6, P < 0.01), and increased median total charges ($32,305.5; IQR: $18,600 - $61,599 vs. $30,782; IQR: $16,847 - $58,667, P < 0.01), compared to CD patients without NAFLD. Non-alcoholic steatohepatitis (NASH) was found to be independently associated with increased mortality (odds ratio (OR): 1.7; 95% confidence interval (CI): 1.1 - 2.6, P = 0.03) and a higher odd for all-cause 30-day non-elective readmission (OR: 1.6: 95% CI: 1.3 - 1.9, P < 0.001). Factors independently associated with NAFLD in patients with CD included portal hypertension (OR: 5.347; 95% CI: 4.604 - 6.211, P < 0.001), vitamin A deficiency (OR: 9.89; 95% CI: 4.49 - 21.76, P < 0.001) and vitamin B12 deficiency (OR: 1.56; 95% CI: 1.098 - 2.209, P = 0.013). Conclusions: NAFLD is associated with worse hospitalization outcomes in patients with CD. Study findings suggest the need for early identification and effective management of NAFLD predictors to reduce complications.

Iron Deficiency Anemia: An Overlooked Complication of Crohn's Disease.

Background: There are few studies to evaluate the association between iron deficiency anemia (IDA) and Crohn's disease (CD). We examined this association in a USA-based cohort of patients with CD. Methods: We queried the Nationwide Readmission Databases 2018 using the International Classification of Disease, 10th Revision, and Clinical Modification (ICD-10-CM) codes to identify all adult patients admitted with a diagnosis of CD. Primary outcomes were the prevalence of IDA among patients with CD. Secondary outcomes included inpatient mortality, the length of stay, all-cause 30-day non-elective readmission rate, and total cost of hospitalization. Multivariate regression analysis was performed to study the impact of IDA on inpatient mortality and non-elective readmissions. Results: Of the 72,076 patients discharged from an index hospitalization for CD, 8.1% had IDA. CD patients with IDA had increased length of stays in days (4, interquartile range (IQR): 2 - 6 vs. 3, IQR: 2 - 5; P < 0.001), increased median total charges ($35,160, IQR: $19,786 - $64,126 vs. $31,299, IQR: $17,226 - $59,561; P < 0.001), and were more common to require blood transfusion during hospitalization (13.6% vs. 3.4%, P < 0.001) compared to CD patients without IDA, respectively. IDA was independently associated with increased odds of all-cause 30-day non-elective readmission (odds ratio (OR): 1.254, 95% confidence interval (CI): 1.154 - 1.363, P < 0.001) and increased odds of all-cause 90-day non-elective readmission (OR: 1.396, 95% CI: 1.302 - 1.498, P < 0.001). Conclusions: In a large nationwide cohort of patients hospitalized for CD, we observed a significant burden of IDA. Additionally, we found a significant association between IDA and worse hospitalization outcomes.

Use of Focus Groups to Inform a New Community-Based Youth Diabetes Prevention Program.

There have been few youth-led diabetes prevention programs. Our objective was to conduct focus groups to explore peer influences on adolescent lifestyle behaviors and strategies for implementing a youth peer education model for diabetes prevention. We conducted six focus groups with 52 youth (ages 13-22; 62% male, 38% female; 64% Hispanic, 36% non-Hispanic Black) from East Harlem, NYC. We used a Thematic Analysis approach to identify major themes, compared findings, and resolved differences through discussion and consensus. Three dominant themes arose: (1) Adolescents generally encounter more unhealthy peer influences on diet and more healthy peer influences on physical activity; (2) Adolescents endorse youth-led diabetes prevention strategies and describe ideal qualities for peer leaders and methods to support and evaluate leaders; (3) Adolescents prefer text messaging to monitor behaviors, track goals, and receive personalized guidance. Using study findings, our Community Action Board developed a peer-led diabetes prevention program for prediabetic adolescents.

Prevalence and Impact of Acute Pancreatitis on Hospitalization Outcomes in a Cohort of Patients With Crohn's Disease.

Background: Few studies evaluated the risk of acute pancreatitis (AP) in patients with Crohn's disease (CD). It's controversial if AP can be considered as an extraintestinal manifestation of CD. We studied this potential association in a retrospective cohort of patients with CD. Methods: We draw our cohort from the Nationwide Readmission Databases 2016 - 2018. We used the International Classification of Diseases, Tenth Revision, Clinical Modification (ICD-10-CM) codes to identify all adult patients admitted with a diagnosis of CD. Patient with a comorbid AP were identified. We analyzed the significant impact of AP on hospitalization outcomes. A multivariate regression analysis was used to identify factors associated with AP. Results: We included 214,622 patients discharged from an index hospitalization for CD, 1.1% had AP. AP was independently associated with higher odds of inpatient mortality (odds ratio (OR): 1.831; 95% confidence interval (CI): 1.345 - 2.492, P < 0.001), gallstone disease (OR: 4.047; 95% CI: 3.343 - 4.9, P < 0.001), nonalcoholic fatty liver disease (NAFLD) (OR: 3.568; 95% CI: 3.08 - 4.133, P < 0.001), and hypercalcemia (OR: 1.964; 95% CI: 1.302 - 2.965, P = 0.001). Thirty-day readmission analysis showed that CD patients with AP were more commonly to be readmitted for AP than for any other reason. Conclusions: In our nationwide cohort of CD patients, there was a significant association between AP and worse hospitalization outcomes. Additionally, we found independent associations for having AP that may help identify patients at high risk.

Cancer Care in the Wake of a Cyberattack: How to Prepare and What to Expect.

PURPOSE: Cyberattacks targeting health care organizations are becoming more frequent and affect all aspects of care delivery. Cancer care is particularly susceptible to major disruptions because of the potential of immediate and long-term consequences for patients who often rely on timely diagnostic testing and regular administration of systemic therapy in addition to other local treatment modalities to cure or control their diseases. On October 28, 2020, a cyberattack was launched on the University of Vermont Health Network with wide-ranging consequences for oncology, including loss of access to all network intranet servers, e-mail communications, and the electronic medical record (EMR). METHODS: This review details the immediate challenges faced by hematology and oncology during the cyberattack. The impact and response on inpatient, outpatient, and special patient populations are described. Steps that other academic- and community-based oncology practices can take to lessen the brunt of such an assault are suggested. RESULTS: The two areas of immediate impact after the cyberattack were communications and lack of EMR access. The oncology-specific impact included loss of the individualized EMR chemotherapy plan templates and electronic safeguards built into multistep treatment preparation and delivery. With loss of access to schedules, basic patient information, encrypted communications platforms and radiology, and laboratory and pharmacy services, clinical outpatient care delivery was reduced by 40%. The infusion visit volume dropped by 52% in the first week and new patients could not access necessary services for timely diagnostic evaluation, requiring the creation of command centers to oversee ethical and transparent triage and allocation of systemic therapies and address new patient referrals. This included appropriate transfer of patients to alternate sites to minimize delays. Inpatient care including transitions of care was particularly challenging and addressing patient populations whose survival might be affected by delays in care. CONCLUSION: Oncology health care leaders and providers should be aware of the potential impact of a cyberattack on cancer care delivery and preventively develop processes to mitigate the impact.

Identification of Mycobacterium tuberculosis genes preferentially expressed during human infection.

The identification of Mycobacterium tuberculosis genes, specifically expressed during infection is a key step in understanding molecular mechanism of mycobacterial pathogenesis. Such genes likely encode proteins required for mycobacterium's survival and progressive infection within the host. In this study, we applied in-vivo-induced antigen technology (IVIAT) to M. tuberculosis and identified 11 putative in-vivo induced genes encoding for immunogenic proteins of diverse functions; these included transcriptional regulators (Rv1460 and Rv2565), biosynthesis and macromolecule metabolism (leuD, guaB1, plcC, hupB and glyS), polyketide synthases (pks6 and pks9), cell processes (ctpA) and one with unknown function (Rv3701c). Quantitative real time-PCR analysis of these genes in the specimens obtained from TB patients demonstrated induced expression of eight genes as compared with bacteria grown in-vitro. In addition, distribution of these genes in different strains of M. tuberculosis was analyzed using PCR and their nucleotide sequence alignments and they were found to be widely distributed among M. tuberculosis isolates including multiple-drug resistant (MDR) and extensively-drug resistant (XDR). This study identified several antigenic determinants of M. tuberculosis expressed during infection, which might help pathogens adapt to or counter hostile environments and suggesting their role during disease process.

In vitro antifungal activity of hydroxychavicol isolated from Piper betle L.

BACKGROUND: Hydroxychavicol, isolated from the chloroform extraction of the aqueous leaf extract of Piper betle L., (Piperaceae) was investigated for its antifungal activity against 124 strains of selected fungi. The leaves of this plant have been long in use tropical countries for the preparation of traditional herbal remedies. METHODS: The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of hydroxychavicol were determined by using broth microdilution method following CLSI guidelines. Time kill curve studies, post-antifungal effects and mutation prevention concentrations were determined against Candida species and Aspergillus species "respectively". Hydroxychavicol was also tested for its potential to inhibit and reduce the formation of Candida albicans biofilms. The membrane permeability was measured by the uptake of propidium iodide. RESULTS: Hydroxychavicol exhibited inhibitory effect on fungal species of clinical significance, with the MICs ranging from 15.62 to 500 microg/ml for yeasts, 125 to 500 microg/ml for Aspergillus species, and 7.81 to 62.5 microg/ml for dermatophytes where as the MFCs were found to be similar or two fold greater than the MICs. There was concentration-dependent killing of Candida albicans and Candida glabrata up to 8 x MIC. Hydroxychavicol also exhibited an extended post antifungal effect of 6.25 to 8.70 h at 4 x MIC for Candida species and suppressed the emergence of mutants of the fungal species tested at 2 x to 8 x MIC concentration. Furthermore, it also inhibited the growth of biofilm generated by C. albicans and reduced the preformed biofilms. There was increased uptake of propidium iodide by C. albicans cells when exposed to hydroxychavicol thus indicating that the membrane disruption could be the probable mode of action of hydroxychavicol. CONCLUSIONS: The antifungal activity exhibited by this compound warrants its use as an antifungal agent particularly for treating topical infections, as well as gargle mouthwash against oral Candida infections.

Molecular cloning and characterization of amylase from soil metagenomic library derived from Northwestern Himalayas.

The increasing demand for novel biocatalysts stimulates exploration of resources from soil. Metagenomics, a culture independent approach, represent a sheer unlimited resource for discovery of novel biocatalysts from uncultured microorganisms. In this study, a soil-derived metagenomic library containing 90,700 recombinants was constructed and screened for lipase, cellulase, protease and amylase activity. A gene (pAMY) of 909 bp encoding for amylase was found after the screening of 35,000 Escherichia coli clones. Amino acid sequence comparison and phylogenetic analysis indicated that pAMY was closely related to uncultured bacteria. The molecular mass of pAMY was estimated about 38 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Amylase activity was determined using soluble starch, amylose, glycogen and maltose as substrates. The maximal activity (2.46 U/mg) was observed at 40 degrees C under nearly neutral pH conditions with amylose; whereas it retains 90% of its activity at low temperature with all the substrates used in this study. The ability of pAMY to work at low temperature is unique for amylases reported so far from microbes of cultured and uncultured division.

Detection of the in vitro modulation of Plasmodium falciparum Arf1 by Sec7 and ArfGAP domains using a colorimetric plate-based assay.

The regulation of human Arf1 GTPase activity by ArfGEFs that stimulate GDP/GTP exchange and ArfGAPs that mediate GTP hydrolysis has attracted attention for the discovery of Arf1 inhibitors as potential anti-cancer agents. The malaria parasite Plasmodium falciparum encodes a Sec7 domain-containing protein - presumably an ArfGEF - and two putative ArfGAPs, as well as an Arf1 homologue (PfArf1) that is essential for blood-stage parasite viability. However, ArfGEF and ArfGAP-mediated activation/deactivation of PfArf1 has not been demonstrated. In this study, we established an in vitro colorimetric microtiter plate-based assay to detect the activation status of truncated human and P. falciparum Arf1 and used it to demonstrate the activation of both proteins by the Sec7 domain of ARNO, their deactivation by the GAP domain of human ArfGAP1 and the inhibition of the respective reactions by the compounds SecinH3 and QS11. In addition, we found that the GAP domains of both P. falciparum ArfGAPs have activities equivalent to that of human ArfGAP1, but are insensitive to QS11. Library screening identified a novel inhibitor which selectively inhibits one of the P. falciparum GAP domains (IC50 4.7 µM), suggesting that the assay format is suitable for screening compound collections for inhibitors of Arf1 regulatory proteins.