Decreased In Vitro Artemisinin Sensitivity of Plasmodium falciparum across India.

Artemisinin-based combination therapy (ACT) has been used to treat uncomplicated Plasmodium falciparum infections in India since 2004. Since 2008, a decrease in artemisinin effectiveness has been seen throughout the Greater Mekong Subregion. The geographic proximity and ecological similarities of northeastern India to Southeast Asia may differentially affect the long-term management and sustainability of ACT in India. In order to collect baseline data on variations in ACT sensitivity in Indian parasites, 12 P. falciparum isolates from northeast India and 10 isolates from southwest India were studied in vitro Ring-stage survival assay (RSA) showed reduced sensitivity to dihydroartemisinin in 50% of the samples collected in northeast India in 2014 and 2015. Two of the 10 assayed samples from the southwest region of India from as far back as 2012 also showed decreased sensitivity to artemisinin. In both these regions, kelch gene sequences were not predictive of reduced artemisinin sensitivity, as measured by RSA. The present data justify future investments in integrated approaches involving clinical follow-up studies, in vitro survival assays, and molecular markers for tracking potential changes in the effectiveness of artemisinin against P. falciparum throughout India.

Hospital-derived antibody profiles of malaria patients in Southwest India.

BACKGROUND: Naturally acquired immunity to malaria across the globe varies in intensity and protective powers. Many of the studies on immunity are from hyperendemic regions of Africa. In Asia, particularly in India, there are unique opportunities for exploring and understanding malaria immunity relative to host age, co-occurrence of Plasmodium falciparum and Plasmodium vivax infections, varying travel history, and varying disease severity. Variation in immunity in hospital settings is particularly understudied. METHODS: A US NIH ICEMR (South Asia) team examined the level of immunity in an Indian malaria patient population visiting or admitted to Goa Medical College and Hospital in Goa, India. Sera from 200 patients of different ages, in different seasons, infected with P. falciparum or P. vivax or both species, and with different clinical severity were applied to an established protein array system with over 1000 P. falciparum and P. vivax antigens. Differential binding of patient IgG to different antigens was measured. RESULTS: Even though Goa itself has much more P. vivax than P. falciparum, IgG reactivity towards P. falciparum antigens was very strong and comparable to that seen in regions of the world with high P. falciparum endemicity. Of 248 seropositive P. falciparum antigens, the strongest were VAR, MSP10, HSP70, PTP5, AP2, AMA1, and SYN6. In P. vivax patients, ETRAMPs, MSPs, and ApiAP2, sexual stage antigen s16, RON3 were the strongest IgG binders. Both P. falciparum and P. vivax patients also revealed strong binding to new antigens with unknown functions. Seropositives showed antigens unique to the young (HSP40, ACS6, GCVH) or to non-severe malaria (MSP3.8 and PHIST). CONCLUSION: Seroreactivity at a major hospital in Southwest India reveals antibody responses to P. falciparum and P. vivax in a low malaria transmission region with much migration. In addition to markers of transmission, the data points to specific leads for possible protective immunity against severe disease. Several, but not all, key antigens overlap with work from different settings around the globe and from other parts of India. Together, these studies confidently help define antigens with the greatest potential chance of universal application for surveillance and possibly for disease protection, in many different parts of India and the world.

Severe adult malaria is associated with specific PfEMP1 adhesion types and high parasite biomass.

The interplay between cellular and molecular determinants that lead to severe malaria in adults is unexplored. Here, we analyzed parasite virulence factors in an infected adult population in India and investigated whether severe malaria isolates impair endothelial protein C receptor (EPCR), a protein involved in coagulation and endothelial barrier permeability. Severe malaria isolates overexpressed specific members of the Plasmodium falciparum var gene/PfEMP1 (P. falciparum erythrocyte membrane protein 1) family that bind EPCR, including DC8 var genes that have previously been linked to severe pediatric malaria. Machine learning analysis revealed that DC6- and DC8-encoding var transcripts in combination with high parasite biomass were the strongest indicators of patient hospitalization and disease severity. We found that DC8 CIDRα1 domains from severe malaria isolates had substantial differences in EPCR binding affinity and blockade activity for its ligand activated protein C. Additionally, even a low level of inhibition exhibited by domains from two cerebral malaria isolates was sufficient to interfere with activated protein C-barrier protective activities in human brain endothelial cells. Our findings demonstrate an interplay between parasite biomass and specific PfEMP1 adhesion types in the development of adult severe malaria, and indicate that low impairment of EPCR function may contribute to parasite virulence.

Reticulocyte Preference and Stage Development of Plasmodium vivax Isolates.

Plasmodium vivax, the most widely distributed human malaria parasite, is restricted to reticulocytes, limiting its asexual proliferation. In recent years, cases of severe and high-level P. vivax parasitemia have been reported, challenging the assumption that all isolates are equally restricted. In this article, we analyze the reticulocyte preference of a large number of Indian P. vivax isolates. Our results show that P. vivax isolates significantly vary in their level of reticulocyte preference. In addition, by carefully staging the parasites, we find that P. vivax schizonts are largely missing in peripheral blood, with the presence of schizonts in circulation correlating with a high reticulocyte preference.

In vitro adaptation of Plasmodium falciparum reveal variations in cultivability.

BACKGROUND: Culture-adapted Plasmodium falciparum parasites can offer deeper understanding of geographic variations in drug resistance, pathogenesis and immune evasion. To help ground population-based calculations and inferences from culture-adapted parasites, the complete range of parasites from a study area must be well represented in any collection. To this end, standardized adaptation methods and determinants of successful in vitro adaption were sought. METHODS: Venous blood was collected from 33 P. falciparum-infected individuals at Goa Medical College and Hospital (Bambolim, Goa, India). Culture variables such as whole blood versus washed blood, heat-inactivated plasma versus Albumax, and different starting haematocrit levels were tested on fresh blood samples from patients. In vitro adaptation was considered successful when two four-fold or greater increases in parasitaemia were observed within, at most, 33 days of attempted culture. Subsequently, parasites from the same patients, which were originally cryopreserved following blood draw, were retested for adaptability for 45 days using identical host red blood cells (RBCs) and culture media. RESULTS: At a new endemic area research site, ~65% of tested patient samples, with varied patient history and clinical presentation, were successfully culture-adapted immediately after blood collection. Cultures set up at 1% haematocrit and 0.5% Albumax adapted most rapidly, but no single test condition was uniformly fatal to culture adaptation. Success was not limited by low patient parasitaemia nor by patient age. Some parasites emerged even after significant delays in sample processing and even after initiation of treatment with anti-malarials. When 'day 0' cryopreserved samples were retested in parallel many months later using identical host RBCs and media, speed to adaptation appeared to be an intrinsic property of the parasites collected from individual patients. CONCLUSIONS: Culture adaptation of P. falciparum in a field setting is formally shown to be robust. Parasites were found to have intrinsic variations in adaptability to culture conditions, with some lines requiring longer attempt periods for successful adaptation. Quantitative approaches described here can help describe phenotypic diversity of field parasite collections with precision. This is expected to improve population-based extrapolations of findings from field-derived fresh culture-adapted parasites to broader questions of public health importance.

Demographic and clinical profiles of Plasmodium falciparum and Plasmodium vivax patients at a tertiary care centre in southwestern India.

BACKGROUND: Malaria remains an important cause of morbidity and mortality in India. Though many comprehensive studies have been carried out in Africa and Southeast Asia to characterize and examine determinants of Plasmodium falciparum and Plasmodium vivax malaria pathogenesis, fewer have been conducted in India. METHODS: A prospective study of malaria-positive individuals was conducted at Goa Medical College and Hospital (GMC) from 2012 to 2015 to identify demographic, diagnostic and clinical indicators associated with P. falciparum and P. vivax infection on univariate analysis. RESULTS: Between 2012 and 2015, 74,571 febrile individuals, 6287 (8.4%) of whom were malaria positive, presented to GMC. The total number of malaria cases at GMC increased more than two-fold over four years, with both P. vivax and P. falciparum cases present year-round. Some 1116 malaria-positive individuals (mean age = 27, 91% male), 88.2% of whom were born outside of Goa and 51% of whom were construction workers, were enroled in the study. Of 1088 confirmed malaria-positive patients, 77.0% had P. vivax, 21.0% had P. falciparum and 2.0% had mixed malaria. Patients over 40 years of age and with P. falciparum infection were significantly (p < 0.001) more likely to be hospitalised than younger and P. vivax patients, respectively. While approximately equal percentages of hospitalised P. falciparum (76.6%) and P. vivax (78.9%) cases presented with at least one WHO severity indicator, a greater percentage of P. falciparum inpatients presented with at least two (43.9%, p < 0.05) and at least three (29.9%, p < 0.01) severity features. There were six deaths among the 182 hospitalised malaria positive patients, all of whom had P. falciparum. CONCLUSION: During the four year study period at GMC, the number of malaria cases increased substantially and the greatest burden of severe disease was contributed by P. falciparum.

Improved light microscopy counting method for accurately counting Plasmodium parasitemia and reticulocytemia.

Even with the advances in molecular or automated methods for detection of red blood cells of interest (such as reticulocytes or parasitized cells), light microscopy continues to be the gold standard especially in laboratories with limited resources. The conventional method for determination of parasitemia and reticulocytemia uses a Miller reticle, a grid with squares of different sizes. However, this method is prone to errors if not used correctly and counts become inaccurate and highly time-consuming at low frequencies of target cells. In this report, we outline the correct guidelines to follow when using a reticle for counting, and present a new counting protocol that is a modified version of the conventional method for increased accuracy in the counting of low parasitemias and reticulocytemias. Am. J. Hematol. 91:852-855, 2016. © 2016 Wiley Periodicals, Inc.

PIN6 is required for nectary auxin response and short stamen development.

The PIN family of proteins is best known for its involvement in polar auxin transport and tropic responses. PIN6 (At1g77110) is one of the remaining PIN family members in Arabidopsis thaliana to which a biological function has not yet been ascribed. Here we report that PIN6 is a nectary-enriched gene whose expression level is positively correlated with total nectar production in Arabidopsis, and whose function is required for the proper development of short stamens. PIN6 accumulates in internal membranes consistent with the ER, and multiple lines of evidence demonstrate that PIN6 is required for auxin-dependent responses in nectaries. Wild-type plants expressing auxin-responsive DR5:GFP or DR5:GUS reporters displayed intense signal in lateral nectaries, but pin6 lateral nectaries showed little or no signal for these reporters. Further, exogenous auxin treatment increased nectar production more than tenfold in wild-type plants, but nectar production was not increased in pin6 mutants when treated with auxin. Conversely, the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) reduced nectar production in wild-type plants by more than twofold, but had no significant effect on pin6 lines. Interestingly, a MYB57 transcription factor mutant, myb57-2, closely phenocopied the loss-of-function mutant pin6-2. However, PIN6 expression was not dependent on MYB57, and RNA-seq analyses of pin6-2 and myb57-2 mutant nectaries showed little overlap in terms of differentially expressed genes. Cumulatively, these results demonstrate that PIN6 is required for proper auxin response and nectary function in Arabidopsis. These results also identify auxin as an important factor in the regulation of nectar production, and implicate short stamens in the maturation of lateral nectaries.

Incidence of narcotic abuse during pregnancy in northwestern Ontario: three-year prospective cohort study.

OBJECTIVE: To document the incidence and outcomes of narcotic use during pregnancy in northwestern Ontario. DESIGN: Three-year prospective cohort study. SETTING: Sioux Lookout and surrounding communities in northwestern Ontario. PARTICIPANTS: A total of 1206 consecutive births in a catchment area of 28 000 First Nations patients. MAIN OUTCOME MEASURES: Incidence of narcotic use, and maternal and neonatal outcomes. RESULTS: Incidence of narcotic use in pregnancy has risen to 28.6% (P < .001) and incidence of neonatal abstinence syndrome has fallen to 18.0% of narcotic-exposed births (P = .003). Daily intravenous drug use is now a common pattern of abuse. CONCLUSION: Narcotic abuse in pregnancy has dramatically increased in northwestern Ontario. Neonatal outcomes have improved as a result of a family medicine-based prenatal and obstetric program that includes a narcotic replacement and tapering program.

A method for CRISPR/Cas9 mutation of genes in fathead minnow (Pimephales promelas).

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing allows for the disruption or modification of genes in a multitude of model organisms. In the present study, we describe and employ the method for use in the fathead minnow (Pimephales promelas), in part, to assist in the development and validation of adverse outcome pathways (AOPs). The gene coding for an enzyme responsible for melanin production, tyrosinase (tyr), was the initial target chosen for development and assessment of the method since its disruption results in abnormal pigmentation, a phenotype obvious within 3-4 d after injection of fathead minnow embryos. Three tyrosinase-targeting guide strands were generated using the fathead minnow sequence in tandem with the CRISPOR guide strand selection tool. The strands targeted two areas: one stretch of sequence in a conserved region that demonstrated homology to EGF-like or laminin-like domains as determined by Protein Basic Local Alignment Search Tool in concert with the Conserved Domain Database, and a second area in the N-terminal region of the tyrosinase domain. To generate one cell embryos, in vitro fertilization was performed, allowing for microinjection of hundreds of developmentally-synchronized embryos with Cas9 proteins complexed to each of the three guide strands. Altered retinal pigmentation was observed in a portion of the tyr guide strand injected population within 3 d post fertilization (dpf). By 14 dpf, fish without skin and swim bladder pigmentation were observed. Among the three guide strands injected, the guide targeting the EGF/laminin-like domain was most effective in generating mutants. CRISPR greatly advances our ability to directly investigate gene function in fathead minnow, allowing for advanced approaches to AOP validation and development.

Facile whole mitochondrial genome resequencing from nipple aspirate fluid using MitoChip v2.0.

BACKGROUND: Mutations in the mitochondrial genome (mtgenome) have been associated with many disorders, including breast cancer. Nipple aspirate fluid (NAF) from symptomatic women could potentially serve as a minimally invasive sample for breast cancer screening by detecting somatic mutations in this biofluid. This study is aimed at 1) demonstrating the feasibility of NAF recovery from symptomatic women, 2) examining the feasibility of sequencing the entire mitochondrial genome from NAF samples, 3) cross validation of the Human mitochondrial resequencing array 2.0 (MCv2), and 4) assessing the somatic mtDNA mutation rate in benign breast diseases as a potential tool for monitoring early somatic mutations associated with breast cancer. METHODS: NAF and blood were obtained from women with symptomatic benign breast conditions, and we successfully assessed the mutation load in the entire mitochondrial genome of 19 of these women. DNA extracts from NAF were sequenced using the mitochondrial resequencing array MCv2 and by capillary electrophoresis (CE) methods as a quality comparison. Sequencing was performed independently at two institutions and the results compared. The germline mtDNA sequence determined using DNA isolated from the patient's blood (control) was compared to the mutations present in cellular mtDNA recovered from patient's NAF. RESULTS: From the cohort of 28 women recruited for this study, NAF was successfully recovered from 23 participants (82%). Twenty two (96%) of the women produced fluids from both breasts. Twenty NAF samples and corresponding blood were chosen for this study. Except for one NAF sample, the whole mtgenome was successfully amplified using a single primer pair, or three pairs of overlapping primers. Comparison of MCv2 data from the two institutions demonstrates 99.200% concordance. Moreover, MCv2 data was 99.999% identical to CE sequencing, indicating that MCv2 is a reliable method to rapidly sequence the entire mtgenome. Four NAF samples contained somatic mutations. CONCLUSION: We have demonstrated that NAF is a suitable material for mtDNA sequence analysis using the rapid and reliable MCv2. Somatic mtDNA mutations present in NAF of women with benign breast diseases could potentially be used as risk factors for progression to breast cancer, but this will require a much larger study with clinical follow up.

Mitochondrial genome deletion aids in the identification of false- and true-negative prostate needle core biopsy specimens.

We report the usefulness of a 3.4-kb mitochondrial genome deletion (3.4 mtdelta) for molecular definition of benign, malignant, and proximal to malignant (PTM) prostate needle biopsy specimens. The 3.4 mtdelta was identified through long-extension polymerase chain reaction (PCR) analysis of frozen prostate cancer samples. A quantitative PCR assay was developed to measure the levels of the 3.4 mtdelta in clinical samples. For normalization, amplifications of a nuclear target and total mitochondrial DNA were included. Cycle threshold data from these targets were used to calculate a score for each biopsy sample. In a pilot study of 38 benign, 29 malignant, and 41 PTM biopsy specimens, the difference between benign and malignant core biopsy specimens was well differentiated (P & .0001), with PTM indistinguishable from malignant samples (P = .833). Results of a larger study were identical. In comparison with histopathologic examination for benign and malignant samples, the sensitivity and specificity were 80% and 71%, respectively, and the area under a receiver operating characteristic (ROC) curve was 0.83 for the deletion. In a blinded external validation study, the sensitivity and specificity were 83% and 79%, and the area under the ROC curve was 0.87. The 3.4 mtdelta may be useful in defining malignant, benign, and PTM prostate tissues.

The pseudo-mitochondrial genome influences mistakes in heteroplasmy interpretation.

BACKGROUND: Nuclear mitochondrial pseudogenes (numts) are a potential source of contamination during mitochondrial DNA PCR amplification. This possibility warrants careful experimental design and cautious interpretation of heteroplasmic results. RESULTS: Here we report the cloning and sequencing of numts loci, amplified from human tissue and rho-zero (rho0) cells (control) with primers known to amplify the mitochondrial genome. This paper is the first to fully sequence 46 paralogous nuclear DNA fragments that represent the entire mitochondrial genome. This is a surprisingly small number due primarily to the primer sets used in this study, because prior to this, BLAST searches have suggested that nuclear DNA harbors between 400 to 1,500 paralogous mitochondrial DNA fragments. Our results indicate that multiple numts were amplified simultaneously with the mitochondrial genome and increased the load of pseudogene signal in PCR reactions. Further, the entire mitochondrial genome was represented by multiple copies of paralogous nuclear sequences. CONCLUSION: These findings suggest that mitochondrial genome disease-associated biomarkers must be rigorously authenticated to preclude any affiliation with paralogous nuclear pseudogenes. Importantly, the common perception that mitochondrial template "swamps" numts loci precluding detectable amplification, depends on the region of the mitochondrial genome targeted by the PCR reaction and the number of pseudogene loci that may co-amplify. Cloning and relevant sequencing data will facilitate the correct interpretation. This is the first complete, wet-lab characterization of numts that represent the entire mitochondrial genome.

Somatic mitochondrial DNA mutations in prostate cancer and normal appearing adjacent glands in comparison to age-matched prostate samples without malignant histology.

Studies of somatic mitochondrial DNA mutations have become an important aspect of cancer research because these mutations might have functional significance and/or serve as a biosensor for tumor detection. Here we report somatic mitochondrial DNA mutations from three specific tissue types (tumor, adjacent benign, and distant benign) recovered from 24 prostatectomy samples. Needle biopsy tissue from 12 individuals referred for prostate biopsy, yet histologically benign (symptomatic benign), were used as among individual control samples. We also sampled blood (germplasm tissue) from each patient to serve as within individual controls relative to the somatic tissues sampled (malignant, adjacent, and distant benign). Complete mitochondrial genome sequencing was attempted on each sample. In contrast to both control groups [within patient (blood) and among patient (symptomatic benign)], all of the tissue types recovered from the malignant group harbored significantly different mitochondrial DNA (mtDNA) mutations. We conclude that mitochondrial genome mutations are an early indicator of malignant transformation in prostate tissue. These mutations occur well before changes in tissue histo-pathology, indicative of prostate cancer, are evident to the pathologist.

Roles of endogenous ascorbate and glutathione in the cellular reduction and cytotoxicity of sulfamethoxazole-nitroso.

Sulfamethoxazole (SMX) is an effective drug for the management of opportunistic infections, but its use is limited by hypersensitivity reactions, particularly in HIV-infected patients. The oxidative metabolite SMX-nitroso (SMX-NO), is thought to be a proximate mediator of SMX hypersensitivity, and can be reduced in vitro by ascorbate or glutathione. Leukocytes from patients with SMX hypersensitivity show enhanced cytotoxicity from SMX metabolites in vitro; this finding has been attributed to a possible "detoxification defect" in some individuals. The purpose of this study was to determine whether variability in endogenous ascorbate or glutathione could be associated with individual differences in SMX-NO cytotoxicity. Thirty HIV-positive patients and 23 healthy control subjects were studied. Both antioxidants were significantly correlated with the reduction of SMX-NO to its hydroxylamine, SMX-HA, by mononuclear leukocytes, and both were linearly depleted during reduction. Controlled ascorbate supplementation in three healthy subjects increased leukocyte ascorbate with no change in glutathione, and significantly enhanced SMX-NO reduction. Ascorbate supplementation also decreased SMX-NO cytotoxicity compared to pre-supplementation values. Rapid reduction of SMX-NO to SMX-HA was associated with enhanced direct cytotoxicity from SMX-NO. When forward oxidation of SMX-HA back to SMX-NO was driven by the superoxide dismutase mimetic, Tempol, SMX-NO cytotoxicity was increased, without enhancement of adduct formation. This suggests that SMX-NO cytotoxicity may be mediated, at least in part, by redox cycling between SMX-HA and SMX-NO. Overall, these data indicate that endogenous ascorbate and glutathione are important for the intracellular reduction of SMX-NO, a proposed mediator of SMX hypersensitivity, and that redox cycling of SMX-HA to SMX-NO may contribute to the cytotoxicity of these metabolites in vitro.

Distinct genomic architecture of Plasmodium falciparum populations from South Asia.

Previous whole genome comparisons of Plasmodium falciparum populations have not included collections from the Indian subcontinent, even though two million Indians contract malaria and about 50,000 die from the disease every year. Stratification of global parasites has revealed spatial relatedness of parasite genotypes on different continents. Here, genomic analysis was further improved to obtain country-level resolution by removing var genes and intergenic regions from distance calculations. P. falciparum genomes from India were found to be most closely related to each other. Their nearest neighbors were from Bangladesh and Myanmar, followed by Thailand. Samples from the rest of Southeast Asia, Africa and South America were increasingly more distant, demonstrating a high-resolution genomic-geographic continuum. Such genome stratification approaches will help monitor variations of malaria parasites within South Asia and future changes in parasite populations that may arise from in-country and cross-border migrations.

Evaluation of the clinical, immunologic, and biochemical effects of nitroso sulfamethoxazole administration to dogs: a pilot study.

Sulfonamide antimicrobials such as sulfamethoxazole (SMX) have been associated in humans with hypersensitivity reactions, to include fever, skin eruptions, hepatotoxicity, and blood dyscrasias. These reactions also occur in dogs, the only non-human species known to develop a similar spectrum of sulfonamide hypersensitivity. Sulfonamide hypersensitivity is not well understood, but has been hypothesized to be due to the generation of the reactive oxidative metabolite, nitroso sulfamethoxazole (SMX-NO). SMX-NO, unlike the parent sulfonamide, is cytotoxic in vitro, haptenizes tissue proteins, and is immunogenic in rodents. The purpose of this pilot study was to determine whether SMX-NO, when administered to dogs, would lead to drug-tissue adducts, anti-drug antibodies, antioxidant depletion, or clinical evidence of drug hypersensitivity. Four dogs were randomized to one of four treatments: SMX-NO 1 mg/kg; SMX-NO 3 mg/kg; SMX-NO 10 mg/kg; or vehicle control. Dosing was by the intraperitoneal route, once daily for four consecutive days per week, for 2 weeks total, followed by a third week of observation. Following this, all dogs were challenged with trimethoprim-sulfamethoxazole, 25 mg/kg for 12 h for 2 weeks. No dog developed clinical or biochemical evidence of drug hypersensitivity. Plasma cysteine and leukocyte reduced glutathione were not depleted during dosing; however, ascorbate was significantly depleted by week 2 following SMX-NO at 10 mg/kg. Anti-SMX antibodies (IgG or IgM by ELISA) were not detected in any dogs at any time points. SMX-hemoglobin adducts were detected in the spleen in SMX-NO dosed dogs; however, these adducts were not accompanied by an immunologic or systemic response. The results of this pilot study indicate that SMX-NO dosing in dogs, using a dosing protocol shown to be immunogenic in other species, produces modest ascorbate depletion and hemoglobin adduct formation, but is insufficient to produce an immunologic response or a clinical syndrome of sulfonamide hypersensitivity in this susceptible species.

The incentives created by a harm reduction approach to smoking cessation: Snus and smoking in Sweden and Finland.

BACKGROUND: Tobacco harm reduction involves advocating the use of a less harmful alternative to smoking for those users who are unwilling or unable to quit. The net effect of such an approach is unclear as it may create opposing incentives. Although some smokers may substitute toward this less harmful alternative, it may reduce the incentive to quit by undermining public health efforts and may act as a gateway to smoking. This research paper aims to answer the question: Does the availability of a less harmful alternative to smoking lead to cessation? To explore the opposing incentives created by a harm reduction approach to smoking cessation, I focus on the role of snus, a popular smokeless tobacco product in Scandinavia that is widely used in Sweden. METHODS: This paper exploits a quasi-natural experiment to examine the net effect resulting from these opposing incentives. While two Scandinavian countries, Sweden and Finland, joined the European Union (EU) in 1995, Finland was subject to a pre-existing EU ban on oral tobacco products while Sweden received an exemption. A difference in differences framework is used to estimate the change in the smoking rate in Finland due to the implementation of the ban. A secondary analysis uses Finnish smoking data to test for a structural break in trend. RESULTS: In the post-ban period, smoking was 3.47 percentage points higher in Finland relative to what it would have been in the absence of the ban. CONCLUSION: The availability of snus, a less harmful alternative to smoking, appears to have had a positive impact (reduction) on the smoking rate. Offering acceptable alternatives to cigarettes is critical in reducing smoking prevalence.

Malaria evolution in South Asia: knowledge for control and elimination.

The study of malaria parasites on the Indian subcontinent should help us understand unexpected disease outbreaks and unpredictable disease presentations from Plasmodium falciparum and Plasmodium vivax infections. The Malaria Evolution in South Asia (MESA) research program is one of ten International Centers of Excellence for Malaria Research (ICEMR) sponsored by the US National Institutes of Health. In this second of two reviews, we describe why population structures of Plasmodia in India will be characterized and how we will determine their consequences on disease presentation, outcome and patterns. Specific projects will determine if genetic diversity, possibly driven by parasites with higher genetic plasticity, plays a role in changing epidemiology, pathogenesis, vector competence of parasite populations and whether innate human genetic traits protect Indians from malaria today. Deep local clinical knowledge of malaria in India will be supplemented by basic scientists who bring new research tools. Such tools will include whole genome sequencing and analysis methods; in vitro assays to measure genome plasticity, RBC cytoadhesion, invasion, and deformability; mosquito infectivity assays to evaluate changing parasite-vector compatibilities; and host genetics to understand protective traits in Indian populations. The MESA-ICEMR study sites span diagonally across India and include a mixture of very urban and rural hospitals, each with very different disease patterns and patient populations. Research partnerships include government-associated research institutes, private medical schools, city and state government hospitals, and hospitals with industry ties. Between 2012 and 2017, in addition to developing clinical research and basic science infrastructure at new clinical sites, our training workshops will engage new scientists and clinicians throughout South Asia in the malaria research field.

Recent developments in factor-facilitated ribosome assembly.

Escherichia coli ribosomal subunits can be reconstituted in vitro under highly optimized conditions. These reconstitution systems have proven invaluable for the study of ribosomal subunit assembly. While E. coli ribosomal subunits can self-assemble in vitro there has been much speculation regarding the existence of extra-ribosomal assembly factors that act in functional subunit formation in vivo. Recently, a biochemical assay has been implemented to identify factors that facilitate a single, critical step in 30S subunit assembly in vitro. These studies have revealed that the DnaK (heat shock protein 70) chaperone system can facilitate 30S subunit assembly in vitro. The 30S subunits, formed in the presence of the chaperones under otherwise non-permissive conditions, are highly similar to 30S subunits formed under standard reconstitution conditions. It has become evident that the manner in which the "factor-assembled" 30S subunits are purified is critical for monitoring formation of functional ribosomal particles. Given that methodologies for in vitro reconstitution and functional analysis of ribosomal subunits have been described in detail previously, this manuscript will focus on isolation of functional 30S subunits that have been assembled in the presence of exogenous factors in vitro. Also, recent efforts toward understanding the roles of exogenous factors in 50S subunit and eukaryotic ribosome assembly will be briefly discussed.