3MM Filter Paper Research Articles

Genetic Polymorphisms in<i> Plasmodium falciparum </i>Chloroquine Resistance Gene<i>, pfcrt </i>in Massakory (Chad)

<i>Background and Objective</i>: In 2005, Chad, like several other WHO countries, withdrew chloroquine as a first-line treatment for <i>Plasmodium falciparum</i> malaria in response to WHO recommendations related to the reason for the increase in treatment failures and the global spread of chloroquine resistance. Artemisinin-based combination therapy (ACTs), Artemether-lumefantrine, has replaced chloroquine as the first-choice treatment for malaria. The present study assessed <i>pfcrt</i> polymorphism in <i>Plasmodium falciparum</i> isolates in Massakory. <i>Methodology and Results</i>: Blood samples for PCR analysis were collected on Whatman 3MM filter paper in Massakory during a therapeutic efficacy study (TES) conducted from December 14, 2019 to March 14, 2020. Genomic DNA was extracted from 113 dried blood spots with the QIAamp DNA Micro Kit (Qiagen, Valencia, CA) as per manufacturer’s protocol and amplified by nested-PCR with <i>pfcrt</i> specific primer. The amplification products were revealed by electrophoresis on 2% agarose gel and then sequenced according to Sanger method. A total of 71 sequences were readable. The <i>pfcrt</i> analysis showed that of the 71 readable sequences, high mutation prevalence: 66 (92.96%) <I>IET</I>, 2 (4.22%) <I>IDT</I> and 3 (4.22%) <I>MNK</I> wild <i>pfcrt</i> isolates. <i>Conclusion</i>: These results challenge the highest health authorities in the country. The government, through the Ministry of Public Health and National Solidarity and the National Malaria Control Program, must raise awareness for the effective withdrawal of chloroquine. This action will promote on the one hand the re-emergence of parasites sensitive to chloroquine, and on the other hand make possible the reintroduction of chloroquine in the treatment of simple malaria after the suppression of drug pressure.

Can malaria rapid diagnostic tests by drug sellers under field conditions classify children 5\xa0years old or less with or without Plasmodium falciparum malaria? Comparison with nested PCR analysis

BackgroundMalaria rapid diagnostic tests (RDTs) available as dipsticks or strips, are simple to perform, easily interpretable and do not require electricity nor infrastructural investment. Correct interpretation of and compliance with the RDT results is a challenge to drug sellers. Thus, drug seller interpretation of RDT strips was compared with laboratory scientist re-reading, and PCR analysis of Plasmodium DNA extracted from RDT nitrocellulose strips and fast transient analysis (FTA) cards. Malaria RDT cassettes were also assessed as a potential source of Plasmodium DNA.MethodsA total of 212 children aged between 2 and 60 months, 199 of whom had complete records at two study drug shops in south western Uganda participated in the study. Duplicate 5 μL samples of capillary blood were picked from the 212 children, dispensed onto the sample well of the CareStart™ Pf-HRP2 RDT cassette and a FTA, Whatman™ 3MM filter paper in parallel. The RDT strip was interpreted by the drug seller within 15–20 min, visually re-read centrally by laboratory scientist and from it; Plasmodium DNA was recovered and detected by PCR, and compared with FTA recovered P. falciparum DNA PCR detection.ResultsMalaria positive samples were 62/199 (31.2%, 95% CI 24.9, 38.3) by drug seller interpretation of RDT strip, 59/212 (27.8%, 95% CI 22.2, 34.3) by laboratory scientist, 55/212 (25.9%, 95% CI 20.0, 32.6) by RDT nitrocellulose strip PCR and 64/212 (30.2%, 95% CI 24.4, 37.7). The overall agreement between the drug seller interpretation and laboratory scientist re-reading of the RDT strip was 93.0% with kappa value of 0.84 (95% CI 0.75, 0.92). The drug seller compliance with the reported RDT results was 92.5%. The performance of the three diagnostic strategies compared with FTA-PCR as the gold standard had sensitivity between 76.6 and 86.9%, specificity above 90%, positive predictive values ranging from 79.0 to 89.8% and negative predictive values above 90%.ConclusionDrug sellers can use RDTs in field conditions and achieve acceptable accuracy for malaria diagnosis, and they comply with the RDT results. Plasmodium DNA can be recovered from RDT nitrocellulose strips even in the context of drug shops. Future malaria surveillance and diagnostic quality control studies with RDT cassette as a source of Plasmodium DNA are recommended.

A novel method for extracting nucleic acids from dried blood spots for ultrasensitive detection of low-density\xa0Plasmodium falciparum and Plasmodium vivax\xa0infections

BackgroundGreater Mekong Subregion countries are committed to eliminating Plasmodium falciparum malaria by 2025. Current elimination interventions target infections at parasite densities that can be detected by standard microscopy or rapid diagnostic tests (RDTs). More sensitive detection methods have been developed to detect lower density “asymptomatic” infections that may represent an important transmission reservoir. These ultrasensitive polymerase chain reaction (usPCR) tests have been used to identify target populations for mass drug administration (MDA). To date, malaria usPCR tests have used either venous or capillary blood sampling, which entails complex sample collection, processing and shipping requirements. An ultrasensitive method performed on standard dried blood spots (DBS) would greatly facilitate the molecular surveillance studies needed for targeting elimination interventions.MethodsA highly sensitive method for detecting Plasmodium falciparum and P. vivax 18S ribosomal RNA from DBS was developed by empirically optimizing nucleic acid extraction conditions. The limit of detection (LoD) was determined using spiked DBS samples that were dried and stored under simulated field conditions. Further, to assess its utility for routine molecular surveillance, two cross-sectional surveys were performed in Myanmar during the wet and dry seasons.ResultsThe lower LoD of the DBS-based ultrasensitive assay was 20 parasites/mL for DBS collected on Whatman 3MM filter paper and 23 parasites/mL for Whatman 903 Protein Saver cards—equivalent to 1 parasite per 50 µL DBS. This is about 5000-fold more sensitive than standard RDTs and similar to the LoD of ≤16–22 parasites/mL reported for other ultrasensitive methods based on whole blood. In two cross-sectional surveys in Myanmar, nearly identical prevalence estimates were obtained from contemporaneous DBS samples and capillary blood samples collected during the wet and dry season.ConclusionsThe DBS-based ultrasensitive method described in this study shows equal sensitivity as previously described methods based on whole blood, both in its limit of detection and prevalence estimates in two field surveys. The reduced cost and complexity of this method will allow for the scale-up of surveillance studies to target MDA and other malaria elimination interventions, and help lead to a better understanding of the epidemiology of low-density malaria infections.

Microscopic and molecular evidence of the presence of asymptomatic Plasmodium falciparum and Plasmodium vivax infections in an area with low, seasonal and unstable malaria transmission in Ethiopia.

BackgroundThe presence of asymptomatic infections has serious implications for malaria elimination campaigns. Since asymptomatic carriers do not seek treatment for their infection and may become gametocyte carriers, they undoubtedly contribute to the persistence of malaria transmission in a population. The presence of asymptomatic parasitemias was noted in areas with seasonal malaria transmission. In Ethiopia there is a paucity of data regarding the prevalence of asymptomatic malaria carriage. This study was undertaken to assess the presence and prevalence of asymptomatic Plasmodium falciparum and Plasmodium vivax infections in south-central Oromia, Ethiopia.MethodsA total of 1094 apparently healthy individuals ≥ 2 years of age in south-central Oromia, Ethiopia, an area with seasonal and unstable malaria transmission, were screened for the presence of asymptomatic plasmodial infections. Finger-prick blood samples were taken from each participant for blood film preparation for microscopy and the rapid diagnostic test (RDT). Blood samples were also spotted on Whatman 3MM filter paper for parasite DNA extraction.ResultsThe prevalence of asymptomatic Plasmodium carriage (P. falciparum, P. vivax and mixed species) was 5.0 % (55/1,094) as determined by microscopy, while the prevalence as determined using RDT was 8.2 % (90/1,094). PCR was done on 47 of 55 microscopy-confirmed and on 79 of 90 RDT-confirmed samples. PCR detected parasite DNA in 89.4 % (42/47) of the microscopy-positive samples and in 77.2 % (61/79) of the RDT-positive samples. No significant difference was observed in the prevalence of asymptomatic P. falciparum or P. vivax infections in the study area (P > 0.1). However, the prevalence of asymptomatic parasitaemia was significantly associated with gender (OR = 0.47, P = 0.015; being higher in males than females) and age (X2 = 25, P < 0.001; being higher in younger than in older individuals). Age and parasite densities had an inverse relationship.ConclusionsThis study confirms the presence of asymptomatic P. falciparum and P. vivax infections in south-central Oromia, an area with low, seasonal and unstable malaria transmission in Ethiopia. Of 55 microscopically confirmed asymptomatic infections, P. falciparum monoinfection accounted for 45.5 % and of 90 RDT positive asymptomatic infections, 66.7 % were P. falciparum. Although not statistically significant, P. falciparum accounted for a relatively large number of the asymptomatic infections as determined by both tests. The prevalence of asymptomatic parasitaemia was highest in the younger age group. HRP-2-based RDTs specific for P. falciparum showed high false positivity rate compared to Plasmodium lactate dehydrogenase (pLDH) specific to P. vivax. Although microscopy and RDT detected substantial numbers of asymptomatic infections in apparently healthy inhabitants, the use of a highly sensitive molecular diagnostics offers a more accurate assessment of the magnitude of asymptomatic infections.

Dried fluid spots for peste des petits ruminants virus load evaluation allowing for non-invasive diagnosis and genotyping.

BackgroundActive surveillance of peste des petits ruminants (PPR) should ease prevention and control of this disease widely present across Africa, Middle East, central and southern Asia. PPR is now present in Turkey at the gateway to the European Union. In Bangladesh, the diagnosis and genotyping of PPR virus (PPRV) may be hampered by inadequate infrastructures and by lack of proper clinical material, which is often not preserved under cold chain up to laboratories. It has been shown previously that Whatman® 3MM filter paper (GE Healthcare, France) preserves the nucleic acid of PPRV for at least 3 months at 32°C.ResultsIn this study, we demonstrate the performances of filter papers for archiving RNA from local PPRV field isolates for further molecular detection and genotyping of PPRV, at −70°C combined with ambient temperature, for periods up to 16 months. PPR-suspected live animals were sampled and their blood and nasal swabs were applied on filter papers then air dried. Immediately after field sampling, RT-PCR amplifying a 448-bp fragment of the F gene appeared positive for both blood and nasal swabs when animals were in febrile stage and only nasal swabs were detected positive in non-febrile stage. Those tested positive were monitored by RT-PCR up to 10 months by storage at −70°C. At 16 months, using real time RT-PCR adapted to amplify the N gene from filter paper, high viral loads could still be detected (~2 x 107 copy numbers), essentially from nasal samples. The material was successfully sequenced and a Bayesian phylogenetic reconstruction achieved adequate resolution to establish temporal relationships within or between the geographical clusters of the PPRV strains.ConclusionsThis clearly reveals the excellent capacity of filter papers to store genetic material that can be sampled using a non-invasive approach.

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Aim Anorexia nervosa (AN) is a complex disorder with many genetic factors implicated in disease onset and pathology. In some cases the presence of a particular allele appears to modulate the immune response to certain exogenous antigens and protects the host from illness, whereas in other cases the host may mount an immune response against self-antigens. In the present study, we examined the possible association of DQB1∗ polymorphism with patients with Anorexia Nervosa, in Castilla y Leon (Spain) population. Methods We took samples of whole blood, 50-200mcl 28 AN,18 restrictive AN,: 10 compulsive AN, and 126 control subjects. Blood spots on 3MM filter paper were dried and stored at room temperature. DNA was extracted by two different procedures: salt precipitation and resin balls with metallic core (kit DNA Direct II from Dynal). We choosed the last one for its facility and rapidity, obtaining 1-4mcg of DNA. Classic SSP DQ “Low resolution” kit of Dynal was employed in this study. We have changed some general conditions of the DQ typing kit from the Dynal SSP method. We have modified the amount of DNA per tube to 2-4 ng and the concentration of the Taq Polymerase to 0,1 U/tube, instead of the 100 ng of DNA and 0,4 U/tube of Taq Polymerase recommended by the kit instructions. The conditions of the PCR had been: 1) 94 °C, 2 min, 1 cycle. 2) 94 °C 10 s, 65 °C 60 s 10 cycles. 3) 94 °C 10 s, 61 °C 50 s, 72 °C 30 s 30 cycles. 4) 72 °C 3 min. Results The allelic distribution in our study are for HLA DQB1∗02, ∗03, ∗04, ∗05, and ∗06 in control group (n = 126), 29,7; 27,3; 1,5; 19,4; 21,8. in AN group (n = 12) 34,3; 24,2; 0,7; 20,3; 20,6. Conclusions The Chi-square test show differences P

Filter paper collection of Plasmodium falciparum mRNA for detecting low-density gametocytes

BackgroundAccurate sampling of sub-microscopic gametocytes is necessary for epidemiological studies to identify the infectious reservoir of Plasmodium falciparum. Detection of gametocyte mRNA achieves sensitive detection, but requires careful handling of samples. Filter papers can be used for collecting RNA samples, but rigorous testing of their capacity to withstand adverse storage conditions has not been fully explored.MethodsThree gametocyte dilutions: 10/μL, 1.0/μL and 0.1/μL were spotted onto Whatman™ 903 Protein Saver Cards, FTA Classic Cards and 3MM filter papers that were stored under frozen, cold chain or tropical conditions for up to 13 weeks . RNA was extracted, then detected by quantitative nucleic acid sequence-based amplification (QT-NASBA) and reverse-transcriptase PCR (RT-PCR).ResultsSuccessful gametocyte detection was more frequently observed from the Whatman 903 Protein Saver Card compared to the Whatman FTA Classic Card, by both techniques (p < 0.0001). When papers were stored at higher temperatures, a loss in sensitivity was experienced for the FTA Classic Card but not the 903 Protein Saver Card or Whatman 3MM filter paper. The sensitivity of gametocyte detection was decreased when papers were stored at high humidity.ConclusionsThis study indicates the Whatman 903 Protein Saver Card is better for Pfs25 mRNA sampling compared to the Whatman FTA Classic Card, and that the Whatman 3MM filter paper may prove to be a satisfactory cheaper option for Pfs25 mRNA sampling. When appropriately dried, filter papers provide a useful approach to Pfs25 mRNA sampling, especially in settings where storage in RNA-protecting buffer is not possible.

Remembering Nutrient Quality of Sugar in Drosophila

Taste is an early stage in food and drink selection for most animals [1, 2]. Detecting sweetness indicates the presence of sugar and possible caloric content. However, sweet taste can be an unreliable predictor of nutrient value because some sugars cannot be metabolized. In addition, discrete sugars are detected by the same sensory neurons in the mammalian [3] and insect [4, 5] gustatory systems, making it difficult for animals to readily distinguish the identity of different sugars using taste alone [6-8]. Here we used an appetitive memory assay in Drosophila [9-11] to investigate the contribution of palatability and relative nutritional value of sugars to memory formation. We show that palatability and nutrient value both contribute to reinforcement of appetitive memory. Nonnutritious sugars formed less robust memory that could be augmented by supplementing with a tasteless but nutritious substance. Nutrient information is conveyed to the brain within minutes of training, when it can be used to guide expression of a sugar-preference memory. Therefore, flies can rapidly learn to discriminate between sugars using a postingestive reward evaluation system, and they preferentially remember nutritious sugars.

Controlled release of d-glucose from starch granules containing 29% free d-glucose and Eudragit L100-55 as a binding and coating agent

Waxy maize starch (100% amylopectin) granules were modified by reaction of the granules with glucoamylase in a minimum amount of water to give 29% (w/w) d-glucose inside the granules [Kim, Y.-K.; Robyt, J. F. Carbohydr. Res. 1999, 318, 129−134]. These granules were made into beads by dropping an ethanol slurry of starch and different amounts of Eudragit L100-55 in a constant ratio of 100:1 from a pipette onto Whatman 3MM filter paper. The starch beads were air dried and then repeatedly sprayed 0–12 times with 2.0% (w/v) Eudragit L100-55 in ethanol, with drying between each spraying, to coat the surface of the starch beads, giving different amounts of Eudragit L100-55 coating. Seven different kinds of beads, with different amounts of Eudragit L100-55 binding and coating agent, were obtained. The rates of release of d-glucose into water from the seven kinds of beads were inversely proportional to the amount of binding and coating agent. Bead type I, which was without any binding and coating gave a fast 100% release of d-glucose in 30 min. Beads II and III also gave a fast 100% release in 60 min and 90 min, respectively. Bead IV gave a near linear release of 97% d-glucose in 150 min; Bead V gave a 50% release in 120 min followed by the remaining 50% in 60 min; and Beads VI and VII gave a slow release of 10% and 4%, respectively, from 0 to 120 min, followed by a rapid 100% release from 120 to 180 min.

Improvement of DNA Extraction Method for Dried Blood Spots and Comparison of Four PCR Methods for Detection of Babesia gibsoni (Asian Genotype) Infection in Canine Blood Samples

To eradicate canine babesiosis in epidemic areas, mass-screening of the infection situation of Babesia gibsoni including occult infection is necessary. The development of cost-effective method for storage and transport of blood samples is required. A highly efficient DNA extraction procedure from dried blood spots (DBS) onto Whatman 3MM filter paper was developed for the diagnosis of B. gibsoni infection in dog by PCR. In 3 extraction methods, Chelex-based method in combination with saponin washing and phenol-chloroform-isoamyl alcohol extraction (Saponin-PCI method) provided the best results. Sensitivity of the 4 previously described PCR methods for detection of B. gibsoni infection was also compared using serially diluted blood samples of B. gibsoni-infected dogs. The PCR method using Gib599F/Gib1270R primer pair provided the best performance. To evaluate the stability of DNA in DBS, DBS of B. gibsoni-infected dogs stored at room temperature for 2 months. The stability was superior to whole blood samples stored at -20 degrees C for 2 months. This highly efficient DNA extraction method on DBS using Whatman 3MM filter paper has potential to be cost-effective and high performance tool for storage, and molecular diagnosis of clinical blood sample from dog. This procedure in combination with the PCR method using Gib599F/Gib1270R primer pair may greatly assist in diagnosis of B. gibsoni infection in dog populations that are geographically distant.

The Drosophila Inhibitor of Apoptosis (IAP) DIAP2 Is Dispensable for Cell Survival, Required for the Innate Immune Response to Gram-negative Bacterial Infection, and Can Be Negatively Regulated by the Reaper/Hid/Grim Family of IAP-binding Apoptosis Inducers

Many inhibitor of apoptosis (IAP) family proteins inhibit apoptosis. IAPs contain N-terminal baculovirus IAP repeat domains and a C-terminal RING ubiquitin ligase domain. Drosophila IAP DIAP1 is essential for the survival of many cells, protecting them from apoptosis by inhibiting active caspases. Apoptosis initiates when proteins such as Reaper, Hid, and Grim bind a surface groove in DIAP1 baculovirus IAP repeat domains via an N-terminal IAP-binding motif. This evolutionarily conserved interaction disrupts DIAP1-caspase interactions, unleashing apoptosis-inducing caspase activity. A second Drosophila IAP, DIAP2, also binds Rpr and Hid and inhibits apoptosis in multiple contexts when overexpressed. However, due to a lack of mutants, little is known about the normal functions of DIAP2. We report the generation of diap2 null mutants. These flies are viable and show no defects in developmental or stress-induced apoptosis. Instead, DIAP2 is required for the innate immune response to Gram-negative bacterial infection. DIAP2 promotes cytoplasmic cleavage and nuclear translocation of the NF-kappaB homolog Relish, and this requires the DIAP2 RING domain. Increasing the genetic dose of diap2 results in an increased immune response, whereas expression of Rpr or Hid results in down-regulation of DIAP2 protein levels. Together these observations suggest that DIAP2 can regulate immune signaling in a dose-dependent manner, and this can be regulated by IBM-containing proteins. Therefore, diap2 may identify a point of convergence between apoptosis and immune signaling pathways.

A simple 'paper smear' method for dry collection, transport and storage of cervical cytological specimens for rapid screening of HPV infection by PCR.

Human papillomaviruses (HPVs) are major pathogens associated with the development of cancer of the uterine cervix, the most common malignant tumour of women worldwide. Reliable diagnosis of HPV infection, particularly the 'high-risk' types (16/18), may facilitate early identification of 'high-risk' populations for developing cervical cancer and may augment the sensitivity and specificity of primary cervical cancer screening programmes by complementing the conventional Pap test. A simple paper smear method has been developed for dry collection, transport and storage of cervical smears/scrapes at room temperature for subsequent detection of HPV DNA by PCR assay. Imprint biopsies, blood and fine-needle aspirates were also collected by this method. The cervical scrapes or other body fluids were smeared (within 0.5-1 cm diameter) and dried on to sterile small slides made of Whatman 3MM filter paper, and stored individually at room temperature or at 4 degrees C. A small piece (2-3 mm) of the paper smear was punched or cut out with a sterile surgical blade, boiled in an eppendorf tube containing 50 microl of distilled water for 5 min and used directly for PCR amplification. The quality and quantity of DNA derived from paper smears and the results of PCR amplifications for HPV type 16, BRCA1 and p53 genes were identical to those obtained from the same samples following collection in PBS, storage (-70 degrees C) and phenol-chloroform-based DNA extraction. DNA was stable in the paper smears for up to a year, whether stored at room temperature or at 4 degrees C. This method is simple, rapid and cost-effective, and can be effectively employed for large-scale population screening, especially for regions where the specimens are to be transported from distant places to the laboratory.

The cloning of sialidase genes from Clostridium indolis and Clostridium cocleatum.

Gut microbial microflora is composed of u10 bacteria per 24 hours. The DNA was partially digested with HindIII; 2to 10kb fragments were isolated from agarose gel. Two-tenths of a mg gram, representing u400 species. It comprises both endogenous (resident) and exogenous (transiting) microbiota, which in healthy of insert DNA was ligated with 1 mg of HindIII-digested and alcaline phosphatase–treated l Express vector (Stratagene, La humans is kept under tight equilibrium. Our laboratory has developed a simplified model in trixenic mouse representing minimal Jolla, CA). Following in vitro packaging (Gigapack, Stratagene), the titer resident flora capable of inhibiting implantation of Clostridium difficile, a pathogenic bacterium responsible for a disease spectrum of the genomic library thus obtained was determined by infecting Escherichia coli XL1-Blue MRF cells (Stratagene) with aliquots ranging from postantibiotic diarrhea to pseudomembranous colitis. This flora comprises three strains: Clostridium indolis, Clostridium of packaged phage; it was found to be 3 1 10 pfu/mL. Plating onto indicator plates containing isopropyl-b-D-thiogalactopyracocleatum, and Eubacterium species, which act in concert to impede colonization by C. difficile. However, the mechanism by noside (IPTG; Promega, Madison, WI) and 5-bromo-4-chloro-3indolyl-b-D-galactopyranoside (X-Gal; Promega) revealed that which this is accomplished remains unknown. To explain the barrier effect, we have focused our attention on 87% of lambda arms carried an insert. The library was amplified by reinfecting E. coli XL1-Blue MRF; its titer was 4.5 1 10 the interactions taking place at the level of the cecal mucus layer pfu/mL. between the three players implicated in the process: the host, the To identify potential sialidase-positive clones, the phage library barrier microbiota, and C. difficile. By scanning electron microswas converted into a plasmid one by in vivo excision of pBKcopy [1], we have observed that the three barrier strains are embedCMV phagemid (Stratagene) carried by l Express, as described ded in the mucus (figure 1); furthermore, they are able to degrade in Stratagene protocols. About 4,000 white colonies were picked mucins [2]. The latter is the result of bacterial osidase activity, from X-Gal/IPTG-containing plates and tested for sialidase activity which first removes blood group residues (fucose, a-galactose, by placement of a 3MM filter paper (Prolabo, Fontenay-sous-Bois, N-acetylgalactosamine) from the mucus surface, followed by the France) soaked with the synthetic sialidase substrate 4-methylumremoval of sialic acids, b-galactose, and N-acetylglucosamine. belliferyl-a-D-N-acetylneuraminic acid (Sigma, St. Louis) in conOur present studies have focused on the sialidase activities of tact with the colonies for 15 minutes at 377C. One clone expressing the barrier strains for two reasons. First, albeit weak, this is the sialidase activity was identified by its blue-white fluorescence unonly mucinolytic activity expressed by C. indolis. In contrast, der ultraviolet light (360 nm). This clone carries an insert of C. cocleatum displays much higher and varied mucin-degrading 2.7 kb. activity. Second, sialic acid residues usually occupy terminal posiThe restriction map is shown in figure 2. E. coli carrying the tions on sialoglycoconjugates such as mucins and thus represent recombinant plasmid expresses a sialidase of identical size (C70 the outermost surface available for interactions between host and kD), detectable in immunoblots by antiserum raised against the microbiota. large isoform of Clostridium perfringens. Two subclones of the The sialidase activity expressed by the barrier strains could play plasmid have been created by digesting the insert with Ssp I (figure a role in the barrier mechanism inasmuch as the removal of sialic 2); nucleotide sequencing is in progress. acids could remove an attachment site for C. difficile. We intend To clone the sialidase gene of C. cocleatum, a genomic library to study the potential role of the sialidase in the barrier function was constructed in l Zap II (Stratagene). The library was conby molecular approach, i.e., by constructing mutant strains devoid of sialidase activity that can be tested in the trixenic mouse model available in our laboratory, in order to elucidate the effect of this mutation on the barrier function. Isolation of the sialidase genes is the first step in this process. To clone the gene encoding the sialidase of C. indolis, a genomic library was constructed. High-molecular-weight DNA was isolated as described [3] from a strain grown in anaerobic conditions in TGY (Tryptone Glucose Yeast broth, Difco, Detroit) at 377C for

Isozyme Analysis ofXanthomonas campestrispv.citri

Isozyme Analysis of<i>Xanthomonas campestris</i>pv.<i>citri</i>

Synthesis of peptidoglycan by high molecular weight penicillin-binding proteins of Bacillus subtilis and Bacillus stearothermophilus.

The high molecular weight penicillin-binding proteins (PBP(s) ) Bacillus subtilis PBPs 1, 2, and 4 and Bacillus stearothermophilus PBPs 1-4 were shown to catalyze peptidoglycan synthesis from the undecaprenol-containing lipid intermediate substrate in two assay systems. In a filter paper assay system, high levels of substrate polymerization occurred when reaction mixtures were incubated on Whatman 3MM filter paper. The pH optimum for peptidoglycan synthesis was 7.5 for B. subtilis PBPs 1, 2, and 4 and 8.5 for B. stearothermophilus PBPs 1-4. Polymerization was Mg2+-independent and was unaffected by sulfhydryl reagents. Reconstitution with membrane lipids or addition of detergent (optimal concentration, 0.1%) was necessary for synthesis to occur. Bacitracin, penicillin, and cephalothin did not affect polymerization while vancomycin, ristocetin, moenomycin, and macarbomycin were strong inhibitors. In a test tube assay system, optimal synthesis occurred either in the presence of 10% ethylene glycol, 10% glycerol, and 8% methanol or in the presence of 10% N-acetylglucosamine. The products of lysozyme digestion of the synthesized peptidoglycan were analyzed by gel filtration and paper chromatography. B. stearothermophilus PBPs 1-4 synthesized a peptidoglycan product that was 5-7% cross-linked. No evidence for cross-linking was apparent in the peptidoglycan product of B. subtilis PBPs 1, 2, and 4.

Fecal contamination in urine amino acid screening: artifactual cause of hyperaminoaciduria.

The analysis of urine in order to identify errors of amino acid metabolism is now a widely employed procedure. Currently the most popular methods utilize urine samples and the technic of paper chromatography, with various modifications of the unidimensional and bidimensional systems originally described by Consden and associates. A handicap in these methods, however, is the frequent difficulty in obtaining urine specimens from neonates or young infants. This problem is especially acute when large numbers of these infants are tested in a screening program. To obviate this, the urine screening program for metabolic disorders currently being conducted in Massachusetts, by the Massachusetts Department of Public Health in a cooperative project with the Massachusetts General Hospital, utilizes a method of urine collection that involves placing a 5by 2.5-cm. strip of Whatman No. 3MM filter paper* inside the diaper. The urine-impregnated strip is then analyzed by unidimensional paper chromatography according to the technic of Efron and colleagues. Since the inception of the urine screening program, an occasional specimen has revealed an increased quantity of many of the amino acids normally present in urine, as well as an increased quantity of the imino acid, proline, but not of hydroxyproline. Repeat specimens from infants with this amino and imino acid pattern have invariably yielded normal chromatograms, and inspection of the original filter papers has

Effect of X-Rays on Trace-Labeled I131-Insulin and Its Relevance to Biologic Studies with I131-Labeled Proteins

In vivo metabolic studies in man and animals have revealed that the major fraction of insulin-I131 is degraded with a half-time of the order of forty to sixty minutes or less following its intravenous administration (1). However, a smaller fraction, the magnitude of which varies with different lots of radioiodinated insulin, is removed from the blood stream at a much slower rate, with a half-time of six to twenty-four hours. It has also been shown by paper electrophoresis and paper chromatography that the slowly degraded moiety is bound to the serum proteins in contrast to the rapidly degraded fraction which is free of such association. Appraisal of the factors influencing the relative amounts of rapidly and slowly degraded fractions led to the conclusion that the serum protein-bound components represent alterations of the native insulin and to the suspicion that these alterations are manifestations of radiation damage to the hormone. The present study shows that similar changes can be produced by external irradiation with x-rays, peak energy 1 Mev, at dosage levels comparable to those received by the I131 trace-labeled proteins of high specific activity which are currently being employed in many biologic investigations. Trace amounts of unaltered insulin-I131 were adsorbed to Whatman No. 3MM filter paper at the site of application. The extent of alteration was then determined from the fraction of radioactivity which migrated during electrophoretic or chromatographic separation. The results show that, in addition to the production of I131-containing products which bind to serum proteins, I131 is also released as iodide ion following irradiation of insulin-I131. The radiation dose required for the alteration of a fixed fraction of insulin-I131 is an inverse function not only of the concentration of stable insulin but also of the concentration of other substances such as purified human serum albumin or cysteine. At insulin concentrations of 30 mg. per liter, irradiation at 600 r/min. for fifteen minutes produces approximately 18 per cent alteration and at 1,800 r/min. for thirty minutes produces 75 per cent alteration. The pH of the solution is another important variable, since irradiation-induced changes are barely detectable at pH7 with dosage levels sufficient to alter almost completely the same lots of insulin-I131 by irradiation at pH3. Hydrogen peroxide, in concentrations far exceeding those produced by irradiation, had no significant effect on the insulin-I131.