Rapid and non-destructive identification of Anopheles gambiae and Anopheles arabiensis mosquito species using Raman spectroscopy via machine learning classification models.

BACKGROUND: Identification of malaria vectors is an important exercise that can result in the deployment of targeted control measures and monitoring the susceptibility of the vectors to control strategies. Although known to possess distinct biting behaviours and habitats, the African malaria vectors Anopheles gambiae and Anopheles arabiensis are morphologically indistinguishable and are known to be discriminated by molecular techniques. In this paper, Raman spectroscopy is proposed to complement the tedious and time-consuming Polymerase Chain Reaction (PCR) method for the rapid screening of mosquito identity. METHODS: A dispersive Raman microscope was used to record spectra from the legs (femurs and tibiae) of fresh anaesthetized laboratory-bred mosquitoes. The scattered Raman intensity signal peaks observed were predominantly centered at approximately 1400 cm-1, 1590 cm-1, and 2067 cm-1. These peaks, which are characteristic signatures of melanin pigment found in the insect cuticle, were important in the discrimination of the two mosquito species. Principal Component Analysis (PCA) was used for dimension reduction. Four classification models were built using the following techniques: Linear Discriminant Analysis (LDA), Logistic Regression (LR), Quadratic Discriminant Analysis (QDA), and Quadratic Support Vector Machine (QSVM). RESULTS: PCA extracted twenty-one features accounting for 95% of the variation in the data. Using the twenty-one principal components, LDA, LR, QDA, and QSVM discriminated and classified the two cryptic species with 86%, 85%, 89%, and 93% accuracy, respectively on cross-validation and 79%, 82%, 81% and 93% respectively on the test data set. CONCLUSION: Raman spectroscopy in combination with machine learning tools is an effective, rapid and non-destructive method for discriminating and classifying two cryptic mosquito species, Anopheles gambiae and Anopheles arabiensis belonging to the Anopheles gambiae complex.

Application of principal component analysis to multispectral-multimodal optical image analysis for malaria diagnostics.

BACKGROUND: Multispectral imaging microscopy is a novel microscopic technique that integrates spectroscopy with optical imaging to record both spectral and spatial information of a specimen. This enables acquisition of a large and more informative dataset than is achievable in conventional optical microscopy. However, such data are characterized by high signal correlation and are difficult to interpret using univariate data analysis techniques. METHODS: In this work, the development and application of a novel method which uses principal component analysis (PCA) in the processing of spectral images obtained from a simple multispectral-multimodal imaging microscope to detect Plasmodium parasites in unstained thin blood smear for malaria diagnostics is reported. The optical microscope used in this work has been modified by replacing the broadband light source (tungsten halogen lamp) with a set of light emitting diodes (LEDs) emitting thirteen different wavelengths of monochromatic light in the UV-vis-NIR range. The LEDs are activated sequentially to illuminate same spot of the unstained thin blood smears on glass slides, and grey level images are recorded at each wavelength. PCA was used to perform data dimensionality reduction and to enhance score images for visualization as well as for feature extraction through clusters in score space. RESULTS: Using this approach, haemozoin was uniquely distinguished from haemoglobin in unstained thin blood smears on glass slides and the 590-700 spectral range identified as an important band for optical imaging of haemozoin as a biomarker for malaria diagnosis. CONCLUSION: This work is of great significance in reducing the time spent on staining malaria specimens and thus drastically reducing diagnosis time duration. The approach has the potential of replacing a trained human eye with a trained computerized vision system for malaria parasite blood screening.

Model studies of polychlorinated dibenzo-p-dioxin formation during municipal refuse incineration.

Toxic chlorinated dibenzo-p-dioxins are known to be formed in incinerators that burn municipal refuse. These compounds were synthesized by surface-catalyzed reactions on fly ash particulates taken from incinerators. Dioxins were produced catalytically from chlorinated phenol precursors, from non-chlorinated compounds that were chemically dissimilar to dioxins, and from reaction of phenol with inorganic chlorides. The relative amounts of dioxins formed from [13C6]pentachlorophenol with different fly ashes that had been cleaned of all organic compounds corresponded well with those amounts originally found on the samples as received from the incinerators. The optimum temperature range for the formation of dioxins from pentachlorophenol was 250 degrees to 350 degrees C.

Thermoregulation in the desert iguana Dipsosaurus dorsalis.

The body temperature of desert iguanas implanted with miniature temperature-sensitive radio transmitters was continuously monitored in their natural habitat. Extensive thermoregulatory behavior occurred in retreat burrows prior to morning emergence. Such behavior permits the igluana to emerge from below ground at its preferred body temperature rather than suboptimal temperature at which activity in the burrow is initiated.

Fasting plasma glucose and risk factor assessment: Comparing sensitivity and specificity in identifying gestational diabetes in urban black African women.

BACKGROUND: Identifying women with gestational diabetes mellitus (GDM) allows interventions to improve perinatal outcomes. A fasting plasma glucose (FPG) level ≥5.1 mmol/L is 100% specific for a diagnosis of GDM. The International Association of Diabetes and Pregnancy Study Groups acknowledges that FPG <4.5 mmol/L is associated with a low probability of GDM. OBJECTIVES: The validity of selective screening based on the presence of risk factors was compared with the universal application of FPG ≥4.5 mmol/L to identify women with GDM. FPG ≥4.5 mmol/L or the presence of one or more risk factors was assumed to indicate an intermediate to high risk of GDM and therefore the need for an oral glucose tolerance test (OGTT). METHODS: Consecutive black South African (SA) women were recruited to a 2-hour 75 g OGTT at 24 - 28 weeks' gestation in an urban community health clinic. Of 969 women recruited, 666 underwent an OGTT, and of these 589 were eligible for analysis. The glucose oxidase laboratory method was used to measure plasma glucose concentrations. The World Health Organization GDM diagnostic criteria were applied. All participants underwent a risk factor assessment. The χ2 test was used to determine associations between risk factors and a positive diagnosis of GDM. The sensitivity and specificity of a positive diagnosis of GDM were calculated for FPG ≥4.5 mmol/L, FPG ≥5.1 mmol/L, and the presence of one or more risk factors. RESULTS: The prevalence of overt diabetes mellitus and GDM was 0.5% and 7.0%, respectively. Risk factor-based selective screening indicated that 204/589 (34.6%) of participants needed an OGTT, but 18/41 (43.9%) of positive GDM diagnoses were missed. Universal screening using the FPG threshold of ≥4.5 mmol/L indicated that 152/589 (25.8%) of participants needed an OGTT, and 1/41 (2.4%) of positive diagnoses were missed. An FPG of ≥5.1 mmol/L identified 36/41 (87.8%) of GDM-positive participants. The sensitivity and specificity of the presence of one or more risk factors were 56% and 67%, respectively. The sensitivity and specificity of FPG ≥4.5 mmol/L were 98% and 80%, respectively. CONCLUSIONS: Universal screening using FPG ≥4.5 mmol/L had greater sensitivity and specificity in identifying GDM-affected women and required fewer women to undergo a resource-intensive diagnostic OGTT than risk factor-based selective screening. A universal screening strategy using FPG ≥4.5 mmol/L may be more efficient and cost-effective than risk factor-based selective screening for GDM in black SA women.

The occurrence of cardiac abnormalities in canine steroid-responsive meningitis arteritis.

OBJECTIVES: To document the prevalence of cardiac abnormalities in dogs with steroid-responsive meningitis arteritis and to assess resolution of these abnormalities following corticosteroid therapy. MATERIALS AND METHODS: Steroid-responsive meningitis arteritis was diagnosed based on signalment, physical examination findings, complete blood count, biochemistry and CSF analysis. Echocardiography, C-reactive protein and cardiac troponin I were measured in all cases before and 10 to 14 days after commencing corticosteroid therapy. Fibrinogen was also measured in a proportion of dogs. RESULTS: Fourteen dogs were prospectively enrolled. Increased cardiac troponin I was identified in five of 14 dogs and echocardiographic abnormalities were detected in 12 of 14 dogs, including spontaneous echo contrast (12 of 14), mild pericardial effusion (five of 14) and mildly decreased fractional shortening (five of 14). All dogs had increased C-reactive protein and fibrinogen was increased in 11 of 12. Corticosteroid treatment was associated with clinical improvement and normalisation of C-reactive protein in all dogs. The cardiac troponin I levels normalised in four of five and fibrinogen had normalised in all five dogs which were retested. Spontaneous echo contrast improved or completely resolved in 12 of 12 and pericardial effusion resolved in five of five dogs. Fractional shortening normalised in two of five dogs. CLINICAL SIGNIFICANCE: Cardiac changes are common in dogs with steroid-responsive meningitis arteritis and most resolve with therapy. Further investigation into the cause and significance of these changes is necessary in determining whether antithrombotic therapy or positive inotropic therapy is indicated.

Multiple homing pathways used by yeast mitochondrial group II introns.

The yeast mitochondrial DNA group II introns aI1 and aI2 are retroelements that insert site specifically into intronless alleles by a process called homing. Here, we used patterns of flanking marker coconversion in crosses with wild-type and mutant aI2 introns to distinguish three coexisting homing pathways: two that were reverse transcriptase (RT) dependent (retrohoming) and one that was RT independent. All three pathways are initiated by cleavage of the recipient DNA target site by the intron-encoded endonuclease, with the sense strand cleaved by partial or complete reverse splicing, and the antisense strand cleaved by the intron-encoded protein. The major retrohoming pathway in standard crosses leads to insertion of the intron with unidirectional coconversion of upstream exon sequences. This pattern of coconversion suggests that the major retrohoming pathway is initiated by target DNA-primed reverse transcription of the reverse-spliced intron RNA and completed by double-strand break repair (DSBR) recombination with the donor allele. The RT-independent pathway leads to insertion of the intron with bidirectional coconversion and presumably occurs by a conventional DSBR recombination mechanism initiated by cleavage of the recipient DNA target site by the intron-encoded endonuclease, as for group I intron homing. Finally, some mutant DNA target sites shift up to 43% of retrohoming to another pathway not previously detected for aI2 in which there is no coconversion of flanking exon sequences. This new pathway presumably involves synthesis of a full-length cDNA copy of the inserted intron RNA, with completion by a repair process independent of homologous recombination, as found for the Lactococcus lactis Ll.LtrB intron. Our results show that group II intron mobility can occur by multiple pathways, the ratios of which depend on the characteristics of both the intron and the DNA target site. This remarkable flexibility enables group II introns to use different recombination and repair enzymes in different host cells.

Rat cholesterol side-chain cleavage enzyme (P-450scc): use of a cDNA probe to study the hormonal regulation of P-450scc mRNA levels in ovarian granulosa cells.

A rat ovarian cDNA library was constructed and screened by differential colony hybridization to detect cDNA clones specific for mRNA induced by follicle-stimulating hormone (FSH). The cDNA clone which demonstrated the greatest degree of induction contained a 766-bp insert which was characterized and sequenced. We conclude that this cDNA is specific for the rat gene coding for cholesterol side-chain cleavage enzyme (P-450scc) by virtue of nucleotide sequence homology to the bovine and human P-450scc cDNA sequences. Southern blotting of rat genomic DNA suggests the presence of a single P-450scc gene. Northern blot analysis indicates that P-450scc mRNA is present in steroidogenic tissues (ovary, adrenal, testis), but not in brain, kidney, liver, lung, or heart. The rat P-450scc mRNA is induced by FSH or pregnant mare's serum gonadotropin in ovaries of estrogen-treated immature rats in vivo. In cultured granulosa cells, estradiol treatment alone did not increase P-450scc mRNA levels, but in combination with FSH or 8-Br-cAMP resulted in three- to four-fold increase in this mRNA.

Treating psychiatric problems in medical students.

The medical school educational experience is very stressful for many students, prompting some to seek formal psychiatric care. The authors describe the Medical Student Support Services program of the University of Kentucky College of Medicine. From July 1983 to June 1985, this program served 66 patients, representing 417 visits. On the basis of retrospective chart review with the examining clinicians, the authors present DSM-III diagnoses, types of problems seen, descriptive profiles of the patients, duration of treatment, types of therapy used, and data on marital issues. They discuss the intricacies of providing psychiatric services to medical students and make recommendations for program development for such patients.

Fatty acid and phorbol ester-mediated interference of mitogenic signaling via novel protein kinase C isoforms in pancreatic beta-cells (INS-1).

It is possible that activation of protein kinase C (PKC) isoforms by free fatty acids (FFA) plays a role in the failure of pancreatic beta-cell mass expansion to compensate for peripheral insulin resistance in the pathogenesis of type-2 diabetes. The effect of lipid moieties on activation of conventional (PKC-alpha and -beta1), novel (PKC-delta) and atypical (PKC-zeta) PKC isoforms was evaluated in an in vitro assay, using biotinylated neurogranin as a substrate. Oleoyl-Coenzyme A (CoA) and palmitoyl-CoA, but not unesterified FFA, significantly increased the activity of all PKC isoforms (P< or =0.05), particularly that for PKC-delta. It was found that FFA (0.4 mM oleate/complexed to 0.5% bovine serum albumin) inhibited IGF-I-induced activation of protein kinase B (PKB) in the pancreatic beta-cell line (INS-1), but this was alleviated in the presence of the general PKC inhibitor (Gö6850; 1 microM). To further investigate whether conventional or novel PKC isoforms adversely affect beta-cell proliferation, the effect of phorbol ester (phorbol 12-myristate 13-acetate; PMA)-mediated activation of these PKC isoforms on glucose/IGF-I-induced INS-1 cell mitogenesis, and insulin receptor substrate (IRS)-mediated signal transduction was investigated. PMA-mediated activation of PKC (100 nM; 4 h) reduced glucose/IGF-I mediated beta-cell mitogenesis (>50%; P< or =0.05), which was reversible by the general PKC inhibitor Gö6850 (1 microM), indicating an effect of PKC and not due to a non-specific PMA toxicity. PMA inhibited IGF-I-induced activation of PKB, correlating with inhibition of IGF-I-induced association of IRS-2 with the p85 regulatory subunit of phosphatidylinositol-3 kinase. However, in contrast, PMA activated the mitogen-activated protein kinases, Erk1/2. Titration inhibition analysis using PKC isoform inhibitors indicated that these PMA-induced effects were via novel PKC isoforms. Thus, FFA/PMA-induced activation of novel PKC isoforms can inhibit glucose/IGF-I-mediated beta-cell mitogenesis, in part by decreasing PKB activation, despite an upregulation of Erk1/2. Thus, activation of novel PKC isoforms by long-chain acyl-CoA may well contribute to decreasing beta-cell mass in the pathogenesis of type-2 diabetes, similar to their inhibition of insulin signal transduction which causes insulin resistance.

Automated entry of hospital infection surveillance data.

OBJECTIVE: To assess the accuracy of an automated data entry system employing optical scanning technology and to provide an analysis of its costs as compared to manual data entry. DESIGN: The accuracy and cost of automated data entry of 100 surgical-wound infection surveillance questionnaires was compared to manual entry. SETTING: The Surgical Directorate, The Royal Hospitals, Belfast, Northern Ireland. RESULTS: The use of optical scanning technology greatly improved the speed and accuracy of data entry. The time spent by the keyboard operator on data entry was reduced substantially. For each surgical-wound infection questionnaire automatically processed, there was a saving in clerical time equivalent to $0.63. The automated data entry process resulted in a 22-fold productivity increase compared to manual data entry with validation. After validation, an error rate of < 0.2 errors per 1,000 responses was detected in automatically entered data compared to a rate of 12.4 errors per 1,000 responses for manually entered data. The automated system, including validation, provided a seven-fold productivity increase compared to "quick-and-dirty" manual data entry without validation. CONCLUSION: Hospital information technology systems may achieve total integration of data management, but realistically this would appear to be very much in the future. Until then, in view of the accuracy and substantial savings in time and money, we recommend the use of automated data entry technology. This system would be especially useful where data are transported from outlying hospitals to a central receiving center for collation and analysis.

Isolation and characterization of the human fibrillar collagen genes.

In order to elucidate some of the mechanisms leading to the pathological expression of the human fibrillar collagens, as well as to understand the evolution of these loci, specific cDNA and genomic clones have been isolated. The primary structure of the COOH-terminal propeptide of the four collagen chains and either part or the entire exon/intron arrangement of the genes have been determined. Interspecies and pairwise comparison revealed that the four loci have evolved at slightly different rates, maintaining, however, remarkably similar exon/intron arrangement. The fibrillar genes, albeit sharing the same elaborate structure, exhibit different sizes that correlate with the average length of their intron sequences, possibly because of their different chromosomal origin.

Phorbol myristate acetate-differentiated THP-1 cells display increased levels of MHC class I and class II mRNA and interferon-gamma-inducible tumoricidal activity.

The protein kinase C activators phorbol 12-myristate 13-acetate (PMA) and mezerein induce differentiation of human monocytic leukemia (THP-1) cells along the monocyte/macrophage pathway of development. The differentiated cells express many important macrophage functions including phagocytosis and the secretion of immunomodulatory cytokines. Mezerein-differentiated THP-1 cells secrete interleukin-1 beta as well as a tumor cell growth inhibitory factor whose basal level is increased in response to interferon-gamma. However, tumoricidal, as opposed to tumoristatic, activity of differentiated THP-1 has not been documented. We report herein that PMA-differentiated THP-1 cells (PD/THP-1) contain elevated levels of MHC class I and class II mRNAs even in the absence of activating factors, and kill HT-29 human colon carcinoma cells when stimulated with recombinant human interferon-gamma. These two characteristics are important components of the macrophage phenotype. The results presented in this study extend previous observations on THP-1 cells by demonstrating that PD/THP-1 cells display a critical, immunologically relevant macrophage function, and therefore, enhance the utility of THP-1 as a model for the in vitro study of immunomodulatory drugs and macrophage-mediated cytocidal processes.

A decrease in glucose 6-phosphate dehydrogenase activity and mRNA is an early event in phorbol ester-induced differentiation of thp-1 promonocytic leukemia cells.

Redox modification of regulatory proteins implicates the glutathione redox system (GRS) in the control of gene expression. Glucose-6-phosphate dehydrogenase (G6PD) provides reducing equivalents for the GRS, and it has been suggested that high levels of G6PD in preneoplastic lesions are directly related to neoplastic transformation. We have used THP-1 human promonocytic leukemia cells, an established model of induced macrophage differentiation, to test an important corollary of this hypothesis, viz., that a decrease in G6PD activity should accompany the loss of the transformed phenotype. Phorbol 12-myristate 13-acetate (PMA) arrests the constitutive cycling of THP-1 and induces a phenotype that approaches normalcy. We measured the specific activities of the GRS enzymes, G6PD, glutathione peroxidase, and glutathione reductase during the early stages of phorbol ester-induced differentiation of THP-1 cells. We observed an 80% decrease in G6PD activity and an increase in the apparent KM for glucose 6-phosphate. In contrast, glutathione peroxidase (GPX) activity increased, while glutathione reductase (GR) activity remained essentially constant. The reduction in G6PD activity, preceding the loss of the transformed phenotype, is accompanied by a fourfold decrease in steady-state levels of G6PD mRNA. These findings are consistent with the hypothesis that high levels of G6PD are causally related to neoplastic transformations.

Gas chromatographic-mass spectrometric determination of sulfolane in wetland vegetation exposed to sour gas-contaminated groundwater.

Described is a GC-MS method for the determination of the levels of sulfolane (tetrahydrothiophene 1,1-dioxide, C4H8O2S; a water miscible chemical used in the sweetening of sour gas) in wetland vegetation (roots, shoots, berries, seeds, grasses, and leaves). The technique was developed to provide positive detection of sulfolane in a variety of wetland vegetation and to determine the extent to which sulfolane may translocate within the plants. Vegetation samples collected at a sour gas processing facility were extracted using a two-stage process which utilized a back extraction of a water extract with toluene. The main advantages of this procedure were: good extraction efficiency (recovery of 80+/-12%), exclusion of most of the highly polar co-extractives during the toluene back extraction step, and a final extract well suited to routine GC-MS selected ion monitoring of sulfolane with a detection limit of 90 ng g(-1) (wet mass). In general, the method was rugged, based on a study period of 18 months in which over 175 runs were conducted.

The use of midazolam for hypnosedative interviews.

The authors describe three cases in which midazolam was successfully used in diagnostic hypnosedative interviews. A discussion comparing and contrasting midazolam to other intravenous hypnosedatives is presented. The authors suggest that midazolam is an effective agent for diagnostic hypnosedative interviews and may be preferable to other agents when posthypnotic recollection of interview content is undesirable.

Inhibition of growth of subcutaneous xenografts and metastasis of human breast carcinoma by swainsonine: modulation of tumor cell HLA class I antigens and host immune effector mechanisms.

Swainsonine, an indolizidine alkaloid, can decrease the organ colonization potential of metastatic murine tumor cells by augmentation of host immune effector mechanisms. In this report the above findings were extended by the demonstration that systemic administration of swainsonine strongly suppressed the growth of human breast carcinoma subcutaneous xenografts and experimentally induced lung metastases. This inhibition was not due to a direct effect of swainsonine on cell growth. However swainsonine treatment of tumor cells resulted in enhanced expression of HLA Class I antigens, and HLA class I mRNA. Swainsonine was a potent immunodulator as evidenced by the increased (a) cytotoxicity of splenocytes and macrophages, and, (b) proliferative potential of splenocytes and bone marrow cells. These data suggest that swainsonine-induced inhibition of tumor growth and metastases may be mediated via activation of host effector cells and/or alteration of tumor cell antigenicity.

Intermediate steps in cellular iron uptake from transferrin. II. A cytoplasmic pool of iron is released from cultured cells via temperature-dependent mechanical wounding.

A previous study described a cytoplasmic, transferrin (Tf)-free, iron (Fe) pool that was detected only when cells were mechanically detached from the culture substratum at 4 degrees C, after initial incubation with 59Fe-125I-Tf at 37 degrees C (Richardson and Baker, 1992a). The release of this internalized 59Fe could be markedly reduced if the cells were treated with proteases or incubated at 37 degrees C prior to detachment. The present study was designed to characterize this Fe pool and understand the mechanism of its release. The results show that cellular 59Fe release increased linearly as a function of preincubation time with 59Fe-Tf subsequent to mechanical detachment at 4 degrees C using a spatula. These data suggest that the 59Fe release was largely composed of end product(s) and was not an "intermediate Fe pool." When the Fe(II) chelator, dipyridyl (DP), was incubated with 59Fe-Tf and the cells, it prevented the accumulation of 59Fe that was released following mechanical detachment at 4 degrees C. Other chelators had much less effect on the proportion of 59Fe released. Examination of the 59Fe released showed that after a 4-h preincubation with 59Fe-Tf, approximately 50% of the 59Fe was present in ferritin. These data indicate that mechanical detachment of cells at 4 degrees C resulted in membrane disruptions that allow the release of high M(r), molecules. Moreover, electron microscopy studies showed that detachment of cells from the substratum at 4 degrees C resulted in pronounced membrane damage. In contrast, when cells were detached at 37 degrees C, or at 4 degrees C after treatment with pronase, membrane damage was minimal or not apparent. These results may imply that temperature-dependent processes prevent the release of intracellular contents on membrane wounding, or alternatively, prevent wounding at 37 degrees C. The evidence also indicates that caution is required when interpreting data from experiments where cells have been mechanically detached at 4 degrees C.

Understanding iron absorption and metabolism, aided by studies of hemochromatosis.

Duodenal iron absorption from food is selectively blocked to prevent iron intoxication. The prime example of pathologic increase in intestinal iron absorption is seen in patients with hemochromatosis. They suffer iron damage to the heart, liver, and other tissues resulting in premature death if the iron is not removed by vigorous phlebotomy. Examples of overcoming the intestinal barrier to iron are alcohol consumption, vitamin preparations with vitamin C, and iron consumed by individuals without anemia. Endogenous generation of excess iron by hemolysis, owing to abnormal hemoglobin or many transfusions, are not controlled by the intestinal barrier.