The Development of Optical Nanosensor Technology for Single Cell Analysis

Advances in modern biosciences and optical biosensor technology have provided exciting new insights and capabilities. The integration of these fields has witnessed revolutionary advances, which include the development of optical nanosensors. Optical nanosensors are devices based on a direct spatial coupling between biologically active molecules and a signal transducer element interfaced to electronic equipment for signal amplification, acquisition and recording. Optical nanosensors consist of biorecognition molecules covalently immobilized onto the nanotips nanoscale optical fiber that serves as the transducing element. By combining the specificity of biorecognition molecules and the excellent sensitivity of laser-based optical detection, optical nanosensors are capable of detecting and differentiating biochemical constituents of complex systems enabling the provision of sensitive and specific identification of specific molecular events inside living cells. This work explores and focuses on the development and application of novel optical nanosensors for single living cell analysis. In this context, single cell analysis involves the application of optical nanosensor technology to observe and possibly map molecular events inside single living cells. Previous studies have focused on the bulk response of cells and this largely increases the probability of missing critical underlying mechanisms specific to the single cell. The ability to perform single cell analysis can dramatically improve our understanding of basic cellular processes e.g., signal transduction as well as improving our knowledge of the intracellular transport and the fate of therapeutic agents at the single cell level. This is important not only because of the capability to perform minimally invasive analysis, but also to overcome the problem of ensemble averaging. This capability to overcome ensemble averaging has the potential to yield new information that is not available from population averaged cellular measurements. This work involves the development and application of optical nanosensors for specific and sensitive chemical and protein analysis within single living cells. The ability of these sensors to successfully perform chemical and protein analysis at the single cell level, lay in their design specifications, size, specificity, sensitivity and eliminating interferences. With regard to their specifications, their size was in the nanometer regime, which is relative to the scale of a single mammalian cell (~ 10 µm) to allow non-invasive-to-minimally-invasive measurements in single living cells. In addition, they incorporated biological recognition molecules to achieve specificity and finally, near-field evanescent wave excitation and detection to achieve high sensitivity. High specificity and sensitivity allowed for precise and accurate identification of physicochemically detectable substances in complex matrices to eliminate any potential interference. The optical nanosensor intracellular measurement process is straightforward and begins with a sparsely distributed cell culture in a petri dish to allow viewing of single cells using an inverted fluorescence microscope. The optical nanosensor is secured onto the manipulating arms of the microscope and gently manipulated toward the single cell, interacting with the cell, penetrating but not disrupting cellular membranes. The optical nanosensor is briefly incubated in a single living cell and the laser is turned on and excitation light is launched into the optical nanosensor and propagated to the near field of the nanotip where the target analyte is excited by evanescent optical waves. The fluorescence signal generated when the target analyte is excited is collected by the optical set-up of the inverted fluorescence microscope, passes through spectral and spatial filters before detection with a sensitive photon counting photomultiplier tube (PMT). The PMT signal is amplified and recorded via a universal counter interfaced to a personal computer (PC). Data acquisition and recording are controlled using an integrated custom-written program, built on LabView platform. During in vitro and in vivo measurements, the optical nanosensor response is determined in terms of the sensitivity, specificity, linear dynamic range, response time, nanosensor stability, and reproducibility. In the course of experimental measurements, it was evident that optical nanosensors have characteristics including fast response times (msec range), sensitivity (pM range), selectivity, and excellent reproducibility. In addition to the above figures of merit, optical nanosensors demonstrated biocompatibility with no observed detrimental effects on the cell under investigation in control growth conditions. This demonstrated the utility of optical nanosensor technology for minimally invasive measurement of cellular reactions without altering or destroying the chemical make-up of the cell. This work also illustrates the potential of optical nanosensors in playing an important role in elucidating and enhancing our understanding of cell signaling and transduction pathways in real-time

Isolation and Characterization of Two siamese Phenotypic Modifiers and Their Role in Endoreplication and Trichome Development

Recessive mutations in the ENS1 gene of Arabidopsis thaliana result in reduced trichome branching without altering the ploidy level of trichomes. This implies that ENS1 regulates trichome branching in an endoreplication-independent manner. Mutations in ENS1 also enhance the multicellularity of sim mutant trichomes indicating that ENS1 plays a role in the regulation of the cell cycle during trichome cell differentiation. We have shown that ENS1 is required by the negative regulators of trichome branching for the trichomes to achieve their supernumerary branching. The interaction between ens1-1 and try-JC suggests that ENS1 is involved in both primary and secondary branching events during trichome development. The ENS1 protein interacts both genetically and physically with the STI protein and double mutant analysis between ens1-1 and sti mutants revealed that they function in the same pathway during trichome development. We propose that ENS1 and STI form a complex that functions to ensure the development of a wild-type trichome phenotype. The ens2-1 mutations result in trichomes with reduced branching and reduced endoreplication. The ens2-1 mutations also enhance the multicellularity of sim mutant trichomes. The ENS2 gene encodes an activator of the APC/C, a multisubunit protein complex that targets proteins for degradation by the 26S proteasome. Overexpression of ENS2 and its close Arabidopsis homologue AtCCS52A2 results in plants with retarded growth that have enlarged leaf epidermal pavement cells containing highly endoreplicated nuclei. These plants also have large multibranched trichomes with highly endoreplicated nuclei. Both ENS2 and AtCCS52A2 suppress the sim mutant trichome phenotype, suggesting these two genes are functionally similar and function in the same pathway in the regulation of the endoreplication cell cycle. The ens2-1 mutation induces multicellular trichomes in plants overexpressing cell cycle regulators cyclin B1;1, cyclin B1;2, cyclin D4;1 and CDKA;1, indicating cyclin D4;1 and CDKA;1 may be involved in the regulation of the G2/M as well as the G1/S phase of the cell cycle. Our research findings indicate that ENS1 and ENS2 are involved in the regulation of the cell cycle during trichome development and that the cell cycle and the cell shape mechanism interact during trichome development to ensure the development of the wild-type trichome architecture

Pengaruh Partisipasi Penyusunan Anggaran dan Peran Kuasa Pengguna Anggaran terhadap Kinerja Pemerintah Daerah Kabupaten Kepulauan Talaud

This research aims to discover the effects of Participation in Budget Preparation and the Role of Authority of the Budget User on the Regional Government Performance. The research was conducted in the Talaud Regency, one of the Autonomy Regions in North Sulawesi. The samples were taken using purposive sampling. The research sample was the Authorized Officials of Budget Users in Satuan Kerja Perangkat Daerah Pemerintah Kabupaten Kepulauan Talaud which were as many as 103 people.The Multiple Linear Regression, descriptive statistics, classic assumption test, correlation coefficient ( R) , determination coefficient ( R² ) and hypothesis testing ( t test and F test ) were used to analyze the data. This method of analysis used the Software Statistical Products and Solution Services ( SPSS) version 20.0.The results of the research shows that the Participation in Budget Preparation and the Role of Authority of the Budget User on the Regional Government Performance both partially and simultaneously. Therefore, the hypotheses proposed in the research were accepted

Attitude and practices of household heads towards leishmaniases infections in marigat sub-county, Baringo County, Kenya

Objective: To assess the attitude and practices of household heads towards  leishmaniases in Marigat division.Design: A cross-sectional study.Setting: Marigat Sub-County, Baringo County, Kenya.Subjects: Four hundred and twenty two participants were enrolled into the study.Results: The belief that Kalaazar is more serious than malaria was reported to be (82.1%) by the household respondents. Majority (92.6%) of respondents reported that they will seek medical advice if they realize they are infected. The fear of death was the  major reason for seeking treatment (68.4%). There were various  leishmaniases preventive practices that were employed by the respondents’  households to prevent them from being infected with kalaazar. The preventive practices included use of bed nets which were being used by (88%) of respondents, spraying their houses with insecticides (16%), observing personal hygiene  (46%), practising proper waste disposal (34%), installed window mesh in the households (5%), use sterilised water (19%) and applying sand fly repellents (14%). The households using bed nets (χ2 =7.397, df = 1, P= 0.007), households spraying their houses with insecticides (χ2= 7.813, df = 1, P =0.005), households observing personal hygiene (χ2 = 10.144, df = 1, P =0.001), and households that were using sterilised water (χ2 = 7.151, df = 1, P =0.007) had significant partial effects to the occurrence of kalaazar disease.Conclusion: There was a strong evidence of association between the isolation of patients and occurrence of kalaazar disease (χ= 12.908, df =1, P<0.001). The study also showed that there was strong evidence of relationship on risk of contracting  leishmaniases in young boys (χ = 19.038, df = 1, P<0.001) followed by young girls (χ = 10.623, df = 1, P = 0.001). The isolation of patients with  leishmaniases infections, fear of death and the negative impacts of the disease are the major issues associated with leishmaniases. The use of bed nets and spraying houses with insecticides are among the preferred methods to prevent the sand fly bites

Transformation and Regeneration Protocol for Two Farmer Preferred Open Pollinated Tropical Maize (Zea Mays) Varieties

Article PurchasedAbstract: In vitro regeneration of open pollinated varieties (OPVs) Kakamega Striga Tolerant Population 94 (KSTP’94) and ‘Namba Nane’ alongside a tropical inbred line (CML144) was evaluated using immature zygotic embryos as explants. Four callus induction media (CIM) regimes; Murashige and Skoog (MS), Linsmaier and Skoog (LS), Chu (N6) and N6*(N6 medium fortified with 0.35 gL-1 L-proline and 0.8 mgL-1 AgNO3) were evaluated for their potential to induce callus in the three genotypes. All the media were supplemented with sucrose and five levels of 2, 4-Dichlorophenoxyacetic acid (2, 4-D) (0.5, 1.0, 1.5, 2.0 and 2.5 mgL-1). Resulting calli were matured on MS and N6 basal media supplemented with 60 g/L sucrose and similar concentration levels (0.5, 1.0, 1.5, 2.0 and 2.5 mgL-1) of 2, 4-D while the subsequent embryogenic calli were regenerated on hormone-free media. Transformability of these varieties was assessed via histochemical analysis of β-glucuronidase (GUS) reporter gene following Agrobacterium-mediated transformation. Statistical analyses were done using Statistical Analysis Software (SAS) and Graphpad Prism softwares with mean separations achieved at 95% confidence intervals. Of the 2 OPVs, KSTP’94 recorded the highest callus induction frequency (84.4%) while Namba Nane (45.6%) had the lowest. Similarly, KSTP, 94 had the highest mean of mature somatic embryos (59.7%) while Namba Nane recorded the lowest (16.4%). Assessment of regeneration frequencies from embryogenic calli revealed no significant differences among the 3 lines although CML 144 had the highest mean number of juvenile plantlets (36.7%). Analysis of transformation frequency (upon selection of calli on media with basta) showed that Namba Nane recorded the lowest transformation frequency (average 13.5%) some words missing. Transformation frequency (based on GUS positive calli) of these varieties ranged from 0.8 to 2.1%. This work therefore provides an empirical platform for potential introduction of useful genes into these varieties

Peningkatan Kemampuan Menulis Surat Pribadi Melalui Metode Latihan Terbimbing Siswa Kelas V SD Inpres Tomoli Selatan

Masalah utama pada penelitian ini adalah rendahnya hasil belajar siswa kelas V SD Inpres Tomoli Selatan pada materi menulis surat pribadi. Ada beberapa hal yang menyebabkan permasalahan tersebut, di antaranya: 1) siswa kurang memahami konsep yang diajarkan dan 2) metode yang digunakan guru dalam melaksanakan pembelajaran membuat siswa kurang aktif. Untuk meningkatkan hasil belajar siswa tersebut, maka peneliti menerapkan metode latihan terbimbing dalam mengajarkan materi menulis surat pribadi. Metode latihan terbimbing memungkinkan adanya peningkatan aktivitas siswa dalam kegiatan pembelajaran, sehingga pengajaran tidak lagi terpusat pada guru tetapi pada siswa. Rumusan masalah dalam penelitian ini adalah apakah dengan melalui metode latihan terbimbing dapat ditingkatkan kemampuan siswa kelas V SD Inpres Tomoli Selatan dalam menulis surat pribadi? Untuk menjawab permasalahan di atas, maka peneliti melakukan penelitian tindakan kelas dengan menggunakan metode deskriptif. Rancangan penelitian ini mengacu pada model Kemmis dan Mc Taggart yang terdiri dari empat komponen, yaitu 1) perencanaan, 2) pelaksanaan tindakan, 3) observasi dan 4) refleksi. Data yang dikumpulkan pada penelitian ini adalah berupa data aktivitas guru dan siswa selama proses pembelajaran yang diperoleh dengan menggunakan lembar observasi dan data hasil belajar siswa yang diperoleh dengan memberikan tes individu kepada siswa. Pada penelitian yang telah dilaksanakan, diperoleh hasil bahwa siswa kelas V SD Inpres Tomoli Selatan mengalami peningkatan daya serap klasikal mencapai 61,5% pada siklus I menjadi 86% pada siklus II. Peningkatan daya serap klasikal pada siklus II disebabkan karena peneliti lebih menekankan pada penguasaan konsep dalam menulis surat dengan menggunakan metode latihan terbimbing. Sementara pada siklus I, peneliti belum menekankan pada penguasaan konsep. Berdasarkan hasil tersebut, dapat disimpulkan bahwa penerapan metode latihan terbimbing dapat meningkatkan hasil belajar siswa

Laboratory and semi-field evaluation of long-lasting insecticidal nets against leishmaniasis vector, Phlebotomus (Phlebotomus) duboscqi in Kenya

Background & objectives: Phlebotomine sandflies are vectors of leishmaniases and other diseases.Long-lasting insecticidal nets (LLINs) as possible tools for control have not been widely testedagainst them. The objective of this study was to determine the efficacy of Olyset® Net and PermaNet®LLINs alongside a local brand, K-O Tab® treated net (Supanet) against Phlebotomus duboscqifemale sandflies.Methods: Four replicates of unwashed and 20x washed Olyset Nets and PermaNets, K-O Tabtreatedand untreated Supanet and ‘no net’ treatments were evaluated against sandflies within thelaboratory by tunnel tests and in semi-field conditions in the greenhouse model for their efficacy.Results: All bednets allowed entry of P. duboscqi sandflies and subsequent blood-feeding. Olysetnet’s blood feeding inhibition was significantly higher than that of Supanet in the laboratory butnot in semi-field condition. Of the LLINs, only Olyset net had sandflies that could not feedsignificantly more than those of Supanet. Additionally, no significant efficacy difference wasobserved between LLINs washed 20x and unwashed ones. The only significant difference noted innumber of sandflies that were found dead or paralyzed within bednets in the semi-field conditionwas between Olyset and K-O Tab treated Supanet. In the laboratory, unwashed Olyset had asignificantly higher number of sandflies killed than all other bednet treatments.Conclusion: Olyset net use in areas where sandflies are nuisance biters and/or disease vectorscould be more beneficial in preventing sandfly bites than other tested bednets. It is recommendedthat mesh sizes of LLINs should be smaller for control of sandflies than those used for control ofmosquitoes

ANTI-INFLAMMATORY PROPERTIES OF METHANOLIC BARK EXTRACTS OF TERMINALIA BROWNII IN WISTAR ALBINO RATS

Objective: This study investigated the anti-inflammatory properties of methanolic bark extract of Terminalia brownii in Wistar albino rats (Rattus novegicus). Methods: The experimental animals were divided into six groups of five rats each; normal control, negative control, positive control and three experimental groups. Carrageenan was used to induce inflammation. Diclofenac was used as the reference drug, and the three experimental groups were treated with the extract at the dose levels of 50, 100 and 150 mg/kg bw. The extract was screened for the presence or absence of selected phytochemical secondary metabolites using standard procedures. Results: The methanolic bark extracts of T. brownii significantly [p Ë‚ 0.05] reduced the carrageenan-induced paw edema by between 1.57%-20.41% while diclofenac reduced it by between 11.12%-25.33%. Phytochemical screening of the extract indicated the presence of alkaloids, flavonoids, phenols, saponins, steroids and terpenoids. Conclusion: The present study revealed that T. brownii bark extract is a potential candidate for the development of a novel anti-inflammatory formulation

Genetic diversity assessment of farmers’ and improved potato (Solanum tuberosum) cultivars from Eritrea using simple sequence repeat (SSR) markers

Sixty three potato clones (51 farmers’ and 12 varieties) from Eritrea, 18 and 12 varieties from Kenya and Rwanda, respectively were characterized using 12 highly polymorphic simple sequence repeat (SSR) markers. The study was designed to assess the genetic diversity and varietal distinctness among the different samples. In total, 91 alleles ranging between 2 (STM1053) to 13 (STM0031) alleles per marker were scored. All but 97.8 SSR markers were highly polymorphic with an average PIC value of 0.87 (0.51 to 0.98). All of the 51 farmers’ cultivars were clearly distinct from each other. Samples from Eritrea showed the highest genetic diversity as explained by the diversity index (h). The principal coordinate analysis (PCoA) revealed that the local farmers’ Eritrean samples are different from the Kenyan, Rwandese and even the imported varieties. Genetic distance analysis generated three clusters correlating with the PCoA findings. Cluster I consisted of 45 samples with 6 sub-clusters; Cluster II consisted of 29 samples with a majority (26) from Eritrea while cluster III consisted of 19 samples. Potato materials from Eritrea appeared to cluster separately from the other samples, which reflects a contribution from the Tuberosum germplasm prominent in temperate regions, unlike from the Andigenum germplasm for Kenyan and Rwandan potato materials. Most of the Eritrean samples in cluster I are farmers’ cultivars with intermediate maturity, good performance and better tuber quality characteristics. Cluster II contains mainly the imported variety from Eritrea characterized by late emergence and late maturity. The Kenyan and Rwandese were grouped mainly in Cluster III. In summary, the farmers’ cultivars are distinct from the Kenyan and Rwandese materials and represent more genetic diversity than the varieties imported into Eritrea. This finding is of interest to national breeding program to use the farmer’s materials as source of genetic variation for traits of interest.Keywords: Potato, simple sequence repeat (SSR), principal coordinate analysis (PCoA), cluster analysis, Eritrea, multivariat

Constitutive Expressor of Pathogenesis-Related Genes5 affects cell wall biogenesis and trichome development

<p>Abstract</p> <p>Background</p> <p>The Arabidopsis thaliana <it>CONSTITUTIVE EXPRESSOR OF PATHOGENESIS-RELATED GENES5 </it>(<it>CPR5</it>) gene has been previously implicated in disease resistance, cell proliferation, cell death, and sugar sensing, and encodes a putative membrane protein of unknown biochemical function. Trichome development is also affected in <it>cpr5 </it>plants, which have leaf trichomes that are reduced in size and branch number.</p> <p>Results</p> <p>In the work presented here, the role of <it>CPR5 </it>in trichome development was examined. Trichomes on <it>cpr5 </it>mutants had reduced birefringence, suggesting a difference in cell wall structure between <it>cpr5 </it>and wild-type trichomes. Consistent with this, leaf cell walls of <it>cpr5 </it>plants contained significantly less paracrystalline cellulose and had an altered wall carbohydrate composition. We also found that the effects of <it>cpr5 </it>on trichome size and endoreplication of trichome nuclear DNA were epistatic to the effects of mutations in <it>triptychon </it>(<it>try</it>) or overexpression of <it>GLABRA3</it>, indicating that these trichome developmental regulators are dependant on <it>CPR5 </it>function for their effects on trichome expansion and endoreplication.</p> <p>Conclusion</p> <p>Our results suggest that <it>CPR5 </it>is unlikely to be a specific regulator of pathogen response pathways or senescence, but rather functions either in cell wall biogenesis or in multiple cell signaling or transcription response pathways.</p