MODEL OF CROATIAN SEA PASSENGER PORTS MANAGEMENT RATIONALIZATION

Predmet istraživanja u ovome znanstvenom radu je razvitak pomorskoputničkih luka u Republici Hrvatskoj do 2012. godine. Za definiranje svojstava i determinanti pomorskoputničkih luka koristilo se modelom na bazi matrice rasta. Analiza i vrednovanje pojedinih elemenata modela i dobivene izravne stope rasta imale su za cilj znanstveno formulirati rezultate istraživanja, prema najvažnijim teorijskim zakonitostima razvitka pomorskoputničkih luka u Republici Hrvatskoj. Autori su se u znanstvenom istraživanju i prezentiranju rezultata istraživanja ovog rada služili kombinaciju znanstvenih metoda kao što su: metoda analize i sinteze, metoda konkretizacije, komparativna metoda i metoda modeliranja (matrica rasta). Glavna znanstvena hipoteza dokazana je izravnim stopama rasta odabranih elemenata modela a ona glasi: Znanstveno utemeljenim spoznajama o funkcioniranju i poslovanju sustava pomorskoputničkih luka moguće je predložiti model, mjere i aktivnosti za racionalno upravljanje tim lukama kako bi se osigurao rast i razvoj sustava pomorskoputničkih luka.This paper analyses the sustainable development of sea passenger ports in the Republic of Croatia until 2012. A model of growth was used in order to define the main characteristics and determinants of sea passenger ports. The purpose of the paper was to present a scientifically-based formulation of sustainable development analysis of sea passenger ports in Croatia, based on the evaluation and analysis of relevant elements and resulting direct rates. The authors in their scientific research and presentation used a various combination of scientific methods like: analysis and syntheses method, concretization method, comparative method and modeling method (growth matrix). The main scientific hypothesis is: By using scientifically based acknowledgments about functioning and management of sea passenger port system it is possible to suggest a model, measurements and activities for the rational management of sea passenger ports in Croatia in order to secure their growth and development. This scientific hypothesis was confirmed by the direct rates of growth of the model elements

Cytogenetic appearance of amplified sequences.

<p>FISH analysis of metaphase spreads prepared from transfected CHO DG44 cells showing dots (A); lines (B); a fine ladder HSR of approximately two (C, I, and J), four (D), or six (E) chromosome widths (cw); and a ladder HSR (F). The frequencies of these structures were determined and are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052990#pone-0052990-g005" target="_blank">Figures 5</a> to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052990#pone-0052990-g007" target="_blank">7</a>. Multiple HSRs in a single cell (G) or ring chromosomes consisting of ladder HSRs (H) were observed in transfectants grown in 500 nM Mtx. The densities of the plasmid signals in the HSR shown in panels I and J are clearly different; however, both were classified as two cw, because the signals are distributed along the length of two cws.</p

Quantitation of <i>d2GFP</i> gene expression by flow cytometry.

<p>CHO DG44 cells were transfected with the pCMV-d2EGFP plasmid and IR/MAR-negative pSFV-V-<i>Dhfr</i> (A, C) or IR/MAR-positive pΔBN AR1-<i>Dhfr</i> (B, D) plasmid. These vector sets are indicated by the double-headed black arrows in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052990#pone-0052990-g002" target="_blank">Figure 2</a>. Cells were then selected with 10 µg/ml blasticidin in αMEM(+)(A and B), or with 10 µg/ml blasticidin and 5 nM Mtx in αMEM(−) (C and D). Stable transfectants produced without gene amplification (A), with IR/MAR-amplification (B), <i>Dhfr</i>/Mtx amplification (C), or IR/MAR-<i>Dhfr</i> fusion amplification (D), were obtained and d2GFP fluorescence was detected using flow cytometry. The number of culture days at the time of analysis is noted in each panel.</p

Clones obtained from plasmid set δ.

<p>Polyclonal transfectants (CN61-1, CN61-3, CN61-5, and CN61-6) that had been adapted to 0 nM (M0) or 5 nM (M5) Mtx in nucleotide-deficient medium (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052990#pone-0052990-g007" target="_blank">Figure 7</a>), and polyclonal transfectant CN49-4 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052990#pone-0052990-g005" target="_blank">Figure 5</a>), were subjected to limiting dilution in 96-well plates. Wells containing a single colony were recorded and the culture medium from 100 (CN61-1, CN61-3, CN61-5, and CN61-6) or 50 (CN49-4) colonies for each transfectant was analyzed by ELISA (1st screening; data not shown). The ten (CN61-1, CN61-3, CN61-5, and CN61-6) or five (CN49-4) highest antibody-producing clones of each transfectant were selected; these cells were cultured in 96-well plates for 3 days in the presence or absence of 10 mM butyrate and the culture medium was analyzed by ELISA. The mean +/− S.D. are indicated in the upper right corner of each panel. Error bars represent mean +/− S.D.</p

<i>FcR</i> plasmids and expression of the <i>FcR</i> gene.

<p>CHO DXB-11 cells were transfected with the IR/MAR-positive pΔBM AR1<i>Dhfr</i>-FcR plasmid or the IR/MAR-negative pECE-FcR <i>Dhfr</i> plasmid [23_ENREF_23] by electroporation, and then cultured for 10 days in α-MEM(−). Clones were obtained by limiting dilution; the highest producer of FcR was determined by ELISA and then cultured in the presence of 25 nM Mtx for 15 days. Surviving cells were subjected to another round of limiting dilution and the highest producers were then cultured in the presence of 125 nM Mtx for 20 days. The same selection process was used for clones obtained using the conventional <i>Dhfr</i>/Mtx method; however, cells were selected and cloned in medium containing 5, 50 and 500 nM Mtx. After each round of selection, the level of FcR protein (µg/ml) was determined by ELISA.</p

Stability of clones obtained using plasmid set δ.

<p>The five highest antibody-producing plasmid set δ clones generated using the IR/MAR-fusion (A to E) and <i>Dhfr</i>/Mtx (F to J) methods were selected and cultured in the presence (red lines) or absence (blue lines) of selective pressure (10 µg/ml blasticidin and 5 nM Mtx in the absence of nucleotides). After the indicated number of days post-transfection, cells were cultured in 96-well plates for 3 days in the presence (solid lines) or absence (dashed lines) of 10 mM butyrate and the culture medium was analyzed by ELISA.</p

Amplification and antibody expression by using plasmid set δ.

<p>Plasmids 1 (indicated) of plasmid set δ were co-transfected and selected by culture in the presence of 10 µg/ml blasticidin, the indicated concentrations of Mtx, and the absence of G418. The transfectant number (No.), Mtx concentration (nM), and the amplification method are indicated at the bottom of the figure. Transfectants CN61-3 and -1, and CN61-5 and -6, differ by whether the selection was started from 0 or 5 nM Mtx in the nucleotide-deficient medium. Cells reached confluence at the indicated number of days after transfection (Days a Trf). Cytogenetic structures were analyzed by FISH (A) (cw: chromowome width). Antibody expression was quantified by real-time PCR (B), and ELISA (C). Cells prepared for ELISA were grown in the presence (+) or absence (−) of 10 mM sodium butyrate (But). Error bars represent mean +/− S.D.</p

Amplification and antibody expression using plasmid set

<p>α<b>.</b> CHO DG44 cells were co-transfected with pMycLH (plasmid 2) and pΔBN AR1-<i>Dhfr</i> or pSFV-V-<i>Dhfr</i> (plasmid 1); cells were then selected by culture in the presence of 500 µg/ml G418, and the indicated concentrations of blasticidin (BS) and Mtx. The transfectant number (No.), Mtx concentration (nM), BS concentration (µg/ml), αMEM used (with (αMEM +) or without (αMEM−) nucleosides and deoxynucleosides), and amplification method, are indicated at the bottom of the figure. IR/MAR: IR/MAR method; Conv: conventional expression plasmid. Cells reached confluence at the indicated number of days after transfection (Days a Trf). Cytogenetic structures were analyzed by FISH (A) (cw: chromosome width). Antibody expression was quantified by real-time PCR (B), and ELISA (C). Cells prepared for ELISA were grown in the presence (+) or absence (−) of 10 mM sodium butyrate (But). Error bars represent mean +/− S.D.</p

Amplification and antibody expression using plasmid sets ß and γ.

<p>CHO DG44 cells were transfected with plasmids 1 and 2 (as indicated) of sets β (A to C) and γ (D to F), and then selected by culture in the presence of 10 µg/ml BS, the indicated concentrations of Mtx, and the absence of G418. The transfectant number (No.), Mtx concentration (nM), and the amplification method, are indicated at the bottom of the figure. Cells reached confluence at the indicated number of days after transfection (Days a Trf). Cytogenetic structures were analyzed by FISH (A) (cw: chromowome width). Antibody expression was quantified by real-time PCR (B), and ELISA (C). Cells prepared for ELISA were grown in the presence (+) or absence (−) of 10 mM sodium butyrate (But). Error bars represent mean +/− S.D.</p

DataSheet_1_Simple promotion of Cas9 and Cas12a expression improves gene targeting via an all-in-one strategy.pdf

Gene targeting (GT) is a promising tool for precise manipulation of genome sequences, however, GT in seed plants remains a challenging task. The simple and direct way to improve the efficiency of GT via homology-directed repair (HDR) is to increase the frequency of double-strand breaks (DSBs) at target sites in plants. Here we report an all-in-one approach of GT in Arabidopsis by combining a transcriptional and a translational enhancer for the Cas expression. We find that facilitating the expression of Cas9 and Cas12a variant by using enhancers can improve DSB and subsequent knock-in efficiency in the Arabidopsis genome. These results indicate that simply increasing Cas protein expression at specific timings - egg cells and early embryos - can improve the establishment of heritable GTs. This simple approach allows for routine genome engineering in plants.</p