Isolasi Dan Identifikasi Bakteri Aerob Yang Berpotensi Menjadi Sumber Penularan Infeksi Nosokomial Di Irina a Rsup Prof. Dr. R. D. Kandou Manado

: Nosocomial infection or Hospital Acquired Infection (HAI) is an infection caused by bacteria, parasite, or virus in the hospital, infection occur at least 72 hours since hospitalized. This infection occurs due to lack of hygiene of the environment causing microorganism infection from environment to human, infection can also occur due to transmission of microorganism from one patient to other patients. Inpatients potentially have very high risk of nosocomial infection occur due to continuous requiring treatment for more than 24 hours. Purpose: To determine the existence of aerobic bacteria that could potentially be the source of transmission of nosocomial infection in Irina A RSUP Prof. Dr. R. D. Kandou Manado. Method: This research was descriptive with cross sectional approach. Fourteen samples were taken from the surface of medical equipment, bed, floor, and wall of the treatment room and eight samples were taken from the air. Identification of bacteria was performed by culture on agar medium, staining gram, and biochemical test. Result: Bacillus subtilis found in nine samples (41%), Serratia liquefaciens found in five samples (22,7%), Lactobacillus found in two samples (9,1%), Staphylococcus found in two samples (9,1%), Coccus Gram negative found in two samples (9,1%), Enterobacter aerogenes found in one sample (4,5%), and Enterobacter agglomerans found in one sample (4,5%). Conclusion: Bacillus subtilis is the most bacteria which had been found in this research

GPR161 and SMO localization.

Representative images from each batch showing variation in A) SMO and B) GPR161 localization. C) Normalized fluorescence intensity (arbitrary units, au) of GPR161 with and without stimulation in an additional passage of hTERT RPE-1 cells. Black bars = median normalized fluorescence intensity. Small symbol = normalized fluorescence intensity of one cilium. Numbers of cilia measured are listed below each column in the graph. Representative cilia are shown next to the graphs for unstimulated (-SAG) and stimulated (+SAG) cells. Scale bars are 5 μm. T-test of equal variances was performed. p-values: * 0.01 to 0.05, ** 0.001 to 0.01, and *** (TIF)</p

HEK293T ciliation data.

A) Proportion of ciliated HEK293T cells at 0, 24, 48, and 72 hours post serum starvation from additional thaws. B) Representative images of monolayer areas of HEK293T cell growth from Fig 1, Batch 3, which had the highest ciliation rates (top row) and repeat experiments with additional thaws of HEK293T cells. Cilia were stained with anti-ARL13B antibody. C) Representative images displaying ciliation in areas with multi-layer growth from additional thaws of HEK293T cells. Cilia were stained with anti-ARL13B antibody. Scale bars are 10μm. (TIF)</p

SMO and GPR161 localization in response to Hh pathway stimulation.

Normalized fluorescence intensity (au) of A) SMO and B) GPR161 with and without pathway stimulation. Large symbol = median normalized fluorescence intensity for individual coverslip data. Small symbol = normalized fluorescence intensity of one cilium. Measurement from three separate batches are indicated by a unique symbol shape and color. Total number of cilia measured listed in the second row below the graph. Representative images are shown below graphs for unstimulated (-SAG) and stimulated (+SAG) cells. Two-tailed Student’s t-test was performed. P-values represented significant differences if they ranged from 0.01 to 0.05 (*), 0.001 to 0.01 (**), and <0.001 (***).</p

Genetic interaction between <i>ninl</i> and <i>cc2d2a</i>.

<p>(<b>a-d</b>) Partial <i>ninl</i> knockdown enhances the cystic kidney phenotype of <i>cc2d2a</i> mutants. (<b>a-c</b>) Glomerulus and proximal pronephric tubules highlighted in the transgenic line Tg(wt1b-EGFP). (<b>a</b>) Injection of a low dose of <i>ninl</i> atgMO (0.75 ng/nl) causes no cysts in wild-type larvae. (<b>b</b>) <i>cc2d2a</i>-/- larvae display small dilatations of the proximal tubules (arrow) in ~40% of cases. (<b>c</b>) Injection of this low dose of <i>ninl</i> atgMO in the <i>cc2d2a</i>-/- background leads to large dilatations of the proximal tubules and glomerular space (arrow) in 89% of mutants. <b><i>g</i></b> glomerulus, <b><i>p</i></b> pancreas. (<b>d</b>) Quantification of the glomerular + proximal tubular area displayed as a scatter plot, demonstrating a significant increase in proximal pronephric area in <i>cc2d2a</i>-/- larvae injected with low-dose <i>ninl</i> atgMO. The bars represent the mean and standard error of the mean (SEM) for each treatment group and each datapoint is an individual fish. (<b>e-g’</b>) Immunohistochemistry with anti-opsin antibody (4D2, green) on retinal cryosections of 4dpf <i>cc2d2a</i>-/- uninjected larvae (<b>f-f”</b>) and <i>cc2d2a</i>-/- larvae injected with subphenotypic doses of <i>ninl</i> MO (<b>g’g”’</b>), that cause no mislocalization in wild-type fish (<b>e-e’</b>), demonstrates that partial <i>ninl</i> knockdown increases the mislocalization of opsins (<b>e’-g’</b>). (<b>h</b>) Quantification of the mean intracellular fluorescence displayed as a scatter plot shows significant increase in intracellular fluorescence in <i>cc2d2a</i>-/- larvae injected with low dose of <i>ninl</i> atgMO. The bars represent the mean and standard error of the mean (SEM) for each treatment group and each datapoint represents the mean intracellular fluorescence from 10 photoreceptors in one individual fish. Cell membrane and outer segments are stained with bodipy (red in <b>e-</b>g). Nuclei are counterstained with DAPI. Scale bars are 100 μm in (a-c) and 4 μm in (e-g’). (<b>i</b>) Pedigree of a consanguineous family with one affected boy (UW48-3) and 4 unaffected siblings. UW48-3 carried a homozygous missense <i>CC2D2A</i> mutation as well as a frameshift mutation in <i>NINL</i> leading to premature truncation. (<b>j</b>) Pedigree of a family where the affected individual (UW36-3) carries the same homozygous <i>CC2D2A</i> mutation as in (<b>i</b>) but no additional rare deleterious variants. (<b>k</b>) Pedigree of a family where the affected individual (UW07-3) carries compound heterozygous <i>C5ORF42</i> frameshift mutations and a nonsense mutation in <i>NINL</i>. (<b>l</b>) Pedigree of a family where the affected individual (UW57-3) carries compound heterozygous <i>TMEM67</i> mutations and a missense <i>NINL</i> mutation. The phenotype of the affected individuals is detailed in <i>italic</i> on each pedigree under the corresponding mutations. <i>MTS</i> Molar Tooth Sign, <i>DD</i> Developmental Delay, <i>ESRF</i> End-Stage Renal Failure.</p

Ninl is required for correct Rab8A localization.

<p>(<b>a-a’</b>) Expression of a rhodopsin-promoter driven cherry-tagged Rab8a in wild-type photoreceptors is mostly concentrated in one or several puncta (arrows <b>a-a’</b>) whereas it is diffuse in the majority of <i>ninl</i> morphant photoreceptors (<b>b-b’</b>). (<b>c</b>) Proportion of Rab8a-cherry expressing photoreceptors with punctate expression versus diffuse expression (bars represent 95% confidence interval; ** <i>P</i><0.001, <i>Fisher’s</i> exact test). (<b>d-d’</b>) Endogenous Rab8a localization as seen by immunohistochemistry using an anti-Rab8a antibody (green) displays similar puncta (arrowheads) in wild-type photoreceptors, while the number of puncta is decreased in <i>ninl</i> morphant photoreceptors (<b>e-e’</b>). (<b>f</b>) Quantification of the number of Rab8a puncta displayed in the form of a scatter plot indicating that significantly fewer endogenous Rab8 puncta per μm² are present in <i>ninl</i> morphants compared to uninjected controls (*<i>P = 0</i>.<i>01</i>, unpaired Student’s <i>t-</i>test; bars represent standard error of the mean). Scoring was performed blinded as to injection status for (<b>c</b>) and (<b>f</b>). Outer segments are counterstained with bodipy in (<b>d-e</b>). Nuclei are counterstained with DAPI. All images are cryosections of 4 dpf larvae. Scale bars are 4 μm in all panels.</p

NINL interactome screen identifies MICAL3.

<p>(<b>a</b>) Strep-SILAC and TAP (tandem affinity purification) experiments show that NINL interacts specifically with MICAL3 (Yellow). The solid line between NINL and MICAL3 symbolizes a direct interaction, whereas the dashed lines indicate interactions determined by IP. (<b>b</b>) Co-immunoprecipitation of eGFP-MICAL3 with FLAG-NINL^isoB, but not with FLAG-STRAD. The immunoblot (IB) in the top panel shows that eGFP-tagged MICAL3 co-immunoprecipitated with FLAG -tagged NINL (lane 2), whereas FLAG-tagged STRAD used as a negative control (lane 3) did not. The anti-GFP immunoprecipitates are shown in the middle panel; protein input is shown in the bottom panel. Reciprocal IP experiments using anti-FLAG antibodies confirmed the co-immunoprecipitation of eGFP-tagged MICAL3 with FLAG-tagged NINL^isoB (lane 2) and not with STRAD (lane 3) shown in the top panel. The anti-FLAG immunoprecipitations are shown in the middle panel; protein input is shown in the bottom panel. A co-immunoprecipitation experiment using untagged eGFP as a negative control (right panel) showed that eGFP-tagged MICAL3 immunoprecipitates with FLAG-tagged NINL^isoB but not with untagged eGFP.</p

Proposed model for CC2D2A and NINL function in trafficking, docking and fusion of rhodopsin-carrier vesicles.

<p>1) CC2D2A binds NINL and thus provides a docking point at the base of the connecting cilium for incoming vesicles. 2) NINL binds MICAL3 which in turn binds RAB8 that is coating the rhodopsin-carrier vesicles. Since NINL also associates with the cytoplasmic dynein1 motor complex, it provides a link between the carrier vesicles and the motor generating the movement along the microtubules. 3). MICAL3 subsequently interacts with ELKS and its redox activity promotes remodeling of the docking complex resulting in fusion of the vesicle at the periciliary region.</p

CC2D2A and NINL co-localize at the ciliary base in hTERT-RPE1 cells and in zebrafish retina.

<p>(<b>a, a’</b> and inset) When expressed alone, eCFP-tagged CC2D2A (green signal) localizes to the ciliary base (basal body, accessory centriole). The cilium is marked by anti-polyglutamylated tubulin (red signal, <b>a’</b> and inset). eCFP-tagged CC2D2A (green signal; <b>b</b>) also (partly) localizes to the ciliary transition zone, which was visualized using anti-RPGRIP1L as a marker (red signal; <b>b</b>). (<b>c, c’</b> and inset) mRFP-tagged NINL isoform B was localized at the ciliary base (cilium in green, <b>c’</b> and inset). (<b>d</b> and inset) mRFP-tagged NINL isoform B (red signal) localizes adjacent to the ciliary transition zone (anti-RPGRIP1L; green signal). (<b>e-e”</b> and inset) Co-expression of mRFP-tagged NINL isoform B (red signal) and eCFP-tagged CC2D2A showed co-localization of both proteins around the ciliary base (yellow signal). (<b>f</b>) In wild-type larval zebrafish retina (4 dpf), Cc2d2a marked by anti-Cc2d2a antibodies (red signal) is localized apically to the photoreceptor basal body (marked by anti-centrin antibodies, green signal). (<b>g</b>) Ninl, stained with anti-Ninl antibodies, (red signal) is localized at the zebrafish photoreceptor ciliary base, partially overlapping with and apical to the green centrin signal. (<b>h</b>) Cc2d2a localization is unaffected by <i>ninl</i> knockdown and (<b>i</b>) Ninl localization is normal in <i>cc2d2a</i>^<b>-/-</b> larvae. (<b>j</b>) Schematic representation of the localization of Ninl and Cc2d2a in zebrafish photoreceptor cells. (<b>f-i</b>) are immunostainings on cryosections from 4 dpf larvae. Nuclei were stained with DAPI (blue signal) in all panels. Scale bars are 10 μm in a-e, and 4 μm in f-i.</p

CC2D2A associates with NINL.

<p>(<b>a</b>) Yeast two-hybrid interaction assays were performed with different fragments of CC2D2A fused to the GAL4 DNA binding domain (BD) and full length NINL isoform A and B, fused to the GAL4 activation domain (AD). Activation of the reporter genes, which indicates a physical interaction, was dependent on coiled-coil (CC) domains 1 and 2 of CC2D2A and either NINL isoform A or B. (<b>b</b>) The top panel of the immunoblot (IB) shows that FLAG-tagged CC2D2A, but not the FLAG-tagged LRRK2 that was included as a negative control, was co-precipitated with HA-tagged NINL isoform B using a rat monoclonal antibody directed against the HA-epitope. Protein input is shown in the lower panel; anti-HA precipitates are shown in the middle panel. (<b>c</b>) In a reciprocal experiment, HA-tagged NINL^isoB was co-precipitated with FLAG-tagged CC2D2A, but not with FLAG-tagged LRRK2. Protein input is shown in the lower panel; anti-FLAG precipitates are shown in the middle panel.</p