Complications of laser enucleation of the prostate: Results at two institutions

Objective: Benign prostate hyperplasia (BPH) is a common disease with bothersome symptoms. Conventional transurethral resection of prostate is the gold standard surgical treatment. Recently, various laser enucleation techniques of the prostate for BPH have been adopted worldwide. We report perioperative and postoperative complications with the modified Clavien classification system at two institutions. Materials and methods: We performed a retrospective analysis through chart review among patients who had undergone laser enucleation of the prostate for BPH in two tertiary referral centers between January, 2009 and December, 2012. The primary outcome was peri- and postoperative complications, whereas secondary outcome was duration of hospital stay and catheterization. The mean age of 271 patients was 72.1 years (range, 51–93 years). Their mean prostate volume was 62.8 mL (range, 22–270 mL). Fifty-seven (21%) patients had prostate volume > 80 mL. There were 101 patients and 169 patients in the diode and thulium groups, respectively. Results: No patients had Grade 5 complications. There were Grade 4a complications in three (1.1%) patients, Grade 3a in 22 (8.1%) patients, Grade 2 in nine (3.3%) patients, and Grade 1 in 62 (22.9%) patients. The prostate volume > 80 mL was not significantly related to higher complication rate (p = 0.456). The average duration of hospital stay and catheterization were 3.05±0.75 days and 2.25±1.29 days. Four patients (1.5%) needed secondary resection for residual prostate tissue. The complication rate of acute urine retention (AUR; p = 0.285), urethral stricture (p = 0.996), minor (p = 0.430), major (p = 0.371), or all complications (p = 0.105) was not statistically different between diode and thulium groups. Conclusion: Laser enucleation of the prostate was a safe treatment with low significant complication rate, even for large-volume prostates. The complication rates between diode and thulium lasers were not significantly different. The reoperation rate of laser enucleation was very low but patients with extremely large prostate volume may have higher risk

Symptoms on squash plants after inoculation with <i>Zucchini yellow mosaic virus</i> (ZYMV) recombinant viruses carrying different point-mutated NSs proteins.

<p><b>(A)</b> Symptoms on squash plants inoculated with individual ZYMV recombinants at 14 days post-infection (dpi). <b>(B)</b> Detection of mutated NSs proteins expressed by individual ZYMV recombinants at 14 dpi using anti-NSs MAb or anti-ZYMV CP PAb. Coomassie blue-stained RuBisCO protein was used as loading controls.</p

Two Novel Motifs of <i>Watermelon Silver Mottle Virus</i> NSs Protein Are Responsible for RNA Silencing Suppression and Pathogenicity

<div><p>The NSs protein of <i>Watermelon silver mottle virus</i> (WSMoV) is the RNA silencing suppressor and pathogenicity determinant. In this study, serial deletion and point-mutation mutagenesis of conserved regions (CR) of NSs protein were performed, and the silencing suppression function was analyzed through agroinfiltration in <i>Nicotiana benthamiana</i> plants. We found two amino acid (aa) residues, H113 and Y398, are novel functional residues for RNA silencing suppression. Our further analyses demonstrated that H113 at the common epitope (CE) (¹⁰⁹KFTMHNQ¹¹⁷), which is highly conserved in Asia type tospoviruses, and the benzene ring of Y398 at the C-terminal <i>β</i>-sheet motif (³⁹⁷IYFL⁴⁰⁰) affect NSs mRNA stability and protein stability, respectively, and are thus critical for NSs RNA silencing suppression. Additionally, protein expression of other six deleted (ΔCR1-ΔCR6) and five point-mutated (Y15A, Y27A, G180A, R181A and R212A) mutants were hampered and their silencing suppression ability was abolished. The accumulation of the mutant mRNAs and proteins, except Y398A, could be rescued or enhanced by co-infiltration with potyviral suppressor HC-Pro. When assayed with the attenuated <i>Zucchini yellow mosaic virus</i> vector in squash plants, the recombinants carrying individual seven point-mutated NSs proteins displayed symptoms much milder than the recombinant carrying the wild type NSs protein, suggesting that these aa residues also affect viral pathogenicity by suppressing the host silencing mechanism.</p></div

Mutated NSs proteins for analyzing RNA silencing suppression function.

<p><b>(A)</b> The maps of different mutants with individual deletions of the highly conserved regions (ΔCR1-ΔCR 6) or the common epitope NSscon (ΔCE) of the NSs protein of <i>Watermelon silver mottle virus</i> (WSMoV). The aa positions of the individual deleted regions are indicated. <b>(B)</b> The aa positions locating on each conserved region chosen for alanine-mutagenesis in this study.</p

Expression levels of deleted and point-mutated NSs proteins analyzed by co-infiltration with potyviral suppressor HC-Pro.

<p><b>(A)</b> Expression levels of protein and mRNA of deleted NSs mutants, following co-infiltration of the empty vector (EV) or HC-Pro (HC), detected at 4 day post agroinfiltration (dpa) by anti-NSs MAb (left panel) or PAb (right panel) and α-<b>32</b>P labeled NSs-probe, respectively. <b>(B)</b> Expression levels of point-mutated NSs proteins, following co-infiltration with the empty vector or HC-Pro, detected at 4 dpa by anti-NSs MAb (left panel) or PAb (right panel). <b>(C)</b> Time-course detection of the protein expression levels of the NSs mutants, G180A and Y398A, at different hours post infiltration (hpi) with EV or HC, detected by anti-NSs MAb (left panel) or anti-HC-Pro PAb (right panel). <b>(D)</b> Expression levels of G180A, R181A and Y398A NSs proteins, mRNA and barstar^R mRNA following co-infiltration of EV or HC construct, detected at 4 dpa. Coomassie Blue stained-RuBisCO proteins or 18S rRNA were used as loading controls.</p

The expression and RNA silencing suppression capability of the mutated NSs proteins of <i>Watermelon silver mottle virus</i>, with modifications in conserved aa residues in the highly conserved (CR1-6) regions or the common epitope (CE), analyzed by agroinfiltration in leaf tissues of <i>N</i>. <i>benthamiana</i> plants.

<p>^a Bold proteins indicate the aa residues are highly conserved in NSs proteins of all tospoviruses of Asia and Euro-America types from databases. The others are only from Asia type tospoviruses.</p><p>The expression and RNA silencing suppression capability of the mutated NSs proteins of <i>Watermelon silver mottle virus</i>, with modifications in conserved aa residues in the highly conserved (CR1-6) regions or the common epitope (CE), analyzed by agroinfiltration in leaf tissues of <i>N</i>. <i>benthamiana</i> plants.</p

Analysis of RNA silencing suppression capability of mutated R181A and Y398A NSs proteins.

<p><b>(A)</b> GFP intensity at leaf areas co-infiltrated with <i>Agrobacterium</i> strain carrying individual point-mutated NSs constructs and the strains separately carrying pBA-GFP and pBA-GFi constructs, recorded under white light or UV light illumination at 4 days post agroinfiltration (dpa). The concentrations of <i>Agrobacterium</i> culture carrying pBA-GFi (left panel) or individual NSs (right panel) constructs are shown on the top of each panel. The concentrations of <i>Agrobacterium</i> carrying pBA-GFP and NSs constructs (left panel) or pBA-GFP and pBA-GFi constructs (right panel) were adjusted to OD₆₀₀ = 1.0. <b>(B)</b> Left panel: silencing suppression analyzed at 4 dpa. The relative proportions of <i>Agrobacterium</i> was 1:0.5:2 for GFP:GFi:NSs. Middle panel: western blotting was conducted for detection of mutated NSs protein and GFP protein from the leaf areas co-infiltrated with <i>Agrobacterium</i> cultures carrying GFP, hairpin GFP and individual NSs constructs. Total protein was extracted at 4 dpa. Coomassie blue stained RuBisCO proteins were used as loading controls. Right panel: The number indicates the relative accumulation of GFP co-infiltrated with individual NSs constructs, as quantified by Kodak image system 4000MM software. Statistically significant difference is indicated by “*”(n = 3, <i>P</i> = 0.012 < 0.05.).</p

Analysis of RNA silencing suppression function of individually deleted or point-mutated NSs proteins by agroinfiltration in <i>N</i>. <i>benthamiana</i> plants.

<p>The leaf areas agroinfiltrated with each deleted <b>(A, upper panel)</b> or point-mutated <b>(B, upper panel)</b> NSs constructs were examined under UV and white light. The GFP fluorescence was recorded at 4 days after co-infiltration (dpa) of <i>Agrobacterium</i> separately carrying pBA-GFP (the expresser), pBA-GFi (the silencing inducer) and individual constructs with deleted or point mutated NSs proteins described in Fig <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126161#pone.0126161.g001" target="_blank">1A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126161#pone.0126161.g001" target="_blank">1B</a>. Western blotting was performed for the detection of individually deleted <b>(A, lower panel)</b> or point-mutated <b>(B, lower panel)</b> NSs proteins expressed at 4 dpa. NSs monoclonal (MAb) and NSs polyclonal antibodies (PAb) were used for detecting NSs protein or NSs protein in which the common epitope was mutated, respectively. Expression levels of point-mutated NSs proteins, NSs mRNA, GFP and GFP mRNA were detected at 4 dpa <b>(C)</b>. Coomassie blue-stained RuBisCO protein was used as loading controls for proteins and 18S rRNA used as loading controls for RNAs.</p

Imprinted NanoVelcro Microchips for Isolation and Characterization of Circulating Fetal Trophoblasts: Toward Noninvasive Prenatal Diagnostics

Circulating fetal nucleated cells (CFNCs) in maternal blood offer an ideal source of fetal genomic DNA for noninvasive prenatal diagnostics (NIPD). We developed a class of nanoVelcro microchips to effectively enrich a subcategory of CFNCs, <i>i</i>.<i>e</i>., circulating trophoblasts (cTBs) from maternal blood, which can then be isolated with single-cell resolution by a laser capture microdissection (LCM) technique for downstream genetic testing. We first established a nanoimprinting fabrication process to prepare the LCM-compatible nanoVelcro substrates. Using an optimized cTB-capture condition and an immunocytochemistry protocol, we were able to identify and isolate single cTBs (Hoechst+/CK7+/HLA-G+/CD45–, 20 μm > sizes > 12 μm) on the imprinted nanoVelcro microchips. Three cTBs were polled to ensure reproducible whole genome amplification on the cTB-derived DNA, paving the way for cTB-based array comparative genomic hybridization (aCGH) and short tandem repeats analysis. Using maternal blood samples collected from expectant mothers carrying a single fetus, the cTB-derived aCGH data were able to detect fetal genders and chromosomal aberrations, which had been confirmed by standard clinical practice. Our results support the use of nanoVelcro microchips for cTB-based noninvasive prenatal genetic testing, which holds potential for further development toward future NIPD solution