Ethical Implications of Cleft Lip and Palate Repair in Patients with Trisomy 13 and Trisomy 18.

BACKGROUND: Children born with Trisomy 13 or 18 (T13/18) often have multiple congenital anomalies, many of which drastically shorten their lifespan. Among these defects are cleft lip and palate, the repair of which presents an ethical dilemma to the surgeon given the underlying comorbidities associated with T13/18. The authors present an ethical discussion and institutional experience in navigating this dilemma. METHODS: The authors analyzed existing literature on T13 and T18 surgery and mortality. A retrospective study over ten years was also conducted to identify pediatric patients who underwent surgical correction of cleft lip and/or palate secondary to a confirmed diagnosis of T13/18. The authors identified two patients and examined their treatment course. RESULTS: The authors' review of literature coupled with their institution's experience builds on the published successes of correcting cleft lip and palate in the setting of T13/18. It was found that both patients identified in the case series underwent successful correction with no surgical complications. CONCLUSION: A careful balance must be struck between improved quality of life, benefits of treatment, and risks of surgery in children with T13/T18. Careful consideration should be given to the medical status of these complex patients. If the remaining medical comorbidities are well managed and under control, there is an ethical precedent for performing cleft lip and palate surgeries on these children. A diagnosis of T13/T18 alone is not enough to disqualify patients from cleft lip/palate surgery.

Assessment of a novel microalgae-cork based technology for removing antibiotics, pesticides and nitrates from groundwater.

Groundwater pollution has increased in recent years due to the intensification of agricultural and livestock activities. This results in a significant reduction in available freshwater resources. Here, we have studied the long term assessment of a green technology (1-4 L/day) based on a photobioreactor (PBR) containing immobilised microalgae-bacteria in polyurethane foam (PF) followed by a cork filter (CF) for removing nitrates, pesticides (atrazine and bromacil), and antibiotics (sulfamethoxazole and sulfacetamide) from groundwater. The prototype was moderately effective for removing nitrates (58%) at an HRT of 8 days, while its efficiency decreased at a HRT of 4 and 2 days (<20% removal). The combined use of PBR-CF enabled antibiotics and pesticides to be attenuated by up to 95% at an HRT of 8 days, but their attenuation decreased with shorter HRT, with pesticides being the compounds most affected (reducing from 97 to 98% at an HRT of 8 days to 23-45% at an HRT of 2 days). Pesticide transformation products were identified after the CF, supporting biodegradation as the main attenuation process. A gene-based metataxonomic assessment linked the attenuation of micropollutants to the presence of specific pesticide biodegradation species (e.g. genus Phenylobacterium, Sphingomonadaceae, and Caulobacteraceae). Therefore, the results highlighted the potential use of microalgae and cork to treat polluted groundwater.

"You can't make me!" Managing adolescent dissent to anesthesia.

When adolescents require health care, the need to obtain consent from the parent/legal guardian and assent from the patient can create the potential for an ethical dilemma when these two parties are not in agreement. Here, we describe a representative and common case scenario in which both parent and adolescent patient gave consent and assent, respectively, with a full understanding of the risks and benefits of the procedure and anesthetic. At the time of anesthetic induction, however, the patient expresses that she no longer wishes to have the procedure. We identify a number of considerations that inform the ethical analysis of such cases and offer recommendations about the most appropriate path forward for a practitioner faced with a difficult decision about how to respond.

A polyphasic approach to the study of the genus Nitzschia (Bacillariophyta): three new planktonic species from the Adriatic Sea.

The paraphyletic diatom genus Nitzschia comprises over 1000 morphologically distinct pennate taxa, known from the benthos and plankton of freshwater, brackish, and marine environments. The principal diagnostic characters for delimitation of Nitzschia species include valve shape, the position and structure of the raphe, presence/absence and shape of the proximal raphe endings and terminal raphe fissures, areola structure, and specific morphometric features such as cell size, and stria and fibula density. In this study, we isolated 12 diatom strains into culture from samples collected at the surface or greater depths of the southeastern Adriatic Sea. Morphological analyses included LM, SEM, and TEM observations, which, along with specific morphometric features, allowed us to distinguish three new Nitzschia species. These findings were congruent with the results of phylogenetic analyses performed on nuclear-encoded SSU (18S) rDNA and chloroplast-encoded rbcL and psbC genes. One of the new species (Nitzschia dalmatica sp. nov.) formed a lineage within a clade of Bacillariaceae containing members of the Nitzschia sect. Dubiae, which was sister to Psammodictyon. A second lineage was part of a novel clade that is significantly distinct from other Nitzschia species sequenced so far and includes Nitzschia adhaerens sp. nov. and N. cf. adhaerens. A further new species was found, Nitzschia inordinata sp. nov., which appeared as the sister group to the N. adhaerens clade and the conopeoid Nitzschia species in our phylogenetic trees. Our findings contribute to the overall diversity of genus Nitzschia, especially in identifying some deep branches within the Bacillariaceae, and highlight under-scoring of this genus in marine plankton.

Surgical interventions and anesthesia in the 1st year of life for lower urinary tract obstruction.

BACKGROUND: Patients with a prenatal diagnosis of lower urinary tract obstruction (LUTO) may undergo prenatal interventions, such as vesicoamniotic shunt (VAS) placement, as a temporary solution for relieving urinary tract obstruction. A recent FDA communication has raised awareness of the potential neurocognitive adverse effects of anesthesia in children. We hypothesized as to whether a prenatal LUTO staging system was predictive of the number of anesthesia events for prenatally diagnosed LUTO patients. METHODS: We retrospectively reviewed the prenatal and postnatal clinical records for patients with prenatally diagnosed LUTO from 2012 to 2015. Patients were stratified by prenatal VAS status and by LUTO disease severity according to Ruano et al. (Ultrasound Obstet Gynecol. 2016). RESULTS: 31 patients were identified with a prenatal LUTO diagnosis, and postnatal records were available for 21 patients (seven patients in each stage). When combining prenatal and postnatal anesthesia, there was a significant difference in the number of anesthesia encounters by stage (1.6, 3.7, and 6.7 for Stage I, II, and III respectively, p = .034). Upon univariate analysis, higher gestational age (GA) at birth was associated with a decreased number of anesthesia events in the first year (p = .031). CONCLUSIONS: The majority of infants with prenatally diagnosed LUTO will undergo postnatal procedures with general anesthesia exposure in the first year of life. Patients with higher prenatal LUTO severity experienced a higher number of both prenatal and postnatal anesthesia encounters. In addition, higher GA at birth was associated with fewer anesthesia encounters in the first year. LEVEL OF EVIDENCE: This is a prognostic study with Level IV evidence.

Fetoscopic Open Neural Tube Defect Repair: Development and Refinement of a Two-Port, Carbon Dioxide Insufflation Technique.

OBJECTIVE: To describe development of a two-port fetoscopic technique for spina bifida repair in the exteriorized, carbon dioxide-filled uterus and report early results of two cohorts of patients: the first 15 treated with an iterative technique and the latter 13 with a standardized technique. METHODS: This was a retrospective cohort study (2014-2016). All patients met Management of Myelomeningocele Study selection criteria. The intraoperative approach was iterative in the first 15 patients and was then standardized. Obstetric, maternal, fetal, and early neonatal outcomes were compared. Standard parametric and nonparametric tests were used as appropriate. RESULTS: Data for 28 patients (22 endoscopic only, four hybrid, two abandoned) are reported, but only those with a complete fetoscopic repair were analyzed (iterative technique [n=10] compared with standardized technique [n=12]). Maternal demographics and gestational age (median [range]) at fetal surgery (25.4 [22.9-25.9] compared with 24.8 [24-25.6] weeks) were similar, but delivery occurred at 35.9 (26-39) weeks of gestation with the iterative technique compared with 39 (35.9-40) weeks of gestation with the standardized technique (P<.01). Duration of surgery (267 [107-434] compared with 246 [206-333] minutes), complication rates, preterm prelabor rupture of membranes rates (4/12 [33%] compared with 1/10 [10%]), and vaginal delivery rates (5/12 [42%] compared with 6/10 [60%]) were not statistically different in the iterative and standardized techniques, respectively. In 6 of 12 (50%) compared with 1 of 10 (10%), respectively (P=.07), there was leakage of cerebrospinal fluid from the repair site at birth. Management of Myelomeningocele Study criteria for hydrocephalus-death at discharge were met in 9 of 12 (75%) and 3 of 10 (30%), respectively, and 7 of 12 (58%) compared with 2 of 10 (20%) have been treated for hydrocephalus to date. These latter differences were not statistically significant. CONCLUSION: Fetoscopic open neural tube defect repair does not appear to increase maternal-fetal complications as compared with repair by hysterotomy, allows for vaginal delivery, and may reduce long-term maternal risks. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, https://clinicaltrials.gov, NCT02230072.

Feasibility and Outcomes of Fetoscopic Tracheal Occlusion for Severe Left Diaphragmatic Hernia.

OBJECTIVE: To evaluate feasibility and initial outcomes of fetoscopic tracheal occlusion for severe diaphragmatic hernia compared with a historical cohort who had not received fetal tracheal occlusion. METHODS: Outcomes in a prospective observational cohort who underwent fetoscopic tracheal occlusion for severe fetal left diaphragmatic hernia without associated anomalies were compared with our historical nontreated cohort of matched fetuses of similar severity. Fetuses were classified using the same ultrasonography and magnetic resonance imaging methodology-prospectively in the fetoscopic tracheal occlusion group and retrospectively in the historical nontreated cohort. Obstetric and postnatal outcomes were evaluated and compared. RESULTS: Between January 2004 and June 2015, 218 fetuses with diaphragmatic hernia were evaluated. Twenty (9%) fetuses had severe left diaphragmatic hernia (lung-head ratio 1.0 or less and liver herniation), of which 9 of 20 were managed without tracheal occlusion. Eleven were offered the procedure and in 10, it was successful. Mean (±standard deviation) gestational age was 27.9±1.1 weeks at attempted balloon placement, 34.1±1.1 weeks at removal, and 35.3±2.2 weeks at delivery. One patient required an ex utero intrapartum treatment procedure at delivery to remove the balloon. There were no maternal complications or fetal deaths. All neonates underwent postnatal repair with a patch. The 6-month, 1-year, and 2-year survival rates were significantly higher in our treated cohort than in our nontreated historical cohort (80% compared with 11%, risk difference 69%, 95% confidence interval [CI] 38-100%, P=.01; 70% compared with 11%, risk difference 59%, 95% CI 24-94%, P=.02; and 67% compared with 11%, risk difference 56%, 95% CI 19-93%, P=.04, respectively) with reduced need for extracorporeal membrane oxygenation (30% compared with 70%, risk difference 40%, 95% CI 10-79%, P=.05). CONCLUSION: Fetoscopic tracheal occlusion is feasible and is associated with improved postnatal outcomes in severe left diaphragmatic hernia. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, https://clinicaltrials.gov, NCT00881660.

Downregulation of a UDP-Arabinomutase Gene in Switchgrass (Panicum virgatum L.) Results in Increased Cell Wall Lignin While Reducing Arabinose-Glycans.

Background: Switchgrass (Panicum virgatum L.) is a C4 perennial prairie grass and a dedicated feedstock for lignocellulosic biofuels. Saccharification and biofuel yields are inhibited by the plant cell wall's natural recalcitrance against enzymatic degradation. Plant hemicellulose polysaccharides such as arabinoxylans structurally support and cross-link other cell wall polymers. Grasses predominately have Type II cell walls that are abundant in arabinoxylan, which comprise nearly 25% of aboveground biomass. A primary component of arabinoxylan synthesis is uridine diphosphate (UDP) linked to arabinofuranose (Araf). A family of UDP-arabinopyranose mutase (UAM)/reversible glycosylated polypeptides catalyze the interconversion between UDP-arabinopyranose (UDP-Arap) and UDP-Araf. Results: The expression of a switchgrass arabinoxylan biosynthesis pathway gene, PvUAM1, was decreased via RNAi to investigate its role in cell wall recalcitrance in the feedstock. PvUAM1 encodes a switchgrass homolog of UDP-arabinose mutase, which converts UDP-Arap to UDP-Araf. Southern blot analysis revealed each transgenic line contained between one to at least seven T-DNA insertions, resulting in some cases, a 95% reduction of native PvUAM1 transcript in stem internodes. Transgenic plants had increased pigmentation in vascular tissues at nodes, but were otherwise similar in morphology to the non-transgenic control. Cell wall-associated arabinose was decreased in leaves and stems by over 50%, but there was an increase in cellulose. In addition, there was a commensurate change in arabinose side chain extension. Cell wall lignin composition was altered with a concurrent increase in lignin content and transcript abundance of lignin biosynthetic genes in mature tillers. Enzymatic saccharification efficiency was unchanged in the transgenic plants relative to the control. Conclusion: Plants with attenuated PvUAM1 transcript had increased cellulose and lignin in cell walls. A decrease in cell wall-associated arabinose was expected, which was likely caused by fewer Araf residues in the arabinoxylan. The decrease in arabinoxylan may cause a compensation response to maintain cell wall integrity by increasing cellulose and lignin biosynthesis. In cases in which increased lignin is desired, e.g., feedstocks for carbon fiber production, downregulated UAM1 coupled with altered expression of other arabinoxylan biosynthesis genes might result in even higher production of lignin in biomass.

Gametogenesis and auxospore development in Actinocyclus (Bacillariophyta).

cGametogenesis and auxospore development have been studied in detail in surprisingly few centric diatoms. We studied the development of sperm, eggs and auxospores in Actinocyclus sp., a radially symmetrical freshwater diatom collected from Japan, using LM and electron microscopy of living cultures and thin sections. Actinocyclus represents a deep branch of the 'radial centric' diatoms and should therefore contribute useful insights into the evolution of sexual reproduction in diatoms. Spermatogenesis was examined by LM and SEM and involved the formation of two spermatogonia (sperm mother-cells) in each spermatogonangium through an equal mitotic division. The spermatogonia produced a reduced 'lid' valve, resembling a large flat scale with irregular radial thickenings. Sperm formation was merogenous, producing four sperm per spermatogonium, which were released by dehiscence of the 'lid' valve. The sperm were spindle-shaped with numerous surface globules and, as usual for diatoms, the single anterior flagellum bore mastigonemes. One egg cell was produced per oogonium. Immature eggs produced a thin layer of circular silica scales before fertilization, while the eggs were still contained within the oogonium. Sperm were attracted in large numbers to each egg and were apparently able to contact the egg surface via a gap formed between the long hypotheca and shorter epitheca of the oogonium and a small underlying hole in the scale-case. Auxospores expanded isodiametrically and many new scales were added to its envelope during expansion. Finally, new slightly-domed initial valves were produced at right angles to the oogonium axis, after a strong contraction of the cell away from the auxospore wall. At different stages, Golgi bodies were associated with chloroplasts or mitochondria, contrasting with the constancy of Golgi-ER-mitochondrion (G-ER-M) units in some other centric diatoms, which has been suggested to have phylogenetic significance. Electron-dense bodies in the vacuole of Actinocyclus are probably acidocalcisomes containing polyphosphate.

Very bright orange fluorescent plants: endoplasmic reticulum targeting of orange fluorescent proteins as visual reporters in transgenic plants.

BACKGROUND: The expression of fluorescent protein (FP) genes as real-time visual markers, both transiently and stably, has revolutionized plant biotechnology. A palette of colors of FPs is now available for use, but the diversity has generally been underutilized in plant biotechnology. Because of the green and far-red autofluorescent properties of many plant tissues and the FPs themselves, red and orange FPs (RFPs, and OFPs, respectfully) appear to be the colors with maximum utility in plant biotechnology. Within the color palette OFPs have emerged as the brightest FP markers in the visible spectra. This study compares several native, near-native and modified OFPs for their "brightness" and fluorescence, therefore, their usability as marker genes in transgenic plant tissues. RESULTS: The OFPs DsRed2, tdTomato, mOrange and pporRFP were all expressed under the control of the CaMV 35S promoter in agroinfiltration-mediated transient assays in Nicotiana benthamiana. Each of these, as well as endoplasmic reticulum (ER)-targeted versions, were stably expressed in transgenic Nicotiana tabacum and Arabidopsis thaliana. Congruent results were observed between transient and stable assays. Our results demonstrated that there are several adequate OFP genes available for plant transformation, including the new pporRFP, an unaltered tetramer from the hard coral Porites porites. When the tandem dimer tdTomato and the monomeric mOrange were targeted to the ER, dramatic, ca. 3-fold, increase in plant fluorescence was observed. CONCLUSIONS: From our empirical data, and a search of the literature, it appears that tdTomato-ER and mOrange-ER are the two highest fluorescing FPs available as reporters for transgenic plants. The pporRFP is a brightly fluorescing tetramer, but all tetramer FPs are far less bright than the ER-targeted monomers we report here.

Functional characterization of the switchgrass (Panicum virgatum) R2R3-MYB transcription factor PvMYB4 for improvement of lignocellulosic feedstocks.

• The major obstacle for bioenergy production from switchgrass biomass is the low saccharification efficiency caused by cell wall recalcitrance. Saccharification efficiency is negatively correlated with both lignin content and cell wall ester-linked p-coumarate: ferulate (p-CA : FA) ratio. In this study, we cloned and functionally characterized an R2R3-MYB transcription factor from switchgrass and evaluated its potential for developing lignocellulosic feedstocks. • The switchgrass PvMYB4 cDNAs were cloned and expressed in Escherichia coli, yeast, tobacco and switchgrass for functional characterization. Analyses included determination of phylogenetic relations, in situ hybridization, electrophoretic mobility shift assays to determine binding sites in target promoters, and protoplast transactivation assays to demonstrate domains active on target promoters. • PvMYB4 binds to the AC-I, AC-II and AC-III elements of monolignol pathway genes and down-regulates these genes in vivo. Ectopic overexpression of PvMYB4 in transgenic switchgrass resulted in reduced lignin content and ester-linked p-CA : FA ratio, reduced plant stature, increased tillering and an approx. threefold increase in sugar release efficiency from cell wall residues. • We describe an alternative strategy for reducing recalcitrance in switchgrass by manipulating the expression of a key transcription factor instead of a lignin biosynthetic gene. PvMYB4-OX transgenic switchgrass lines can be used as potential germplasm for improvement of lignocellulosic feedstocks and provide a platform for further understanding gene regulatory networks underlying switchgrass cell wall recalcitrance.

Switchgrass (Panicum virgatum L.) polyubiquitin gene (PvUbi1 and PvUbi2) promoters for use in plant transformation.

BACKGROUND: The ubiquitin protein is present in all eukaryotic cells and promoters from ubiquitin genes are good candidates to regulate the constitutive expression of transgenes in plants. Therefore, two switchgrass (Panicum virgatum L.) ubiquitin genes (PvUbi1 and PvUbi2) were cloned and characterized. Reporter constructs were produced containing the isolated 5' upstream regulatory regions of the coding sequences (i.e. PvUbi1 and PvUbi2 promoters) fused to the uidA coding region (GUS) and tested for transient and stable expression in a variety of plant species and tissues. RESULTS: PvUbi1 consists of 607 bp containing cis-acting regulatory elements, a 5' untranslated region (UTR) containing a 93 bp non-coding exon and a 1291 bp intron, and a 918 bp open reading frame (ORF) that encodes four tandem, head -to-tail ubiquitin monomer repeats followed by a 191 bp 3' UTR. PvUbi2 consists of 692 bp containing cis-acting regulatory elements, a 5' UTR containing a 97 bp non-coding exon and a 1072 bp intron, a 1146 bp ORF that encodes five tandem ubiquitin monomer repeats and a 183 bp 3' UTR. PvUbi1 and PvUbi2 were expressed in all examined switchgrass tissues as measured by qRT-PCR. Using biolistic bombardment, PvUbi1 and PvUbi2 promoters showed strong expression in switchgrass and rice callus, equaling or surpassing the expression levels of the CaMV 35S, 2x35S, ZmUbi1, and OsAct1 promoters. GUS staining following stable transformation in rice demonstrated that the PvUbi1 and PvUbi2 promoters drove expression in all examined tissues. When stably transformed into tobacco (Nicotiana tabacum), the PvUbi2+3 and PvUbi2+9 promoter fusion variants showed expression in vascular and reproductive tissues. CONCLUSIONS: The PvUbi1 and PvUbi2 promoters drive expression in switchgrass, rice and tobacco and are strong constitutive promoter candidates that will be useful in genetic transformation of monocots and dicots.

A MOLECULAR SYSTEMATIC APPROACH TO EXPLORE DIVERSITY WITHIN THE SELLAPHORA PUPULA SPECIES COMPLEX (BACILLARIOPHYTA)(1).

The common and cosmopolitan freshwater benthic diatom Sellaphora pupula (Kütz.) Mereschk. is a model system for studying the nature of species in microalgae; the biological significance of morphological variation in this species complex has been widely demonstrated. The aim of this study was to establish a two-gene phylogeny (18S rDNA and rbcL) for 23 Sellaphora taxa, including 19 S. pupula aggregate (agg.) demes or species, S. bacillum (Ehrenb.) D. G. Mann, and S. laevissima (Kütz.) D. G. Mann. A range of analyses on separate and combined data sets indicated that Sellaphora is a monophyletic group containing four major clades. Of the traditionally recognized species, S. bacillum and S. laevissima are natural groups, but S. pupula is paraphyletic or polyphyletic because S. bacillum groups with S. pupula"small lanceolate" and S. lanceolata. Thickened bars at the poles of valves within the core "pupula-bacillum" group may be a morphological synapomorphy; the fossil record suggests that this group is at least 12 million years old. Otherwise, there was no clear pattern in the distribution of different morphologies among the major clades; each clade was also heterogeneous with respect to mating system. More intensive and extensive sampling will doubtless uncover even greater diversity; the challenge lies in its interpretation. Our results demonstrate the limitations of paleoecological, ecological, and biogeographical research based on morphospecies.