Alar Asymmetry in Patients with Unilateral Cleft Lip: Implications for Secondary Rhinoplasty.

OBJECTIVE: Alar asymmetry in unilateral cleft lip (UCL) nasal deformity is a well-recognized clinical feature. However, there is a lack of comprehensive quantitative analysis of this asymmetry. This study compares the shape, volume, and axis rotation between the cleft and non-cleft ala in skeletally mature patients with UCL. DESIGN: A retrospective comparative study utilizing three-dimensional rendered CT scans. SETTING: Tertiary care pediatric institution. PATIENTS, PARTICIPANTS: This study included 18 patients with UCL nasal deformity at skeletal maturity. MAIN OUTCOME MEASURE(S): Cleft and non-cleft side ala volume, surface area, and axis to the midsagittal plane. RESULTS: The cleft-side ala was significantly lesser in volume by 27.3%, significantly lesser in surface area by 17.6%, and significantly greater in surface area to volume ratio by 14.6% than the non-cleft ala. The cleft-side ala was significantly greater by 43.1% horizontal axis to the midsagittal plane. In patients with primary rhinoplasty, the cleft-side ala had 28.0% less volume and 18.7% less surface area. In intermediate rhinoplasty, the cleft-side ala had 39.1% less volume and 23.5% less surface area than the non-cleft ala. CONCLUSIONS: Significant asymmetry exists between the cleft-side and non-cleft ala in patients with UCL. The cleft-side ala is significantly smaller in volume and surface area than the non-cleft ala. Additionally, the cleft-side ala demonstrates a significantly greater horizontal axis that contributes considerably to nasal asymmetry, supporting the need to restore a normal vertical axis to the clef-side ala.

Ice-POP: Ice Packs for Postoperative Pain: A Randomized Controlled Trial.

STUDY OBJECTIVE: To evaluate the benefit of ice packs as a supplement to standard pain management following laparoscopic hysterectomy (LH). DESIGN: This Institutional Review Board-approved randomized controlled trial involved patients undergoing LH for benign conditions. Subjects were randomized to receive standard enhanced recovery after surgery pain management or standard enhanced recovery after surgery plus ice packs. SETTING: Two academic tertiary care centers PATIENTS: Patients undergoing planned outpatient LH with the minimally invasive gynecologic surgery team between February 2019 and November 2020 were considered. Patients with chronic pain, current opioid use ≥1 week, or planned overnight hospitalizations were excluded. Primary outcome data were available for 51 subjects (24 control, 27 intervention). INTERVENTIONS: Ice packs were placed on the abdomen in the operating room. MEASUREMENTS AND MAIN RESULTS: Pain was assessed at multiple time points throughout the study using a visual analogue scale (VAS). Opioid requirement was assessed using morphine milligram equivalent. There was no difference between the groups on any demographic variables. Morphine milligram equivalent requirements were also not different between the groups (p = .63). Postoperative day 1 (POD#1) VAS scores were not different (p = .89). Eighty-five percent of subjects reported feeling that their pain was controlled. Subjects who reported that they did not feel their pain was controlled did not use more opioids on POD#1 (p = .37), nor did they have higher POD#1 VAS scores (p = .55). Eighty-seven percent of the intervention subjects said they would use ice again, and 82.6% of them said they would recommend ice to others. There were no adverse events related to ice. All subjects were prescribed 20 tablets oxycodone and averaged 2.9 (SD 3.4) tablets used after discharge. CONCLUSION: Ice packs are an acceptable supplement for postoperative pain control, but they do not reduce postoperative pain or opioid usage compared to standard pain management without ice packs.

Microbiome reduction prevents lipid accumulation during early diapause in the northern house mosquito, Culex pipiens pipiens.

The mosquito microbiome is critical to multiple facets of their biology, including larval development and disease transmission. For mosquitoes that reside in temperate regions, periods of diapause are critical to overwintering survival, but how the microbiome impacts this state is unknown. In this study, we compared the midgut microbial communities of diapausing and non-diapausing Culex pipiens and assessed how a reduced midgut microbiome influences diapause preparation. High community variability was found within and between non-diapausing and diapausing individuals, but no specific diapause-based microbiome was noted. Emergence of adult, diapausing mosquitoes under sterile conditions generated low bacterial load (LBL) lines with nearly a 1000-fold reduction in bacteria levels. This reduction in bacterial content resulted in significantly lower survival of diapausing females after two weeks, indicating acquisition of the microbiome in adult females is critical for survival throughout diapause. LBL diapausing females had high carbohydrate levels, but did not accumulate lipid reserves, suggesting an inability to process ingested sugars necessary for diapause-associated lipid accumulation. Expression patterns of select genes associated with mosquito lipid metabolism during diapause showed no significant differences between LBL and control lines, suggesting transcriptional changes may not underlie impaired lipid accumulation. Overall, a diverse, adult-acquired microbiome is critical for diapause in C. pipiens to process sugar reserves and accumulate lipids that are necessary to survive prolonged overwintering.

Three-Dimensional Treatment Outcomes of a Virtual Helmet Design Protocol for Sagittal Strip Craniectomy.

BACKGROUND: The helmet worn after sagittal strip craniectomy must be customized to the surgical procedure and the patient's anatomy to achieve optimal outcomes. This study compares three-dimensional head shape outcomes obtained from a novel virtual helmet design and from a traditional helmet design. METHODS: Twenty-four patients underwent extended sagittal strip craniectomy performed by a single surgeon and helmet management performed by a single orthotist. Eleven patients constitute the traditional helmet group, with helmet design based on laser scans. Thirteen patients constitute the virtual helmet group, with helmet design based on an overlay of a three-dimensional volume rendering of a low-radiation protocol computed tomographic scan and three-dimensional photograph. Cephalic index and vertical height were recorded from three-dimensional photographs. Three-dimensional whole-head composite images were generated to compare global head shape outcomes to those of age-matched controls. RESULTS: There was no significant difference in mean cephalic index between the virtual helmet group (83.70 ± 2.33) and controls (83.53 ± 2.40). The differences in mean cephalic index between the traditional helmet group (81.07 ± 3.37) and controls and in mean vertical height were each significant (p < 0.05). Three-dimensional analysis demonstrated normal biparietal and vertical dimensions in the virtual helmet group compared to controls. The traditional helmet group exhibited narrower biparietal dimension and greater vertical dimension compared to controls. CONCLUSIONS: Traditional and virtual helmet protocols improved mean cephalic index, but the virtual helmet group yielded more consistent and greater change in cephalic index. The virtual helmet design protocol yielded three-dimensional outcomes similar to those of age-matched controls. Traditional helmet design yielded a narrower biparietal dimension and greater vertical dimension to the cranial vault compared to the virtual helmet group and controls. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, III.

Urology Program Directors' Perception of Pregnancy During Residency.

OBJECTIVE: To assess urology program directors' (PDs) perception of pregnancy during residency training. METHODS: A 30 question anonymous survey was sent to 142 urology PDs regarding their demographics, program information, institution policies, and self-reported opinions. Results were assessed via descriptive analysis. RESULTS: A total of 63 PDs responded with a response rate of 44%: 19% were female, 73% between 40 and 59 years of age, and 91% had children. A minority (17%) of programs had 40% or more female residents. 37% of PDs had never had a pregnant resident during their time as PD while 57% had 1 to 5 pregnant residents. On multivariate analysis, PDs age > 60 years or PD having their first child when > 30 years old were predictors for poor support of maternity leave. The majority of PDs felt their program was better/much better at being supportive toward maternity leave compared to other surgical specialties at their institution. Only 21% of PDs felt that taking maternity leave burdened other residents unfairly. Of respondents, 62% felt prepared/completely prepared to advise residents on pregnancy during residency. However, 91% of PDs affirmed it would be helpful to have formal policies in place regarding maternity/paternity leave. CONCLUSION: While the majority of PDs do not have a negative perception of pregnancy during residency, a small portion feels that pregnancy during residency is a burden on other residents. More than half of PDs feel prepared to discuss this issue with their residents. However, a large majority would find formal policies helpful.

Analysis of the Whole CDR3 T Cell Receptor Repertoire after Hematopoietic Stem Cell Transplantation in 2 Clinical Cohorts.

A major cause of morbidity and mortality for patients who undergo hematologic stem cell transplantation (HSCT) is acute graft-versus-host disease (aGVHD), a mostly T cell-mediated disease. Examination of the T cell receptor (TCR) repertoire of HSCT recipients and the use of next-generation nucleotide sequencing have raised the question of whether features of TCR repertoire reconstitution might reproducibly associate with aGVHD. We hypothesized that the peripheral blood TCR repertoire of patients with steroid-nonresponsive aGVHD would be less diverse. We also hypothesized that patients with GVHD who shared HLA might also share common clones at the time of GVHD diagnosis, thereby potentially providing potential clinical indicators for treatment stratification. We further hypothesized that HSCT recipients with the same HLA mismatch might share a more similar TCR repertoire based on a potentially shared focus of alloreactive responses. We studied 2 separate patient cohorts and 2 separate platforms for measuring TCR repertoire. The first cohort of patients was from a multicenter Phase III randomized double-blinded clinical trial of patients who developed aGVHD (NCT01002742). The second cohort comprised samples from biobanks from 2 transplantation centers and the Center for International Blood and Marrow Transplant Research of patients who underwent mismatched HSCT. There were no statistically significant differences in the TCR diversity of steroid responders and nonresponders among patients with aGVHD on the day of diagnosis. Most clones in the repertoire were unique to each patient, but a small number of clones were found to be both exclusive to and shared among aGVHD nonresponders. We were also able to show a strong correlation between the presence of Vß20 and Vß29 and steroid responsiveness. Using the Bhattacharya coefficient, those patients who shared the same HLA mismatch were shown to be no more similar to one another than to those who had a completely different mismatch. Using 2 separate clinical cohorts and 2 separate platforms for analyzing the TCR repertoire, we have shown that the sampled human TCR repertoire is largely unique to each patient but contains glimmers of common clones of subsets of clones based on responsiveness to steroids in aGVHD on the day of diagnosis. These studies are informative for future strategies to assess for reproducible TCR responses in human alloreactivity and possible markers of GVHD responsiveness to therapy.

High-parametric evaluation of human invariant natural killer T cells to delineate heterogeneity in allo- and autoimmunity.

Human invariant natural killer T (iNKT) cells are a rare innate-like lymphocyte population that recognizes glycolipids presented on CD1d. Studies in mice have shown that these cells are heterogeneous and are capable of enacting diverse functions, and the composition of iNKT cell subsets can alter disease outcomes. In contrast, far less is known about how heterogeneity in human iNKT cells relates to disease. To address this, we used a high-dimensional, data-driven approach to devise a framework for parsing human iNKT heterogeneity. Our data revealed novel and previously described iNKT cell phenotypes with distinct functions. In particular, we found 2 phenotypes of interest: (1) a population with T helper 1 function that was increased with iNKT activation characterized by HLA-II+CD161- expression, and (2) a population with enhanced cytotoxic function characterized by CD4-CD94+ expression. These populations correlate with acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation and with new onset type 1 diabetes, respectively. Our study identifies human iNKT cell phenotypes associated with human disease that could aid in the development of biomarkers or therapeutics targeting iNKT cells.

Phase-Separated Liposomes Enhance the Efficiency of Macromolecular Delivery to the Cellular Cytoplasm.

INTRODUCTION: From viruses to organelles, fusion of biological membranes is used by diverse biological systems to deliver macromolecules across membrane barriers. Membrane fusion is also a potentially efficient mechanism for the delivery of macromolecular therapeutics to the cellular cytoplasm. However, a key shortcoming of existing fusogenic liposomal systems is that they are inefficient, requiring a high concentration of fusion-promoting lipids in order to cross cellular membrane barriers. OBJECTIVES: Toward addressing this limitation, our experiments explore the extent to which membrane fusion can be amplified by using the process of lipid membrane phase separation to concentrate fusion-promoting lipids within distinct regions of the membrane surface. METHODS: We used confocal fluorescence microscopy to investigate the integration of fusion-promoting lipids into a ternary lipid membrane system that separated into liquid-ordered and liquid-disordered membrane phases. Additionally, we quantified the impact of membrane phase separation on the efficiency with which liposomes transferred lipids and encapsulated macromolecules to cells, using a combination of confocal fluorescence imaging and flow cytometry. RESULTS: Here we report that concentrating fusion-promoting lipids within phase-separated lipid domains on the surfaces of liposomes significantly increases the efficiency of liposome fusion with model membranes and cells. In particular, membrane phase separation enhanced the delivery of lipids and model macromolecules to the cytoplasm of tumor cells by at least 4-fold in comparison to homogenous liposomes. CONCLUSIONS: Our findings demonstrate that phase separation can enhance membrane fusion by locally concentrating fusion-promoting lipids on the surface of liposomes. This work represents the first application of lipid membrane phase separation in the design of biomaterials-based delivery systems. Additionally, these results lay the ground work for developing fusogenic liposomes that are triggered by physical and molecular cues associated with target cells.

Erratum to: Phase-Separated Liposomes Enhance the Efficiency of Macromolecular Delivery to the Cellular Cytoplasm.

[This corrects the article DOI: 10.1007/s12195-017-0489-4.].

Steric Pressure among Membrane-Bound Polymers Opposes Lipid Phase Separation.

Lipid rafts are thought to be key organizers of membrane-protein complexes in cells. Many proteins that interact with rafts have bulky polymeric components such as intrinsically disordered protein domains and polysaccharide chains. Therefore, understanding the interaction between membrane domains and membrane-bound polymers provides insights into the roles rafts play in cells. Multiple studies have demonstrated that high concentrations of membrane-bound polymeric domains create significant lateral steric pressure at membrane surfaces. Furthermore, our recent work has shown that lateral steric pressure at membrane surfaces opposes the assembly of membrane domains. Building on these findings, here we report that membrane-bound polymers are potent suppressors of membrane phase separation, which can destabilize lipid domains with substantially greater efficiency than globular domains such as membrane-bound proteins. Specifically, we created giant vesicles with a ternary lipid composition, which separated into coexisting liquid ordered and disordered phases. Lipids with saturated tails and poly(ethylene glycol) (PEG) chains conjugated to their head groups were included at increasing molar concentrations. When these lipids were sparse on the membrane surface they partitioned to the liquid ordered phase. However, as they became more concentrated, the fraction of GUVs that were phase-separated decreased dramatically, ultimately yielding a population of homogeneous membrane vesicles. Experiments and physical modeling using compositions of increasing PEG molecular weight and lipid miscibility phase transition temperature demonstrate that longer polymers are the most efficient suppressors of membrane phase separation when the energetic barrier to lipid mixing is low. In contrast, as the miscibility transition temperature increases, longer polymers are more readily driven out of domains by the increased steric pressure. Therefore, the concentration of shorter polymers required to suppress phase separation decreases relative to longer polymers. Collectively, our results demonstrate that crowded, membrane-bound polymers are highly efficient suppressors of phase separation and suggest that the ability of lipid domains to resist steric pressure depends on both their lipid composition and the size and concentration of the membrane-bound polymers they incorporate.

Combination of 13 cis-retinoic acid and tolfenamic acid induces apoptosis and effectively inhibits high-risk neuroblastoma cell proliferation.

Chemotherapeutic regimens used for the treatment of Neuroblastoma (NB) cause long-term side effects in pediatric patients. NB arises in immature sympathetic nerve cells and primarily affects infants and children. A high rate of relapse in high-risk neuroblastoma (HRNB) necessitates the development of alternative strategies for effective treatment. This study investigated the efficacy of a small molecule, tolfenamic acid (TA), for enhancing the anti-proliferative effect of 13 cis-retinoic acid (RA) in HRNB cell lines. LA1-55n and SH-SY5Y cells were treated with TA (30µM) or RA (20µM) or both (optimized doses, derived from dose curves) for 48h and tested the effect on cell viability, apoptosis and selected molecular markers (Sp1, survivin, AKT and ERK1/2). Cell viability and caspase activity were measured using the CellTiter-Glo and Caspase-Glo kits. The apoptotic cell population was determined by flow cytometry with Annexin-V staining. The expression of Sp1, survivin, AKT, ERK1/2 and c-PARP was evaluated by Western blots. The combination therapy of TA and RA resulted in significant inhibition of cell viability (p<0.0001) when compared to individual agents. The anti-proliferative effect is accompanied by a decrease in Sp1 and survivin expression and an increase in apoptotic markers, Annexin-V positive cells, caspase 3/7 activity and c-PARP levels. Notably, TA+RA combination also caused down regulation of AKT and ERK1/2 suggesting a distinct impact on survival and proliferation pathways via signaling cascades. This study demonstrates that the TA mediated inhibition of Sp1 in combination with RA provides a novel therapeutic strategy for the effective treatment of HRNB in children.

Habitat visualization and genomic analysis of "Candidatus Pantoea carbekii," the primary symbiont of the brown marmorated stink bug.

Phytophagous pentatomid insects can negatively impact agricultural productivity and the brown marmorated stink bug (Halyomorpha halys) is an emerging invasive pest responsible for damage to many fruit crops and ornamental plants in North America. Many phytophagous stink bugs, including H. halys, harbor gammaproteobacterial symbionts that likely contribute to host development, and characterization of symbiont transmission/acquisition and their contribution to host fitness may offer alternative strategies for managing pest species. "Candidatus Pantoea carbekii" is the primary occupant of gastric ceca lumina flanking the distal midgut of H. halys insects and it is acquired each generation when nymphs feed on maternal extrachorion secretions following hatching. Insects prevented from symbiont uptake exhibit developmental delays and aberrant behaviors. To infer contributions of Ca. P. carbekii to H. halys, the complete genome was sequenced and annotated from a North American H. halys population. Overall, the Ca. P. carbekii genome is nearly one-fourth (1.2 Mb) that of free-living congenerics, and retains genes encoding many functions that are potentially host-supportive. Gene content reflects patterns of gene loss/retention typical of intracellular mutualists of plant-feeding insects. Electron and fluorescence in situ microscopic imaging of H. halys egg surfaces revealed that maternal extrachorion secretions were populated with Ca. P. carbekii cells. The reported findings detail a transgenerational mode of symbiont transmission distinct from that observed for intracellular insect mutualists and illustrate the potential additive functions contributed by the bacterial symbiont to this important agricultural pest.

Evolutionary basis of mitonuclear discordance between sister species of mole salamanders (Ambystoma sp.).

Distinct genetic markers should show similar patterns of differentiation between species reflecting their common evolutionary histories, yet there are increasing examples of differences in the biogeographic distribution of species-specific nuclear (nuDNA) and mitochondrial DNA (mtDNA) variants within and between species. Identifying the evolutionary processes that underlie these anomalous patterns of genetic differentiation is an important goal. Here, we analyse the putative mitonuclear discordance observed between sister species of mole salamanders (Ambystoma barbouri and A. texanum) in which A. barbouri-specific mtDNA is found in animals located within the range of A. texanum. We test three hypotheses for this discordance (undetected range expansion, mtDNA introgression, and hybridization) using nuDNA and mtDNA data analysed with methods that varied in the parameters estimated and the timescales measured. Results from a Bayesian clustering technique (structure), bidirectional estimates of gene flow (migrate-n and IMa2) and phylogeny-based methods (*beast, bucky) all support the conclusion that the discordance is due to geographically restricted mtDNA introgression from A. barbouri into A. texanum. Limited data on species-specific tooth morphology match this conclusion. Significant differences in environmental conditions exist between sites where A. texanum with and without A. barbouri-like mtDNA occur, suggesting a possible role for selection in the process of introgression. Overall, our study provides a general example of the value of using complimentary analyses to make inferences of the directionality, timescale, and source of mtDNA introgression in animals.

Obligate insect endosymbionts exhibit increased ortholog length variation and loss of large accessory proteins concurrent with genome shrinkage.

Extreme genome reduction has been observed in obligate intracellular insect mutualists and is an assumed consequence of fixed, long-term host isolation. Rapid accumulation of mutations and pseudogenization of genes no longer vital for an intracellular lifestyle, followed by deletion of many genes, are factors that lead to genome reduction. Size reductions in individual genes due to small-scale deletions have also been implicated in contributing to overall genome shrinkage. Conserved protein functional domains are expected to exhibit low tolerance for mutations and therefore remain relatively unchanged throughout protein length reduction while nondomain regions, presumably under less selective pressures, would shorten. This hypothesis was tested using orthologous protein sets from the Flavobacteriaceae (phylum: Bacteroidetes) and Enterobacteriaceae (subphylum: Gammaproteobacteria) families, each of which includes some of the smallest known genomes. Upon examination of protein, functional domain, and nondomain region lengths, we found that proteins were not uniformly shrinking with genome reduction, but instead increased length variability and variability was observed in both the functional domain and nondomain regions. Additionally, as complete gene loss also contributes to overall genome shrinkage, we found that the largest proteins in the proteomes of nonhost-restricted bacteroidetial and gammaproteobacterial species often were inferred to be involved in secondary metabolic processes, extracellular sensing, or of unknown function. These proteins were absent in the proteomes of obligate insect endosymbionts. Therefore, loss of genes encoding large proteins not required for host-restricted lifestyles in obligate endosymbiont proteomes likely contributes to extreme genome reduction to a greater degree than gene shrinkage.