Predictive Factors for Chemoradiation-Induced Oral Mucositis and Dysphagia in Head and Neck Cancer: A Scoping Review.

Despite advances in head and neck cancer treatment, virtually all patients experience chemoradiation-induced toxicities. Oral mucositis (OM) and dysphagia are among the most prevalent and have a systemic impact on patients, hampering treatment outcome and harming quality of life. Accurate prediction of severe cases is crucial for improving management strategies and, ultimately, patient outcomes. This scoping review comprehensively maps the reported predictors and critically evaluates the performance, methodology, and reporting of predictive models for these conditions. A total of 174 studies were identified from database searches, with 73 reporting OM predictors, 97 reporting dysphagia predictors, and 4 reporting both OM and dysphagia predictors. These predictors included patient demographics, tumor classification, chemoradiotherapy regimen, radiation dose to organs-at-risk, genetic factors, and results of clinical laboratory tests. Notably, many studies only conducted univariate analysis or focused exclusively on certain predictor types. Among the included studies, numerous predictive models were reported: eight for acute OM, five for acute dysphagia, and nine for late dysphagia. The area under the receiver operating characteristic curve (AUC) ranged between 0.65 and 0.81, 0.60 and 0.82, and 0.70 and 0.85 for acute oral mucositis, acute dysphagia, and late dysphagia predictive models, respectively. Several areas for improvement were identified, including the need for external validation with sufficiently large sample sizes, further standardization of predictor and outcome definitions, and more comprehensive reporting to facilitate reproducibility.

The evolution of synaptic and cognitive capacity: Insights from the nervous system transcriptome of Aplysia.

The gastropod mollusk Aplysia is an important model for cellular and molecular neurobiological studies, particularly for investigations of molecular mechanisms of learning and memory. We developed an optimized assembly pipeline to generate an improved Aplysia nervous system transcriptome. This improved transcriptome enabled us to explore the evolution of cognitive capacity at the molecular level. Were there evolutionary expansions of neuronal genes between this relatively simple gastropod Aplysia (20,000 neurons) and Octopus (500 million neurons), the invertebrate with the most elaborate neuronal circuitry and greatest behavioral complexity? Are the tremendous advances in cognitive power in vertebrates explained by expansion of the synaptic proteome that resulted from multiple rounds of whole genome duplication in this clade? Overall, the complement of genes linked to neuronal function is similar between Octopus and Aplysia. As expected, a number of synaptic scaffold proteins have more isoforms in humans than in Aplysia or Octopus. However, several scaffold families present in mollusks and other protostomes are absent in vertebrates, including the Fifes, Lev10s, SOLs, and a NETO family. Thus, whereas vertebrates have more scaffold isoforms from select families, invertebrates have additional scaffold protein families not found in vertebrates. This analysis provides insights into the evolution of the synaptic proteome. Both synaptic proteins and synaptic plasticity evolved gradually, yet the last deuterostome-protostome common ancestor already possessed an elaborate suite of genes associated with synaptic function, and critical for synaptic plasticity.

Molecular profiling of individual FDA-approved clinical drugs identifies modulators of nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) degrades transcripts with premature stop codons. Given the prevalence of nonsense single nucleotide polymorphisms (SNPs) in the general population, it is urgent to catalog the effects of clinically approved drugs on NMD activity: any interference could alter the expression of nonsense SNPs, inadvertently inducing adverse effects. This risk is higher for patients with disease-causing nonsense mutations or an illness linked to dysregulated nonsense transcripts. On the other hand, hundreds of disorders are affected by cellular NMD efficiency and may benefit from NMD-modulatory drugs. Here, we profiled individual FDA-approved drugs for their impact on cellular NMD efficiency using a sensitive method that directly probes multiple endogenous NMD targets for a robust readout of NMD modulation. We found most FDA-approved drugs cause unremarkable effects on NMD, while many elicit clear transcriptional responses. Besides several potential mild NMD modulators, the anticancer drug homoharringtonine (HHT or omacetaxine mepesuccinate) consistently upregulates various endogenous NMD substrates in a dose-dependent manner in multiple cell types. We further showed translation inhibition mediates HHT's NMD effect. In summary, many FDA drugs induce transcriptional changes, and a few impact global NMD, and direct measurement of endogenous NMD substrate expression is robust to monitor cellular NMD.

Sex chromosome transformation and the origin of a male-specific X chromosome in the creeping vole.

The mammalian sex chromosome system (XX female/XY male) is ancient and highly conserved. The sex chromosome karyotype of the creeping vole (Microtus oregoni) represents a long-standing anomaly, with an X chromosome that is unpaired in females (X0) and exclusively maternally transmitted. We produced a highly contiguous male genome assembly, together with short-read genomes and transcriptomes for both sexes. We show that M. oregoni has lost an independently segregating Y chromosome and that the male-specific sex chromosome is a second X chromosome that is largely homologous to the maternally transmitted X. Both maternally inherited and male-specific sex chromosomes carry fragments of the ancestral Y chromosome. Consequences of this recently transformed sex chromosome system include Y-like degeneration and gene amplification on the male-specific X, expression of ancestral Y-linked genes in females, and X inactivation of the male-specific chromosome in male somatic cells. The genome of M. oregoni elucidates the processes that shape the gene content and dosage of mammalian sex chromosomes and exemplifies a rare case of plasticity in an ancient sex chromosome system.

Nanopore sequencing and the Shasta toolkit enable efficient de novo assembly of eleven human genomes.

De novo assembly of a human genome using nanopore long-read sequences has been reported, but it used more than 150,000 CPU hours and weeks of wall-clock time. To enable rapid human genome assembly, we present Shasta, a de novo long-read assembler, and polishing algorithms named MarginPolish and HELEN. Using a single PromethION nanopore sequencer and our toolkit, we assembled 11 highly contiguous human genomes de novo in 9 d. We achieved roughly 63× coverage, 42-kb read N50 values and 6.5× coverage in reads >100 kb using three flow cells per sample. Shasta produced a complete haploid human genome assembly in under 6 h on a single commercial compute node. MarginPolish and HELEN polished haploid assemblies to more than 99.9% identity (Phred quality score QV = 30) with nanopore reads alone. Addition of proximity-ligation sequencing enabled near chromosome-level scaffolds for all 11 genomes. We compare our assembly performance to existing methods for diploid, haploid and trio-binned human samples and report superior accuracy and speed.

Comparison of scrotal antegrade sclerotherapy and laparoscopic Palomo surgery in treatment of adolescent varicocele: A 15-year review.

INTRODUCTION/OBJECTIVE: Varicocele is a common condition seen in adolescence and associated with adult subfertility. Numerous types of intervention have been described with differences in success and complication rates. This study aims to review and compare the surgical outcomes of laparoscopic Palomo surgery and scrotal antegrade sclerotherapy at our center. STUDY DESIGN: A retrospective analysis was done of all patients younger than 18 years old with idiopathic varicocele treated operatively between February 2001 and December 2016. Demographics such as age at operation, side, varicocele grade, operative date, and types of operation were collected. Primary outcomes were clinical recurrence, defined as any grading noted on follow-up within 1 year post-op and post-operative hydrocele. Secondary outcomes were operative time, length of stay, and other surgical complications. Mann-Whitney U test, independent t test and chi-square tests were used for analysis. All p-values were two-sided and considered statistically significant at p ≤ 0.05. RESULTS: A total of 119 patients fit the criteria, of whom 117 patients were included in data analysis (Table). Sixty-two patients had laparoscopic Palomo surgery (LPS) and 55 had scrotal antegrade sclerotherapy (SAS). Clinical recurrence (grade 2-3) within 1 year was similar between the two groups, with four out of 48 patients in the SAS group and six out of 62 patients in the LPS group (8.4% in SAS vs. 9.7% in LPS, p = 1.00). Of the 11 patients who had recurrence in the SAS group, seven had grade 1 recurrence (14.5%), two (4.2%) had grade 2 recurrence, and two (4.2%) had grade 3 recurrence. For the LPS group, 17 out of 62 patients had clinical recurrence within 1 year, of whom 11 (17.7%) had grade 1 recurrence, one (1.6%) had grade 2, and five (8.1%) had grade 3 recurrence. Post-operative hydrocele was significantly higher in the LPS group; there was none in the SAS group but 11 patients in the LPS group (0% in SAS vs. 17.7%, p = 0.002). Three patients had clinically significant hydrocele requiring Jaboulays' procedure. Complications other than hydrocele were noted in three patients in the SAS group and one patient in the LPS group (5.5% in SAS vs. 1.6% in LPS, p = 0.158). None required operative intervention. No conversion to open procedure was seen in the LPS group and there were no perioperative complications. Mean operative time was 62.9 min in the SAS group and 60.7 min in the LPS group (p = 0.624). Mean length of stay was 17.5 h in the SAS group and 31.7 h in the LPS group (p < 0.001). CONCLUSION: Both SAS and LPS are safe procedures for treatment of adolescent varicocele with similar clinical recurrence rate within 1 year. SAS has a significantly lower rate of post-operative hydrocele.

Surgical Outcome Analysis of Pneumovesicoscopic Ureteral Reimplantation and Endoscopic Dextranomer/Hyaluronic Acid Injection for Primary Vesicoureteral Reflux in Children: A Multicenter 12-Year Review.

OBJECTIVE: Vesicoureteral reflux (VUR) is a common condition associated with childhood urinary tract infection (UTI), which may lead to chronic renal failure and hypertension. Different antireflux approaches were advocated with differences in morbidity and success. The aim of this study is to review and analyze the surgical outcomes of pneumovesicoscopic ureteral reimplantation and endoscopic injection of dextranomer/hyaluronic acid (Dx/HA) in three tertiary centers. MATERIALS AND METHODS: The medical records of 215 patients (159 boys and 56 girls) for a total of 323 ureters underwent surgical interventions for primary VUR from February 2002 to August 2014 were reviewed. Data on baseline demographics, preoperative symptoms, radiological imaging studies, and postoperative outcomes were analyzed. VUR resolution was defined as when no VUR was detected by micturating cystourethrogram at 3 months or later after the intervention. Independent t-test, Mann-Whitney U test, Fisher's Exact test, and Chi-Square test were used for different parameters. All results with P value ≤.05 were regarded as statistically significant. RESULTS: The mean age at operation was 3.33 and 4.63 for reimplantation and Dx/HA injection respectively. A total of 234 ureters underwent Dx/HA injection and 92 ureters underwent pneumovesicoscopic ureteral reimplantation with mean preoperative VUR grading of 3.1 and 4.2 respectively (P = .0001). The overall VUR downgrading and resolution rates were both significantly higher in reimplantation than Dx/HA injection (97.8% versus 78.6% P = .0001 and 84.3% versus 65% P = .0011). Further subgroup analyses across the different VUR gradings showed higher downgrading and resolution rates in reimplantation group than Dx/HA injection for grade 4 (100% versus 81% P = .0147 and 82.4% versus 63% P = .0411) and grade 5 VUR (97.3% versus 50% P = .0022 and 81.6% versus 40% P = .0256). Dx/HA injection was associated with shorter operation time (41.5 minutes versus 147.5 minutes, P < .001), less postoperative analgesic usage (P = .049), and shorter hospital stay (1.06 days versus 4.44 days P < .0001). No major complications were identified in both groups. The mean follow-up time was significantly longer in reimplantation group than Dx/HA group (57.25 months versus 37.85 months, P = .002). There was no significant difference in the rate of subsequent UTI development during follow-up (P = .8). CONCLUSIONS: Both Dx/HA injection and pneumovesicoscopic ureteral reimplantation are safe and effective treatments for VUR. Reimplantation is associated with significantly higher VUR downgrading and resolution rates than Dx/HA injection especially in the higher grade VUR while Dx/HA injection has significantly shorter operation time, lower postoperative analgesic usage, and shorter hospital stay. Dx/HA injection can be considered as the first line surgical treatment especially for lower grade VUR. Pneumovesicoscopic ureteral reimplantation can be used for higher grade reflux or those who failed Dx/HA treatments.

D-dimer level correlation with treatment response in children with venous malformations.

AIM: Localized intravascular coagulopathy is present in children with venous malformations (VMs) as evidenced by elevated D-dimer levels. Few studies have looked into the changes in D-dimer after sclerotherapy and its correlation with treatment outcome and complications. Our study aims to investigate changes in D-dimer in children with VMs undergoing alcohol sclerotherapy. METHODS: A prospective cohort study from 2014 to 2016, which included children (<18years) with VM undergoing alcohol sclerotherapy, was completed. Demographics and lesion characteristics were recorded. Perioperative D-dimer levels were collected 2weeks prior to treatment (baseline) and on postoperative days 1, 2, 5, and 14, respectively. A raised postoperative D-dimer was defined as a peak level of at least 50% increase of baseline D-dimer. Children were followed up with documentation of lesional size at 6months and long-term recurrence beyond 6months of treatment. RESULTS: Eighteen children were identified (10 females, 8 males) with a median follow up of 21months. Overall, 15 patients (83%) had a satisfactory outcome. Baseline D-dimer levels were high in 8 patients (44%). Postoperative D-dimer level was raised in 12 patients irrespective of their baseline levels, with 92% peaking on postoperative day one (n=11). In the elevated D-dimer group, 11 patients had a satisfactory outcome, and 10 patients did not have long-term recurrence. We did not encounter any complications in our cohort. CONCLUSION: Changes in perioperative D-dimer levels may predict early treatment response and long-term recurrence after alcohol sclerotherapy. With a standardized protocol, alcohol sclerotherapy for venous malformation is safe with minimal complications. TYPE OF STUDY: Prognosis study. LEVEL OF EVIDENCE: IV.

Are all patients with short segment Hirschsprung's disease equal? A retrospective multicenter study.

BACKGROUND/PURPOSE: Short segment Hirschsprung's disease (HSCR) carries a better prognosis than long segment disease, but the definition of short is controversial. The objective of this study is to determine anatomically the extent of disease involvement that would be associated with a better functional outcome. METHODS: This is a retrospective multicenter (n = 3) study with patients (≥ 3 years) who had transanal pullthrough operation done for aganglionosis limited to the recto-sigmoid colon were reviewed. The extent of disease involvement and bowel resection was retrieved by reviewing the operative records as well as histopathological reports of the resected specimens. Clinical assessment was performed according to the criteria of a seven-itemed bowel function score (BFS) (maximum score = 20). Manometric assessment was performed with anorectal manometry. RESULTS: The study period started from 2003 to 45 patients were studied with median age at assessment = 52.0 months and operation = 3.0 months. The disease involvement was categorized into upper sigmoid-descending colon (DC) (n = 8), sigmoid colon (SC) (n = 12), upper rectum (UR) (n = 14) and lower rectum (LR) (n = 11) according to the level of normal biopsy result. There was no significant difference in the age of assessment between the four groups. The median BFSs in the DC, SC, UR and LR were 13, 15, 17 and 17, respectively (p = 0.01). Nine patients from the DC and SC groups reported soiling for more than twice per week. Sub-group analysis comparing patients with and without the entire sigmoid colon resected revealed worse functional outcomes in terms of the incidence of soiling (40.7 vs 22.2%, p = 0.05) and the BFS (14 vs 18, p = 0.04) in the former group. Anorectal manometry did not reveal any significant difference between the four groups, but a higher proportion of patients in the UR and LR groups appeared to have a normal sphincter resting pressure (DC vs SC vs UR vs LR = 62.5 vs 75.0 vs 85.7 vs 80.0%, p = 0.10). CONCLUSION: Patients with short segment HSCR are not equal at all. HSCR patients with aganglionosis limited to the rectum without the need of removing the entire sigmoid colon have a better bowel control and overall functional score. Less bowel loss and colonic dissection maybe the underlying reasons. Although future studies with a larger sample size and a longer follow-up period are required to validate the results of this study, it has provided a new insight to the current understanding of short segment disease in HSCR.

Molecular rheotaxis directs DNA migration and concentration against a pressure-driven flow.

In-line preconcentration techniques are used to improve the sensitivity of microfluidic DNA analysis platforms. The most common methods are electrokinetic and require an externally applied electric field. Here we describe a microfluidic DNA preconcentration technique that does not require an external field. Instead, pressure-driven flow from a fluid-filled microcapillary into a lower ionic strength DNA sample reservoir induces spontaneous DNA migration against the direction of flow. This migratory phenomenon that we call Molecular Rheotaxis initiates in seconds and results in a concentrated DNA bolus at the capillary orifice. We demonstrate the ease with which this concentration method can be integrated into a microfluidic total analysis system composed of in-line DNA preconcentration, size separation, and single-molecule detection. Paired experimental and numerical simulation results are used to delineate the parameters required to induce Molecular Rheotaxis, elucidate the underlying mechanism, and optimize conditions to achieve DNA concentration factors exceeding 10,000 fold.

Determination of absolute expression profiles using multiplexed miRNA analysis.

Accurate measurement of miRNA expression is critical to understanding their role in gene expression as well as their application as disease biomarkers. Correct identification of changes in miRNA expression rests on reliable normalization to account for biological and technological variance between samples. Ligo-miR is a multiplex assay designed to rapidly measure absolute miRNA copy numbers, thus reducing dependence on biological controls. It uses a simple 2-step ligation process to generate length coded products that can be quantified using a variety of DNA sizing methods. We demonstrate Ligo-miR's ability to quantify miRNA expression down to 20 copies per cell sensitivity, accurately discriminate between closely related miRNA, and reliably measure differential changes as small as 1.2-fold. Then, benchmarking studies were performed to show the high correlation between Ligo-miR, microarray, and TaqMan qRT-PCR. Finally, Ligo-miR was used to determine copy number profiles in a number of breast, esophageal, and pancreatic cell lines and to demonstrate the utility of copy number analysis for providing layered insight into expression profile changes.

A Multiplex Ligation Assay for miRNA Copy Number Profiling.

Ligo-miR is an assay technology that can perform multiplexed detection of miRNAs from a wide range of biological sources. At its core are two sequential ligation steps. First in the capture ligation, template molecules are created by ligating a DNA adapter to the 3' end of all miRNA molecules. Then in the coding ligation these templates are used to generate, linearly amplified, DNA products encoded by length. The resultant number of each DNA product is proportional to the original number of miRNA molecules. The products and their corresponding miRNA can be identified and quantified using common DNA sizing methods such as electrophoresis.

Venous Malformation and Localized Intravascular Coagulopathy in Children.

Aim Localized intravascular coagulopathy (LIC) has been described in adults with venous malformation (VM) but rarely reported in children. This study aims to determine the prevalence of LIC in children with VM and associated risk factors. Methods Patients younger than 18 years with VM from 2010 to 2014 were reviewed. Diagnosis was confirmed by Doppler ultrasound and/or magnetic resonance imaging. Demographics data and VM characteristics including volume, site, extension, painful symptoms, and palpable phleboliths were studied. Plasma D-dimer level of greater than 500 ng/mL was considered as abnormal. Results Total 24 children were included, of whom 8 were boys. Median age of presentation was 9 months (range: 0-12 years). Head-and-neck VM occurred in 17 (70.8%) patients and 3 (12.5%) had multifocal lesions. Seven (29.2%) patients had VM volume greater than 10 mL. Five (20.8%) patients had painful symptoms. Palpable phleboliths were found in two patients. Plasma D-dimer was raised in eight cases (33.3%). One patient with Klippel-Trenaunay syndrome (KTS) had D-dimer level of 5,000 ng/mL. Raised D-dimer was found in 23.5% of small VM (volume < 10 mL) and 57.1% of large VM (p = 0.167). D-dimer was significantly raised in multifocal VM (p = 0.028) and showed increasing trend in lesions with palpable phleboliths (p = 0.101). All patients had sclerotherapy performed with indications (cosmesis 41.7%, enlarging lesion 29.2%, pain 20.8%, bleeding 8.3%). Perioperatively, bolus intravenous fluid and mannitol were given to selected patients. All patients had VM volume reduction after sclerotherapy. There were no major thromboembolic complications. Conclusion LIC with raised D-dimer level occurred in one-third of pediatric VM. It was more common in large, multifocal VM and in those with palpable phleboliths or KTS.

A Simple Thermoplastic Substrate Containing Hierarchical Silica Lamellae for High-Molecular-Weight DNA Extraction.

An inexpensive, magnetic thermoplastic nanomaterial is developed utilizing a hierarchical layering of micro- and nanoscale silica lamellae to create a high-surface-area and low-shear substrate capable of capturing vast amounts of ultrahigh-molecular-weight DNA. Extraction is performed via a simple 45 min process and is capable of achieving binding capacities up to 1 000 000 times greater than silica microparticles.

Single Molecule Hydrodynamic Separation Allows Sensitive and Quantitative Analysis of DNA Conformation and Binding Interactions in Free Solution.

Limited tools exist that are capable of monitoring nucleic acid conformations, fluctuations, and distributions in free solution environments. Single molecule free solution hydrodynamic separation enables the unique ability to quantitatively analyze nucleic acid biophysics in free solution. Single molecule fluorescent burst data and separation chromatograms can give layered insight into global DNA conformation, binding interactions, and molecular distributions. First, we show that global conformation of individual DNA molecules can be directly visualized by examining single molecule fluorescent burst shapes and that DNA exists in a dynamic equilibrium of fluctuating conformations as it is driven by Poiseuille flow through micron-sized channels. We then show that this dynamic equilibrium of DNA conformations is reflected as shifts in hydrodynamic mobility that can be perturbed using salt and ionic strength to affect packing density. Next, we demonstrate that these shifts in hydrodynamic mobility can be used to investigate hybridization thermodynamics and binding interactions. We distinguish and classify multiple interactions within a single sample, and demonstrate quantification amidst large concentration differences for the detection of rare species. Finally, we demonstrate that these differences can resolve perfect complement, 2 bp mismatched, and 3 bp mismatched sequences. Such a system can be used to garner diverse information about DNA conformation and structure, and potentially be extended to other molecules and mixed-species interactions, such as between nucleic acids and proteins or synthetic polymers.

Efficient synthesis of stably adenylated DNA and RNA adapters for microRNA capture using T4 RNA ligase 1.

MicroRNA profiling methods have become increasingly important due to the rapid rise of microRNA in both basic and translational sciences. A critical step in many microRNA profiling assays is adapter ligation using pre-adenylated adapters. While pre-adenylated adapters can be chemically or enzymatically prepared, enzymatic adenylation is preferred due to its ease and high yield. However, previously reported enzymatic methods either require tedious purification steps or use thermostable ligases that can generate side products during the subsequent ligation step. We have developed a highly efficient, template- and purification-free, adapter adenylation method using T4 RNA ligase 1. This method is capable of adenylating large amounts of adapter at ~100% efficiency and can efficiently adenylate both DNA and RNA bases. We find that the adenylation reaction speed can differ between DNA and RNA and between terminal nucleotides, leading to bias if reactions are not allowed to run to completion. We further find that the addition of high PEG levels can effectively suppress these differences.

From observation to aetiology: a case report of a twin fetus-in-fetu and a revisit of the known rarity.

A baby girl presented with an antenatal diagnosis of a retroperitoneal tumour. Postnatal imaging suggested that this mass contained two fetiform structures with spine and long bone formation. This teratomatous mass was completely excised at 3 weeks of age. Histology was consistent with twin fetuses-in-fetu, revealing two fetiform masses each with an umbilical cord connecting to a common placenta-like mass. Despite a difference in the weight of the twin fetuses-in-fetu, the level of organogenesis was identical and corresponded to fetuses of 10 weeks of gestation. Each mass had four limbs, intact skin, rib cage, intestines, anus, ambiguous genitalia, primitive brain tissue and a spine with ganglion cells in the cord. Although considered a mature teratoma in the current World Health Organization classification, the theory of formation from multiple pregnancies has been commonly implied in more recent literature. The true aetiology of this rare condition remains unclear.

Direct interrogation of DNA content distribution in nanoparticles by a novel microfluidics-based single-particle analysis.

Nonviral gene delivery holds great promise not just as a safer alternative to viral vectors in traditional gene therapy applications, but also for regenerative medicine, induction of pluripotency in somatic cells, and RNA interference for gene silencing. Although it continues to be an active area of research, there remain many challenges to the rational design of vectors. Among these, the inability to characterize the composition of nanoparticles and its distribution has made it difficult to probe the mechanism of gene transfection process, since differences in the nanoparticle-mediated transfection exist even when the same vector is used. There is a lack of sensitive methods that allow for full characterization of DNA content in single nanoparticles and its distribution among particles in the same preparation. Here we report a novel spectroscopic approach that is capable of interrogating nanoparticles on a particle-by-particle basis. Using PEI/DNA and PEI-g-PEG/DNA nanoparticles as examples, we have shown that the distribution of DNA content among these nanoparticles was relatively narrow, with the average numbers of DNA of 4.8 and 6.7 per particle, respectively, in PEI/DNA and PEI-g-PEG/DNA nanoparticles. This analysis enables a more accurate description of DNA content in polycation/DNA nanoparticles. It paves the way toward comparative assessments of various types of gene carriers and provides insights into bridging the efficiency gap between viral and nonviral vehicles.

Elimination of ligation dependent artifacts in T4 RNA ligase to achieve high efficiency and low bias microRNA capture.

Adapter ligation is a critical first step in many microRNA analysis methods including microarray, qPCR, and sequencing. Previous studies have shown that ligation bias can have dramatic effects on both the fidelity of expression profiles and reproducibility across samples. We have developed a method for high efficiency and low bias microRNA capture by 3' adapter ligation using T4 RNA ligase that does not require pooled adapters. Using a panel of 20 microRNA, we investigated the effects of ligase type, PEG concentration, ligase amount, adapter concentration, incubation time, incubation temperature, and adapter design on capture efficiency and bias. Of these factors, high PEG% was found to be critical in suppressing ligation bias. We obtained high average capture efficiency and low CV across the 20 microRNA panel, both in idealized buffer conditions (86% ± 10%) and total RNA spiking conditions (64% ± 17%). We demonstrate that this method is reliable across microRNA species that previous studies have had difficulty capturing and that our adapter design performs significantly better than the common adapter designs. Further, we demonstrate that the optimization methodology must be specifically designed for minimizing bias in order to obtain the ideal reaction parameters.

Identification of the major sex pheromone component of the scale insect, Aulacaspis murrayae Takahashi.

(R)-Solanone was identified as a female-specific compound from aerations of virgin females of the scale insect, Aulacaspis murrayae Takahashi. The stereochemistry of the insect-produced solanone was confirmed to be (R) by comparison with synthesized racemic and chiral standards on a chiral stationary phase GC column. In bioassays, males were strongly attracted to a synthesized sample of (R)-solanone, demonstrating that this compound is a sex pheromone component for this species.