Recent advances in high-resolution traveling wave-based ion mobility separations coupled to mass spectrometry.

Recently, ion mobility spectrometry-mass spectrometry (IMS-MS) has become more readily incorporated into various omics-based workflows. These growing applications are due to developments in instrumentation within the last decade that have enabled higher-resolution ion mobility separations. Two such platforms are the cyclic (cIMS) and structures for lossless ion manipulations (SLIM), both of which use traveling wave ion mobility spectrometry (TWIMS). High-resolution separations achieved with these techniques stem from the drastically increased pathlengths, on the order of 10 s of meters to >1 km, in both cIMS-MS and SLIM IMS-MS, respectively. Herein, we highlight recent developments and advances, for the period 2019-2023, in high-resolution traveling wave-based IMS-MS through instrumentation, calibration strategies, hyphenated techniques, and applications. Specifically, we will discuss applications including CCS calculations in multipass IMS-MS separations, coupling of IMS-MS with chromatography, imaging, and cryogenic infrared spectroscopy, and isomeric separations of glycans, lipids, and other small metabolites.

Sequencing Sialic Acid Positioning in Gangliosides by High-Resolution Cyclic Ion Mobility Separations Coupled with Multiple Collision-Induced Dissociation-Based Tandem Mass Spectrometry Strategies.

Gangliosides, a diverse class of glycosphingolipids, are highly abundant in neural tissue and have been implicated in numerous aging-related diseases. Their characterization with methods such as liquid chromatography-tandem mass spectrometry is often precluded by their structural complexity, isomeric heterogeneity, and lack of commercially available authentic standards. In this work, we coupled high-resolution cyclic ion mobility spectrometry with multiple collision-induced dissociation-based tandem mass spectrometry strategies to sequence the sialic acid positions in various ganglioside isomers. Initially, as a proof-of-concept demonstration, we were able to characterize the sialic acid positions in several GD1 and GT1 species. From there, we extended our approach to identify the location of N-glycolylneuraminic acid (NeuGc) residues in previously uncharacterized GD1 and GQ1 isomers. Our results highlight the potential of this presented methodology for the de novo characterization of gangliosides within complex biological matrices without the need for authentic standards.

Permethylation and Metal Adduction: A Toolbox for the Improved Characterization of Glycolipids with Cyclic Ion Mobility Separations Coupled to Mass Spectrometry.

Lipids are an important class of molecules involved in various biological functions but remain difficult to characterize through mass-spectrometry-based methods because of their many possible isomers. Glycolipids, specifically, play important roles in cell signaling but display an even greater level of isomeric heterogeneity as compared to other lipid classes stemming from the introduction of a carbohydrate and its corresponding linkage position and α/ß anomericity at the headgroup. While liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) remains the gold standard technique in lipidomics, it is still unable to characterize all isomeric species, thus presenting the need for new, orthogonal, methodologies. Ion mobility spectrometry-mass spectrometry (IMS-MS) can provide an additional dimension of information that supplements LC-MS/MS workflows, but has seen little use for glycolipid analyses. Herein, we present an analytical toolbox that enables the characterization of various glycolipid isomer sets using high-resolution cyclic ion mobility separations coupled with mass spectrometry (cIMS-MS). Specifically, we utilized a combination of both permethylation and metal adduction to fully resolve isomeric sphingolipids and ceramides with our cIMS-MS platform. We also introduce a new metric that can enable comparing peak-to-peak resolution across varying cIMS-MS pathlengths. Overall, we envision that our presented methodologies are highly amenable to existing LC-MS/MS-based workflows and can also have broad utility toward other omics-based analyses.

Ultra-Fast Ion Mobility Spectrometer for High-Throughput Chromatography.

Fast chromatography systems especially developed for high sample throughput applications require sensitive detectors with a high repetition rate. These high throughput techniques, including various chip-based microfluidic designs, often benefit from detectors providing subsequent separation in another dimension, such as mass spectrometry or ion mobility spectrometry (IMS), giving additional information about the analytes or monitoring reaction kinetics. However, subsequent separation is required at a high repetition rate. Here, we therefore present an ultra-fast drift tube IMS operating at ambient pressure. Short drift times while maintaining high resolving power are reached by several key instrumental design features: short length of the drift tube, resistor network of the drift tube, tristate ion shutter, and improved data acquisition electronics. With these design improvements, even slow ions with a reduced mobility of just 0.94 cm2/(V s) have a drift time below 1.6 ms. Such short drift times allow for a significantly increased repetition rate of 600 Hz compared with previously reported values. To further reduce drift times and thus increase the repetition rate, helium can be used as the drift gas, which allows repetition rates of up to 2 kHz. Finally, these significant improvements enable IMS to be used as a detector following ultra-fast separation including chip-based chromatographic systems or droplet microfluidic applications requiring high repetition rates.

The dependence of reduced mobility, ion-neutral collisional cross sections, and alpha values on reduced electric field strengths in ion mobility.

As ion mobility spectrometry (IMS) is used with mass spectrometry in more applications, increased emphasis is placed on the ion-neutral collisional cross sections (CCS) to identify unknown analytes in complex matrices. While CCS values can provide useful information about relative analyte size, several critical assumptions are inherent in the most common method of calculating CCS values, the Mason-Schamp equation. The largest source of error in the Mason-Schamp equation originates from not accounting for higher reduced electric field strengths, which are present in low-pressure instruments that require calibration. Previous corrections based on field strength have been proposed in literature, but their data used atomic ions in atomic gases, whereas most applications examine molecules measured in nitrogen. Here, we use a series of halogenated anilines measured in air and nitrogen between 6-120 Td on a first principles ion mobility instrument (HiKE-IMS). With this series of measurements, the average velocity of the ion packet is known allowing for direct calculation of reduced mobilities (K0), alpha functions, and finally, a detailed examination of CCS as a function of E/N. In the worst-case scenario, there is over a 55% difference in CCS values for molecular ions measured at high fields depending on the method used. When comparing CCS values to those in a database for unknown identification, this difference can lead to misidentification. To immediately alleviate some of the error in calibration procedures, we propose an alternative method using K0 and alpha functions that simulate first principles mobilities at higher fields.

The origin of isomerization of aniline revealed by high kinetic energy ion mobility spectrometry (HiKE-IMS).

Although aniline is a relatively simple small molecule, the origin of its two peaks observed in ion mobility spectrometry (IMS) has remained under debate for at least 30 years. First hypothesized as a difference in protonation site (amine vs. benzene ring), each ion mobility peak differs by one Dalton when coupled with mass spectrometry where the faster mobility peak is the molecular ion peak, and the slower mobility peak is protonated. To complicate the deconvolution of structures, some previous literature shows the peaks as unresolved and thus proposes these species exist in equilibrium. In this work, we show that when measured with high kinetic energy ion mobility spectrometry (HiKE-IMS), the two peaks observed in spectra of both aniline and all n-fluoroanilines are fully separated (chromatographic resolution from 2-7, Rp > 110) and therefore not in equilibrium. The HiKE-IMS is capable of changing ionization conditions independently of drift region conditions, and our results agree with previous literature showing that ionization source settings (including possible fragmentation at this stage) are the only influence determining the speciation of the two aniline peaks. Finally, when the drift and reactant gas are changed to nitrogen, a third peak appears at high E/N for 2-fluoroaniline and 4-fluoroaniline for the first time in reported literature. As observed by HiKE-IMS-MS, the new third peak is also protonated showing that the para-protonated aniline and resulting fragment ion, molecular ion aniline, can be fully separated in the mobility domain for the first time. The appearance of the third peak is only possible due to the increased separation of the other two peaks within the HiKE-IMS.

Sparse Lissajous scanning reflectance confocal microscope with an adjustable field of view and fast iterative Fourier filtering reconstruction.

Medical imaging devices are becoming increasingly compact, necessitating optimization research into different methods of actuation. Actuation influences important parameters of the imaging device such as size, weight, frame rate, field of view (FOV), and image reconstruction for imaging devices point scanning techniques. Current literature around piezoelectric fiber cantilever actuators focuses on device optimization with a fixed FOV but neglects adjustability. In this paper, we introduce an adjustable FOV piezoelectric fiber cantilever microscope and provide a characterization and optimization procedure. To overcome calibration challenges, we utilize a position sensitive detector (PSD) and address trade-offs between FOV and sparsity with a novel inpainting technique. Our work demonstrates the potential for scanner operation when sparsity and distortion dominate the FOV, extending the usable FOV for this form of actuation and others that currently only operate under ideal imaging conditions.

Long-Term Outcomes in Patients With Peripheral Arterial Disease Who Undergo Free Flap Reconstruction for Chronic Lower Extremity Wounds.

BACKGROUND: Free tissue transfer (FTT) is critical for limb salvage of chronic lower extremity (LE) wounds. In patients with peripheral arterial disease (PAD), FTT LE reconstruction can be challenging due to limited vessel selection for anastomosis. The study aims to evaluate our surgical and functional outcomes after FTT to LE in patients with PAD. METHODS: A retrospective review identified patients who underwent LE free flap reconstruction between 2011 and 2021. All patients underwent preoperative arteriogram and subsequent FTT. Patients were classified into PAD or non-PAD cohorts, based on the presence of LE arterial stenoses or occlusions identified on arteriogram. Primary outcomes included complications, flap success, need for post-FTT vascular reintervention, limb salvage, and ambulatory status. RESULTS: A total of 253 patients underwent FTT to LE, with 84 patients (33.2%) in the PAD cohort. Patients with PAD had a higher prevalence of diabetes (83.3% vs 39.1%, P < 0.001) and end-stage renal disease (8.3% vs 2.4%, P = 0.028). Osteomyelitis was more common in the PAD group (73.8% vs 55.0%, P = 0.004). Free tissue transfer donor sites and flap composition were similar between cohorts. At a mean follow-up of 21.1 months, limb salvage rates were similar between non-PAD and PAD cohorts (90.5% vs 84.5%, P = 0.158), with no significant differences in ambulatory status or mortality. Higher complication rates occurred in the PAD cohort (38.1% vs 20.7%, P = 0.003), of which partial flap necrosis was more prevalent in the PAD group (6.0% vs 0.6%, P = 0.016). There was no difference in flap success rates between groups (P = 0.430). More postflap angiograms were performed in the PAD group (29.8% vs 7.1%, P < 0.001), with repeat percutaneous endovascular intervention performed in 68.0% of the PAD group versus 33.3% of the non-PAD group (P < 0.001). CONCLUSIONS: This is the largest study to demonstrate excellent long-term limb salvage outcomes in patients with PAD who undergo FTT to LE. Percutaneous endovascular intervention and FTT are effective methods to achieve limb salvage in vasculopathic patients with chronic LE wounds.

Functional and Phenotypic Characterisations of Common Syngeneic Tumour Cell Lines as Estrogen Receptor-Positive Breast Cancer Models.

Estrogen receptor-positive breast cancers (ER+ BCas) are the most common form of BCa and are increasing in incidence, largely due to changes in reproductive practices in recent decades. Tamoxifen is prescribed as a component of standard-of-care endocrine therapy for the treatment and prevention of ER+ BCa. However, it is poorly tolerated, leading to low uptake of the drug in the preventative setting. Alternative therapies and preventatives for ER+ BCa are needed but development is hampered due to a paucity of syngeneic ER+ preclinical mouse models that allow pre-clinical experimentation in immunocompetent mice. Two ER-positive models, J110 and SSM3, have been reported in addition to other tumour models occasionally shown to express ER (for example 4T1.2, 67NR, EO771, D2.0R and D2A1). Here, we have assessed ER expression and protein levels in seven mouse mammary tumour cell lines and their corresponding tumours, in addition to their cellular composition, tamoxifen sensitivity and molecular phenotype. By immunohistochemical assessment, SSM3 and, to a lesser extent, 67NR cells are ER+. Using flow cytometry and transcript expression we show that SSM3 cells are luminal in nature, whilst D2.0R and J110 cells are stromal/basal. The remainder are also stromal/basal in nature; displaying a stromal or basal Epcam/CD49f FACS phenotype and stromal and basal gene expression signatures are overrepresented in their transcript profile. Consistent with a luminal identity for SSM3 cells, they also show sensitivity to tamoxifen in vitro and in vivo. In conclusion, the data indicate that the SSM3 syngeneic cell line is the only definitively ER+ mouse mammary tumour cell line widely available for pre-clinical research.

Heterotypic Stressors Unmask Behavioral Influences of PMAT Deficiency in Mice.

Certain life stressors having enduring physiological and behavioral consequences, in part by eliciting dramatic signaling shifts in monoamine neurotransmitters. High monoamine levels can overwhelm selective transporters like the serotonin transporter. This is when polyspecific transporters like plasma membrane monoamine transporter (PMAT, Slc29a4) are hypothesized to contribute most to monoaminergic signaling regulation. Here, we employed two distinct counterbalanced stressors-fear conditioning and swim stress-in mice to systematically determine how reductions in PMAT function affect heterotypic stressor responsivity. We hypothesized that male heterozygotes would exhibit augmented stressor responses relative to female heterozygotes. Decreased PMAT function enhanced context fear expression, an effect unexpectedly obscured by a sham stress condition. Impaired cued fear extinction retention and enhanced context fear expression in males were conversely unmasked by a sham swim condition. Abrogated corticosterone levels in male heterozygotes that underwent swim stress after context fear conditioning did not map onto any measured behaviors. In sum, male heterozygous mouse fear behaviors proved malleable in response to preceding stressor or sham stress exposure. Combined, these data indicate that reduced male PMAT function elicits a form of stress-responsive plasticity. Future studies should assess how PMAT is differentially affected across sexes and identify downstream consequences of the stress-shifted corticosterone dynamics.

Agroforestry perennials reduce nitrous oxide emissions and their live and dead trees increase ecosystem carbon storage.

Agroforestry systems (AFS) contribute to carbon (C) sequestration and reduction in greenhouse gas emissions from agricultural lands. However, previously understudied differences among AFS may underestimate their climate change mitigation potential. In this 3-year field study, we assessed various C stocks and greenhouse gas emissions across two common AFS (hedgerows and shelterbelts) and their component land uses: perennial vegetated areas with and without trees (woodland and grassland, respectively), newly planted saplings in grassland, and adjacent annual cropland in central Alberta, Canada. Between 2018 and 2020 (~April-October), nitrous oxide emissions were 89% lower under perennial vegetation relative to the cropland (0.02 and 0.18 g N m-2  year-1 , respectively). In 2020, heterotrophic respiration in the woodland was 53% lower in shelterbelts relative to hedgerows (279 and 600 g C m-2  year-1 , respectively). Within the woodland, deadwood C stock was particularly important in hedgerows (35 Mg C ha-1 or 7% of ecosystem C) relative to shelterbelts (2 Mg C ha-1 or <1% of ecosystem C), and likely affected C cycling differences between the woodland types by enhancing soil labile C and microbial biomass in hedgerows. Deadwood C stock was positively correlated with annual heterotrophic respiration and total (to ~100 cm depth) soil organic C, water-soluble organic C, and microbial biomass C. Total ecosystem C was 1.90-2.55 times greater within the woodland than all other land uses, with 176, 234, 237, and 449 Mg C ha-1 found in the cropland, grassland, planted saplings treatment, and woodland, respectively. Shelterbelt and hedgerow woodlands contained 2.09 and 3.03 times more C, respectively, than adjacent cropland. Our findings emphasize the importance of AFS for fostering C sequestration and reducing greenhouse gas emissions and, in particular, retaining hedgerows (legacy woodland) and their associated deadwood across temperate agroecosystems will help mitigate climate change.

Robotic Assistance for Intraocular Microsurgery: Challenges and Perspectives.

Intraocular surgery, one of the most challenging discipline of microsurgery, requires sensory and motor skills at the limits of human physiological capabilities combined with tremendously difficult requirements for accuracy and steadiness. Nowadays, robotics combined with advanced imaging has opened conspicuous and significant directions in advancing the field of intraocular microsurgery. Having patient treatment with greater safety and efficiency as the final goal, similar to other medical applications, robotics has a real potential to fundamentally change microsurgery by combining human strengths with computer and sensor-based technology in an information-driven environment. Still in its early stages, robotic assistance for intraocular microsurgery has been accepted with precaution in the operating room and successfully tested in a limited number of clinical trials. However, owing to its demonstrated capabilities including hand tremor reduction, haptic feedback, steadiness, enhanced dexterity, micrometer-scale accuracy, and others, microsurgery robotics has evolved as a very promising trend in advancing retinal surgery. This paper will analyze the advances in retinal robotic microsurgery, its current drawbacks and limitations, as well as the possible new directions to expand retinal microsurgery to techniques currently beyond human boundaries or infeasible without robotics.

The Need for Innovation in Rhinoplasty.

Rhinoplasty is a challenging surgery and results are not always perfect. There are many obstacles to achieving optimal results. Among these are inadequate instrumentation, the unpredictability of healing, imprecise planning, and many more. Furthermore, selecting patients who can most benefit from surgery is equally important. In this article, some of the more pressing areas of rhinoplasty that need innovation are discussed. From proper patient selection, to advances in education, to the standardization of training programs, to the development of sophisticated implants, the future of rhinoplasty surgery lies in continued creativity and innovation.

Timing and duration of access mat use impacts their mitigation of compaction effects from industrial traffic.

Grasslands are declining worldwide and are often impacted by industrial activities, including infrastructure development. Current best management practices for low-disturbance development on grasslands include the use of wooden access mats as temporary work platforms and roadways to mitigate soil compaction and rutting due to heavy traffic. We assessed the impacts of heavy traffic (TON), and the impacts of the same heavy equipment driven over top of access mats (AM), on soil physical, hydrological, and nutrient responses in sandy and loamy soils in the Dry Mixedgrass prairies over a 2-year period. We also assessed how the timing (early vs. late in the growing season) and duration (6 vs. 12 vs. 24 weeks) of AM and TON affected the same metrics. Compared to undisturbed soils, TON increased soil penetration resistance (15 cm depth) up to 93% in loamy and up to 101% in sandy soils, and decreased water infiltration rates from 53 to 71%, respectively. Notably, the negative impacts of TON on soil physical characteristics and hydrology were larger in sandy vs. loamy soils, and when moist soils were exposed to traffic early in the growing season. AMs were effective at mitigating soil compaction from industrial traffic when used on sandy soils. However, AM use increased the supply of total nitrogen and other plant macro- and micro-nutrients, particularly in soils subject to longer (12-24 wk) mat placement. Results indicate TON may have long-lasting effects on grassland (particularly sandy) soils, and that AM use represents an effective tool to mitigate traffic impacts. Further, early-season traffic should be avoided when soils are moist (whether with AM or not), and AMs should be placed on soils for limited durations (≤6 wk) to minimize potential nutrient losses.

Reproducibility of bone age assessment from DXA hand scans: expert versus novice.

Dual-energy x-ray absorptiometry (DXA) scans are frequently used in human biological research to study bone health and body composition. Hand-wrist scans for the assessment of skeletal maturity can also be easily obtained in immature research participants who are being scanned to assess bone health. Whilst assessment by an expert is the desired arrangement such expertise may not be available, and thus knowledge of the relative reproducibility of a trained novice and an acknowledged expert is pertinent. Here we compare the relative reproducibility of an expert and a trained novice on 41 DXA left-hand scans of adolescent males using the Tanner-Whitehouse 3 (TW3) RUS method. The trained novice showed almost perfect reproducibility when evaluating bone age from DXA hand scans compared to an expert in skeletal maturity assessment. Both observers demonstrated reproducibility good enough to suggest that the TW3 method is appropriate to use with DXA hand scans by a trained researcher.

FGF13 promotes metastasis of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) represents 10-20% of all human ductal adenocarcinomas and has a poor prognosis relative to other subtypes, due to the high propensity to develop distant metastases. Hence, new molecular targets for therapeutic intervention are needed for TNBC. We recently conducted a rigorous phenotypic and genomic characterization of four isogenic populations of MDA-MB-231 human triple-negative breast cancer cells that possess a range of intrinsic spontaneous metastatic capacities in vivo, ranging from nonmetastatic (MDA-MB-231_ATCC) to highly metastatic to lung, liver, spleen and spine (MDA-MB-231_HM). Gene expression profiling of primary tumours by RNA-Seq identified the fibroblast growth factor homologous factor, FGF13, as highly upregulated in aggressively metastatic MDA-MB-231_HM tumours. Clinically, higher FGF13 mRNA expression was associated with significantly worse relapse free survival in both luminal A and basal-like human breast cancers but was not associated with other clinical variables and was not upregulated in primary tumours relative to normal mammary gland. Stable FGF13 depletion restricted in vitro colony forming ability in MDA-MB-231_HM TNBC cells but not in oestrogen receptor (ER)-positive MCF-7 or MDA-MB-361 cells. However, despite augmenting MDA-MB-231_HM cell migration and invasion in vitro, FGF13 suppression almost completely blocked the spontaneous metastasis of MDA-MB-231_HM orthotopic xenografts to both lung and liver while having negligible impact on primary tumour growth. Together, these data indicate that FGF13 may represent a therapeutic target for blocking metastatic outgrowth of certain TNBCs. Further evaluation of the roles of individual FGF13 protein isoforms in progression of the different subtypes of breast cancer is warranted.

Assessing the Impact of Drift Gas Polarizability in Polyatomic Ion Mobility Experiments.

Due to the core assumptions of kinetic theory and the drive toward realizing reproducible gas-phase measurements, ion mobility experiments are commonly conducted in the presence of an inert, neat buffer gas, usually nitrogen or helium. Mixing drift gases in defined, static ratios can provide useful information not only for optimizing the separation of analytes but also for defining the interaction between the ion and neutral particle. In a foundational effort, we seek to validate the role of the drift gas polarizability on the observed mobility of the ions by systematically mixing drift gases to discretely access a range of bulk gas polarizabilities not given by pure drift gases. Compared to historical efforts to probe the role of polarizability on the ion-neutral collisional cross section where a linear relationship was assumed, the data collected in the present effort clearly illustrate a quadratic dependency of the ion-neutral particle collision cross section and polarizability (R2 > 0.999). When translating these data into the mobility dimension, we illustrate that the gas-phase mobility of polyatomic ions conforms to Blanc's law. These observations combined with considerations related to Langevin's polarization limit provide an experimental mechanism to estimate to what degree an ion-neutral interaction conforms to either the hard-sphere or induced-dipole model. To support these observations, additional comparisons are made with the respective reduced masses, polarizabilities, and mobilities of ions in mixtures where different degrees of hard-sphere interactions are present.

Current Management of Extraperitoneal Bladder Injuries: Results from the Multi-Institutional Genito-Urinary Trauma Study (MiGUTS).

PURPOSE: We studied the current management trends for extraperitoneal bladder injuries and evaluated the use of operative repair versus catheter drainage, and the associated complications with each approach. MATERIALS AND METHODS: We prospectively collected data on bladder trauma from 20 level 1 trauma centers across the United States from 2013 to 2018. We excluded patients with intraperitoneal bladder injury and those who died within 24 hours of hospital arrival. We separated patients with extraperitoneal bladder injuries into 2 groups (catheter drainage vs operative repair) based on their initial management within the first 4 days and compared the rates of bladder injury related complications among them. Regression analyses were used to identify potential predictors of complications. RESULTS: From 323 bladder injuries we included 157 patients with extraperitoneal bladder injuries. Concomitant injuries occurred in 139 (88%) patients with pelvic fracture seen in 79%. Sixty-seven patients (43%) initially underwent operative repair for their extraperitoneal bladder injuries. The 3 most common reasons for operative repair were severity of injury or bladder neck injury (40%), injury found during laparotomy (39%) and concern for pelvic hardware contamination (28%). Significant complications were identified in 23% and 19% of the catheter drainage and operative repair groups, respectively (p=0.55). The only statistically significant predictor for complications was bladder neck or urethral injury (RR 2.69, 95% 1.21-5.97, p=0.01). CONCLUSIONS: In this large multi-institutional cohort, 43% of patients underwent surgical repair for initial management of extraperitoneal bladder injuries. We found no significant difference in complications between the initial management strategies of catheter drainage and operative repair. The most significant predictor for complications was concomitant urethral or bladder neck injury.

Cumulative live birth rates following blastocyst- versus cleavage-stage embryo transfer in the first complete cycle of IVF: a population-based retrospective cohort study.

STUDY QUESTION: Is there a difference in the odds of a live birth following blastocyst- versus cleavage-stage embryo transfer in the first complete cycle of IVF? SUMMARY ANSWER: After adjusting for indication bias, there was not enough evidence to suggest a difference in the odds of live birth following blastocyst- versus cleavage-stage embryo transfer in the first complete cycle of IVF. WHAT IS KNOWN ALREADY: Replacement of blastocyst-stage embryos has become the dominant practice in IVF but there is uncertainty about whether this technique offers an improved chance of cumulative live birth over all fresh and frozen-thawed embryo transfer attempts associated with a single oocyte retrieval. STUDY DESIGN, SIZE, DURATION: National population-based retrospective cohort study of 100 610 couples who began their first IVF/ICSI treatment at a licenced UK clinic between 1 January 1999 and 30 July 2010. PARTICIPANTS/MATERIALS, SETTING, METHODS: Data from the Human Fertilisation and Embryology Authority (HFEA) register on IVF/ICSI treatments using autologous gametes between 1999 and 2010 were analysed. The primary outcome was the live birth rate over the first complete cycle of IVF. Cumulative live birth rates (CLBR) were compared for couples who underwent blastocyst and cleavage transfer, and the adjusted odds of live birth over the first complete cycle were estimated for each group using binary logistic regression. This analysis was repeated within groups of female age, oocytes collected and primary versus secondary infertility. Inverse probability of treatment weighting was used to account for the imbalance in couple characteristics between treatment groups. MAIN RESULTS AND THE ROLE OF CHANCE: In total, 94 294 (93.7%) couples had a cleavage-stage embryo transfer while 6316 (6.3%) received blastocysts. Over the first complete cycle of IVF/ICSI (incorporating all fresh and frozen-thawed embryo transfers associated with the first oocyte retrieval), the CLBR was increased in those who underwent blastocyst transfer (56.5%) compared to cleavage-stage embryo transfer (34.8%). However, after accounting for the imbalance between exposures, blastocyst transfer did not significantly influence the odds of live birth over the first complete cycle (adjusted odds ratio: 1.03 (0.96, 1.10)). LIMITATIONS, REASONS FOR CAUTION: Limitations of our study include the retrospective nature of the HFEA dataset and availability of linked data up until 2010. We were unable to adjust for some confounders, such as smoking status, BMI and embryo quality, as these data are not collected at national level by the HFEA. Similarly, there may be unknown couple, treatment or clinic variables that may influence our results. We were unable to assess the intended stage of embryo transfer for women who did not have an embryo replaced, and therefore excluded them from our study. Perinatal outcomes were not included in our analyses and would be a useful basis for future study. WIDER IMPLICATIONS OF THE FINDINGS: Our findings show that blastocyst-stage embryo transfer may offer an improved chance of live birth in both the first fresh and the first complete cycle of IVF/ICSI compared to cleavage-stage transfer, even in couples with typically poorer prognoses. Where possible, offering blastocyst transfer to a wider range of couples may increase cumulative success rates. STUDY FUNDING/COMPETING INTEREST(S): N.J.C. received a Wolfson Foundation Intercalated Degree Research Fellowship funded by the Wolfson Foundation, through the Royal College of Physicians. This work was supported by a Chief Scientist Office Postdoctoral Training Fellowship in Health Services Research and Health of the Public Research (Ref PDF/12/06) held by D.J.M. The views expressed here are those of the authors and not necessarily those of the Chief Scientist Office or the Wolfson Foundation. The funders did not have any role in the study design; in the collection, analysis and interpretation of data; in the writing of the report; nor in the decision to submit the paper for publication. None of the authors has any conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Characterization of a novel breast cancer cell line derived from a metastatic bone lesion of a breast cancer patient.

PURPOSE: We aimed to generate and characterize a novel cell line from a breast cancer bone metastasis to better study the progression of the disease. METHODS: The cell line, P7731, was derived from a metastatic bone lesion of a breast cancer patient and assessed for marker expression. P7731 was analyzed for DNA copy number variation, somatic mutations, and gene expression and was compared with the primary tumor. RESULTS: P7731 cells are negative for estrogen receptor alpha (ERα), progesterone receptor (PR), and HER2 (triple-negative); strongly express vimentin (100% of cells positive) and also express cytokeratins 8/18 and 19 but at lower frequencies. Flow cytometry indicates P7731 cells are predominantly CD44+/CD49f+/EpCAM-, consistent with a primitive, mesenchymal-like phenotype. The cell line is tumorigenic in immunocompromised mice. Exome sequencing identified a total of 45 and 76 somatic mutations in the primary tumor and cell line, respectively, of which 32 were identified in both samples and included mutations in known driver genes PIK3CA, TP53, and ARID1A. P7731 retains the DNA copy number alterations present in the matching primary tumor. Homozygous deletions detected in the cell line and in the primary tumor were found in regions containing three known (CDKN2A, CDKN2B, and CDKN1B) and 23 putative tumor suppressor genes. Cell line-specific gene amplification coupled with mRNA expression analysis revealed genes and pathways with potential pro-metastatic functions. CONCLUSION: This novel human breast cancer-bone metastasis cell line will be a useful model to study aspects of breast cancer biology, particularly metastasis-related changes from breast to bone.