Exploration of fs-laser ablation parameter space for 2D/3D imaging of soft and hard materials by tri-beam microscopy.

Tri-beam microscopes comprising a fs-laser beam, a Xe+ plasma focused ion beam (PFIB) and an electron beam all in one chamber open up exciting opportunities for site-specific correlative microscopy. They offer the possibility of rapid ablation and material removal by fs-laser, subsequent polishing by Xe-PFIB milling and electron imaging of the same area. While tri-beam systems are capable of probing large (mm) volumes providing high resolution microscopical characterisation of 2D and 3D images across exceptionally wide range of materials and biomaterials applications, presenting high quality/low damage surfaces to the electron beam can present a significant challenge, especially given the large parameter space for optimisation. Here the optimal conditions and artefacts associated with large scale volume milling, mini test piece manufacture, serial sectioning and surface polishing are investigated, both in terms of surface roughness and surface quality for metallic, ceramic, mixed complex phase, carbonaceous, and biological materials. This provides a good starting place for those wishing to examine large areas or volumes by tri-beam microscopy across a range of materials.

A molecular staging model for accurately dating the endometrial biopsy.

Natural variability in menstrual cycle length, coupled with rapid changes in endometrial gene expression, makes it difficult to accurately define and compare different stages of the endometrial cycle. Here we develop and validate a method for precisely determining endometrial cycle stage based on global gene expression. Our 'molecular staging model' reveals significant and remarkably synchronised daily changes in expression for over 3400 endometrial genes throughout the cycle, with the most dramatic changes occurring during the secretory phase. Our study significantly extends existing data on the endometrial transcriptome, and for the first time enables identification of differentially expressed endometrial genes with increasing age and different ethnicities. It also allows reinterpretation of all endometrial RNA-seq and array data that has been published to date. Our molecular staging model will significantly advance understanding of endometrial-related disorders that affect nearly all women at some stage of their lives, such as heavy menstrual bleeding, endometriosis, adenomyosis, and recurrent implantation failure.

Using a Quantitative High-Throughput Screening Platform to Identify Molecular Targets and Compounds as Repurposing Candidates for Endometriosis.

Endometriosis, defined as the growth of hormonally responsive endometrial-like tissue outside of the uterine cavity, is an estrogen-dependent, chronic, pro-inflammatory disease that affects up to 11.4% of women of reproductive age and gender-diverse people with a uterus. At present, there is no long-term cure, and the identification of new therapies that provide a high level of efficacy and favourable long-term safety profiles with rapid clinical access are a priority. In this study, quantitative high-throughput compound screens of 3517 clinically approved compounds were performed on patient-derived immortalized human endometrial stromal cell lines. Following assay optimization and compound criteria selection, a high-throughput screening protocol was developed to enable the identification of compounds that interfered with estrogen-stimulated cell growth. From these screens, 23 novel compounds were identified, in addition to their molecular targets and in silico cell-signalling pathways, which included the neuroactive ligand-receptor interaction pathway, metabolic pathways, and cancer-associated pathways. This study demonstrates for the first time the feasibility of performing large compound screens for the identification of new translatable therapeutics and the improved characterization of endometriosis molecular pathophysiology. Further investigation of the molecular targets identified herein will help uncover new mechanisms involved in the establishment, symptomology, and progression of endometriosis.

Reducing donor-site complications in DIEP flap breast reconstruction with closed incisional negative pressure therapy: A cost-benefit analysis.

INTRODUCTION: Deep inferior epigastric perforator (DIEP) flaps are considered the gold standard for autologous breast reconstruction but create large abdominal incisions that risk donor-site morbidity during harvest. Closed incision negative pressure therapy (ciNPT) is emerging as an effective alternative to standard postoperative dressings, but there is a paucity of data in DIEP flap donor sites. METHODS: We conducted a retrospective case-control study investigating the use of ciNPT in DIEP flap donor sites at a single institution between March 2017 and September 2021. Patients who underwent microsurgical autologous breast reconstruction with DIEP flaps were included. Patients were divided into those with donor incision sites managed with ciNPT (n = 24) and those with conventional postoperative wound dressings (n = 20). We compared patient demographics, wound drainage volumes and postoperative outcomes between the two groups. A cost-benefit analysis was employed to compare the overall costs associated with each complication and differences in length of stay between the two groups. RESULTS: There was no statistically significant difference in age, body mass index (BMI), comorbidity burden or smoking status between the two groups. Both groups had similar lengths of stay and wound drainage volumes with no readmissions or reoperations in either group. There was a statistically significant reduction in donor-site complications (p = 0.018), surgical site infections (p = 0.014) and seroma formation (p = 0.016) in those with ciNPT. Upon cost-benefit analysis, the ciNPT group had a mean reduction in cost-per-patient associated with postoperative complications of £420.77 (p = 0.031) and £446.47 (p = 0.049) when also accounting for postoperative length of stay CONCLUSION: ciNPT appears to be an effective alternative incision management system with the potential to improve complication rates and postoperative morbidity in DIEP flap donor sites. Our analysis demonstrates improved cost-benefit outweighing the increase in costs associated with ciNPT. We recommend a multicentre prospective trial with formal cost-utility analysis to strengthen these findings.

UK Guidelines for Lipomodelling of the Breast on behalf of Plastic, Reconstructive and Aesthetic Surgery and Association of Breast Surgery Expert Advisory Group.

Lipomodelling has become increasingly popular for reconstructive, aesthetic and therapeutic indications. The guidelines summarise available evidence for indications, training, technique, audit and outcomes in lipomodelling and also highlight areas for further research.

Global upper-atmospheric heating on Jupiter by the polar aurorae.

Jupiter's upper atmosphere is considerably hotter than expected from the amount of sunlight that it receives1-3. Processes that couple the magnetosphere to the atmosphere give rise to intense auroral emissions and enormous deposition of energy in the magnetic polar regions, so it has been presumed that redistribution of this energy could heat the rest of the planet4-6. Instead, most thermospheric global circulation models demonstrate that auroral energy is trapped at high latitudes by the strong winds on this rapidly rotating planet3,5,7-10. Consequently, other possible heat sources have continued to be studied, such as heating by gravity waves and acoustic waves emanating from the lower atmosphere2,11-13. Each mechanism would imprint a unique signature on the global Jovian temperature gradients, thus revealing the dominant heat source, but a lack of planet-wide, high-resolution data has meant that these gradients have not been determined. Here we report infrared spectroscopy of Jupiter with a spatial resolution of 2 degrees in longitude and latitude, extending from pole to equator. We find that temperatures decrease steadily from the auroral polar regions to the equator. Furthermore, during a period of enhanced activity possibly driven by a solar wind compression, a high-temperature planetary-scale structure was observed that may be propagating from the aurora. These observations indicate that Jupiter's upper atmosphere is predominantly heated by the redistribution of auroral energy.

Revision Surgery to Improve Cosmesis with Immediate Implant-Based Breast Reconstruction.

BACKGROUND: Following mastectomy for breast cancer, patients may be presented with a range of reconstructive options. The most popular being immediate implant-based reconstruction (IBR). OBJECTIVE: To determine the rate of revision surgery to improve cosmesis following IBR. DESIGN: Retrospective cohort study. SETTING/PATIENTS: All patients who underwent IBR at a single UK-based specialist breast reconstructive centre between June 2012 and June 2013. MEASUREMENTS: The authors collected data, including demographics, original surgery, revision surgeries and factors likely to influence the cosmetic result. RESULTS: A total of 88 procedures were included in the study and follow up was performed for a mean duration of 1125 days. In all, 39 breasts required further revision to improve cosmesis to undergo a total of 53 additional procedures. Lipomodelling was the most frequently performed revision (n = 18), whilst implant exchange (n = 16), implant removal (n = 11) and other minor revisions (n = 8) made up the remainder. An early (<3 months) complication, adjuvant radiotherapy and capsular contracture significantly increased the chance of revision surgery (p = 0.018, p = 0.04 and p = 0.009, respectively). Revision surgery added an additional monetary cost of 27.1%-74.1%, which depends on the original procedure performed. LIMITATIONS: The risk of further revision surgery is likely to be higher in those who are followed up for longer periods. CONCLUSIONS: Following immediate IBR, revision surgery represents a substantial burden to the patient including healthcare costs.

Atmospheric implications of the lack of H3+ detection at Neptune.

H3+ has been detected at all of the solar system giant planets aside from Neptune. Current observational upper limits imply that there is far less H3+ emission at Neptune than rudimentary modelling would suggest. Here, we explore via modelling a range of atmospheric conditions in order to find some that could be consistent with observational constraints. In particular, we consider that the upper atmosphere might be much cooler than it was during the 1989 Voyager 2 encounter, and we examine the impact of an enhanced influx of external material that could act to reduce H3+ density. Resulting ionosphere models that are consistent with existing H3+ observational constraints have an exospheric temperature of 450 K or less, 300 K lower than the Voyager 2 value. Alternatively, if a topside CO influx of 2 × 108 cm-2 s-1 is imposed, the upper atmospheric temperature can be higher, up to 550 K. The potential cooling of Neptune's atmosphere is relevant for poorly understood giant planet thermospheric energetics, and would also impact aerobreaking manoeuvers for any future spacecraft. Such a large CO influx, if present, could imply Triton is a very active moon with prominent atmospheric escape, and/or that Neptune's rings significantly modify its upper atmosphere, and the introduction of so much exogenic material would complicate interpretation of the origin of species observed in Neptune's lower atmosphere. This article is part a discussion meeting issue 'Future exploration of ice giant systems'.

Endometrial uNK cell counts do not predict successful implantation in an IVF population.

STUDY QUESTION: Are uterine natural killer (uNK) cell numbers and their distribution relative to endometrial arterioles altered in women with recurrent implantation failure (RIF) compared to women with embryo implantation success (IS)? SUMMARY ANSWER: uNK cell numbers and their distribution relative to endometrial arterioles are not significantly different in women with RIF compared to women in whom embryo implantation occurs successfully following IVF. WHAT IS ALREADY KNOWN: uNK cells are regulators of decidual angiogenesis and spiral arteriole remodelling during early pregnancy. Although some studies have shown that uNK cell numbers may be altered in women with RIF, the methods used to measure uNK cell numbers have proven inconsistent, making reproduction of these results difficult. It is unclear, therefore, whether the results reported so far are reproducible. Moreover, it is not known how uNK cell numbers may impact IVF outcomes. Despite the lack of conclusive evidence, uNK cell numbers are often evaluated as a prognostic criterion in women undergoing assisted reproductive procedures. STUDY DESIGN, SIZE, DURATION: Endometrial pipelle biopsies were collected 6-8 days post-LH surge in natural cycles from women with RIF (n = 14), women with IS (n = 11) and women with potential RIF at the time of the study (PRIF; n = 9) from 2013 to 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS: uNK cells (i.e. CD56+ and/or CD16+ phenotypes) and their distribution relative to endometrial arterioles were investigated by standard immunohistochemistry protocols and quantified using Aperio ScanScopeXT images digitized by ImageJ and deconvoluted into binary images for single cell quantification using a Gaussian Blur and Yen algorithm. MAIN RESULTS AND THE ROLE OF CHANCE: There was no significant difference in the cell density of CD56+ or CD16+ uNK cells in women with RIF compared to women with IS or PRIF. There was a higher proportion of uNK cells in the distal regions compared to the regions closest to the arterioles in all patient groups. Further, we identified a significant reduction in uNK cell density in women who had a previous pregnancy compared to those who had not, regardless of their current implantation status. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: Spiral arterioles could not always be accurately identified by digital image analysis; therefore, all endometrial arterioles were selected and analysed. Patient numbers for the study were low. However, as the clinical phenotypes of each patient were well defined, and endometrial dating was accurately determined by three independent pathologists, differences between patient groups with respect to the uNK numbers and distribution should have been measurable if uNK cell counts were to be useful as a prognostic marker of RIF. WIDER IMPLICATIONS OF THE FINDINGS: Our findings demonstrate that CD56+ and CD16+ uNK cell numbers are not significantly different in women with RIF in a typical cohort of women undergoing IVF. Further, prior pregnancy was associated with a significantly reduced number of uNK cells in both the RIF and IS patient groups, suggestive of a long-term pregnancy induced suppression of uNK cells. Combined, these findings do not support the clinical value of using uNK cell numbers as a prognostic indicator of implantation success with IVF treatment. STUDY FUNDING/COMPETING INTEREST(S): Funding for this work was provided by Royal Women's Hospital Foundation. P.P. was supported by an NHMRC Early Career Fellowship [TF 11/14] and W.T.T. was supported by an NHMRC Postgraduate Scholarship [1055814]. The authors do not have any competing interests with this study.

Why is the H3+ hot spot above Jupiter's Great Red Spot so hot?

Recent observations of Jupiter's Great Red Spot indicate that the thermosphere above the storm is hotter than its surroundings by more than 700 K. Possible suggested sources for this heating have thus far included atmospheric gravity waves and lightning-driven acoustic waves. Here, we propose that Joule heating, driven by Great Red Spot vorticity penetrating up into the lower stratosphere and coupling to the thermosphere, may contribute to the large observed temperatures. The strength of Joule heating will depend on the local inclination angle of the magnetic field and thus the observed emissions and inferred temperatures should vary with planetary longitude as the Great Red Spot tracks across the planet. This article is part of a discussion meeting issue 'Advances in hydrogen molecular ions: H3+, H5+ and beyond'.

Modelling H3+ in planetary atmospheres: effects of vertical gradients on observed quantities.

Since its detection in the aurorae of Jupiter approximately 30 years ago, the H3+ ion has served as an invaluable probe of giant planet upper atmospheres. However, the vast majority of monitoring of planetary H3+ radiation has followed from observations that rely on deriving parameters from column-integrated paths through the emitting layer. Here, we investigate the effects of density and temperature gradients along such paths on the measured H3+ spectrum and its resulting interpretation. In a non-isothermal atmosphere, H3+ column densities retrieved from such observations are found to represent a lower limit, reduced by 20% or more from the true atmospheric value. Global simulations of Uranus' ionosphere reveal that measured H3+ temperature variations are often attributable to well-understood solar zenith angle effects rather than indications of real atmospheric variability. Finally, based on these insights, a preliminary method of deriving vertical temperature structure is demonstrated at Jupiter using model reproductions of electron density and H3+ measurements. The sheer diversity and uncertainty of conditions in planetary atmospheres prohibits this work from providing blanket quantitative correction factors; nonetheless, we illustrate a few simple ways in which the already formidable utility of H3+ observations in understanding planetary atmospheres can be enhanced. This article is part of a discussion meeting issue 'Advances in hydrogen molecular ions: H3+, H5+ and beyond'.

The H3+ ionosphere of Uranus: decades-long cooling and local-time morphology.

The upper atmosphere of Uranus has been observed to be slowly cooling between 1993 and 2011. New analysis of near-infrared observations of emission from H3+ obtained between 2012 and 2018 reveals that this cooling trend has continued, showing that the upper atmosphere has cooled for 27 years, longer than the length of a nominal season of 21 years. The new observations have offered greater spatial resolution and higher sensitivity than previous ones, enabling the characterization of the H3+ intensity as a function of local time. These profiles peak between 13 and 15 h local time, later than models suggest. The NASA Infrared Telescope Facility iSHELL instrument also provides the detection of a bright H3+ signal on 16 October 2016, rotating into view from the dawn sector. This feature is consistent with an auroral signal, but is the only of its kind present in this comprehensive dataset. This article is part of a discussion meeting issue 'Advances in hydrogen molecular ions: H3+, H5+ and beyond'.

Additive manufacturing titanium components with isotropic or graded properties by hybrid electron beam melting/hot isostatic pressing powder processing.

A methodology has been demonstrated to consolidate Ti-6Al-4V powder without taking it to the liquid state by novel combination of the electron beam melting additive manufacture and hot isostatic pressing processes. This results in improved static mechanical properties (both strength and yield) in comparison to standard EBM processed material. In addition, the ability to generate microstructurally graded components has been demonstrated by generating a component with a significant change in both microstructure and mechanical properties. This is revealed by the use of electron backscattered diffraction and micro hardness testing to produce maps showing a clear distinction between materials consolidated in different ways. The variation in microstructure and mechanical properties is attributed to the different thermal history experienced by the material at different locations. In particular, it is found that the rapid cooling experienced during EBM leads to a typical fine α lath structure, whereas a more equiaxed α grains generated by diffusion is found in HIP consolidated powder.

Generation of immortalized human endometrial stromal cell lines with different endometriosis risk genotypes.

Endometriotic lesions are composed in part of endometrial-like stromal cells, however, there is a shortage of immortalized human endometrial stromal cultures available for research. As genetic factors play a role in endometriosis risk, it is important that genotype is also incorporated into analysis of pathological mechanisms. Human telomerase reverse transcriptase (hTERT) immortalization (using Lenti-hTERT-green fluorescent protein virus) took place following genotype selection; 13 patients homozygous for either the risk or non-risk 'other' allele for one or more important endometriosis risk single nucleotide polymorphism on chromosome 1p36.12 (rs3820282, rs56318008, rs55938609, rs12037376, rs7521902 or rs12061255). Short tandem repeat DNA profiling validated that donor tissue matched that of the immortalized cell lines and confirmed that cultures were genetically novel. Expression of morphological markers (vimentin and cytokeratin) and key genes of interest (telomerase, estrogen and progesterone receptors and LINC00339) were examined and functional assays for cell proliferation, steroid hormone and inflammatory responses were performed for 7/13 cultures. All endometrial stromal cell lines maintained their fibroblast-like morphology (vimentin-positive) and homozygous endometriosis-risk genotype following introduction of hTERT. Furthermore, the new stromal cultures demonstrated positive and diverse responses to hormones (proliferation and decidualisation changes) and inflammation (dose-dependent response), while maintaining hormone receptor expression. In conclusion, we successfully developed a range of human endometrial stromal cell lines that carry important endometriosis-risk alleles. The wider implications of this approach go beyond advancing endometriosis research; these cell lines will be valuable tools for multiple endometrial pathologies offering a level of genetic and phenotypic diversity not previously available.

Characterization of Diffuse Intrinsic Pontine Glioma Radiosensitivity using Synchrotron Microbeam Radiotherapy and Conventional Radiation Therapy In Vitro.

Synchrotron microbeam radiation therapy is a promising preclinical radiotherapy modality that has been proposed as an alternative to conventional radiation therapy for diseases such as diffuse intrinsic pontine glioma (DIPG), a devastating pediatric tumor of the brainstem. The primary goal of this study was to characterize and compare the radiosensitivity of two DIPG cell lines (SF7761 and JHH-DIPG-1) to microbeam and conventional radiation. We hypothesized that these DIPG cell lines would exhibit differential responses to each radiation modality. Single cell suspensions were exposed to microbeam (112, 250, 560, 1,180 Gy peak dose) or conventional (2, 4, 6 and 8 Gy) radiation to produce clonogenic cell-survival curves. Apoptosis induction and the cell cycle were also analyzed five days postirradiation using flow cytometry. JHH-DIPG-1 cells displayed greater radioresistance than SF7761 to both microbeam and conventional radiation, with higher colony formation and increased accumulation of G2/M-phase cells. Apoptosis was significantly increased in SF7761 cells compared to JHH-DIPG-1 after microbeam irradiation, demonstrating cell-line specific differential radiosensitivity to microbeam radiation. Additionally, biologically equivalent doses to microbeam and conventional radiation were calculated based on clonogenic survival, furthering our understanding of the response of cancer cells to these two radiotherapy modalities.

Radioactive seed localization compared with wire-guided localization of non-palpable breast carcinoma in breast conservation surgery- the first experience in the United Kingdom.

OBJECTIVE: In the UK, guidewires have traditionally been used for localization of non-palpable breast lesions in patients undergoing breast conservation surgery (BCS). Radioactive seed localization (RSL) using Iodine-125 seeds is an alternative localization method and involves inserting a titanium capsule, containing radioactive Iodine-125, into the breast lesion. We aim to demonstrate feasibility of RSL compared with guidewire-localization (GWL) for BCS in the UK. METHODS: Data were collected on 100 patients with non-palpable unifocal invasive carcinoma of the breast undergoing GWL WLE prior to the introduction of RSL and the first 100 patients treated with RSL WLE. Statistical comparisons were made using Χ2-squared analysis or unpaired two-sample t-test. Significance was determined to be at p ≤ 0.05. RESULTS: Mean total tumour size was 19.44 mm (range: 5-55) in the GWL group and 18.61 mm (range: 3.8-59) in the RSL group (p = 0.548), while mean total specimen excision weight was significantly lower in the RSL group; 31.55 g (range: 4.5-112) vs 37.42 g (range: 7.8-157.1) (p = 0.018). Although 15 patients had inadequate surgical resection margins in the GWL group compared the 13 in the RSL group (15 vs 13%, respectively, p = 0.684), 10 of the patients in the GWL group had invasive carcinoma present resulting in at least one positive margin compared with only 3 patients in the RSL group (10 vs 3%, respectively, p = 0.045). CONCLUSION: In this study, RSL is shown to be non-inferior to the use of GWL for non-palpable carcinoma in patients undergoing BCS and we suggest that it could be introduced successfully in other breast units. Advances in knowledge: Here we have demonstrated the use of RSL localization results in significant lower weight resection specimens of breast carcinoma when compared with a matched group using GWL, without any significant differences in oncological outcome between the groups.

Characterization of Diffuse Intrinsic Pontine Glioma Radiosensitivity using Synchrotron Microbeam Radiotherapy and Conventional Radiation Therapy In Vitro.

Synchrotron microbeam radiation therapy is a promising preclinical radiotherapy modality that has been proposed as an alternative to conventional radiation therapy for diseases such as diffuse intrinsic pontine glioma (DIPG), a devastating pediatric tumor of the brainstem. The primary goal of this study was to characterize and compare the radiosensitivity of two DIPG cell lines (SF7761 and JHH-DIPG-1) to microbeam and conventional radiation. We hypothesized that these DIPG cell lines would exhibit differential responses to each radiation modality. Single cell suspensions were exposed to microbeam (112, 250, 560, 1,180 Gy peak dose) or conventional (2, 4, 6 and 8 Gy) radiation to produce clonogenic cell-survival curves. Apoptosis induction and the cell cycle were also analyzed five days postirradiation using flow cytometry. JHH-DIPG-1 cells displayed greater radioresistance than SF7761 to both microbeam and conventional radiation, with higher colony formation and increased accumulation of G2/M-phase cells. Apoptosis was significantly increased in SF7761 cells compared to JHH-DIPG-1 after microbeam irradiation, demonstrating cell-line specific differential radiosensitivity to microbeam radiation. Additionally, biologically equivalent doses to microbeam and conventional radiation were calculated based on clonogenic survival, furthering our understanding of the response of cancer cells to these two radiotherapy modalities.

The introduction of radioactive seed localisation improves the oncological outcome of image guided breast conservation surgery.

INTRODUCTION: Radioactive seed localisation (RSL) has become increasingly popular for localisation of non-palpable breast tumours. This is largely due to advantages it offers in terms of practicality and convenience when compared to guide wire localisation (WL). This institute switched from using WL to RSL in September 2014. The primary aim was to assess whether this change improved the accuracy of excision with regards to inadequate margin rates and weight of excision specimens. The secondary aim was to establish whether there is a "learning curve" associated with RSL technique. METHODS: Retrospective data collection was performed for 333 consecutive cases of unifocal non-palpable invasive breast cancers undergoing excision with WL or RSL. An inadequate margin was defined as tumour <1 mm from an inked radial margin. Patient demographics, tumour characteristics and clinical outcomes were compared between WL and RSL cases. RESULTS: 100 WL and 233 RSL cases were included. Patient demographics and tumour characteristics were similar for both groups. Inadequate margin rates were 18% with WL and 8.6% with RSL (p = 0.013). Median specimen weights were 33.3 g with WL and 28.7 g with RSL (p = 0.014). Subdividing the RSL group into the first 100 cases performed (RSL1) and the subsequent 133 cases (RSL2), inadequate margin rates were 13.0% and 5.3% respectively (p = 0.037). Mean specimen weights were similar. CONCLUSION: Switching from WL to RSL results in a significant reduction in both inadequate margin rates and specimen weights. A procedure-specific learning curve is present on first implementation of RSL and following this, inadequate margin rates are further reduced.