Stream-disk shocks as the origins of peak light in tidal disruption events.

Tidal disruption events (TDEs) occur when stars are ripped apart1,2 by massive black holes and result in highly luminous, multi-wavelength flares3-5. Optical-ultraviolet observations5-7 of TDEs contradict simple models of TDE emission2,8, but the debate between alternative models (for example, shock power9,10 or reprocessed accretion power11-16) remains unsettled, as the dynamic range of the problem has so far prevented ab initio hydrodynamical simulations17. Consequently, past simulations have resorted to unrealistic parameter choices10,12,18-21, artificial mass injection schemes22,23 or very short run-times24. Here we present a three-dimensional radiation-hydrodynamic simulation of a TDE flare from disruption to peak emission, with typical astrophysical parameters. At early times, shocks near pericentre power the light curve and a previously unknown source of X-ray emission, but circularization and outflows are inefficient. Near peak light, stream-disk shocks efficiently circularize returning debris, power stronger outflows and reproduce observed peak optical-ultraviolet luminosities25,26. Peak emission in this simulation is shock-powered, but upper limits on accretion power become competitive near peak light as circularization runs away. This simulation shows how deterministic predictions of TDE light curves and spectra can be calculated using moving-mesh hydrodynamics algorithms.

A statistical solution to the chaotic, non-hierarchical three-body problem.

The three-body problem is arguably the oldest open question in astrophysics and has resisted a general analytic solution for centuries. Various implementations of perturbation theory provide solutions in portions of parameter space, but only where hierarchies of masses or separations exist. Numerical integrations1 show that bound, non-hierarchical triple systems of Newtonian point particles will almost2 always disintegrate into a single escaping star and a stable bound binary3,4, but the chaotic nature of the three-body problem5 prevents the derivation of tractable6 analytic formulae that deterministically map initial conditions to final outcomes. Chaos, however, also motivates the assumption of ergodicity7-9, implying that the distribution of outcomes is uniform across the accessible phase volume. Here we report a statistical solution to the non-hierarchical three-body problem that is derived using the ergodic hypothesis and that provides closed-form distributions of outcomes (for example, binary orbital elements) when given the conserved integrals of motion. We compare our outcome distributions to large ensembles of numerical three-body integrations and find good agreement, so long as we restrict ourselves to 'resonant' encounters10 (the roughly 50 per cent of scatterings that undergo chaotic evolution). In analysing our scattering experiments, we identify 'scrambles' (periods of time in which no pairwise binaries exist) as the key dynamical state that ergodicizes a non-hierarchical triple system. The generally super-thermal distributions of survivor binary eccentricity that we predict have notable applications to many astrophysical scenarios. For example, non-hierarchical triple systems produced dynamically in globular clusters are a primary formation channel for black-hole mergers11-13, but the rates and properties14,15 of the resulting gravitational waves depend on the distribution of post-disintegration eccentricities.

CGMP Compliant Microfluidic Transfection of Induced Pluripotent Stem Cells for CRISPR-Mediated Genome Editing.

Inherited retinal degeneration is a term used to describe heritable disorders that result from the death of light sensing photoreceptor cells. Although we and others believe that it will be possible to use gene therapy to halt disease progression early in its course, photoreceptor cell replacement will likely be required for patients who have already lost their sight. While advances in autologous photoreceptor cell manufacturing have been encouraging, development of technologies capable of efficiently delivering genome editing reagents to stem cells using current good manufacturing practices (cGMP) are needed. Gene editing reagents were delivered to induced pluripotent stem cells (iPSCs) using a Zephyr microfluidic transfection platform (CellFE). CRISPR-mediated cutting was quantified using an endonuclease assay. CRISPR correction was confirmed via digital PCR and Sanger sequencing. The resulting corrected cells were also karyotyped and differentiated into retinal organoids. We describe use of a novel microfluidic transfection platform to correct, via CRISPR-mediated homology-dependent repair (HDR), a disease-causing NR2E3 mutation in patient-derived iPSCs using cGMP compatible reagents and approaches. We show that the resulting cell lines have a corrected genotype, exhibit no off-target cutting, retain pluripotency and a normal karyotype and can be differentiated into retinal tissue suitable for transplantation. The ability to codeliver CRISPR/Cas9 and HDR templates to patient-derived iPSCs without using proprietary transfection reagents will streamline manufacturing protocols, increase the safety of resulting cell therapies, and greatly reduce the regulatory burden of clinical trials.

Automating iPSC generation to enable autologous photoreceptor cell replacement therapy.

BACKGROUND: Inherited retinal degeneration is a leading cause of incurable vision loss in the developed world. While autologous iPSC mediated photoreceptor cell replacement is theoretically possible, the lack of commercially available technologies designed to enable high throughput parallel production of patient specific therapeutics has hindered clinical translation. METHODS: In this study, we describe the use of the Cell X precision robotic cell culture platform to enable parallel production of clinical grade patient specific iPSCs. The Cell X is housed within an ISO Class 5 cGMP compliant closed aseptic isolator (Biospherix XVivo X2), where all procedures from fibroblast culture to iPSC generation, clonal expansion and retinal differentiation were performed. RESULTS: Patient iPSCs generated using the Cell X platform were determined to be pluripotent via score card analysis and genetically stable via karyotyping. As determined via immunostaining and confocal microscopy, iPSCs generated using the Cell X platform gave rise to retinal organoids that were indistinguishable from organoids derived from manually generated iPSCs. In addition, at 120 days post-differentiation, single-cell RNA sequencing analysis revealed that cells generated using the Cell X platform were comparable to those generated under manual conditions in a separate laboratory. CONCLUSION: We have successfully developed a robotic iPSC generation platform and standard operating procedures for production of high-quality photoreceptor precursor cells that are compatible with current good manufacturing practices. This system will enable clinical grade production of iPSCs for autologous retinal cell replacement.

Guided principal component analysis (GPCA): a simple method for improving detection of a known analyte.

There is increasing interest in the application of Raman spectroscopy in a medical setting, ranging from supporting real-time clinical decisions e.g. surgical margins to assisting pathologists with disease classification. However, there remain a number of barriers for adoption in the medical setting due to the increased complexity of probing highly heterogeneous, dynamic biological materials. This inherent challenge can also limit the deployment of higher level analytical approaches such as Artificial Intelligence (AI) including convolutional neural networks (CNN), as there is a lack of a ground truth required for training purposes i.e. in complex clinical samples. Principal component analysis (PCA) is an unsupervised data reduction approach (orthogonal linear transformation) that has been used extensively in spectroscopy for 30+ years, due to its capability to simplify analysis of complex spectroscopic data. However, due to PCA being unsupervised features will inherently appear mixed and their rank may vary between experiments. Here we propose Guided PCA (GPCA), a simple approach that allows PCA to be guided with spectral data to ensure a consistent rank of a key target moiety by the inclusion of a reference (guiding) spectrum to the data set. This simplifies analysis, increases robustness of PCA analysis and improves quantification and the limits of detection and decreases RMSE.

Structure of the human clamp loader reveals an autoinhibited conformation of a substrate-bound AAA+ switch.

DNA replication requires the sliding clamp, a ring-shaped protein complex that encircles DNA, where it acts as an essential cofactor for DNA polymerases and other proteins. The sliding clamp needs to be opened and installed onto DNA by a clamp loader ATPase of the AAA+ family. The human clamp loader replication factor C (RFC) and sliding clamp proliferating cell nuclear antigen (PCNA) are both essential and play critical roles in several diseases. Despite decades of study, no structure of human RFC has been resolved. Here, we report the structure of human RFC bound to PCNA by cryogenic electron microscopy to an overall resolution of ∼3.4 Å. The active sites of RFC are fully bound to adenosine 5'-triphosphate (ATP) analogs, which is expected to induce opening of the sliding clamp. However, we observe the complex in a conformation before PCNA opening, with the clamp loader ATPase modules forming an overtwisted spiral that is incapable of binding DNA or hydrolyzing ATP. The autoinhibited conformation observed here has many similarities to a previous yeast RFC:PCNA crystal structure, suggesting that eukaryotic clamp loaders adopt a similar autoinhibited state early on in clamp loading. Our results point to a "limited change/induced fit" mechanism in which the clamp first opens, followed by DNA binding, inducing opening of the loader to release autoinhibition. The proposed change from an overtwisted to an active conformation reveals an additional regulatory mechanism for AAA+ ATPases. Finally, our structural analysis of disease mutations leads to a mechanistic explanation for the role of RFC in human health.

Analysis of the Chemical Distribution of Self-Assembled Microdomains with the Selective Localization of Amine-Functionalized Graphene Nanoplatelets by Optical Photothermal Infrared Microspectroscopy.

By incorporating 1-(2-aminoethyl)piperazine (AEPIP) into a commercial epoxy blend, a bicontinuous microstructure is produced with the selective localization of amine-functionalized graphene nanoplatelets (A-GNPs). This cured blend underwent self-assembly, and the morphology and topology were observed via spectral imaging techniques. As the selective localization of nanofillers in thermoset blends is rarely achieved, and the mechanism remains largely unknown, the optical photothermal infrared (O-PTIR) spectroscopy technique was employed to identify the compositions of microdomains. The A-GNP tends to be located in the region containing higher concentrations of both secondary amine and secondary alcohol; additionally, the phase morphology was found to be influenced by the amine concentration. With the addition of AEPIP, the size of the graphene domains becomes smaller and secondary phase separation is detected within the graphene domain evidenced by the chemical contrast shown in the high-resolution chemical map. The corresponding chemical mapping clearly shows that this phenomenon was mainly induced by the chemical contrast in related regions. The findings reported here provide new insight into a complicated, self-assembled nanofiller domain formed in a multicomponent epoxy blend, demonstrating the potential of O-PTIR as a powerful and useful approach for assessing the mechanism of selectively locating nanofillers in the phase structure of complex thermoset systems.

Prediction of Upstaging in Ductal Carcinoma in Situ Based on Mammographic Radiomic Features.

Background Improving diagnosis of ductal carcinoma in situ (DCIS) before surgery is important in choosing optimal patient management strategies. However, patients may harbor occult invasive disease not detected until definitive surgery. Purpose To assess the performance and clinical utility of mammographic radiomic features in the prediction of occult invasive cancer among women diagnosed with DCIS on the basis of core biopsy findings. Materials and Methods In this Health Insurance Portability and Accountability Act-compliant retrospective study, digital magnification mammographic images were collected from women who underwent breast core-needle biopsy for calcifications that was performed at a single institution between September 2008 and April 2017 and yielded a diagnosis of DCIS. The database query was directed at asymptomatic women with calcifications without a mass, architectural distortion, asymmetric density, or palpable disease. Logistic regression with regularization was used. Differences across training and internal test set by upstaging rate, age, lesion size, and estrogen and progesterone receptor status were assessed by using the Kruskal-Wallis or χ2 test. Results The study consisted of 700 women with DCIS (age range, 40-89 years; mean age, 59 years ± 10 [standard deviation]), including 114 with lesions (16.3%) upstaged to invasive cancer at subsequent surgery. The sample was split randomly into 400 women for the training set and 300 for the testing set (mean ages: training set, 59 years ± 10; test set, 59 years ± 10; P = .85). A total of 109 radiomic and four clinical features were extracted. The best model on the test set by using all radiomic and clinical features helped predict upstaging with an area under the receiver operating characteristic curve of 0.71 (95% CI: 0.62, 0.79). For a fixed high sensitivity (90%), the model yielded a specificity of 22%, a negative predictive value of 92%, and an odds ratio of 2.4 (95% CI: 1.8, 3.2). High specificity (90%) corresponded to a sensitivity of 37%, positive predictive value of 41%, and odds ratio of 5.0 (95% CI: 2.8, 9.0). Conclusion Machine learning models that use radiomic features applied to mammographic calcifications may help predict upstaging of ductal carcinoma in situ, which can refine clinical decision making and treatment planning. © RSNA, 2022.

A multi-modal exploration of heterogeneous physico-chemical properties of DCIS breast microcalcifications.

Ductal carcinoma in situ (DCIS) is frequently associated with breast calcification. This study combines multiple analytical techniques to investigate the heterogeneity of these calcifications at the micrometre scale. X-ray diffraction, scanning electron microscopy and Raman and Fourier-transform infrared spectroscopy were used to determine the physicochemical and crystallographic properties of type II breast calcifications located in formalin fixed paraffin embedded DCIS breast tissue samples. Multiple calcium phosphate phases were identified across the calcifications, distributed in different patterns. Hydroxyapatite was the dominant mineral, with magnesium whitlockite found at the calcification edge. Amorphous calcium phosphate and octacalcium phosphate were also identified close to the calcification edge at the apparent mineral/matrix barrier. Crystallographic features of hydroxyapatite also varied across the calcifications, with higher crystallinity centrally, and highest carbonate substitution at the calcification edge. Protein was also differentially distributed across the calcification and the surrounding soft tissue, with collagen and ß-pleated protein features present to differing extents. Combination of analytical techniques in this study was essential to understand the heterogeneity of breast calcifications and how this may link crystallographic and physicochemical properties of calcifications to the surrounding tissue microenvironment.

A thermophilic phage uses a small terminase protein with a fixed helix-turn-helix geometry.

Tailed bacteriophages use a DNA-packaging motor to encapsulate their genome during viral particle assembly. The small terminase (TerS) component of this DNA-packaging machinery acts as a molecular matchmaker that recognizes both the viral genome and the main motor component, the large terminase (TerL). However, how TerS binds DNA and the TerL protein remains unclear. Here we identified gp83 of the thermophilic bacteriophage P74-26 as the TerS protein. We found that TerSP76-26 oligomerizes into a nonamer that binds DNA, stimulates TerL ATPase activity, and inhibits TerL nuclease activity. A cryo-EM structure of TerSP76-26 revealed that it forms a ring with a wide central pore and radially arrayed helix-turn-helix domains. The structure further showed that these helix-turn-helix domains, which are thought to bind DNA by wrapping the double helix around the ring, are rigidly held in an orientation distinct from that seen in other TerS proteins. This rigid arrangement of the putative DNA-binding domain imposed strong constraints on how TerSP76-26 can bind DNA. Finally, the TerSP76-26 structure lacked the conserved C-terminal ß-barrel domain used by other TerS proteins for binding TerL. This suggests that a well-ordered C-terminal ß-barrel domain is not required for TerSP76-26 to carry out its matchmaking function. Our work highlights a thermophilic system for studying the role of small terminase proteins in viral maturation and presents the structure of TerSP76-26, revealing key differences between this thermophilic phage and its mesophilic counterparts.

A time-course Raman spectroscopic analysis of spontaneous in vitro microcalcifications in a breast cancer cell line.

Microcalcifications are early markers of breast cancer and can provide valuable prognostic information to support clinical decision-making. Current detection of calcifications in breast tissue is based on X-ray mammography, which involves the use of ionizing radiation with potentially detrimental effects, or MRI scans, which have limited spatial resolution. Additionally, these techniques are not capable of discriminating between microcalcifications from benign and malignant lesions. Several studies show that vibrational spectroscopic techniques are capable of discriminating and classifying breast lesions, with a pathology grade based on the chemical composition of the microcalcifications. However, the occurrence of microcalcifications in the breast and the underlying mineralization process are still not fully understood. Using a previously established model of in vitro mineralization, the MDA-MB-231 human breast cancer cell line was induced using two osteogenic agents, inorganic phosphate (Pi) and ß-glycerophosphate (ßG), and direct monitoring of the mineralization process was conducted using Raman micro-spectroscopy. MDA-MB-231 cells cultured in a medium supplemented with Pi presented more rapid mineralization (by day 3) than cells exposed to ßG (by day 11). A redshift of the phosphate stretching peak for cells supplemented with ßG revealed the presence of different precursor phases (octacalcium phosphate) during apatite crystal formation. These results demonstrate that Raman micro-spectroscopy is a powerful tool for nondestructive analysis of mineral species and can provide valuable information for evaluating mineralization dynamics and any associated breast cancer progression, if utilized in pathological samples.

Self-absorption corrected non-invasive transmission Raman spectroscopy (of biological tissue).

The first near infrared window in biological tissue (λ∼ 700-950 nm) is of great interest for its potential to safely deliver light based diagnosis and therapeutic interventions, especially in the burgeoning field of nano-theranostics. In this context, Raman spectroscopy is increasingly being used to provide rapid non-invasive chemical molecular analysis, including bulk tissue analysis by exploiting the near infrared window, with transmission Raman spectroscopy (TRS). The disadvantage of this approach, is that when probing depths of several centimetres self-attenuation artefacts are typically exhibited, whereby TRS spectra can suffer from relative changes in the "spectral features" due to differential absorption of Raman photons by the various constituents of biological tissues. Simply put, for a homogenous substance with increasing thickness, spectral variances occur due to the optical properties of the material and not through changes in the chemical environment. This can lead to misinterpretation of data, or features of interest become obscured due to the unwanted variance. Here we demonstrate a method to correct TRS data for this effect, which estimates the pathlengths derived from peak attenuation and uses expected optical properties to transform the data. In a validation experiment, the method reduced total Raman spectral intensity variances >5 fold, and improved specific peak ratio distortions 35×. This is an important development for TRS, Spatially Offset Raman Spectroscopy (SORS) and related techniques operating at depth in the near IR window; applicable to samples where there is large sample thickness and inter- and intra-sample thickness is variable i.e. clinical specimens from surgical procedures such as breast cancer. This solution is expected to yield lower detection limits and larger depths in future applications such as non-invasive breast cancer diagnosis in vivo.

An experimental and numerical modelling investigation of the optical properties of Intralipid using deep Raman spectroscopy.

In this study, Monte Carlo simulations were created to investigate the distribution of Raman signals in tissue phantoms and to validate the arctk code that was used. The aim was to show our code is capable of replicating experimental results in order to use it to advise similar future studies and to predict the outcomes. The experiment performed to benchmark our code used large volume liquid tissue phantoms to simulate the scattering properties of human tissue. The scattering agent used was Intralipid (IL), of various concentrations, filling a small quartz tank. A thin sample of PTFE was made to act as a distinct layer in the tank; this was our Raman signal source. We studied experimentally, and then reproduced via simulations, the variation in Raman signal strength in a transmission geometry as a function of the optical properties of the scattering agent and the location of the Raman material in the volume. We have also found that a direct linear extrapolation of scattering coefficients between concentrations of Intralipid is an incorrect assumption at lower concentrations when determining the optical properties. By combining experimental and simulation results, we have calculated different estimates of these scattering coefficients. The results of this study give insight into light propagation and Raman transport in scattering media and show how the location of maximum Raman signal varies as the optical properties change. The success of arctk in reproducing observed experimental signal behaviour will allow us in future to inform the development of noninvasive cancer screening applications (such as breast and prostate cancers) in vivo.

Comparing digital replantation versus revision amputation patient reported outcomes for traumatic digital amputations of the hand: A systematic review and meta-analysis.

PURPOSE: Adults with traumatic digital amputation (TDA) of the hand may be managed with replantation or revision amputation. To date, there is no systematic review evaluating patient reported outcomes (PROs) comparing replantation versus revision amputation. METHODS: Three databases (MEDLINE, EMBASE, and PubMed) were systematically searched in duplicate from inception until June 13, 2019 using Covidence software. Studies comparing replantation versus revision amputation outcomes were considered for inclusion. Methodological quality was assessed using Methodologic Index for Nonrandomized Studies (MINORS) criteria. Data were pooled in a random-effects meta-analysis model using Revman software. Certainty of evidence was evaluated using Grading of Recommendations, Assessment, Development, and Evaluations (GRADE). RESULTS: Of 4350 studies identified, 12 retrospective cohort studies met inclusion criteria and compared TDA outcomes for replantation (n = 717; 82.9% male; mean age 40.3) versus revision amputation (n = 1046; 79.8% male; mean age 41.7). The overall replantation survival rate was 85.3%. The average MINORS score was 57% (13.75/24). Replantation of the thumb had a superior Michigan Hand Questionnaire (MHQ) score (+11.88, 95% CI [7.78-15.99], I2 = 21%) compared with revision amputation. Replantation of single non-thumb digits had a superior MHQ score (+5.31, 95% CI [3.10-7.51], I2 = 67%) and Disability of Arm, Shoulder, and Hand (DASH) score (-5.16, 95% CI [-8.27 to -2.06], I2 = 0%) compared with revision amputation. Most patients in the meta-analysis were from Asian populations (87.9%). CONCLUSION: There is low-quality evidence that thumb replantation achieves superior PROs compared with revision amputation, which may be clinically important. Replantation of single non-thumb digits also yielded superior PROs, which is likely not clinically important and based on very low-quality evidence. Future studies with populations outside Asia are required to determine if PROs vary based on cultural differences toward digital amputation.

Noninvasive Detection of Differential Water Content Inside Biological Samples Using Deep Raman Spectroscopy.

Here we conceptually demonstrate the capability of deep Raman spectroscopy to noninvasively monitor changes in the water content within biological tissues. Water was added by injection into an isolated tissue volume (a 20 mm diameter disk of 5 mm thickness) representing a 20% increase in the overall mass, which was equivalent to a 5% increase in the water/tissue content. The elevated water content was detected through a larger volume of tissue with a total thickness of approximately 12 mm and a spiked tissue segment located in its center using transmission Raman spectroscopy (TRS) by monitoring the change of the OH (∼3390 cm-1) Raman band area (3350-3550 cm-1 spectral region) after being normalized to the neighboring CH stretching band. The tissue sample was raster scanned with TRS to yield a spatial map of the water concentration within the sample encompassing the spiked tissue zone. The mapping revealed the presence and location of the spiked region. The results provide the first conceptual demonstration using a deep Raman-based architecture, which can be used noninvasively for the detection of an elevated water content deep within biological tissues. It is envisaged that this concept could play a role in rapid in vivo detection and localization of cancerous lesions (generally exhibiting a higher water content) beneath the tissue surface.

Plasmonic Nanoassemblies: Tentacles Beat Satellites for Boosting Broadband NIR Plasmon Coupling Providing a Novel Candidate for SERS and Photothermal Therapy.

Optical theranostic applications demand near-infrared (NIR) localized surface plasmon resonance (LSPR) and maximized electric field at nanosurfaces and nanojunctions, aiding diagnosis via Raman or optoacoustic imaging, and photothermal-based therapies. To this end, multiple permutations and combinations of plasmonic nanostructures and molecular "glues" or linkers are employed to obtain nanoassemblies, such as nanobranches and core-satellite morphologies. An advanced nanoassembly morphology comprising multiple linear tentacles anchored onto a spherical core is reported here. Importantly, this core-multi-tentacle-nanoassembly (CMT) benefits from numerous plasmonic interactions between multiple 5 nm gold nanoparticles (NPs) forming each tentacle as well as tentacle to core (15 nm) coupling. This results in an intense LSPR across the "biological optical window" of 650-1100 nm. It is shown that the combined interactions are responsible for the broadband LSPR and the intense electric field, otherwise not achievable with core-satellite morphologies. Further the sub 80 nm CMTs boosted NIR-surface-enhanced Raman scattering (SERS), with detection of SERS labels at 47 × 10-9 m, as well as lower toxicity to noncancerous cell lines (human fibroblast Wi38) than observed for cancerous cell lines (human breast cancer MCF7), presents itself as an attractive candidate for use as biomedical theranostics agents.

Cell Mechanical and Physiological Behavior in the Regime of Rapid Mechanical Compressions that Lead to Cell Volume Change.

Cells respond to mechanical forces by deforming in accordance with viscoelastic solid behavior. Studies of microscale cell deformation observed by high speed video microscopy have elucidated a new cell behavior in which sufficiently rapid mechanical compression of cells can lead to transient cell volume loss and then recovery. This work has discovered that the resulting volume exchange between the cell interior and the surrounding fluid can be utilized for efficient, convective delivery of large macromolecules (2000 kDa) to the cell interior. However, many fundamental questions remain about this cell behavior, including the range of deformation time scales that result in cell volume loss and the physiological effects experienced by the cell. In this study, a relationship is established between cell viscoelastic properties and the inertial forces imposed on the cell that serves as a predictor of cell volume loss across human cell types. It is determined that cells maintain nuclear envelope integrity and demonstrate low protein loss after the volume exchange process. These results define a highly controlled cell volume exchange mechanism for intracellular delivery of large macromolecules that maintains cell viability and function for invaluable downstream research and clinical applications.

Label-free microfluidic enrichment of photoreceptor cells.

Inherited retinal degenerative disorders such as retinitis pigmentosa and Usher syndrome are characterized by progressive death of photoreceptor cells. To restore vision to patients blinded by these diseases, a stem cell-based photoreceptor cell replacement strategy will likely be required. Although retinal stem cell differentiation protocols suitable for generating photoreceptor cells exist, they often yield a rather heterogenous mixture of cell types. To enrich the donor cell population for one or a few cell types, scientists have traditionally relied upon the use of antibody-based selection approaches. However, these strategies are quite labor intensive and require animal derived reagents and equipment that are not well suited to current good manufacturing practices (cGMP). The purpose of this study was to develop and evaluate a microfluidic cell sorting device capable of exploiting the physical and mechanical differences between retinal cell types to enrich specific donor cell populations such as Retinal Pigment Epithelial (RPE) cells and photoreceptor cells. Using this device, we were able to separate a mixture of RPE and iPSC-derived photoreceptor precursor cell lines into two substantially enriched fractions. The enrichment factor of the RPE fraction was 2 and that of the photoreceptor precursor cell fraction was 2.7. Similarly, when human retina, obtained from 3 independent donors, was dissociated and passed through the sorting device, the heterogeneous mixture could be reliably sorted into RPE and photoreceptor cell rich fractions. In summary, microfluidic cell sorting is a promising approach for antibody free enrichment of retinal cell populations.

Correction: Prospective on using fibre mid-infrared supercontinuum laser sources for in vivo spectral discrimination of disease.

Correction for 'Prospective on using fibre mid-infrared supercontinuum laser sources for in vivo spectral discrimination of disease' by Angela B. Seddon et al., Analyst, 2018, 143, 5874-5887.

Calcification Microstructure Reflects Breast Tissue Microenvironment.

Microcalcifications are important diagnostic indicators of disease in breast tissue. Tissue microenvironments differ in many aspects between normal and cancerous cells, notably extracellular pH and glycolytic respiration. Hydroxyapatite microcalcification microstructure is also found to differ between tissue pathologies, including differential ion substitutions and the presence of additional crystallographic phases. Distinguishing between tissue pathologies at an early stage is essential to improve patient experience and diagnostic accuracy, leading to better disease outcome. This study explores the hypothesis that microenvironment features may become immortalised within calcification crystallite characteristics thus becoming indicators of tissue pathology. In total, 55 breast calcifications incorporating 3 tissue pathologies (benign - B2, ductal carcinoma in-situ - B5a and invasive malignancy - B5b) from archive formalin-fixed paraffin-embedded core needle breast biopsies were analysed using X-ray diffraction. Crystallite size and strain were determined from 548 diffractograms using Williamson-Hall analysis. There was an increased crystallinity of hydroxyapatite with tissue malignancy compared to benign tissue. Coherence length was significantly correlated with pathology grade in all basis crystallographic directions (P < 0.01), with a greater difference between benign and in situ disease compared to in-situ disease and invasive malignancy. Crystallite size and non-uniform strain contributed to peak broadening in all three pathologies. Furthermore, crystallite size and non-uniform strain normal to the basal planes increased significantly with malignancy (P < 0.05). Our findings support the view that tissue microenvironments can influence differing formation mechanisms of hydroxyapatite through acidic precursors, leading to differential substitution of carbonate into the hydroxide and phosphate sites, causing significant changes in crystallite size and non-uniform strain.