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Interactions between the immune and central nervous systems strongly influence brain health. Although the blood-brain barrier restricts this crosstalk, we now know that meningeal gateways through brain border tissues facilitate intersystem communication. Cerebrospinal fluid (CSF), which interfaces with the glymphatic system and thereby drains the brain's interstitial and perivascular spaces, facilitates outward signaling beyond the blood-brain barrier. In the present study, we report that CSF can exit into the skull bone marrow. Fluorescent tracers injected into the cisterna magna of mice migrate along perivascular spaces of dural blood vessels and then travel through hundreds of sub-millimeter skull channels into the calvarial marrow. During meningitis, bacteria hijack this route to invade the skull's hematopoietic niches and initiate cranial hematopoiesis ahead of remote tibial sites. As skull channels also directly provide leukocytes to meninges, the privileged sampling of brain-derived danger signals in CSF by regional marrow may have broad implications for inflammatory neurological disorders.

In newborn humans, and up to approximately 2 y of age, calvarial bone defects can naturally regenerate. This remarkable regeneration potential is also found in newborn mice and is absent in adult mice. Since previous studies showed that the mouse calvarial sutures are reservoirs of calvarial skeletal stem cells (cSSCs), which are the cells responsible for calvarial bone regeneration, here we hypothesized that the regenerative potential of the newborn mouse calvaria is due to a significant amount of cSSCs present in the newborn expanding sutures. Thus, we tested whether such regenerative potential can be reverse engineered in adult mice by artificially inducing an increase of the cSSCs resident within the adult calvarial sutures. First, we analyzed the cellular composition of the calvarial sutures in newborn and in older mice, up to 14-mo-old mice, showing that the sutures of the younger mice are enriched in cSSCs. Then, we demonstrated that a controlled mechanical expansion of the functionally closed sagittal sutures of adult mice induces a significant increase of the cSSCs. Finally, we showed that if a calvarial critical size bone defect is created simultaneously to the mechanical expansion of the sagittal suture, it fully regenerates without the need for additional therapeutic aids. Using a genetic blockade system, we further demonstrate that this endogenous regeneration is mediated by the canonical Wnt signaling. This study shows that controlled mechanical forces can harness the cSSCs and induce calvarial bone regeneration. Similar harnessing strategies may be used to develop novel and more effective bone regeneration autotherapies.

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The fate of circulating tumor cells is an important determinant of their ability to form distant metastasis. Here, we demonstrate the use of in vivo flow cytometry as a powerful new method for detecting quantitatively circulating cancer cells. We specifically examine the circulation kinetics of two prostate cancer cell lines with different metastatic potential in mice and rats. We find that the cell line and the host environment affect the circulation kinetics of prostate cancer cells, with the intrinsic cell line properties determining the initial rate of cell depletion from the circulation and the host affecting cell circulation at later time points.

The specific functional properties of a tissue are distributed amongst its component cell types. The various cells act coherently, as an ensemble, in order to execute a physiologic response. Modern approaches for identifying and dissecting novel physiologic mechanisms would benefit from an ability to identify specific cell types in live tissues that could then be imaged in real time. Current techniques require the use of fluorescent genetic reporters that are not only cumbersome, but which only allow the study of three or four cell types at a time. We report a non-invasive imaging modality that capitalizes on the endogenous autofluorescence signatures of the metabolic cofactors NAD(P)H and FAD. By marrying morphological characteristics with autofluorescence signatures, all seven of the airway epithelial cell types can be distinguished simultaneously in mouse tracheal explants in real time. Furthermore, we find that this methodology for direct cell type-specific identification avoids pitfalls associated with the use of ostensibly cell type-specific markers that are, in fact, altered by clinically relevant physiologic stimuli. Finally, we utilize this methodology to interrogate real-time physiology and identify dynamic secretory cell associated antigen passages (SAPs) that form in response to cholinergic stimulus. The identical process has been well documented in the intestine where the dynamic formation of SAPs and goblet cell associated antigen passages (GAPs) enable luminal antigen sampling. Airway secretory cells with SAPs are frequently juxtaposed to antigen presenting cells, suggesting that airway SAPs, like their intestinal counterparts, not only sample antigen but convey their cargo for immune cell processing.

Abnormal hematopoiesis advances cardiovascular disease by generating excess inflammatory leukocytes that attack the arteries and the heart. The bone marrow niche regulates hematopoietic stem cell proliferation and hence the systemic leukocyte pool, but whether cardiovascular disease affects the hematopoietic organ’s microvasculature is unknown. Here we show that hypertension, atherosclerosis and myocardial infarction (MI) instigate endothelial dysfunction, leakage, vascular fibrosis and angiogenesis in the bone marrow, altogether leading to overproduction of inflammatory myeloid cells and systemic leukocytosis. Limiting angiogenesis with endothelial deletion of Vegfr2 (encoding vascular endothelial growth factor (VEGF) receptor 2) curbed emergency hematopoiesis after MI. We noted that bone marrow endothelial cells assumed inflammatory transcriptional phenotypes in all examined stages of cardiovascular disease. Endothelial deletion of Il6 or Vcan (encoding versican), genes shown to be highly expressed in mice with atherosclerosis or MI, reduced hematopoiesis and systemic myeloid cell numbers in these conditions. Our findings establish that cardiovascular disease remodels the vascular bone marrow niche, stimulating hematopoiesis and production of inflammatory leukocytes.

The specific functional properties of a tissue are distributed amongst its component cell types. The various cells act coherently, as an ensemble, in order to execute a properly orchestrated physiologic response. Thus, modern approaches to dissect physiologic mechanism would benefit from an ability to identify specific cell types in live tissues and image them in real time. Current techniques require the use of fluorescent genetic reporters that are not only cumbersome, but which only allow the simultaneous study of 2 or 3 cell types. We report a non-invasive imaging modality that capitalizes on the endogenous autofluorescence signatures of the metabolic cofactors NAD(P)H and FAD. By marrying morphological characteristics with autofluorescence signatures, all seven of the airway epithelial cell types can be distinguished simultaneously in real time. Furthermore, we find that this methodology for direct cell type specific identification avoid potential pitfalls with the use of ostensibly cell type-specific markers that can be altered by clinically relevant physiologic stimuli. Finally, we utilize this methodology to interrogate real-time physiology using a clinically relevant model of cholinergic stimulation and identify dynamic secretory cell associated antigen passages (SAPs) that are highly reminiscent of previously reported goblet cell associated antigen passages (GAPs) in the intestine.eLife’s Review ProcesseLife works to improve the process of peer review so that it more effectively conveys the assessment of expert reviewers to authors, readers and other interested parties. In the future we envision a system in which research is first published as a preprint and the outputs of peer review are the primary way research is assessed, rather than journal title.Our editorial process produces two outputs: i) an assessment by peers designed to be posted alongside a preprint for the benefit of the readers;i) detailed feedback on the manuscript for the authors, including requests for revisions and suggestions for improvement.Therefore we want to change how we construct and write peer reviews to make themuseful to both authors and readers in a way that better reflects the work you put into reading and thinking about a paper.eLife reviews now have three parts:Anevaluation summary(in two or three sentences) that captures the major conclusions of the review in a concise manner, accessible to a wide audience.Apublic reviewthat details the strengths and weaknesses of the manuscript before you, and discusses whether the authors’ claims and conclusions are justified by their data.A set of privaterecommendations for the authorsthat outline how you think the science and its presentation could be strengthened.All three sections will be used as the basis for an eLife publishing decision, which will, as always, be made after a consultation among the reviewers and editor. Each of thepublic reviewswill be published (anonymously) alongside the preprint, together with a response from the authors if they choose. In the case of papers we reject after review, the authors can choose to delay posting until their paper has been published elsewhere.If this is your first time going through this new process, we ask that you take some time to read ourReviewer Guide, which discusses how we see each section will be used, what it should contain, and what we hope it accomplishes. And we remind you that, with the shift of reviews from private correspondence to public discourse, it is more important than ever that reviews are written in aclear and constructive mannerappropriate for a public audience and mindful of the impact language choices might have on the authors.

Purpose To reduce the total clinic visit duration among retina providers in an academic ophthalmology department. Methods All patient encounters across all providers in the department were analyzed to determine baseline clinic visit duration time, defined as the elapsed time between appointment time and checkout. To increase photography capacity, a major bottleneck identified through root cause analysis, four interventions were implemented: training ophthalmic technicians to perform fundus photography in addition to optical coherence tomographies, relocating photography equipment to be adjacent to examination rooms, procuring three additional Optos widefield retinal photography units, and shifting staff schedules to better align with that of the providers. These interventions were implemented in the clinics of two retina providers. Results The average baseline visit duration for all patients across all providers was 87 minutes (19,550 patient visits). The previous average visit duration was 80 minutes for Provider 1 (557 patient visits) and 81 minutes for Provider 2 (1,246 patient visits). In the 4 weeks after interventions were implemented, the average visit duration decreased to 60 minutes for Provider 1 and 57 minutes for Provider 2. Conclusion A systematic approach and a multidisciplinary team resulted in targeted, cost-effective interventions that reduced total visit durations.

Purpose To review the published literature assessing the efficacy and safety of presbyopia-correcting intraocular lenses (IOLs) for the treatment of presbyopia after cataract removal. Methods Literature searches were undertaken in January 2018 and September 2020 in the PubMed, Medline, and Cochrane Library databases. This yielded 761 articles, of which 34 met the criteria for inclusion in this assessment and were assigned a level of evidence rating by the panel methodologist. Thirteen studies were rated level I and 21 studies were rated level II. Results Presbyopia-correcting lenses were effective at improving distance and near visual acuity after cataract surgery. Near acuity at different focal lengths was related directly to the effective add power of multifocal and extended depth-of-focus (EDOF) IOLs. Most multifocal and EDOF lenses that were compared with a control monofocal lens demonstrated that patient-reported spectacle independence was superior to the monofocal lens. All patients who had multifocal and EDOF lenses implanted showed decreased contrast sensitivity and reported more visual phenomena as compared with control participants who received monofocal lenses. Conclusions Presbyopia-correcting lenses improve uncorrected near and distance visual acuity and decrease spectacle dependence after cataract surgery. Mesopic contrast sensitivity is decreased compared with monofocal lenses, and patient-reported visual phenomena are more likely in patients receiving multifocal or EDOF lenses.

Given the existing literature on the subject, there is obviously a need for specific advice on quality assurance (QA) tolerances for departments using or implementing 3D printed bolus for radiotherapy treatments. With a view to providing initial suggested QA tolerances for 3D printed bolus, this study evaluated the dosimetric effects of changes in bolus geometry and density, for a particularly common and challenging clinical situation: specifically, volumetric modulated arc therapy (VMAT) treatment of the nose. Film-based dose verification measurements demonstrated that both the AAA and the AXB algorithms used by the Varian Eclipse treatment planning system (Varian Medical Systems, Palo Alto, USA) were capable of providing sufficiently accurate dose calculations to allow this planning system to be used to evaluate the effects of bolus errors on dose distributions from VMAT treatments of the nose. Thereafter, the AAA and AXB algorithms were used to calculate the dosimetric effects of applying a range of simulated errors to the design of a virtual bolus, to identify QA tolerances that could be used to avoid clinically significant effects from common printing errors. Results were generally consistent, whether the treatment target was superficial and treated with counter-rotating coplanar arcs or more-penetrating and treated with noncoplanar arcs, and whether the dose was calculated using the AAA algorithm or the AXB algorithm. The results of this study suggest the following QA tolerances are advisable, when 3D printed bolus is fabricated for use in photon VMAT treatments of the nose: bolus relative electron density variation within [Formula: see text] (although an action level at [Formula: see text] may be permissible); bolus thickness variation within [Formula: see text] mm (or 0.5 mm variation on opposite sides); and air gap between bolus and skin [Formula: see text] mm. These tolerances should be investigated for validity with respect to other treatment modalities and anatomical sites. This study provides a set of baselines for future comparisons and a useful method for identifying additional or alternative 3D printed bolus QA tolerances.

[18F]PSMA-1007 has potential advantages over [68 Ga]Ga-PSMA-11, although limited prospective data evaluating diagnostic performance exist. The aims of this study are to describe the concordance of [18FPSMA-1007 and [68 Ga]Ga-PSMA-11 for TNM with the American Joint Committee on Cancer (AJCC) prognostic stage and assess differences in tracer uptake. Fifty men (mean age 71.8) were imaged with [68 Ga]Ga-PSMA-11 and [18F]PSMA-1007

The bone marrow (BM) has traditionally been a difficult tissue to access because it is embedded deep within the bone matrix. It is home to the hematopoietic stem cells (HSCs) that give rise to all blood cells in the body. It is also the site of origin for malignant blood cells such as leukemia and multiple myeloma, as well as a frequent site of metastasis for many solid tumors including prostate and breast cancer. The following chapter describes how laser micromachining of bone can be used to improve both optical and physical access to the BM. For example, laser thinning of the overlying bone can improve optical access, enabling deeper imaging into the BM as well as enhancing optical resolution by reducing scattering and aberration. Laser micromachining can also be used to provide physical access into the BM by creating access ports for micropipette insertion and delivery of cells to precise locations in the BM, as well as for the extraction of BM cells and interstitial fluid, all under image guidance. This chapter provides a detailed protocol for installing a laser-micromachining capability for users with an existing multiphoton microscope. Additionally, we briefly outline how such a system improves the optical resolution during imaging as well as its potential use to study injury response.

Thymic atrophy and the progressive immune decline that accompanies it is a major health problem, chronically with age and acutely with immune injury. No solution has been defined. Here we demonstrate that one of the three mesenchymal cell subsets identified by single-cell analysis of human and mouse thymic stroma is a critical niche component for T lymphopoiesis. The Postn+ subset is located perivascularly in the cortical-medullary junction, medulla and subcapsular regions. Cell depletion demonstrated that it recruits T competent cells to the thymus and initiates T lymphopoiesisin vivo. This subset distinctively expresses the chemokineCcl19necessary for niche functions. It markedly declines with age and in the acute setting of hematopoietic stem cell transplant conditioning. When isolated and adoptively transferred, these cells durably engrafted the atrophic thymus, recruited early T progenitors, increased T cell neogenesis, expanded TCR complexity and enhanced T cell response to vaccination. These data define a thymus lymphopoietic niche cell type that may be manipulated therapeutically to regenerate T lymphopoiesis.

Tissue function depends on proper cellular organization. While the properties of individual cells are increasingly being deciphered using powerful single-cell sequencing technologies, understanding their spatial organization and temporal evolution remains a major challenge. Here, we present Image-seq, a technology that provides single-cell transcriptional data on cells that are isolated from specific spatial locations under image guidance, thus preserving the spatial information of the target cells. It is compatible with in situ and in vivo imaging and can document the temporal and dynamic history of the cells being analyzed. Cell samples are isolated from intact tissue and processed with state-of-the-art library preparation protocols. The technique therefore combines spatial information with highly sensitive RNA sequencing readouts from individual, intact cells. We have used both high-throughput, droplet-based sequencing, as well as SMARTseq-v4 library preparation to demonstrate its application to bone marrow and leukemia biology. We discovered that DPP4 is a highly upregulated gene during early AML progression and that it marks a more proliferative subpopulation which is confined to specific bone marrow microenvironments. Furthermore, Image-seq’s ability to isolate viable, intact cells should make it compatible with a range of downstream single-cell analysis tools including multi-omics protocols.

[18F]PSMA-1007 offers advantages of low urinary tracer excretion and theoretical improved spatial resolution for imaging prostate cancer. However, non-specific bone lesions (NSBLs), defined as mild to moderate focal bone uptake without a typical morphological correlate on CT, are a common finding on [18F]PSMA-1007 PET/CT. The purpose of this study was to investigate the clinical outcomes of patients with [18F]PSMA-1007 avid NSBLs, to determine whether patients with NSBLs represent a higher risk clinical cohort, and to determine whether SUVmax can be used as a classifier of bone metastasis. A retrospective audit of 214 men with prostate cancer was performed to investigate the clinical outcomes of [18F]PSMA-1007 avid NSBLs according to defined criteria. We also compared the serum PSA, Gleason score, and uptake time of patients with [18F]PSMA-1007 avid NSBLs to patients without [18F]PSMA-1007 avid bone lesions. Finally, we analysed an SUVmax threshold to identify bone metastases using ROC curve analysis. Ninety-four of 214 patients (43.9%) demonstrated at least one NSBL. No [18F]PSMA-1007 avid NSBLs met criteria for a likely malignant or definitely malignant lesion after a median 15.8-month follow-up interval (11.9% definitely benign, 50.3% likely benign, and 37.7% equivocal). There were no statistically significant differences in serum PSA, Gleason score, and uptake time between patients with [18F]PSMA-1007 avid NSBLs and those without [18F]PSMA-1007 avid bone lesions. All NSBLs with adequate follow-up had SUVmax ≤ 11.1. The value of the highest SUVmax distinguished between NSBLs and definite prostate cancer bone metastases, whereby an SUVmax threshold of ≥ 7.2 maximized the Youden’s index. [18F]PSMA-1007 avid NSBLs rarely represent prostate cancer bone metastases. When identified in the absence of definite metastatic disease elsewhere, it is appropriate to classify those with SUVmax

As head-and-neck radiotherapy treatments become more complex and sophisticated, and the need to control and stabilise the positioning of intra-oral anatomy becomes more important, leading the increasing use of oral positioning stents during head-and-neck radiotherapy simulation and delivery. As an alternative to the established practice of creating oral positioning stents using wax, this study investigated the use of a 3D printing technique. An Ender 5 3D printer (Creality 3D, Shenzhen, China) was used, with PLA+ “food-safe” polylactic acid filament (3D Fillies, Dandenong South, Australia), to produce a low-density 3D printed duplicate of a conventional wax stent. The physical and dosimetric effects of the two stents were evaluated using radiochromic film in a solid head phantom that was modified to include flexible parts. The Varian Eclipse treatment planning system (Varian Medical Systems, Palo Alto, USA) was used to calculate the dose from two different head-and-neck treatment plans for the phantom with each of the two stents. Examination of the resulting four dose distributions showed that both stents effectively pushed sensitive oral tissues away from the treatment targets, even though most of the phantom was solid. Film measurements confirmed the accuracy of the dose calculations from the treatment planning system, despite the steep density gradients in the treated volume, and demonstrated that the 3D print could be a suitable replacement for the wax stent. This study demonstrated a useful method for dosimetrically testing novel oral positioning stents. We recommend the development of flexible phantoms for future studies.

Background Natural killer T (NKT) cells are unconventional T cells that bridge innate and adaptive immunity. NKT cells have been implicated in the development of atopic dermatitis (AD). Objective We aimed to investigate the role of NKT cells in AD development, especially in skin. Methods Global proteomic and transcriptomic analyses were performed by using skin and blood from human healthy–controls and patients with AD. Levels of CXCR4 and CXCL12 expression in skin NKT cells were analyzed in human AD and mouse AD models. By using parabiosis and intravital imaging, the role of skin CXCR4+ NKT cells was further evaluated in models of mice with AD by using CXCR4–conditionally deficient or CXCL12 transgenic mice. Results CXCR4 and its cognate ligand CXCL12 were significantly upregulated in the skin of humans with AD by global transcriptomic and proteomic analyses. CXCR4+ NKT cells were enriched in AD skin, and their levels were consistently elevated in our models of mice with AD. Allergen-induced NKT cells participate in cutaneous allergic inflammation. Similar to tissue-resident memory T cells, the predominant skin NKT cells were CXCR4+ and CD69+. Skin-resident NKT cells uniquely expressed CXCR4, unlike NKT cells in the liver, spleen, and lymph nodes. Skin fibroblasts were the main source of CXCL12. CXCR4+ NKT cells preferentially trafficked to CXCL12-rich areas, forming an enriched CXCR4+ tissue-resident NKT cells/CXCL12+ cell cluster that developed in acute and chronic allergic inflammation in our models of mice with AD. Conclusions CXCR4+ tissue-resident NKT cells may form a niche that contributes to AD, in which CXCL12 is highly expressed.

Purpose To review the literature to evaluate the outcomes of intraocular lens (IOL) power calculation in eyes with a history of myopic LASIK or photorefractive keratectomy (PRK). Methods Literature searches were conducted in the PubMed database in January 2020. Separate searches relevant to cataract surgery outcomes and corneal refractive surgery returned 1169 and 162 relevant citations, respectively, and the full text of 24 was reviewed. Eleven studies met the inclusion criteria for this assessment; all were assigned a level III rating of evidence by the panel methodologist. Results When automated keratometry was used with a theoretical formula designed for eyes without previous laser vision correction, the mean prediction error (MPE) was universally positive (hyperopic), the mean absolute errors (MAEs) and median absolute errors (MedAEs) were relatively high (0.72–1.9 diopters [D] and 0.65–1.73 D, respectively), and a low (8%–40%) proportion of eyes were within 0.5 D of target spherical equivalent (SE). Formulas developed specifically for this population requiring both prerefractive surgery keratometry and manifest refraction (i.e., clinical history, corneal bypass, and Feiz-Mannis) produced a proportion of eyes within 0.5 D of target SE between 26% and 44%. Formulas requiring only preoperative keratometry or no history at all had lower MAEs (0.42–0.94 D) and MedAEs (0.30–0.81 D) and higher (30%–68%) proportions within 0.5 D of target SE. Strategies that averaged several methods yielded the lowest reported MedAEs (0.31–0.35 D) and highest (66%–68%) proportions within 0.5 D of target SE. Even after using the best-known methods, refractive outcomes were less accurate in eyes that had previous excimer laser surgery for myopia compared with those that did not have it. Conclusions Calculation methods requiring both prerefractive surgery keratometry and manifest refraction are no longer considered the gold standard. Refractive outcomes of cataract surgery in eyes that had previous excimer laser surgery are less accurate than in eyes that did not. Patients should be advised of this refractive limitation when considering cataract surgery in the setting of previous corneal refractive surgery. Conclusions are limited by the small sample sizes and retrospective nature of nearly all existing literature in this domain.

Background Patients with unresectable advanced cutaneous squamous cell carcinoma (cSCC) are generally treated with palliative intent. Immune checkpoint blockade has significant activity in the palliative setting in patients with recurrent or metastatic cSCC. This single arm phase 2 prospective study aims to investigate the combination of curative intent chemoradiation and durvalumab (anti-PD-L1 checkpoint inhibitor) for this patient cohort. Methods Patients with unresectable locally and or regionally advanced pathologically confirmed cSCC (stage III-IVa) deemed fit for CRIO by consensus of the Multidisciplinary meeting will be eligible. In the first stage of a two-stage minimax design, we aim to recruit a total of 15 patients. If fewer than 7 patients achieved a complete response in the first stage, we will conclude the treatment is not more effective than standard treatment. The co-primary endpoints of CRIO are the safety of treatment (acute and late toxicities) and the rate of complete response. Secondary endpoints would include overall survival, progression free survival, and locoregional control. Translational research endpoints including biomarkers (CD73, CD39, PD-1, PD-L1) will also be explored utilising multiplex immunohistochemistry on tumour biopsy samples obtained prior to commencing treatment and during treatment (week 2). In addition, the utility of CXCR-4 PET/CT scan will be explored. Discussion CRIO is a novel trial evaluating the combination of curative intent chemoradiotherapy with concurrent and adjuvant durvalumab for patients with unresectable stage III-IVa cSCC. Trial registration: Trial registered with the Australian New Zealand Clinical Trial Registry (ACTRN12618001573246)

Background and purpose: Radiopacifiers are introduced to bone cements to provide the appearance of bone in kilovoltage (kV) radiographic images. For higher energy megavoltage (MV) radiotherapy treatment beams, however, these radiopacifiers do not cause a bone-like perturbation of dose. This study therefore aimed to determine the impact of the barium-contrasted plastic-based cement materials on radiotherapy dose calculations. Materials and methods: The radiological properties of a physical sample of bone cement were characterised by computed tomography (CT) imaging and transmission measurements. Monte Carlo simulations of percentage depth-dose profiles were performed to determine the possible dose error for MV treatment beams. Dose differences were then investigated for clinical volumetric modulated radiotherapy treatment plans, with and without density overrides applied. Results: Differences of up to 7% were observed at the downstream interface of a 0.6 cm thick bone cement layer, compared to bone. Differences in planning target volume dose-volume metrics varied between −0.5% and 2.0%. Conclusion: Before planning radiotherapy treatments for patients who have undergone cranioplasty, every effort should be made to identify whether a radiopacified bone cement has been implanted. Density overrides should be applied to minimise dose calculation errors, whenever bone cement is used.

The biology of hematopoietic stem cells (HSCs) has predominantly been studied under transplantation conditions1,2. Particularly challenging has been the study of dynamic HSC behaviors given that live animal HSC visualization in the native niche still represents an elusive goal in the field. Here, we describe a dual genetic strategy in mice that restricts reporter labeling to a subset of the most quiescent long-term HSCs (LT-HSCs) and that is compatible with current intravital imaging approaches in the calvarial bone marrow (BM)3–5. We find that this subset of LT-HSCs resides in close proximity to both sinusoidal blood vessels and the endosteal surface. In contrast, multipotent progenitor cells (MPPs) display a broader distance distribution from the endosteum and are more likely to be associated with transition zone vessels. LT-HSCs are not found in BM niches with the deepest hypoxia and instead are found in similar hypoxic environments as MPPs. In vivo time-lapse imaging reveals that LT-HSCs display limited motility at steady-state. Following activation, LT-HSCs display heterogenous responses, with some cells becoming highly motile and a fraction of HSCs expanding clonally within spatially restricted domains. These domains have defined characteristics, as HSC expansion is found almost exclusively in a subset of BM cavities exhibiting bone-remodeling activities. In contrast, cavities with low bone-resorbing activities do not harbor expanding HSCs. These findings point to a new degree of heterogeneity within the BM microenvironment, imposed by the stages of bone turnover. Overall, our approach enables direct visualization of HSC behaviors and dissection of heterogeneity in HSC niches.

We have developed a new technique for micron scale resolution cross-sectional imaging in biological systems called Optical Coherence Tomography (OCT)1,2 In OCT, low-coherence optical interferometry3,4 is used to resolve the position of reflective or optical backscattering sites within a sample. Two-dimensional tomographic images of a thin, optical slice of tissue may be obtained with 10 μm longitudinal and lateral resolution. Narrowband optical heterodyne detection achieves a sensitivity to reflected light as small as 10-10 of the incident optical power. OCT is non-contact, non-invasive, and has superior resolution to conventional clinical ultrasound. Unlike scanning laser ophthalmoscopy and scanning laser tomography, the optical sectioning capability of OCT is not restricted by the pupil-limited numerical aperture of the eye or ocular aberrations. OCT may be implemented in a compact, low-cost, fiber-optic based interferometer that is easily coupled to existing ophthalmic instrumentation. We demonstrate high-speed, in vivo OCT imaging in both the anterior and posterior eye.

This article reviews the important role of radiotherapy in the management of early non-melanoma skin cancer, in the definitive and adjuvant settings. Therapeutic considerations and appropriate patient selection will be discussed, as well as evidence for efficacy and potential side-effects. Additionally, we present some recent advances which may improve accessibility and quality of radiotherapy, such as more convenient dose-fractionation, wide-field treatments, electronic brachytherapy and 3D printed bolus.

Tissue function depends on cellular organization. While the properties of individual cells are increasingly being deciphered using powerful single-cell sequencing technologies, understanding their spatial organization and temporal evolution remains a major challenge. Here, we present Image-seq, a technology that provides single-cell transcriptional data on cells that are isolated from specific spatial locations under image guidance, thus preserving the spatial information of the target cells. It is compatible with in situ and in vivo imaging and can document the temporal and dynamic history of the cells being analyzed. Cell samples are isolated from intact tissue and processed with state-of-the-art library preparation protocols. The technique therefore combines spatial information with highly sensitive RNA sequencing readouts from individual, intact cells. We have used both high-throughput, droplet-based sequencing as well as SMARTseq-v4 library preparation to demonstrate its application to bone marrow and leukemia biology. We discovered that DPP4 is a highly upregulated gene during early progression of acute myeloid leukemia and that it marks a more proliferative subpopulation that is confined to specific bone marrow microenvironments. Furthermore, the ability of Image-seq to isolate viable, intact cells should make it compatible with a range of downstream single-cell analysis tools including multi-omics protocols.

Some osteoblasts embed within bone matrix, change shape, and become dendrite-bearing osteocytes. The circuitry that drives dendrite formation during “osteocytogenesis” is poorly understood. Here we show that deletion of Sp7 in osteoblasts and osteocytes causes defects in osteocyte dendrites. Profiling of Sp7 target genes and binding sites reveals unexpected repurposing of this transcription factor to drive dendrite formation. Osteocrin is a Sp7 target gene that promotes osteocyte dendrite formation and rescues defects in Sp7-deficient mice. Single-cell RNA-sequencing demonstrates defects in osteocyte maturation in the absence of Sp7. Sp7-dependent osteocyte gene networks are associated with human skeletal diseases. Moreover, humans with a SP7R316C mutation show defective osteocyte morphology. Sp7-dependent genes that mark osteocytes are enriched in neurons, highlighting shared features between osteocytic and neuronal connectivity. These findings reveal a role for Sp7 and its target gene Osteocrin in osteocytogenesis, revealing that pathways that control osteocyte development influence human bone diseases., The molecular circuitry that drives dendrite formation during osteocytogenesis remains poorly understood. Here the authors show that deletion of Sp7, a gene linked to rare and common skeletal disease, in mature osteoblasts and osteocytes causes severe defects in osteocyte dendrites.

AIMS Weight loss and malnutrition occur frequently in patients with head and neck cancer and are associated with reduced survival. This pragmatic study aimed to determine the effect of a novel pre-treatment model of nutrition care on nutrition outcomes for patients with head and neck cancer receiving chemoradiotherapy. METHODS This health service evaluation consisted of an evaluation of the new model of care implementation (Phase 1) and an evaluation of patient outcomes (Phase 2) in pre- and post-implementation cohorts (n = 64 and n = 47, respectively). All Phase 2 patients received a prophylactic gastrostomy. The new model of care consisted of dietary counselling and commencement of proactive supplementary enteral nutrition via a prophylactic gastrostomy, in addition to normal oral intake, prior to treatment commencement. Nutrition outcomes were collected at baseline (pre-treatment) and 3 months post-radiotherapy completion. RESULTS The new model of care was successfully incorporated into practice with high referral (96.5%) and attendance (91.5%) rates to the counselling session, and high adherence rates to proactive tube feeding (80.9%). Patients in the post-implementation cohort had less weight-loss (1.2%; p = 0.338) and saw less of a decline in nutritional status compared to patients in the pre-implementation cohort (23% vs. 30%, respectively; p = 0.572), deemed clinically important. However, patients still experienced critical weight loss overall (mean 9.9%). CONCLUSION Pre-treatment nutrition care was feasible in standard clinical practice and demonstrated clinically relevant outcome improvements for patients. Future high-quality research is warranted to investigate further multidisciplinary strategies to attenuate weight-loss further, inclusive of patient-reported barriers and enablers to nutrition interventions.