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Background: Prior studies in breast cancer (BC) have demonstrated variability in the immune microenvironment of BCs across subtypes, with higher levels of immune infiltration observed in triple-negative (TN) BC. Clinical trials of immunotherapy in BC have largely targeted patients with TNBC, however many of these patients do not respond to treatment, suggesting heterogeneity across TN tumors. Further, immune infiltrated hormone receptor–positive, HER2-negative (HR+/HER2-) tumors have been observed. Patients with these tumors may benefit from immune checkpoint blockade (ICB). Identification of patients with immunogenic tumors would be valuable for future patient selection and stratification in clinical trials. In this study, we conducted comprehensive molecular profiling of a set of commercially procured BC samples to characterize the tumor microenvironment (TME) of HR+/HER2- and TN tumors and to identify patient or tumor features associated with immune infiltration. Methods: 163 surgically resected BC samples were used for this study (93 HR+/HER2-, 70 TN). Clinically validated immunohistochemistry (IHC) assays were used to evaluate the expression of two immune checkpoint (IC) molecules, PD-L1 (by VENTANA PD-L1 (SP142) Assay and Agilent PD-L1 IHC 28-8 pharmDx) and LAG3 (antibody clone 17B4). The presence of CD8+ cells was measured by IHC (Dako/Agilent clone C8/144B) and used to derive the spatial location and density (topology) of CD8+ cells within the TME. RNA sequencing (RNA-seq) was used to construct gene expression signatures representative of immune cell types. Furthermore, RNAseq data was used to identify expressed somatic mutations and to calculate the total expressed tumor mutational burden (‘eTMB’). Results: As previously reported, the prevalence of PD-L1 + tumors was higher in TN vs HR+/HER2− BC, however close to half (44.1% SP142 IC> 1%) of HR+/HER2- tumors showed some degree of PD-L1 expression. Similar patterns were observed for LAG3 IHC expression (32.3% and 64.7% of HR+ and TN tumors respectively with >1% expression). Concordant expression of the two markers (SP142 IC> 1% and LAG3 >=1% or < 1% and < 1%) was observed in approximately 80% of tumors in both subtypes. Less than 10% of tumors in both subtypes had high LAG3 (>1%) and low PD-L1(< 1%) expression. Most (82.4%) HR+/HER2- tumors in the dataset were deficient in CD8+ cells in both stroma and tumor parenchyma, however a subset (12.9%) were identified as having an ‘excluded’ CD8 pattern, and four (4.7%) were classified as ‘inflamed’. The frequency of both the excluded and inflamed states was slightly higher among the TN tumors (20.6% and 15.9% respectively). Expression of PD-L1+ occurred more frequently among tumors with higher CD8 expression. Among both TN and HR+ tumors, most ‘inflamed’ tumors were grade 3. We also examined a range of gene expression signatures representative of various immune cell subpopulations, their distribution within each subtype, and relationships with other molecular data. For example, expression of a CXCL13+ T cell signature, a cell type that has been shown to be predictive of response to anti-PD-L1 + chemo combo therapy in TNBC, was significantly higher in TN vs HR+ tumors, however, a large subset of HR+ tumors had levels of expression of the signature above the median observed in TN tumors. In addition, we noted that eTMB, while low overall, was positively correlated with certain immune-related expression signatures across HR+/HER2− samples, including signatures previously associated with improved response to ICB, as well as PD-L1 positivity. Conclusions: We observed heterogeneity in the immune profile of the real-world HR+ and TN tumors in this cohort. A subset of tumors in both subtypes expressed markers or signatures previously reported to be associated with response to ICB. (1) Cancer Cell. 2021 Dec 13;39(12):1578-1593.e8. Citation Format: Aparna Chhibber, Lloye M. Dillon, John B. Wojcik, Vishantie Dostal, George Lee, Fayaz Seifuddin, Scott Ely, Mark D. Stern, Charlie Benson-Garnett, Mustimbo Roberts, Jenny Wu. Characterization of Immune Contexture in HR+/HER2- and Triple Negative Breast Cancer in a Real-world Cohort [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-09-07.

Somites are transient structures derived from the pre-somitic mesoderm (PSM), involving mesenchyme-to-epithelial transition (MET) where the cells change their shape and polarize. Using Scanning electron microscopy (SEM), immunocytochemistry and confocal microscopy, we study the progression of these events along the tail-to-head axis of the embryo, which mirrors the progression of somitogenesis (younger cells located more caudally). SEM revealed that PSM epithelialization is a gradual process, which begins much earlier than previously thought, starting with the dorsalmost cells, then the medial ones, and then, simultaneously, the ventral and lateral cells, before a somite fully separates from the PSM. The core (internal) cells of the PSM and somites never epithelialize, which suggests that the core cells could be 'trapped' within the somitocoele after cells at the surfaces of the PSM undergo MET. Three-dimensional imaging of the distribution of the cell polarity markers PKCζ, PAR3, ZO1, the Golgi marker GM130 and the apical marker N-cadherin reveal that the pattern of polarization is distinctive for each marker and for each surface of the PSM, but the order of these events is not the same as the progression of cell elongation. These observations challenge some assumptions underlying existing models of somite formation.

During primitive streak formation in the chick embryo, cells undergo mesendoderm specification and convergent extension at the same time and in the same cells. Previous work has implicated cVG1 (GDF3) as a key factor for induction of primitive streak identity and positioning the primitive streak, whereas FGF signalling was implicated in regulating cell intercalation via regulation of components of the WNT-planar cell polarity (PCP) pathway. FGF has also been reported to be able to induce a primitive streak (but lacking the most axial derivatives such as notochord/prechordal mesendoderm). These signals emanate from different cell populations in the embryo, so how do they interact to ensure that the same cells undergo both cell intercalation and acquire primitive streak identity? Here we begin to address this question by examining in more detail the ability of the two classes of signals in regulating the two developmental events. Using misexpression of inducers and/or exposure to inhibitors and in situ hybridisation, we study how these two signals regulate expression of Brachyury (TBXT) and PRICKLE1 as markers for the primitive streak and the PCP, respectively. We find that both signals can induce both properties, but while FGF seems to be required for induction of the streak by cVG1, it is not necessary for induction of PRICKLE1. The results are consistent with cVG1 being a common regulator for both primitive streak identity and the initiation of convergent extension that leads to streak elongation.

During preimplantation development, contractile forces generated at the apical cortex segregate cells into inner and outer positions of the embryo, establishing the inner cell mass (ICM) and trophectoderm. To which extent these forces influence ICM-trophectoderm fate remains unresolved. Here, we found that the nuclear lamina is coupled to the cortex via an F-actin meshwork in mouse and human embryos. Actomyosin contractility increases during development, upregulating Lamin-A levels, but upon internalization cells lose their apical cortex and downregulate Lamin-A. Low Lamin-A shifts the localization of actin nucleators from nucleus to cytoplasm increasing cytoplasmic F-actin abundance. This results in stabilization of Amot, Yap phosphorylation and acquisition of ICM over trophectoderm fate. By contrast, in outer cells, Lamin-A levels increase with contractility. This prevents Yap phosphorylation enabling Cdx2 to specify the trophectoderm. Thus, forces transmitted to the nuclear lamina control actin organization to differentially regulate the factors specifying lineage identity.

Background Cardiovascular magnetic resonance (CMR) is increasingly used in newborns with congenital heart disease. However, reporting on ventricular volumes and mass is hindered by an absence of normative data in this population. Design/methods Healthy term (37–41 weeks gestation) newborns underwent non-sedated, free-breathing CMR within the first week of life using the ‘feed and wrap’ technique. End-diastolic volume (EDV), end-systolic volume (ESV) stroke volume (SV) and ejection fraction (EF) were calculated for both left ventricle (LV) and right ventricle (RV). Papillary muscles were separately contoured and included in the myocardial volume. Myocardial mass was calculated by multiplying myocardial volume by 1.05 g/ml. All data were indexed to weight and body surface area (BSA). Inter-observer variability (IOV) was performed on data from 10 randomly chosen infants. Results Twenty healthy newborns (65% male) with a mean (SD) birth weight of 3.54 (0.46) kg and BSA of 0.23 (0.02) m2 were included. Normative LV parameters were indexed EDV 39.0 (4.1) ml/m2, ESV 14.5 (2.5) ml/m2 and ejection fraction (EF) 63.2 (3.4)%. Normative RV indexed EDV, ESV and EF were 47.4 (4.5) ml/m2, 22.6 (2.9) ml/m2 and 52.5 (3.3)% respectively. Mean LV and RV indexed mass were 26.4 (2.8) g/m2 and 12.5 (2.0) g/m2, respectively. There was no difference in ventricular volumes by gender. IOV was excellent with an intra-class coefficient > 0.95 except for RV mass (0.94). Conclusion This study provides normative data on LV and RV parameters in healthy newborns, providing a novel resource for comparison with newborns with structural and functional heart disease.

Rehabilitation professionals prescribe exercise regularly with the goals of decreasing pain, increasing function, and returning athletes to competition. To maximize the effect of an exercise intervention, the program must be individualized and in context for the athlete considering biopsychosocial aspects of care. Current models of exercise prescription may not be ideal considering that less than 50% of injured athletes return to their pre-injury level. Advice on exercise prescription has been offered in the past, but the paradigms are either not user friendly or user friendly but linear, based on phases of recovery. As such, there is a need for a more flexible exercise prescription paradigm that should improve the individuality of exercise prescription. In this Current Opinion, we offer a user-friendly construct-oriented paradigm designed to facilitate the creation of individualized exercise programs for athletes.

During early vertebrate development, signals from a special region of the embryo, the organizer, can redirect the fate of non-neural ectoderm cells to form a complete, patterned nervous system. This is called neural induction and has generally been imagined as a single signalling event, causing a switch of fate. Here, we undertake a comprehensive analysis, in very fine time course, of the events following exposure of competent ectoderm of the chick to the organizer (the tip of the primitive streak, Hensen’s node). Using transcriptomics and epigenomics we generate a gene regulatory network comprising 175 transcriptional regulators and 5614 predicted interactions between them, with fine temporal dynamics from initial exposure to the signals to expression of mature neural plate markers. Using in situ hybridization, single-cell RNA-sequencing, and reporter assays, we show that the gene regulatory hierarchy of responses to a grafted organizer closely resembles the events of normal neural plate development. The study is accompanied by an extensive resource, including information about conservation of the predicted enhancers in other vertebrates.

Digital Health Technologies (DHTs) such as connected sensors offer particular promise for improving data collection and patient empowerment in neurology research and care. This study analyzed the recent evolution of the use of DHTs in trials registered on ClinicalTrials.gov for four chronic neurological disorders: epilepsy, multiple sclerosis, Alzheimer’s, and Parkinson’s disease. We document growth in the collection of both more established digital measures (e.g., motor function) and more novel digital measures (e.g., speech) over recent years, highlighting contexts of use and key trends.

Purpose/Objective(s) Preoperative (pre-op) SRS has been demonstrated to be a feasible alternative to postoperative (post-op) SRS primarily based on single institution studies with potential benefits in adverse radiation effect (ARE) and leptomeningeal disease (LMD) compared to post-op SRS. This study (Preoperative Radiosurgery for Brain Metastases – PROPS-BM) reports pre-op SRS treatment details and outcomes from a large multicenter cohort. Materials/Methods Patients (pts) with brain metastases (BM) from solid cancers, of which at least 1 lesion was treated with pre-op SRS and underwent planned resection were included from 5 institutions. SRS to synchronous intact BM was allowed. Exclusion criteria included classically radiosensitive or non-solid cancers, prior or planned whole brain radiotherapy (WBRT), and Results The cohort included 242 pts with 253 pre-op treated index lesions. The majority of pts (62.4%) had a single BM, 93.7% underwent gross total resection (GTR), and 98.8% were treated with a single fraction to a median dose of 15 Gy to a median gross tumor volume (GTV) of 9.9 cc. Median interval between pre-op SRS and surgery was 1 day. Most pts had non-small cell lung (43.4%), breast (20.2%), or melanoma (12.8%) cancer. The table below details 1 and 2-year outcomes. Extent of resection was a strong independent predictor of cavity local recurrence (LR) with LR occurring in 7 of 16 cavities (43.8%) status post subtotal resection (STR) compared with 32 of 237 (13.5%) cavities status post GTR (P = 0.005). The majority of LMD was cLMD type (13 of 19 pts with LMD, 68.4%). Ten of 242 pts (4.1%) experienced grade ≥3 post-op surgical complications. Conclusion This multicenter cohort study represents the largest population of pts treated with pre-op SRS to our knowledge. Approximately half of the pts included are from previously unpublished cohorts. The favorable oncologic outcomes, especially the notably low rates of LMD and ARE, previously demonstrated in single institution studies are confirmed in this expanded multicenter analysis without evidence of excessive post-op surgical complications. STR, though infrequent, is associated with significantly worse cavity LR in this setting. A randomized trial between pre-op and post-op SRS is warranted and is currently being designed.

Framework materials constitute a broad family of solids that range from zeolites and metal-organic frameworks (MOFs) to coordination polymers. The synthesis of such network structures typically rely on precursor molecular building blocks. As an example, the UiO-66 MOF series is constructed of hexanuclear [Zr6O4(OH)4(CO2)12] cluster nodes and linear carboxylate linkers. Unfortunately, these Zr MOF cluster nodes cannot currently be manufactured in a sustainable way, motivating a search for "green" alternative synthesis methods. Stabilizing the hexanuclear Zr(IV) cluster (i.e., the hexamer, {Zr612+}) without the use of organic ligation would enable the use of environmentally friendly solvents such as water. The Zr(IV) tetranuclear cluster (i.e., the tetramer, {Zr48+}) can be stabilized in solution with or without organic ligands, yet the hexamer has yet to be synthesized without supporting ligands. The reasons why certain zirconium clusters are favored in aqueous solution over others are not well understood. This study reports the relative thermodynamic instability of the hypothetical hexamer {Zr612+} compared to the ubiquitous {Zr48+} tetramer. Density functional theory calculations were performed to obtain the hydrolysis Gibbs free energy of these species and used to construct Zr Pourbaix diagrams that illustrate the effects of electrochemical potential, pH, and Zr(IV) concentration. It was found that the aqueous {Zr612+} hexamer is ∼17.8 kcal/mol less stable than the aqueous {Zr48+} tetramer at pH = 0, V = 0, and [Zr(IV)] = 1 M, which is an energy difference on the order of counterion interactions. Electronic structure analyses were used to explore trends in the highest occupied molecular orbital-lowest unoccupied molecular orbital gap, frontier molecular orbitals, and electrostatic potential distribution of these clusters. The evidence suggests that the aqueous {Zr612+} hexamer may be promoted with more strategic syntheses incorporating minimal ligands and counterions.

Stress fractures likely have a 1-2% incidence in athletes in general. In runners, a more vulnerable population, incidence rates likely range between 3.2 and 21% with female runners having greater susceptibility. The incidence of femoral shaft stress fractures is less well known. New basic and translational science research may impact the way clinicians diagnose and treat femoral stress fractures. By using a fictitious case study, this paper applies bone science to suggest new approaches to evaluating and treating femoral shaft stress fractures in the running population.

The current dogma about the heartbeat origin is based on “the pacemaker cell,” a specialized cell residing in the sinoatrial node (SAN) that exhibits spontaneous diastolic depolarization triggering rhythmic action potentials (APs). Recent high-resolution imaging, however, demonstrated that Ca signals and APs in the SAN are heterogeneous, with many cells generating APs of different rates and rhythms or even remaining non-firing (dormant cells), i.e., generating only subthreshold signals. Here we numerically tested a hypothesis that a community of dormant cells can generate normal automaticity, i.e., “the pacemaker cell” is not required to initiate rhythmic cardiac impulses. Our model includes 1) non-excitable cells generating oscillatory local Ca releases and 2) an excitable cell lacking automaticity. While each cell in isolation was not “the pacemaker cell”, the cell system generated rhythmic APs: The subthreshold signals of non-excitable cells were transformed into respective membrane potential oscillations via electrogenic Na/Ca exchange and further transferred and integrated (computed) by the excitable cells to reach its AP threshold, generating rhythmic pacemaking. Cardiac impulse is an emergent property of the SAN cellular network and can be initiated by cells lacking intrinsic automaticity. Cell heterogeneity, weak coupling, subthreshold signals, and their summation are critical properties of the new pacemaker mechanism, i.e., cardiac pacemaker can operate via a signaling process basically similar to that of “temporal summation” happening in a neuron with input from multiple presynaptic cells. The new mechanism, however, does not refute the classical pacemaker cell-based mechanism: both mechanisms can co-exist and interact within SAN tissue.

Ruth Bellairs passed away on 30 December 2021 at the age of 95. Ruth was a legendary figure in chick embryology; she pioneered many new techniques and made some important discoveries, and we were both extremely fortunate to benefit from her extraordinary mentorship, C.W. as final honours project undergraduate in her lab and then as her PhD student (1967-70), and C.D.S. as her postdoc (1978-1984).