Mature Population Research Articles

Преднамеренная искусственная деформация у населения позднесарматского времени (палеопатологический аспект)

Introduction. Intentional artificial head deformation is one of the most striking features of the late Sarmatian nomads from the Eastern European steppes. For quite a long time, archaeologists and anthropologists tried to solve the issues associated with this custom’s emergence and existence in the population of the 2nd – 4 th centuries AD. Later, the question of the deforming structure’s impact on the normal life of a person became a separate research line for the phenomenon of artificial cranial deformation in the late Sarmatians. Methods and materials. 155 craniums of sexually mature late Sarmatian populations (2nd – 4th centuries AD) originating from under the burial mounds in the territory of the Lower Volga region were the material for the study. Comparison of pathological conditions occurrence between the deformed and undeformed skull groups was carried out using nonparametric tests: Pearson’s chi-squared test (χ2) and Fisher’s exact test. Results. Comparative analysis of pathological conditions occurrence on deformed and undeformed skulls in late Sarmatian time has shown that the only deviation in which the compared series differ significantly is temporomandibular joint arthritis. Discussion. The frequency and degree of articular disease development in late Sarmatians with intentional artificial head deformities increase with age. The age-related variability of arthrotic changes in the mandibular joint area indicates the involutionary nature of this condition. At the same time, the absence of statistical differences between age groups in the distribution frequencies of this pathology and its wide distribution in young individuals under 35 years of age suggest that artificial skull deformation is one of the factors stimulating its development. Conclusions. Degenerative changes in the temporomandibular joint area are a statistically more widespread pathology in deformed skulls. The absence of gender differences between the deformed and undeformed skulls in the late Sarmatians, as well as the groups of pathologies associated with diet and the degree of negative factors impacting the natural and social environment, indicates that this custom in the late Sarmatian society did not carry the function of gender or social differentiation. The use of a modifying structure for deliberate artificial cranial deformation is quite compatible with normal human life, and its use did not subsequently lead to the development of serious skull and brain pathologies.

Pheromone and Host Plant Odor Detection in Eastern Spruce Budworm, Choristoneura fumiferana Clemens (Lepidoptera: Tortricidae).

Spruce budworm, Choristoneura fumiferana Clemens, is an ecologically significant defoliator of spruce and balsam fir in North America. Optimization of semiochemical-mediated control is needed to improve the existing integrated pest management systems such as mating disruption and population estimation. This study used single sensillum recordings (SSR) to identify the responses of olfactory receptor neurons (ORNs) in the antennal sensilla of adult male and female C. fumiferana to host plant volatiles, and female sex pheromones. There have been few SSR studies done on spruce budworm, and to our knowledge, the present study represents the first attempt to examine the responses of ORNs from antennal sensilla in response to a range of host and conspecific stimuli. A total of 86 sensilla were characterized and sorted into 15 possible sensillum categories based on odor responses. We observed that specialist sensilla responding to few ligands were more abundant in both male and female than sensilla exhibiting more generalized odorant responses. (E/Z)-11-tetradecenal elicited responses from ORNs from any sensilla which were sensitive to pheromones in both males and females. Female C. fumiferana ORNs were able to detect and physiologically respond to female-produced sex pheromones with the same degree of sensitivity as their male counterparts. Together, these data improve our knowledge of mechanisms by which adult budworms respond to pheromone and host plant volatiles and provide insights that may be complementary to existing integrated pest management (IPM) strategies based on the chemical ecology of spruce budworm.

Pharmacological profiling identifies divergent chemosensitivities of differentiating and maturing iPSC-derived human cortical neuron populations.

Neuronal differentiation and maturation are extended developmental processes. To determine whether neurons at different developmental stages have divergent chemosensitivities, we screened differentiating and maturing neuronal populations using a small compound library comprising FDA-approved and investigational drugs. Using a neurotoxicity assay format, both respective neuronal population-based screening campaigns performed robustly (Z-factors = 0.7-0.8), although the hit rate for the differentiating neurons (2.8%) was slightly higher than for maturing neurons (1.9%). While the majority of hits were toxic to both neuronal populations, these hits predominantly represented promiscuous drugs. Other drugs were selectively neurotoxic, with receptor tyrosine kinase inhibitors disproportionally represented after confirmation. Ponatinib and amuvatinib were neuroinhibitory for differentiating and maturing neurons, respectively. Chemoinformatic analyses confirmed differences in potential drug targets that may be differentially expressed during neuronal development. Subsequent studies demonstrated neuronal expression of AXL, an amuvatinib target, in both neuronal populations. However, functional AXL activity was confirmed only in the maturing neuronal population as determined by AXL phosphorylation in response to GAS6, the cognate ligand of AXL, and concurrent STAT3Y705 phosphorylation. Differentiating neurons were unresponsive to the effects of GAS6 suggesting that the AXL-STAT3 signaling axis was nonfunctional. Amuvatinib treatment of maturing neuronal cultures significantly reduced pAXL levels. These studies indicate that neuronal developmental states may exhibit unique chemosensitivities and that drugs may have different neuro-inhibitory effects depending upon the developmental stage of the neuronal population.

Natural killer cell subsets in endometrial fluid: a pilot study of their association with the endometrial cycle and reproductive parameters.

To investigate if there are natural killer (NK) cells in endometrial fluid (EF) and their relationship with the endometrial cycle and reproductive parameters. The population under study consisted of 43 women aged 18-40 undergoing infertility workup at our University Hospital in 2021-2022. The EF samples were obtained at the first visit to our unit, on occasion of the mock embryo transfer. The day of the cycle was considered only in cycles of 27-29 days. An immunophenotype study of NK in EF was performed by flow cytometry analysis. In a subgroup of women, on the same day, NK was studied in EF and peripheral blood. Our study is the first to evidence NK cells in EF. None of the NK cells observed corresponded to a mature peripheral blood NK cell population (stages 4-5), and neither endometrial nor decidual uNK cells were detected. Nevertheless, we found 2 patient groups with an NK cell subset with a higher expression of CD16+, which could belong to an intermediate or transient stage between the uNK and pbNK NK cell population in the EF. We found that CD16 was significantly increased in the mid-late luteal phase and its correlation with the day of the cycle. The NK immunophenotype was different in EF and peripheral blood. We described a new component of the EF, the NK cells, whose CD16 activity is closely correlated with the day of the cycle. These cells could play a role in implantation/implantation failure.

The recombinase activating genes: architects of immune diversity during lymphocyte development.

The mature lymphocyte population of a healthy individual has the remarkable ability to recognise an immense variety of antigens. Instead of encoding a unique gene for each potential antigen receptor, evolution has used gene rearrangements, also known as variable, diversity, and joining gene segment (V(D)J) recombination. This process is critical for lymphocyte development and relies on recombination-activating genes-1 (RAG1) and RAG2, here collectively referred to as RAG. RAG serves as powerful genome editing tools for lymphocytes and is strictly regulated to prevent dysregulation. However, in the case of dysregulation, RAG has been implicated in cases of cancer, autoimmunity and severe combined immunodeficiency (SCID). This review examines functional protein domains and motifs of RAG, describes advances in our understanding of the function and (dys)regulation of RAG, discuss new therapeutic options, such as gene therapy, for RAG deficiencies, and explore in vitro and in vivo methods for determining RAG activity and target specificity.

LGG-22. CHARACTERIZATION OF THE TUMOR MICROENVIRONMENT IN PEDIATRIC LOW-GRADE GLIOMA

Abstract BACKGROUND Pediatric low-grade gliomas (pLGGs) are the most common brain tumors in children. Although pLGGs represent a significant number of patients with slow-growing, often curable tumors, they are also affected by neurologic injuries, multiple relapses and requiring repeated courses of therapy, including surgery, chemotherapy, and radiotherapy. Novel, more effective therapies are urgently needed. METHODS We used a multipronged approach to better understand the complexity of the tumor microenvironment (TME) within pLGG. We utilized scRNA sequencing, spatial transcriptomics, and cytokine analyses on 23 patient-derived pLGG samples including different tumor types (pilocytic astrocytoma (PA), ganglioglioma (GG), and undefined low-grade glioma (LGG)) and mutation profiles (BRAF V600E, BRAF fusions, and BRAF wild-type) to identify cell types, their spatial arrangement within a tissue, and their underlying functional status. RESULTS scRNA seq revealed both tumor and immune cells within the TME. Analyses of tumor cell subsets revealed a developmental hierarchy identifying progenitor and mature cell populations. Immune cells comprised both myeloid and T lineage cells, with myeloid cells comprising > 50% of the entire population. Interestingly, there was a significant difference between the prevalence of two major myeloid subclusters between two pLGG subtypes (PA and GG). To better understand the underlying difference, we performed bulk and single-cell cytokine analyses to identify immune signals via which these cells communicate. Cytokine analyses revealed distinct immune phenotypes among tumor samples. Notably, BRAF fusion tumors appeared more immunogenic, especially by secreting higher levels of immune cell activators, chemokines (including CCL3 and CCL4), compared to BRAF V600E tumors. Additionally, spatial transcriptomics revealed the differential expression of these chemokines between PA and GG tumors. CONCLUSIONS Characterization of pLGG demonstrated the complexity of tumor microenvironment among different pLGG subtypes. Further investigation of the immune cells and tumor-immune cell interactions has the potential to unravel novel therapeutic targets against pLGG.

Transcriptional programming of CD4+ TRM differentiation in viral infection balances effector- and memory-associated gene expression.

After resolution of infection, T cells differentiate into long-lived memory cells that recirculate through secondary lymphoid organs or establish residence in tissues. In contrast to CD8+ tissue-resident memory T cells (TRM), the developmental origins and transcriptional regulation of CD4+ TRM remain largely undefined. Here, we investigated the phenotypic, functional, and transcriptional profiles of CD4+ TRM in the small intestine (SI) responding to acute viral infection, revealing a shared gene expression program and chromatin accessibility profile with circulating TH1 and the progressive acquisition of a mature TRM program. Single-cell RNA sequencing identified heterogeneity among established CD4+ TRM, which were predominantly located in the lamina propria, and revealed a population of cells that coexpressed both effector- and memory-associated genes, including the transcriptional regulators Blimp1, Id2, and Bcl6. TH1-associated Blimp1 and Id2 and TFH-associated Bcl6 were required for early TRM formation and development of a mature TRM population in the SI. These results demonstrate a developmental relationship between TH1 effector cells and the establishment of early TRM, as well as highlighted differences in CD4+ versus CD8+ TRM populations, providing insights into the mechanisms underlying the origins, differentiation, and persistence of CD4+ TRM in response to viral infection.

Phenotypic trait differences betweenIris pseudacorusin native and introduced ranges support greater capacity of invasive populations to withstand sea level rise

AbstractAimTidal wetlands are greatly impacted by climate change, and by the invasion of alien plant species that are being exposed to salinity changes and longer inundation periods resulting from sea level rise. To explore the capacity for the invasion ofIris pseudacorusto persist with sea level rise, we initiated an intercontinental study along estuarine gradients in the invaded North American range and the native European range.LocationSan Francisco Bay‐Delta Estuary; California, USA and Guadalquivir River Estuary; Andalusia, Spain.MethodsWe compared 15 morphological, biochemical, and reproductive plant traits within populations in both ranges to determine if specific functional traits can predict invasion success and if environmental factors explain observed phenotypic differences.ResultsAlienI. pseudacorusplants in the introduced range had more robust growth than plants in the native range. The vigour of the alien plants was reflected by expression of higher leaf water content, fewer senescent leaves per leaf fan, and more carbohydrate storage reserves in rhizomes than plants in the native range. Moreover, alien plants tended to show higher specific leaf area and seed production than native plants.I. pseudacorusplants in the introduced range were less affected by increasing salinity and were exposed to deeper inundation water along the estuarine gradient than those in the native range.Main ConclusionsFunctional trait differences suggest mature populations ofI. pseudacorusin the introduced range have greater adapted capacity to adjust to environmental stresses induced by rising sea level than those in the native range. Knowledge of these trait responses can be applied to improve risk assessments in invaded estuaries and to achieve climate‐adapted conservation goals for conservation of the species in its native range.

Neutrophil developmental defects cause hyperinflammation in cherubism

Cherubism is a rare autosomal dominant disease caused mainly by a P416R point mutation of Src homology 3 Domain Binding Protein 2 (SH3BP2). Though macrophages and osteoblasts were thought to be the major contributors to the pathology in the past, the involvement of neutrophils in cherubism has been demonstrated by the increasing neutrophil infiltration and extensive neutrophil extracellular trap formation in the lesion recently. Here, we observed decreased expression of lineage marker Ly6G and impaired integrin activation on neutrophils, indicating a developmental defect of neutrophils in cherubism mice carrying the SH3BP2 P416R mutation. Furthermore, we performed single-cell RNA sequencing and compared transcriptomes between blood neutrophils, bone marrow neutrophils, and bone marrow leukocyte progenitors from wildtype and SH3BP2 P416R mice. RNA velocity and pathway analysis show that mutated neutrophils have stalled in the early stage of neutrophil development and failed to mature into granzyme pathway-enhanced, NET-formation-restrained mature neutrophil population in wildtype. Together, our study first illustrates the neutrophil development defects in cherubism as a mechanism for hyperinflammation characterized in cherubism. Ziming Cao and Keaton Karlinsey contributed equally. Beiyan Zhou, I-Ping Chen and Zhichao Fan are co-corresponding authors. This research was supported by grants from the National Institutes of Health, National Heart, Lung, and Blood Institute, USA (R01HL145454), and a startup fund from UConn Health. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Normative Values of Tibial Tubercle–Trochlear Groove Distance and Tibial Tubercle–Posterior Cruciate Ligament Distance in Children

Background: Patellar instability is a common knee pathology in skeletally immature patients. In skeletally mature populations, a tibial tubercle–trochlear groove (TT-TG) distance of ≥20 mm is generally considered a pathological value. However, as pediatric patients grow and as the TT-TG distance varies with age, applying the same cutoff value as adult patients to them is unreasonable. Purpose/Hypothesis: This study aimed to analyze the normative values of the TT-TG and tibial tubercle–posterior cruciate ligament (TT-PCL) distances in children with no patellofemoral instability and to propose the cutoff value of the TT-TG and TT-PCL distances predictive of increased risk of patellofemoral instability in pediatric patients. We hypothesized that the TT-TG and TT-PCL distances increase with age in children and adolescents. Study Design: Cross-sectional study; Level of evidence, 3. Methods: Magnetic resonance imaging scans of the knee were collected from the patellar instability group and the control group. The TT-TG and TT-PCL distances were measured. The normalized values of the TT-TG and TT-PCL distances were calculated by dividing them by the femoral width. Segmented analysis with 1 breakpoint was performed for both the TT-TG and the TT-PCL distances. The optimal cutoff values of the TT-TG and TT-PCL distances were calculated by maximizing the sum of the sensitivity and specificity. Results: A total of 87 patients had patellar instability, and 509 patients did not. The median values of TT-TG and TT-PCL distances in the control group (8.18 mm and 19.48 mm, respectively) were significantly smaller than those in the instability group (16.10 mm and 24.41 mm, respectively). For those aged <15 years, the TT-TG distance significantly increased by 0.39 mm as the age increased by 1 year. The TT-PCL distance increased by 1.14 mm/year until the age of 11 years. In our cohort, the cutoff value of the TT-TG distance of 14.90 mm yielded 66% sensitivity and 81.9% specificity for predicting an increased risk of patellar instability. The cutoff value of the TT-PCL distance of 23.68 mm yielded 63.9% sensitivity and 65.3% specificity for predicting an increased risk of patellar instability. Conclusion: In our cohort of 596 participants, we have documented the normative values of the TT-TG and TT-PCL distances in children aged <20 years. During the surgery for patellar instability in pediatric patients, orthopaedic surgeons should consider the normative values of the TT-TG and TT-PCL distances according to age to determine which patients need distal realignment surgery.

Modelling the erythroblastic island niche of dyserythropoietic anaemia type IV patients using induced pluripotent stem cells.

Introduction: Congenital dyserythropoietic anaemia (CDA) type IV has been associated with an amino acid substitution, Glu325Lys (E325K), in the transcription factor KLF1. These patients present with a range of symptoms, including the persistence of nucleated red blood cells (RBCs) in the peripheral blood which reflects the known role for KLF1 within the erythroid cell lineage. The final stages of RBCs maturation and enucleation take place within the erythroblastic island (EBI) niche in close association with EBI macrophages. It is not known whether the detrimental effects of the E325K mutation in KLF1 are restricted to the erythroid lineage or whether deficiencies in macrophages associated with their niche also contribute to the disease pathology. Methods: To address this question, we generated an in vitro model of the human EBI niche using induced pluripotent stem cells (iPSCs) derived from one CDA type IV patient as well as two iPSC lines genetically modified to express an KLF1-E325K-ERT2 protein that could be activated with 4OH-tamoxifen. The one patient iPSC line was compared to control lines from two healthy donors and the KLF1-E325K-ERT2 iPSC line to one inducible KLF1-ERT2 line generated from the same parental iPSCS. Results: The CDA patient-derived iPSCs and iPSCs expressing the activated KLF1-E325K-ERT2 protein showed significant deficiencies in the production of erythroid cells with associated disruption of some known KLF1 target genes. Macrophages could be generated from all iPSC lines but when the E325K-ERT2 fusion protein was activated, we noted the generation of a slightly less mature macrophage population marked by CD93. A subtle trend in their reduced ability to support RBC enucleation was also associated with macrophages carrying the E325K-ERT2 transgene. Discussion: Taken together these data support the notion that the clinically significant effects of the KLF1-E325K mutation are primarily associated with deficiencies in the erythroid lineage but it is possible that deficiencies in the niche might have the potential to exacerbate the condition. The strategy we describe provides a powerful approach to assess the effects of other mutations in KLF1 as well as other factors associated with the EBI niche.

Distribution of genetic variability in mature and progeny populations of Abies alba Mill. from the Polish Western and Eastern Carpathians

This study aimed to compare patterns in the genetic structure of 27 mature stands and the natural regeneration of A. alba in the Eastern and Western Carpathians within the introgression zone of two refugial lineages from the Apennine and Balkan peninsulas. The distribution of the genetic diversity of fir stands was analysed using paternally inherited chloroplast DNA and five nuclear dominant inter simple sequence repeat markers (ISSRs). The study showed that the Balkan haplotype prevailed in both parental and progeny populations, and this haplotype was found in many mature Eastern Carpathian stands. Relatively high levels of genetic diversity were found in the mature stands (effective number of alleles Ne = 1.517, Shannon index I = 0.436, expected heterozygosity He = 0.295) and progeny (Ne = 1.515, I = 0.436 and He = 0.294) of silver fir. The analysis of molecular variance (AMOVA) revealed slight differences among the mature fir stands from the Western and Eastern Carpathians, with a value of 1.1%. According to principal coordinates analysis (PCoA) and STRUCTURE analyses, the populations, including stands of mature and progeny trees, were genetically separated into two groups. Slight genetic differences between the mature and progeny populations in the Polish Carpathians indicate sound gene pool transmission, which is essential for creating new selection and breeding programmes.

Abstract 5192: NCG-MHC-dKO mice: an excellent model for PBMC reconstitution and pharmacodynamic evaluation in the absence of GvHD

Abstract Human immune cell reconstitution (PBMC or CD34+ hematopoietic stem cell; HSC) is commonly performed in immunodeficient mouse strains such as NCG, NSG, or NOG. The use of PBMC is preferred for multiple reasons, including faster engraftment, lower cost, and the presence of mature immune cell populations. Although there are donor-to-donor variations in GvHD occurrence, and specific donors can be screened, the symptoms of GvHD typically occur within 4 weeks. GvHD significantly limits the study window required to evaluate the efficacy of therapeutic agents, such as cancer immunotherapies. GvHD is an immune reaction triggered mainly by donor T cells identifying the mouse MHC class I and class II as foreign and attacking the host cells and organs. Deletion of β2m (NCG- β2m-KO) reduces the occurrence of GvHD; however, β2m is not only in the MHC class I subunit but also in the FcRn subunit, of which deletion shortens the half-life of IgG, making it unsuitable for IgG antibody agent evaluation. In addition, the deletion of MHC class I or class II alone results in an imbalanced CD4/CD8 ratio. To solve these problems, we developed the NCG-MHC-dKO mouse model by knocking out the H2K1, H2D1, and H2Ab1 genes. Compared with the existing NCG- β2m-KO or other β2m null mouse models, the NCG-MHC-dKO significantly prolonged survival, reduced GvHD occurrence, and did not affect the CD4/CD8 ratio when reconstituted with PBMC. Furthermore, treatment with anti-PD-L1 antibodies significantly inhibited MDA-MB-231 tumor cell growth in PBMC-reconstituted NCG-MHC-dKO mice, similar to NCG mice. Based on our preliminary data, the NCG-MHC-dKO mouse is a promising model for antibody and cell therapy agent evaluation, assessing treatment-related cytokine release syndrome, and evaluating long-term toxicities of cell therapies. Citation Format: Huiyi Wang, Jun Xing, Jialu Fan, Huanhuan Hou, Santi Suryani Chen, Zhiying Li, Mark Wade Moore, Jing Zhao, Xiang Gao, Cunxiang Ju. NCG-MHC-dKO mice: an excellent model for PBMC reconstitution and pharmacodynamic evaluation in the absence of GvHD. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5192.

Abstract 4628: Profiling the tumor microenvironments of triple- and quadruple-negative breast cancers

Abstract Triple-negative breast cancer (TNBC) is a heterogeneous breast cancer subtype that lacks expression of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Approximately 15%-20% of breast cancers are triple-negative. Based on expression of the androgen receptor (AR), TNBC can be categorized as AR-positive (AR+ TNBC) or AR-negative (quadruple-negative breast cancer, QNBC). Studies have reported that the loss of AR expression is associated with poor prognoses. Recent evidence suggests that QNBCs disproportionately affect women of African descent and contributes to poor overall survival. QNBC is a more aggressive disease compared to TNBC, and patients with QNBC currently have no treatment options beyond chemotherapy. Data from interrogating public databases such as TCGA indicates that QNBC is distinct from TNBC in molecular makeup, particularly in immune tumor microenvironment. Currently, QNBC is not considered distinct from the broader TNBC subtype, hence a comprehensive molecular characterization is lacking. We aim to examine differences between the TNBC and QNBC tumor microenvironments that consist of both immune and stromal components. We analyzed RNA expression data from the METABRIC (TNBC= 11, QNBC= 185) and TCGA (TNBC= 29, QNBC= 103) datasets. Based on AR transcript expression, patients with TNBC were stratified into AR-low (considered QNBC) and AR-high (deemed ‘true’ TNBC) groups. The analytical immune tools - CIBERSORTx and xCELL were used to analyze the RNA-Seq data. CIBERSORTx can distinguish 22 mature human hematopoietic populations and provides abundance of member cell types in a mixed cell population, from gene expression data. xCELL is a gene signature-based method that can perform cell type enrichment analysis from gene expression for 64 immune and stromal cell types. Thus, xCell can discriminate between closely related cell types. Using these immune tools, the tumor microenvironments of the TNBC and QNBC groups were compared. Correlations between individual immune and stromal components with patient survival probability were assessed. Significant differences (p-value &amp;lt;0.05) in several immune and stromal cell types were noted when comparing the TNBC and QNBC groups within these datasets. CIBERSORTx and xCELL yielded both unique and common sets of cell types. Notably, significant differences in the stroma score (a composite score of stromal cell types provided by xCELL) were observed between TNBC and QNBC in both the METABRIC and TCGA datasets. These immune tools are based on different methodologies and our results show that they can provide complementary information. Our data suggest that QNBC and TNBC have distinct tumor microenvironments, and that QNBC should be considered as an independent breast cancer subtype that warrants comprehensive characterization. Citation Format: Geetanjali Saini, Manali Rupji, Mahak Bhargava, Sunil Badve, Ritu Aneja. Profiling the tumor microenvironments of triple- and quadruple-negative breast cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4628.

Esthetic considerations for treating the patient of European descent: Thriving in diversity international roundtable series

The European esthetic experience is informative for understanding both innovation as well as how to care for patients of various backgrounds and ages. To discuss best practices for treating the European population and how these approaches may be applied to patient populations across the globe. In support of clinicians who wish to serve a diverse patient population, a 6-part, international roundtable series focused on diversity in esthetics was conducted from August 24, 2021 to May 16, 2022. In each roundtable, expert clinicians were invited to contribute and share best practices. The results of the fifth roundtable in the series, the European Patient, are described here. Key ideas include the growing number of people over the age of 65 years in Europe and the management of this more mature patient population; the role of functional anatomy in treating patients with both fillers and botulinum toxin; and the role of ultrasound in clinical practice for mapping vasculature. While there is no typical European face, there is much to be learned from thoughtful consideration of how to best manage more mature patients as well as how to use minimally invasive modalities, such as injectables, efficiently to achieve natural-looking results.

Pregnenolone enhances the proliferation of mouse neural stem cells and promotes oligodendrogenesis, together with Sox10, and neurogenesis, along with Notch1 and Pax6.

Pregnenolone is a precursor of various steroid hormones involved in osteoblast proliferation, microtubules polymerization and cell survival protection. Previous reports focused on the effects of pregnenolone metabolites on stem cell proliferation and differentiation; however, the effects of pregnenolone itself has not been well explored. The present study aimed to investigate the role of pregnenolone on NSC proliferation and to determine the doses required for NSC differentiation as well as the various genes involved in its mechanism of action. NSCs were isolated from the embryonic cortex of E14 mice, incubated for 5 days, and then treated with pregnenolone doses of 2, 5, 10, 15 and 20μM for another 5 days. The number of neurospheres and neurosphere derived cells were then counted. Flow cytometry was used to evaluate the differentiation of NSCs into oligodendrocytes, astrocytes, and neurons. The expression level of Notch1, Pax6 and Sox10 genes were also measured by Real Time PCR after 5 days of treatment. Our data suggest that treatment with 10μM pregnenolone is optimal for NSC proliferation. In fact, this concentration caused the highest increase in the number of neurospheres and neurosphere derived cells, compared to the control group. In addition, treatment with low doses of pregnenolone (5 and 10μM) caused a significant increase in NSC differentiation towards immature (Olig2+) and mature (MBP+) oligodendrocyte cell populations, compared to controls. However, NSC differentiation into neurons (beta III tubulin+cells) increased in all treatment groups, with the highest and most significant increase obtained at 15μM concentration. It is worth noting that pregnenolone at the highest concentration of 15μM decreased the number of astrocytes (GFAP+). Furthermore, there was an increase of Sox10 expression with low pregnenolone doses, leading to oligodendrogenesis, whereas Notch1 and Pax6 gene expression increased in pregnenolone groups with more neurogenesis. Pregnenolone regulates NSCs proliferation in vitro. Treatment with low doses of pregnenolone caused an increase in the differentiation of NSCs into mature oligodendrocytes while higher doses increased the differentiation of NSCs into neurons. Oligodendrogenesis was accompanied by Sox10 while neurogenesis occurred together with Notch1 and Pax6 expression.

Intravascular large B-cell lymphoma diagnosed at autopsy

Background: Intravascular large B-cell lymphoma is a rare, extranodal lymphoma characterised by intravascular growth of a malignant mature B-cell population, primarily affecting capillaries. Pre-mortem diagnosis can be challenging as patients often present with non-specific clinical findings including fever of unknown origin, decreased general condition and neurological abnormalities. Radiological findings may be non-specific or falsely negative. Aims: We present the case of an 87-year-old lady who presented to hospital with cognitive and functional decline, headache and presumed transient ischaemic attacks. Despite extensive investigations a definitive cause for her symptoms was unable to be established prior to her death. Autopsy revealed multiorgan involvement by intravascular large B-cell lymphoma. Methods: The patient underwent a full non-coronial hospital autopsy. With consent, the patient’s brain was retained for extended fixation and neuropathological assessment which also revealed widespread lymphomatous infiltration. Results and conclusions: This case demonstrates a rare case of intravascular large B-cell lymphoma diagnosed at autopsy and highlights the difficulties in pre-mortem diagnosis due to the non-specific clinical presentation and lack of diagnostic radiological features.

Single cell cortical bone transcriptomics define novel osteolineage gene sets altered in chronic kidney disease.

Due to a lack of spatial-temporal resolution at the single cell level, the etiologies of the bone dysfunction caused by diseases such as normal aging, osteoporosis, and the metabolic bone disease associated with chronic kidney disease (CKD) remain largely unknown. To this end, flow cytometry and scRNAseq were performed on long bone cells from Sost-cre/Ai9+ mice, and pure osteolineage transcriptomes were identified, including novel osteocyte-specific gene sets. Clustering analysis isolated osteoblast precursors that expressed Tnc, Mmp13, and Spp1, and a mature osteoblast population defined by Smpd3, Col1a1, and Col11a1. Osteocytes were demarcated by Cd109, Ptprz1, Ramp1, Bambi, Adamts14, Spns2, Bmp2, WasI, and Phex. We validated our in vivo scRNAseq using integrative in vitro promoter occupancy via ATACseq coupled with transcriptomic analyses of a conditional, temporally differentiated MSC cell line. Further, trajectory analyses predicted osteoblast-to-osteocyte transitions via defined pathways associated with a distinct metabolic shift as determined by single-cell flux estimation analysis (scFEA). Using the adenine mouse model of CKD, at a time point prior to major skeletal alterations, we found that gene expression within all stages of the osteolineage was disturbed. In sum, distinct populations of osteoblasts/osteocytes were defined at the single cell level. Using this roadmap of gene assembly, we demonstrated unrealized molecular defects across multiple bone cell populations in a mouse model of CKD, and our collective results suggest a potentially earlier and more broad bone pathology in this disease than previously recognized.

Living in an Age-Friendly Community: Evidence from a Masterplanned Development in Southwest Sydney.

This paper is situated at the interface of a growing urban studies literature concerned with 'masterplanning' practices in urban planning and another, hitherto relatively discrete, body of research concerned with age-friendly cities and communities. The authors are interested in exploring a gap in aging in place literature around how neighbourhoods and residential settings developed with aging in place principles in mind are experienced and perceived by residents. To explore this research gap, the authors analyse qualitative (primarily interview and focus group) data collected in Park Central, a masterplanned development located in the Campbelltown suburb in the southwest region of the Sydney metropolitan area, Australia. This development was delivered in response to a need identified by the state's land and property development agency, Landcom, for more diverse and affordable medium-density housing in Campbelltown. In particular, a need was identified for housing developments that would be able to sustainably accommodate the changing lifestyle needs of a maturing population in the region. Drawing on our thematic analysis of our data, we discern three key themes in research participants' experiences of Park Central as a place for aging. These themes are elaborated via recourse to excerpts from our data and discussed with a view to informing how the conception, development and practice of further age-friendly communities is approached.

Spatial differences in female lobster mating success and population characteristics in southern Massachusetts

The Southern New England stock of American lobster Homarus americanus is currently severely depleted and experiencing recruitment failure, potentially resulting from increased larval or juvenile mortality, decreased larval production, or both. We examined one aspect of larval production, female mating success, in 2 regions of Buzzards Bay, Massachusetts. The inner Bay areas are typically male-dominated, and the deeper regions at the mouth of the Bay have higher proportions of females. These locations were specifically selected to test our primary hypothesis that female-skewed sex ratios would decrease female mating success. Using a non-invasive method to detect ejaculates stored within females’ seminal receptacles, mating success was determined for 3851 females. Contrary to our initial hypothesis, mating success was higher in the outer areas where the sex ratio was female-skewed. We then explored 2 alternative explanations for this spatial pattern in mating success. First, using the ratio of abdominal width to carapace length (ABD:CL) to approximate maturity stage, a large proportion of females from the inner Bay appeared to be immature or primiparous, while females from the outer regions (with a larger ABD:CL ratio) were likely multiparous. Thus, lower mating activity inside Buzzards Bay corresponded with a higher proportion of immature or newly matured females for any given size. Second, low densities of lobsters inside Buzzards Bay may have resulted in a mate-finding Allee effect, leaving some mature females unmated. These 2 alternative explanations for the spatial pattern in mating success are not mutually exclusive; both may be impacting female mating success in the region.