Abstract: Studies of the impact of increased hemoglobin on spontaneous intracerebral hemorrhage (ICH) are limited. The present study aimed to explore the effect of increased hemoglobin on ICH. Methods A retrospective single-center study using medical records from a database processed by univariate and multivariate analyses was performed in the People’s Hospital of Tibet Autonomous Region in Lhasa, Tibet, China. Results The mean hemoglobin level in 211 patients with ICH was 165.03 ± 34.12 g/l, and a median hematoma volume was 18.5 ml. Eighty-eight (41.7%) patients had large hematomas (supratentorial hematoma ≥ 30 ml; infratentorial hematoma ≥ 10 ml). No differences in ICH risk factors between the groups with different hemoglobin levels were detected. Increased hemoglobin was independently associated with large hematomas [odds ratio (OR) 1.013, P  = 0.023]. Increased hemoglobin was independently associated with ICH with subarachnoid hemorrhage (OR 1.014, P  = 0.016), which was more pronounced in men (OR 1.027, P  = 0.002). Increased hemoglobin was independently associated with basal ganglia hemorrhage and lobar hemorrhage in men (OR 0.986, P  = 0.022; OR 1.013, P  = 0.044, respectively) but not in women ( P   〉  0.1). Conclusions Increased hemoglobin was independently associated with large hemorrhage volume. Increased hemoglobin was independently associated with lobar hemorrhage in men and ICH with subarachnoid hemorrhage, which was more pronounced in men. Additional studies are needed to confirm our findings and explore potential mechanisms.

Abstract: Numerous studies on acute ischemic stroke (AIS) have been conducted at low-altitude regions, and the related findings have been used to guide clinical management. However, corresponding studies at high altitude are few. This study aimed to analyse the clinical characteristics of AIS patients at high-altitude regions through a hospital-based comparative study between Tibet and Beijing. Methods This study included the diagnoses of AIS patients from People’s Hospital of Tibet Autonomous Region (PHOTAR) and Peking University First Hospital (PUFH) between 1 January 2014 and 31 December 2017, where data including patient demographics, treatment time, onset season, risk factors, infarction location, laboratory data, image examination results, treatments, and AIS subtype were collected and compared. Continuous and categorical variables were analysed with a two-sample t-test or Wilcoxon rank sum test and chi-square test, respectively. Significant risk factors were examined with binary logistic regression analysis. Results In total, 236 and 1021 inpatients from PHOTAR and PUFH were included, respectively. The PHOTAR patients were younger than the PUFH patients ( P   〈  0.001). Young adult stroke, erythrocytosis, and hyperhomocysteinemia were more frequent in PHOTAR patients (all P   〈  0.001). Other vascular risk factors, including hypertension, diabetes mellitus, hyperlipidaemia, smoking and alcohol consumption history, were less prevalent in PHOTAR patients than in PUFH patients. The rate of intravenous thrombolysis and the rate of within intravenous thrombolysis window time were also lower in PHOTAR patients (both P   〈  0.001). The PHOTAR group also tended to have anterior circulation infarction. Erythrocytosis and hyperhomocysteinemia were independent risk factors in PHOTAR, and young adults accounted for a larger proportion of stroke cases. Conclusion In Tibet, AIS patients were relatively younger, and anterior circulation infarctions were more common. Erythrocytosis and hyperhomocysteinemia may contribute to these differences. Here, young adult stroke also accounted for a higher proportion, and this may be associated with erythrocytosis. Our findings present the first hospital-based comparative study in Tibet and may contribute to policies for stroke prevention in this region.

Abstract: Although many cancer cells have significantly higher copper concentrations compared with normal cells and tissues, the role of copper in cancer biology and metastatic disease remains poorly understood. Here, we study the importance of copper in osteosarcoma, which frequently metastasizes to the lungs and is often chemoresistant. K12 and K7M2 are murine OS cells with differing metastatic phenotypes: K7M2 is highly metastatic, whereas K12 is much less so. Intracellular copper levels were determined using atomic absorption. Copper transporters were quantified by qPCR. Cytotoxicity of doxorubicin, disulfiram, and copper(II) chloride was assessed with a cell viability fluorescence stain. Additionally, K7M2 viable cell counts were determined by trypan blue exclusion staining after 72 hours of treatment. Copper levels were found to be significantly higher in K12 OS cells than in K7M2 cells. qPCR showed that K12 cells upregulate the copper influx pump CTR1 and downregulate the copper efflux pump ATP7A compared to K7M2 OS cells. Combination treatment of copper chloride (50 nM) with disulfiram (80 nM) was only cytotoxic to K12 cells. Triple treatment with doxorubicin, disulfiram, and copper displayed potent and durable cytotoxicity of highly metastatic K7M2 cells. We demonstrate here that murine OS cell lines differing in metastatic potential also vary in endogenous copper levels and regulation. Additionally, these differences in copper regulation may contribute to selective cytotoxicity of K12 cells by extremely low doses of copper-potentiated disulfiram. The combination of doxorubicin, disulfiram, and copper should be explored as a therapeutic strategy against OS metastases.

Abstract: The Shenzhen ageing-related disorder cohort was designed to detect the associations of lifestyle, environmental and genetic factors with major ageing-related disorders, especially neurological and mental disorders. Participants The cohort was a community-dwelling prospective study of 9411 elderly adults aged 60 to 92 years from 51 community health service centres in Luohu district of Shenzhen, China. The baseline data were collected between 2017 and 2018, including demographics and socioeconomics, lifestyles, medical history, family history of major non-communicable chronic disease, environmental exposures, clinical analysis of blood and urine, clinical imaging measurements, anthropometric measures and neurological function and mental health assessments. Blood and urinary samples were collected at baseline. All participants will be followed for physiological and psychological disorders and updated lifestyle and environmental exposures every 5 years. Findings to date The mean age of the participants was 67.73 years at baseline, and 42.74% were males. The prevalences of individuals with unhealthy conditions were as follows: overweight/obesity (54.38%), hypertension (58.24%), diabetes mellitus (22.30%), dyslipidaemia (75.69%), chronic bronchitis (1.45%), myocardial infarction (0.55%), coronary heart disease (5.69%), stroke (1.10%), cancer (2.18%), arthritis (5.04%), Alzheimer’s disease (0.18%), Parkinson’s disease (0.23%), brain injury (5.75%), cognitive impairment (5.39%) and depression status (3.28%). The mean scores for the Lawton-Brody Activities of Daily Living Scale and the Social Support Rate Scale were 14.15 and 39.54, respectively. Future plans 2000 new entrants from Luohu district will be recruited every year until 2028. The data collection is expected to be ended at the end of 2030. The data will be used to assess the causality of ageing-related disorders, especially neurological and mental disorders through integrating environmental, genetic and lifestyle factors. The data sets generated and/or analysed during the current study are not publicly available at this stage, but are available from the corresponding author on reasonable request.

Abstract: Progressive distal symmetrical axonal neuropathy, a complication of diabetes mellitus (DM), has an unknown cause. Normal physiological metabolism and diabetic dysmetabolism are associated with the generation of γ-diketones. γ-Diketones form pyrroles with protein amines, notably with axonal proteins required for the maintenance of nerve fiber integrity, especially elongate, large-diameter peripheral nerve fibers innervating the extremities. We tested the hypothesis that neuropathy-associated γ-diketone pyrroles are elevated in DM. Research design and methods We measured the urinary concentration of γ-diketone pyrroles in age-matched and gender-matched elderly (60–84 years) persons with (n=267) or without (n=267) indicators of DM based in a community population (9411 community older adults aged ≥60 years) in Shenzhen city, Guangdong, China. We used statistical methods, including a generalized linear model, multivariate logistic regression analysis and restricted cubic splines, to assess linear and nonlinear relationships between urinary γ-diketone pyrroles and indicators of DM. Results Compared with healthy controls, those with DM had significantly higher levels of fasting blood glucose, glycated hemoglobin A1c, urinary ketone bodies and urinary γ-diketone pyrroles. The median concentration of urinary γ-diketone pyrrole adducts was significantly higher (p 〈 0.0001) in individuals with DM (7.5 (5.4) μM) compared with healthy controls (5.9 (4.3) μM). Both linear and non-linear relations were found between urinary γ-diketone pyrroles and indicators of DM. Conclusions Diabetic dysmetabolism includes increased generation and excretion of neuropathy-associated γ-diketone pyrroles. These findings form the foundation for studies to test whether γ-diketone pyrrole concentration correlates with quantitative sensory (vibration and temperature) and electrodiagnostic testing.

Abstract: In high-grade chondrosarcoma, 5-year survival is lower than 50%. Therefore, it is important that preclinical models that mimic the disease with the greatest possible fidelity are used to potentially develop new treatments. Accumulating evidence suggests that two-dimensional (2-D) cell culture may not accurately represent the tumor’s biology. It has been demonstrated in other cancers that three-dimensional (3-D) cancer cell spheroids may recapitulate tumor biology and response to treatment with greater fidelity than traditional 2-D techniques. To our knowledge, the formation of patient-derived chondrosarcoma spheroids has not been described. Questions/purposes (1) Can patient-derived chondrosarcoma spheroids be produced? (2) Do spheroids recapitulate human chondrosarcoma better than 2-D cultures, both morphologically and molecularly? (3) Can chondrosarcoma spheroids provide an accurate model to test novel treatments? Methods Experiments to test the feasibility of spheroid formation of chondrosarcoma cells were performed using HT-1080, an established chondrosarcoma cell line, and two patient-derived populations, TP19-S26 and TP19-S115. Cells were cultured in flasks, trypsinized, and seeded into 96-well ultra-low attachment plates with culture media. After spheroids formed, they were monitored daily by bright-field microscopy. Spheroids were fixed using paraformaldehyde and embedded in agarose. After dehydration with isopropanol, paraffin-embedded spheroids were sectioned, and slides were stained with hematoxylin and eosin. To compare differences and similarities in gene expression between 2-D and 3-D chondrosarcoma cultures and primary tumors, and to determine whether these spheroids recapitulated the biology of chondrosarcoma, RNA was extracted from 2-D cultures, spheroids, and tumors. Quantitative polymerase chain reaction was performed to detect chondrosarcoma markers of interest, including vascular endothelial growth factor alpha, hypoxia-inducible factor 1α, COL2A1, and COL10A1. To determine whether 2-D and 3-D cultures responded differently to novel chondrosarcoma treatments, we compared their sensitivities to disulfiram and copper chloride treatment. To test their sensitivity to disulfiram and copper chloride treatment, 10,000 cells were seeded into 96-well plates for 2-D culturing and 3000 cells in each well for 3-D culturing. After treating the cells with disulfiram and copper for 48 hours, we detected cell viability using quantitative presto-blue staining and measured via plate reader. Results Cell-line and patient-derived spheroids were cultured and monitored over 12 days. Qualitatively, we observed that HT-1080 demonstrated unlimited growth, while TP19-S26 and TP19-S115 contracted during culturing relative to their initial size. Hematoxylin and eosin staining of HT-1080 spheroids revealed that cell-cell attachments were more pronounced at the periphery of the spheroid structure than at the core, while the core was less dense. Spheroids derived from the intermediate-grade chondrosarcoma TP19-S26 were abundant in extracellular matrix, and spheroids derived from the dedifferentiated chondrosarcoma TP19-S115 had a higher cellularity and heterogeneity with spindle cells at the periphery. In the HT-1080 cells, differences in gene expression were appreciated with spheroids demonstrating greater expressions of VEGF-α (1.01 ± 0.16 versus 6.48 ± 0.55; p = 0.003), COL2A1 (1.00 ± 0.10 versus 7.46 ± 2.52; p 〈 0.001), and COL10A1 (1.01 ± 0.19 versus 22.53 ± 4.91; p 〈 0.001). Differences in gene expressions were also noted between primary tumors, spheroids, and 2-D cultures in the patient-derived samples TP19-S26 and TP19-S115. TP19-S26 is an intermediate-grade chondrosarcoma. With the numbers we had, we could not detect a difference in VEGF-α and HIF1α gene expression compared with the primary tumor. COL2A1 (1.00 ± 0.14 versus 1.76 ± 0.10 versus 335.66 ± 31.13) and COL10A1 (1.06 ± 0.378 versus 5.98 ± 0.45 versus 138.82 ± 23.4) expressions were both greater in the tumor (p (COL2A1) 〈 0.001; p (COL10A1) 〈 0.0001) and 3-D cultures (p (COL2A1) = 0.004; p (COL10A1) 〈 0.0001) compared with 2-D cultures. We could not demonstrate a difference in VEGF-α and HIF1α expressions in TP19-S115, a dedifferentiated chondrosarcoma, in the tumor compared with 2-D and 3-D cultures. COL2A1 (1.00 ± 0.02 versus 1.86 ± 0.18 versus 2.95 ± 0.56) and COL10A1 (1.00 ± 0.03 versus 5.52 ± 0.66 versus 3.79 ± 0.36) expressions were both greater in spheroids (p (COL2A1) = 0.003; p (COL10A1) 〈 0.0001) and tumors (p (COL2A1) 〈 0.001; p (COL10A1) 〈 0.0001) compared with 2-D cultures. Disulfiram-copper chloride treatment demonstrated high cytotoxicity in HT-1080 and SW-1353 chondrosarcoma cells grown in the 2-D monolayer, but 3-D spheroids were highly resistant to this treatment. Conclusion We provide preliminary findings that it is possible to generate 3-D spheroids from chondrosarcoma cell lines and two human chondrosarcomas (one dedifferentiated chondrosarcoma and one intermediate-grade chondrosarcoma). Chondrosarcoma spheroids derived from human tumors demonstrated morphology more reminiscent of primary tumors than cells grown in 2-D culture. Spheroids displayed similar expressions of cartilage markers as the primary tumor, and we observed a higher expression of collagen markers in the spheroids compared with cells grown in monolayer. Spheroids also demonstrated greater chemotherapy resistance than monolayer cells, but more patient-derived spheroids are needed to further conclude that 3-D cultures may mimic the chemoresistance that chondrosarcomas demonstrate clinically. Additional studies on patient-derived chondrosarcoma spheroids are warranted. Clinical Relevance Chondrosarcomas demonstrate resistance to chemotherapy and radiation, and we believe that if they can be replicated, models such as 3-D spheroids may provide a method to test novel treatments for human chondrosarcoma. Additional comprehensive genomic studies are required to compare 2-D and 3-D models with the primary tumor to determine the most effective way to study this disease in vitro.

Abstract: The steroid receptor coactivator-1 (SRC-1) is a nuclear receptor co-activator, known to play key roles in both estrogen response in bone and in breast cancer metastases. We previously demonstrated that the P1272S single nucleotide polymorphism (SNP; P1272S; rs1804645) in SRC-1 decreases the activity of estrogen receptor in the presence of selective estrogen receptor modulators (SERMs) and that it is associated with a decrease in bone mineral density (BMD) after tamoxifen therapy, suggesting it may disrupt the agonist action of tamoxifen. Given such dual roles of SRC-1 in the bone microenvironment and in tumor cell-intrinsic phenotypes, we hypothesized that SRC-1 and a naturally occurring genetic variant, P1272S, may promote breast cancer bone metastases. We developed a syngeneic, knock-in mouse model to study if the SRC-1 SNP is critical for normal bone homeostasis and bone metastasis. Our data surprisingly reveal that the homozygous SRC-1 SNP knock-in increases tamoxifen-induced bone protection after ovariectomy. The presence of the SRC-1 SNP in mammary glands resulted in decreased expression levels of SRC-1 and reduced tumor burden after orthotopic injection of breast cancer cells not bearing the SRC-1 SNP, but increased metastases to the lungs in our syngeneic mouse model. Interestingly, the P1272S SNP identified in a small, exploratory cohort of bone metastases from breast cancer patients was significantly associated with earlier development of bone metastasis. This study demonstrates the importance of the P1272S SNP in both the effect of SERMs on BMD and the development of tumor in the bone.