Tissue-Specific Content of Polyunsaturated Fatty Acids in (n-3) Deficiency State of Rats

The dietary intake of fatty acids (FAs) affects the composition and distribution of FAs in the body. Here, a first-generation (n-3)-deficiency study was conducted by keeping young (age 21 ± 2 days) Sprague–Dawley male rats on a peanut-oil-based diet for 33 days after weaning in order to compare the effect of mild (n-3)-deficiency on the lipid composition of different organs and feces. Soybean-oil-based diet was used as a control. The plasma FA levels corresponded to FAs levels in the organs. Lower docosahexaenoic acid (DHA) content was detected in the plasma, brain, testis, visceral fat, heart, and lungs of the (n-3)-deficient group, whereas the DHA content of the eye and feces did not differ between the experimental groups. The DHA content of the brains of the (n-3)-deficient group was 86% of the DHA content of the brains of the (n-3)-adequate group. The DHA level of the organs was affected in the order of visceral fat > liver triacylglycerols > lung > heart > liver phospholipids > testis > eye > brain, with brain being least affected. The low levels of (n-3) FAs in the liver, brain, eye, heart, and lung were offset by an increase in the (n-6) FAs, mainly arachidonic acid. These results indicate that, in rats, adequate maternal nutrition during pregnancy and weaning does not provide enough (n-3) FAs for 33 days of an (n-3)-deficient diet. Results of this study can be used also to evaluate the conditions needed to reach mild (n-3) deficiency in the first generation of rats and to evaluate the feasibility to collect data from a variety of organs or only selected ones.

Effect of serum vitamin D level before ovarian stimulation on the cumulative live birth rate of women undergoing in vitro fertilization: a retrospective analysis

Objective: Vitamin D receptors are present in the female reproductive tract. Studies on the association between serum vitamin D level and pregnancy rate of in vitro fertilization (IVF) showed inconsistent results and focused on a single fresh or frozen embryo transfer cycle. The objective of our study was to evaluate if serum vitamin D level before ovarian stimulation was associated with the cumulative live birth rate (CLBR) of the first IVF cycle. Design: Retrospective cohort study. Methods: Women who underwent the first IVF cycle from 2012 to 2016 at a university-affiliated reproductive medicine center were included. Archived serum samples taken before ovarian stimulation were analyzed for 25(OH)D levels using liquid chromatography-mass spectrometry. Results: 1,113 had pregnancy outcome from the completed IVF cycle. The median age (25th-75th percentile) of the women was 36 (34-38) years and serum 25(OH)D level was 53.4 (41.9-66.6)nmol/L. The prevalence of vitamin D deficiency (less than 50nmol/L) was 42.2%. The CLBR in the vitamin D deficient group was significantly lower compared to the non-deficient group (43.9%,208/474 vs 50.9%,325/639, p=0.021, unadjusted), and after controlling for women’s age, body mass index, antral follicle count, type and duration of infertility. There were no differences in the clinical/ongoing pregnancy rate, live birth rate and miscarriage rate in the fresh cycle between the vitamin D deficient and non-deficient groups. Conclusions: Vitamin D deficiency was prevalent in infertile women in subtropical Hong Kong. The CLBR of the first IVF cycle in the vitamin D deficient group was significantly lower compared to the non-deficient group.

Preoperative Vitamin D Deficiency is Associated with Postoperative Functional Recovery and Complications after Hip Fracture Surgery

Background: Low concentrations of vitamin D are considered one of the risk factors for hip fracture and are associated with worse outcomes. The purpose of this retrospective study was to compare vitamin D deficient group and vitamin D sufficient group and assess the association preoperative vitamin D deficiency and postoperative walking ability after hip fracture surgery.Methods: Between January 2014 and January 2020, 1,029 elderly patients with hip fracture (243 in men and 785 in women) were measured preoperative serum 25-hydroxy-vitamin D3 levels. Among 1,029 elderly patients, 702 patients were classified as Vitamin D deficient group (<20 ng/mL). Outcome parameters for functional recovery were the length of the hospital stay and KOVAL score, and those for complications were delirium, pneumonia, and thromboembolism.Results: The mean length of the hospital stay in the vitamin D deficient group was significantly longer than in the vitamin D sufficient group (27.7±17.8 vs. 2.9±11.8 days; odds ratio [OR], 1.03; 95% confidence interval [CI], 1.02-1.05; P=0.001). The mean postoperative KOVAL score in the deficient group was significantly higher than in the sufficient group (4.0±2.1 vs. 3.1±1.9 days; OR, 1.21; 95% CI, 1.11-1.32; P=0.001). Vitamin D deficiency was significantly associated with a higher risk of delirium and pneumonia in deficiency group.Conclusions: Preoperative vitamin D deficiency in hip fractures patients was associated with prolonged duration of hospital stay and decrease of postoperative ambulatory status, and may increase the risk of delirium and pneumonia. Therefore, it is necessary to evaluate the preoperative vitamin D level and recommend vitamin D supplementation in elderly patients with a high probability of hip fracture.

Association of SNPs within TMPRSS6 and BMP2 genes with iron deficiency status in Saudi Arabia

Background Globally, iron-deficiency anemia (IDA) remains a major health obstacle. This health condition has been identified in 47% of pre-school students (aged 0 to 5 years), 42% of pregnant females, and 30% of non-pregnant females (aged 15 to 50 years) worldwide according to the WHO. Environmental and genetic factors play a crucial role in the development of IDA; genetic testing has revealed the association of a number of polymorphisms with iron status and serum ferritin. Aim The current study aims to reveal the association of TMPRSS6 rs141312 and BMP2 rs235756 with the iron status of females in Saudi Arabia. Methods A cohort of 108 female university students aged 18–25 years was randomly selected to participate: 50 healthy and 58 classified as iron deficient. A 3–5 mL sample of blood was collected from each one and analyzed based on hematological and biochemical iron status followed by genotyping by PCR. Results The genotype distribution of TMPRSS6 rs141312 was 8% (TT), 88% (TC) and 4% (CC) in the healthy group compared with 3.45% (TT), 89.66% (TC) and 6.89% (CC) in the iron-deficient group (P = 0.492), an insignificant difference in the allelic distribution. The genotype distribution of BMP2 rs235756 was 8% (TT), 90% (TC) and 2% (CC) in the healthy group compared with 3.45% (TT), 82.76% (TC) and 13.79% (CC) in iron-deficient group (P = 0.050) and was significantly associated with decreased ferritin status (P = 0.050). In addition, TMPRSS6 rs141312 is significantly (P<0.001) associated with dominant genotypes (TC+CC) and increased risk of IDA while BMP2 rs235756 is significantly (P<0.026) associated with recessive homozygote CC genotypes and increased risk of IDA. Conclusion Our finding potentially helps in the early prediction of iron deficiency in females through the genetic testing.

S100a8/S100a9-Emmprin-Vegfa Axis Initiated By Tet2-Deficient Immune Cells Exacerbates Lung Cancer Progression through Promotion of Angiogenesis

Introductions: Loss-of-function TET2 mutations are frequent in clonal hematopoiesis in patients with solid cancers as well as that in healthy individuals. It remains to be elucidated whether and how TET2-mutated immune cells affect cancer progression in patients with TET2-mutated clonal hematopoiesis. Here, we assessed activity of Tet2-deficient immune cells using a mouse lung cancer model. Methods: Lewis Lung Carcinoma (LLC) cells were subcutaneously transplanted into blood-specific Mx-Cre or myeloid-specific LysM-Cre x Tet2 f/f mice (Tet2 -/- or Tet2 mye-) or control mice (CT). Single-cell RNA sequencing (scRNA-seq) was performed to determine the immune-cell profiles and mediators in tumors of Tet2 -/- mice (Tet2 -/- tumors). Whole transcriptome analysis (WTA) was also performed for granulocytic myeloid-derived cells (GMD), monocytic myeloid-derived cells (MMD), and tumor associated macrophages (TAM), as well as LLC cells sorted from Tet2 -/- tumors and CT tumors. Results: We found that tumor growth was enhanced in both Tet2 -/- and Tet2 mye- comparing to CT. Unsupervised clustering of scRNA-seq data identified 14 cell clusters: GMD into 3 (GMD1, GMD2, and GMD3), MMD into 5 (MMD1, MMD2, MMD3, MMD4, and MMD5), TAMs into 4 (TAM1, TAM2, TAM3, and TAM4), and DCs into 2 (DC1 and DC2). Notably, among all subclusters, the proportions of GMD1, GMD3, TAM3 and TAM4 were markedly expanded in Tet2 -/- tumors comparing to CT. Differentially expressed gene (DEG) analysis of scRNA-seq data found that S100a8 and S100a9 were highly expressed in Tet2-deficient GMD1 compared to CT. Furthermore, S100a8/S100a9 proteins were elevated in plasmas of Tet2 -/- comparing to those of CT. Pathway analysis using DEGs (p &lt; 0.05) from WTA of GMD determined interleukin 1b (Il1b) signaling as upstream of S100a8/S100a9 activity. Gene set enrichment analysis (GSEA) also showed that 6 pathways related to Il1b were enriched in Tet2-deficient group compared to CT group. Gene ontology analysis (GO) for DEGs of GMD, MMD, and TAMs by WTA as well as 13 subclusters by scRNA-seq revealed that the "cellular response to IL-1" pathway was enriched in Tet2-deficient group compared to CT group. To define the downstream effectors in LLC cells, we performed WTA for LLC cells sorted from Tet2 -/- and CT tumors. We found that Vegfa, encoding a mediator for angiogenesis was highly upregulated in LLC cells sorted from Tet2 -/- tumors comparing to CT tumors. GSEA for WTA further identified that multiple Vegfa-related pathways as well as MAPK cascade were enriched in LLC cells from Tet2 -/- tumors comparing to those from CT tumors . Furthermore, S100a8/S100a9 induced Vegfa secretion from LLC cells in vitro. Remarkably, the area of blood vessels was increased in Tet2 -/- tumors comparing to CT tumors. Immunostaining exhibited that the number of Ly6g +GMD foci (&gt;1000 px 2) expressing S100a8/S100a9 was increased in Tet2 -/- tumors comparing to CT tumors. Furthermore, LLC cells surrounding GMD foci highly expressed Vegfa in Tet2 -/- tumors. Finally, administration of an antibody against Emmprin, a receptor for S100a8/S100a9 inhibited the tumor growth in Tet2 -/-. Notably, the area of blood vessels in Tet2 -/- tumors with anti-Emmprin group was decreased at 2-fold compared to that seen in isotype group (p &lt; 0.05). Consistently, S100A8/S100A9 induced VEGFA production in human lung cancer cells in vitro. Conclusions: Tet2-deificient immune cells promote lung cancer progression through S100a8/S100a9-Emmprin-Vegfa axis. Our study suggests a novel role of TET2-mutated clonal hematopoiesis in cancer progression and even provides a novel therapeutic target. Disclosures No relevant conflicts of interest to declare.

Vitamin D Levels: Associations with Acute Pain Events and Self-Reported Pain in Children with Sickle Cell Disease

Background/Aims: Individuals with sickle cell disease (SCD) are at high risk for vitamin D deficiency due to dark skin color, limited physical activity, poor nutrition, and renal dysfunction. In smaller retrospective studies, vitamin D deficiency in SCD was associated with increased frequency of acute pain events and higher opioid use, however the impact of vitamin D deficiency on pain related patient reported outcomes has not been described . Supplementation with high dose vitamin D is shown to be associated with fewer pain days per week and higher physical activity scores in patients with chronic pain. Thus, the objective of our study was to evaluate relationship between vitamin D levels, acute health care utilization and self-reported pain among children with SCD. We hypothesized that vitamin D deficiency is associated with increase health care utilization for pain and patient-reported outcomes for pain. Methods: Patients with SCD (1 to 18 years of age), enrolled in Sickle Cell Clinical Research and Intervention Program (SCCRIP), at St. Jude Children's Research Hospital were included in the study (Hankins et.al., Pediatric Blood and Cancer, 2018). Demographic, clinical and laboratory data were extracted from SCCRIP database. Prospective pain event leading to a hospital visit was analyzed. We analyzed pain-related hospitalizations in two ways: the number of pain-related hospitalizations within 2 years prior to and after vitamin D measurement in a cross-sectional design and longitudinally. Generalized linear regression model was used to examine associations between vitamin D and cross-sectional pain events with adjusting for hydroxyurea use. Generalized linear mixed effect model was used to assess the associations between vitamin D and longitudinal pain events, with adjusting for SCD genotype, sex, age, hydroxyurea use and interaction between age and hydroxyurea use. Pediatric Quality of Life Inventory™ Sickle Cell Disease Module (PedsQL™ SCD) was used to measure pain and pain interference using 1) Pain and Hurt and 2) Pain Impact scales. PedsQL™ SCD assessments include a Likert response scale. Items are reverse -scored and transformed to a 0-100 scale where higher scores indicate better health related quality of life in that scale. Two sample t-test or Wilcoxon rank sum test was used to compare mean scores between the two groups. Results: A total of 799 patients (females, n=398; males, n=401) were included in the study. Mean age (Standard Deviation [SD]), range at time of first vitamin D measurement for entire cohort was 8.7 (4.7), (0.8-18 years). Mean (SD) initial vitamin D level for entire cohort was 19.0 (9.4) ng/dl. Mean (SD) number of pain-related hospitalizations within two years of first vitamin D measurement was higher in the vitamin D deficient group as compared to non deficient group [0.81(1.97) vs 0.66 (1.2), (p=0.0034)]. Longitudinal data analyses showed vitamin D deficiency was associated with increased number of pain related hospitalizations (p=0.0091), after adjusting for covariates listed above (Figure 1a). Mean (SD) scores for Pain and Hurt in vitamin D deficient versus non deficient group were [74.5.7(18.2) versus 83.5(15.6), p &lt; 0.001]. Mean (SD) scores for Pain Impact in vitamin D deficient group as compared to non deficient group were [(66.7 (22.7) versus 75.5 (22.3), p &lt;0.001] (Figure 1b) Conclusion: Results indicated that low vitamin D levels predicted higher frequency of painful events leading to a hospital visit and were associated with higher prevalence of self-reported pain and pain interference. Further studies evaluating mechanisms by which vitamin D influences sickle cell pain are warranted and larger controlled trials can help evaluate the therapeutic efficacy of vitamin D for sickle cell pain. Figure 1 Figure 1. Disclosures Hankins: UpToDate: Consultancy; Bluebird Bio: Consultancy; Vindico Medical Education: Consultancy; Global Blood Therapeutics: Consultancy.

Association of vitamin B12 deficiency with metformin use in patients with type 2 diabetes treated in the largest tertiary care hospital in Qatar

Background: Data on the effect of metformin on serum vitamin B12 (VitB12) level in patients with type 2 diabetes mellitus (T2DM) in Qatar are limited; therefore, we aimed to assess the prevalence of VitB12 deficiency and its related factors among patients with T2DM treated with metformin at Hamad General Hospital in Doha, Qatar, from January 1, 2017, to December 31, 2017. Methods: This cross-sectional analytical study involved patients with T2DM aged ≥ 18 years who used metformin for at least 3 months. The serum VitB12 was quantified on a chemiluminescent enzyme immunoassay analyzer using Cobas e 801 module, Roche, and VitB12 deficiency was defined as serum VitB12 level of ≤ 145 pmol/L. All data were obtained from the patients’ electronic medical records. Results: The study recruited 3124 eligible patients with T2DM. The overall prevalence of metformin-associated VitB12 deficiency was 30.7% [95% confidence of interval, 0.290–0.323]. A significant difference exists in the median VitB12 levels between the VitB12-normal and VitB12-deficient groups [129 vs. 286; p < 0.001]. Compared with the VitB12-normal group, the VitB12-deficient group had higher mean body mass index (BMI) (p < 0.001) and consumed higher doses of metformin (p = 0.001). They also more often used sulfonylurea (p = 0.004), dipeptidyl peptidase-4 inhibitor (p < 0.001), thiazolidinediones (p < 0.001), glucagon-like peptide 1 [GLP-1] receptor agonists (p < 0.001), alpha-glucosidase inhibitor (p < 0.001), and H2 blocker/proton pump inhibitors [PPI] (p < 0.001) than the VitB12-normal group. Moreover, the VitB12-normal group consumed more calcium supplements (p < 0.001) than the VitB12-deficient group. In the multivariate analysis, independent risk factors for metformin-associated VitB12 deficiency in patients with T2DM include high daily dose of metformin >2000 mg, male gender, high BMI, smoking, sulfonylurea, dipeptidyl peptidase-4 inhibitor, H2 blockers/PPI, low fasting blood glucose, and low hemoglobin. Conclusion: This study showed a high prevalence of VitB12 deficiency in patients with T2DM taking metformin and a significant negative correlation between the daily dose of metformin and serum VitB12 level. Therefore, regular screening for serum VitB12 is necessary in patients with T2DM on metformin treatment, especially those who have the abovementioned risk factors.

Study of metabolomics in selenium deprived Przewalski’s Gazelles (P. przewalskii)

To understand why P. przewalskii does not show the same white myopathy as sheep in Se-deficient regions, and to provide reference for feeding nutrition level of artificial population and selection of wild reintroduction areas in the later period, a Se-deficient model was established. The mineral elements content, physiological and biochemical parameters in blood, and serum metabonomics were determined. In the Se-deficient group compared with the control group, the Se content was highly significantly lower (P<0.01), and the Cu content was significantly higher (P<0.05). The activity of glutathione peroxidase (GSH-Px) was significantly lower (P<0.05), but total superoxide dismutase (T-SOD) was significantly higher (P<0.05). Principal component analysis (PCA) revealed significant differences in serum metabolic profiling between the Se-deficient group and control group, orthogonal partial least squares discriminant analysis (OPLS-DA) indicated significant differences in metabolite distribution in the serum between the two groups (P<0.05, variable importance in projection VIP>1). By matching the mass spectrum data of compounds with the Kyoto Encyclopedia of Genes and Genomes (KEGG database), identified 86 types of differential metabolites in the serum. The main metabolic pathways included secondary bile acid biosynthesis, biosynthesis of unsaturated fatty acids and pyrimidine metabolism. Further analysis showed that there were 7 different metabolites in pyrimidine metabolism pathway between the Se-deficient and control group. And there was no significant difference in red blood cell (RBC), hemoglobin (Hb) and total antioxidant capacity (T-AOC) between the two groups (P>0.05). The above results showed that the differential metabolism of substances exhibited complementary functions, thus alleviating some adverse effects and resulting normal activities of P. przewalskii can be carried out under the condition of dietary Se content lower than 0.05mg/kg.

Patient-acceptable symptom state for reporting outcomes following unicompartmental knee arthroplasty

Aims The patient-acceptable symptom state (PASS) is a level of wellbeing, which is measured by the patient. The aim of this study was to determine if the proportion of patients who achieved an acceptable level of function (PASS) after medial unicompartmental knee arthroplasty (UKA) was different based on the status of the anterior cruciate ligament (ACL) at the time of surgery. Methods A total of 114 patients who underwent UKA for isolated medial osteoarthritis (OA) of the knee were included in the study. Their mean age was 65 years (SD 10). No patient underwent a bilateral procedure. Those who had undergone ACL reconstruction during the previous five years were excluded. The Knee injury Osteoarthritis Outcome Score Activities of Daily Living (KOOS ADL) function score was used as the primary outcome measure with a PASS of 87.5, as described for total knee arthroplasty (TKA). Patients completed all other KOOS subscales, Lysholm score, the Western Ontario and McMaster Universities Osteoarthritis Index, and the Veterans Rand 12-item health survey score. Failure was defined as conversion to TKA. Results Survivorship at ten years was 97% in both the ACL-deficient and ACL-intact groups. The mean survival was 16.1 years (95% confidence interval (CI) 15.3 to 16.8) for the ACL-deficient group and 15.6 years (95% CI 14.8 to 16.361) for the ACL-intact group (p = 0.878). At a mean of nine years (SD 3.5) in the ACL-deficient group, 32 patients (87%) reached the PASS for the KOOS ADL. In the ACL-intact group, at a mean of 8.6 years (SD 3) follow-up, 63 patients (85%) reached PASS for the KOOS ADL. There was no significant difference in the percentage of patients who reached PASS for all KOOS subscales and Lysholm between the two groups. Conclusion PASS was achieved in 85% of all UKAs for KOOS ADL, similar to reports for TKA. Fixed-bearing, medial, non-robotically-assisted UKA resulted in 97% survival at ten years in both the ACL-deficient and ACL-intact groups. There was no significant difference in all outcomes between the two groups. Understanding PASS will allow better communication between surgeons and patients to improve the surgical management of patients with single compartment OA of the knee. This study provides mid- to long-term data supporting the use of PASS to document outcomes following UKA. PASS was met in more than 85% of patients with no differences between ACL-deficient and ACL-intact knees at a mean follow-up of nine years. Cite this article: Bone Joint J 2021;103-B(8):1367–1372.

RBM10 Deficiency Is Associated With Increased Immune Activity in Lung Adenocarcinoma

IntroductionRBM10 is one of the frequently mutated genes in lung adenocarcinoma (LUAD). Previous studies have confirmed that RBM10 could suppress the disease progression and cell proliferation in LUAD, but its loss-of-function mutations are more frequent in early-stage disease and decrease with the advancement of the clinical stage. This is contradictory to its role of tumor suppressor. Here, we conducted a systematic analysis to elucidate whether there was other potential biological significance of RBM10 deficiency during the progression of LUAD.Materials and MethodsThe whole exome sequencing data of 39 tumor samples from early-stage LUADs (GGN cohort) and genomic and transcriptome data of the Cancer Genome Atlas (TCGA) LUAD cohort (TCGA_LUAD cohort) and a Chinese LUAD cohort (CHOICE_ADC cohort) were first obtained. Systematic bioinformatic analyses were then conducted to determine gene expression signature, immune infiltration levels and predicted immunotherapy response. Immunohistochemistry (IHC) was also conducted to validate the result of immune infiltration.ResultsThe mutation rate of RBM10 was significantly higher in the GGN cohort than that in the TCGA_LUAD and CHOICE_ADC cohorts. In both TCGA_LUAD and CHOICE_ADC cohorts, multiple immune related pathways were markedly enriched in RBM10 deficient group. Further analyses showed that tumors with RBM10 mutations displayed higher TMB, and LUADs with RBM10 deficiency also showed higher HLA expression levels, including many HLA class I and II molecules. Additionally, many immune cells, including myeloid dendritic cells, macrophages, neutrophils and CD8+T cells, showed higher infiltration levels in LUADs with RBM10 deficiency. Finally, some immune checkpoint molecules, such as PD-L1 and TIM-3, were highly expressed in RBM10 deficient population and the predicted immunotherapy response was calculated through TIDE algorithm, showing that IFNG expression, MSI score and CD8 expression were higher in RBM10 deficient group, while MDSC and M2 macrophage were lower in RBM10 deficient group.ConclusionOur study demonstrates that RBM10 deficient LUADs show higher HLA expression and immune cell infiltration, and some immune checkpoint molecules are also highly expressed. In brief, RBM10 deficiency could enhance anti-tumor immunity in LUAD.