Comparison of Target Enrichment Platforms for Circulating Tumor DNA Detection.

Cancer-related mortality of solid tumors remains the major cause of death worldwide. Circulating tumor DNA (ctDNA) released from cancer cells harbors specific somatic mutations. Sequencing ctDNA opens opportunities to non-invasive population screening and lays foundations for personalized therapy. In this study, two commercially available platforms, Roche's Avenio ctDNA Expanded panel and QIAgen's QIAseq Human Comprehensive Cancer  panel were compared for (1) panel coverage of clinically relevant variants; (2) target enrichment specificity and sequencing performance; (3) the sensitivity; (4) concordance and (5) sequencing coverage using the same human blood sample with ultra-deep next-generation sequencing. Our finding suggests that Avenio detected somatic mutations in common cancers in over 70% of patients while QIAseq covered nearly 90% with a higher average number of variants per patient (Avenio: 3; QIAseq: 8 variants per patient). Both panels demonstrated similar on-target rate and percentage of reads mapped. However, Avenio had more uniform sequencing coverage across regions with different GC content. Avenio had a higher sensitivity and concordance compared with QIAseq at the same sequencing depth. This study identifies a unique niche for the application of each of the panel and allows the scientific community to make an informed decision on the technologies to meet research or application needs.

Plasma C-terminal cross-linking telopeptide of type II collagen as a biomarker in advanced stages of femoral head osteonecrosis.

AIM: The aim of this study is to investigate the potential role of C-terminal cross-linking telopeptide of type II collagen (CTX-II) in the development of femoral head osteonecrosis (FHN).Materials and methodsOne hundred and thirty-two patients diagnosed with FHN and 66 age- and sex-matched healthy subjects were included in this cross-sectional case-control study between May 2016 and November 2016. Bone histomorphology, cartilaginous immunohistochemistry, Western blotting, and level of plasma CTX-II, as well as the receiver operating characteristic (ROC) curve, were evaluated. ResultsImmunohistochemistry and Western blotting showed that CTX-II was abundantly detected in the cartilage of FHN samples in stages III & IV. Plasma CTX-II levels were significantly higher in FHN patients in the advanced stages (III & IV) than was the level in the control group (164.81% and 198.15% higher in stages III & IV, respectively). However, the levels of CTX-II did not differ significantly among the FHN patients with different etiology or other clinical data. ConclusionOur result suggests that the degeneration of cartilage has already begun in stage III, even though a relatively normal articular gap can be found in the X-ray. Plasma CTX-II level may be a biomarker of the development of FHN.

Association of reduced sclerostin expression with collapse process in patients with osteonecrosis of the femoral head.

PURPOSE: Sclerostin is an osteocyte-derived protein that has a potent inhibitory effect on osteoblast activity. The osteocyte apoptosis induced by various causes of osteonecrosis of the femoral head (ONFH) plays a key role in the promotion of femoral head collapse. But the effect of altering sclerostin level on the collapse of ONFH has not been studied. Our aim was to assess the role of sclerostin level in the collapse of ONFH. METHODS: Between May 2016 and November 2016, 236 subjects were enrolled in the present study. The patients were classified according to the Association Research Circulation Osseous (ARCO) classification. The clinical bone histomorphology, the expression position, and level of sclerostin as well as the plasma sclerostin level were evaluated. RESULTS: The sclerostin level was significantly lower in the non-traumatic ONFH group than those in the healthy control group (P = 0.002). The sclerostin level was negatively associated with ARCO stages (r = - 0.239, P = 0.009) and significantly lower in the postcollapse group (P = 0.025). CONCLUSIONS: The reduced expression of sclerostin may play a key role in the collapse process of ONFH and be predictive of the disease progression of ONFH.

Membrane Trafficking Protein CDP138 Regulates Fat Browning and Insulin Sensitivity through Controlling Catecholamine Release.

CDP138 is a calcium- and lipid-binding protein that is involved in membrane trafficking. Here, we report that mice without CDP138 develop obesity under normal chow diet (NCD) or high-fat diet (HFD) conditions. CDP138-/- mice have lower energy expenditure, oxygen consumption, and body temperature than wild-type (WT) mice. CDP138 is exclusively expressed in adrenal medulla and is colocalized with tyrosine hydroxylase (TH), a marker of sympathetic nervous terminals, in the inguinal fat. Compared with WT controls, CDP138-/- mice had altered catecholamine levels in circulation, adrenal gland, and inguinal fat. Adrenergic signaling on cyclic AMP (cAMP) formation and hormone-sensitive lipase (HSL) phosphorylation induced by cold challenge but not by an exogenous ß3 adrenoceptor against CL316243 were decreased in adipose tissues of CDP138-/- mice. Cold-induced beige fat browning, fatty acid oxidation, thermogenesis, and related gene expression were reduced in CDP138-/- mice. CDP138-/- mice are also prone to HFD-induced insulin resistance, as assessed by Akt phosphorylation and glucose transport in skeletal muscles. Our data indicate that CDP138 is a regulator of stress response and plays a significant role in adipose tissue browning, energy balance, and insulin sensitivity through regulating catecholamine secretion from the sympathetic nervous terminals and adrenal gland.

Neutrophil Elastase Regulates Emergency Myelopoiesis Preceding Systemic Inflammation in Diet-induced Obesity.

Inflammation plays a significant role in the development of obesity-related complications, but the molecular events that initiate and propagate such inflammation remain unclear. Here, we report that mice fed a high-fat diet (HFD) for as little as 1-3 days show increased differentiation of myeloid progenitors into neutrophils and monocytes but reduced B lymphocyte production in the bone marrow. Levels of neutrophil elastase (NE) and the nuclear factors CCAAT/enhancer-binding protein α (C/EBPα) and growth factor-independent 1 (GFI-1) are elevated in hematopoietic stem and progenitor cells from HFD-fed mice, but mice lacking either NE or C/EBPα are resistant to HFD-induced myelopoiesis. NE deletion increases expression of the inhibitory isoform of p30 C/EBPα, impairs the transcriptional activity of p42 C/EBPα, and reduces expression of the C/EBPα target gene GFI-1 in hematopoietic stem and progenitor cells, suggesting a mechanism by which NE regulates myelopoiesis. Furthermore, NE deletion prevents HFD-induced vascular leakage. Thus, HFD feeding rapidly activates bone marrow myelopoiesis through the NE-dependent C/EBPα-GFI-1 pathway preceding vascular damage and systemic inflammation.

Imbalance between neutrophil elastase and its inhibitor α1-antitrypsin in obesity alters insulin sensitivity, inflammation, and energy expenditure.

The molecular mechanisms involved in the development of obesity and related complications remain unclear. Here, we report that obese mice and human subjects have increased activity of neutrophil elastase (NE) and decreased serum levels of the NE inhibitor α1-antitrypsin (A1AT, SerpinA1). NE null (Ela2(-/-)) mice and A1AT transgenic mice were resistant to high-fat diet (HFD)-induced body weight gain, insulin resistance, inflammation, and fatty liver. NE inhibitor GW311616A reversed insulin resistance and body weight gain in HFD-fed mice. Ela2(-/-) mice also augmented circulating high molecular weight (HMW) adiponectin levels, phosphorylation of AMP-activated protein kinase (AMPK), and fatty acid oxidation (FAO) in the liver and brown adipose tissue (BAT) and uncoupling protein (UCP1) levels in the BAT. These data suggest that the A1AT-NE system regulates AMPK signaling, FAO, and energy expenditure. The imbalance between A1AT and NE contributes to the development of obesity and related inflammation, insulin resistance, and liver steatosis.

Adipose overexpression of heme oxygenase-1 does not protect against high fat diet-induced insulin resistance in mice.

Heme oxygenase-1 (HO-1) is a stress-responsive enzyme with potent anti-oxidant and anti-inflammatory activities. Previous studies have shown that systemic induction of HO-1 by chemical inducers reduces adiposity and improves insulin sensitivity. To dissect the specific function of HO-1 in adipose tissue, we generated transgenic mice with adipose HO-1 overexpression using the adipocyte-specific aP2 promoter. The transgenic (Tg) mice exhibit similar metabolic phenotype as wild type (WT) control under chow-fed condition. High fat diet (HFD) challenge significantly increased the body weights of WT and Tg mice to a similar extent. Likewise, HFD-induced glucose intolerance and insulin resistance were not much different between WT and Tg mice. Analysis of the adipose tissue gene expression revealed that the mRNA levels of adiponectin and interleukin-10 were significantly higher in chow diet-fed Tg mice as compared to WT counterparts, whereas HFD induced downregulation of adiponectin gene expression in both Tg and WT mice to a similar level. HFD-induced proinflammatory cytokine expression in adipose tissues were comparable between WT and transgenic mice. Nevertheless, immunohistochemistry and gene expression analysis showed that the number of infiltrating macrophages with preferential expression of M2 markers was significantly higher in the adipose tissue of obese Tg mice than WT mice. Further experiment demonstrated that myeloid cells from Tg mice expressed higher level of HO-1 and exhibited greater migration response toward chemoattractant in vitro. Collectively, these data indicate that HO-1 overexpression in adipocytes does not protect against HFD-induced obesity and the development of insulin resistance in mice.

Characterization of a small cryptic plasmid pK50-2 isolated from Lactobacillus reuteri K50.

The complete nucleotide sequence of a cryptic plasmid, pK50-2, from Lactobacillus reuteri K50 had been determined. It consisted of an 1866 bp circular molecule with a G+C content of 35%, from which two putative open reading frames (orfs) could be predicted. Based on sequence similarity, the orf1 was not homologous to any known protein, while the N-terminus of the orf2 shared 56% and 64% identities with RepB proteins of plasmid pAR141 and an unnamed plasmid in L. reuteri strain PA-16, members of the rolling-circle replication (RCR) pMV158 family, respectively. Downstream of orf2, a sequence containing two conserved regions (i.e., bind and nick), possibly involved in the binding and nicking of Rep protein, similar to the dso (double strand origin) of RCR-pMV158 family was also identified. Furthermore, a sequence capable of forming the characteristic secondary structure of ssoT (single-strand origin type T) was subsequently determined upstream of the orf2 gene. Thus, the three elements essential for a RCR plasmid (i.e., dso, sso, and rep gene) were all deducible in the pK50-2. Noteworthy was that a conserved alpha helix-turn-alpha helix (HTH) motif, thus far only seen in theta-type plasmids, was for the first time identified in Rep protein of RCR plasmid, pK50-2. To estimate the pK50-2 could be an expression vector to deliver exogenous antigens, a shuttle vector pK50-S containing both pK50-2 and pUE80 (-) was used to analyze the segregational stability and copy-number, which were shown that pK50-S in L. reuteri DSM 20016 were estimated to be 98%, 77%, and 75% after 36, 72, and 100 generations and about 50 copies per chromosome equivalent by real-time PCR.

Myeloid heme oxygenase-1 haploinsufficiency reduces high fat diet-induced insulin resistance by affecting adipose macrophage infiltration in mice.

Increased adipose tissue macrophages contribute to obesity-induced metabolic syndrome. Heme oxygenase-1 (HO-1) is a stress-inducible enzyme with potent anti-inflammatory and proangiogenic activities in macrophages. However, the role of macrophage HO-1 on obesity-induced adipose inflammation and metabolic syndrome remains unclear. Here we show that high-fat diet (HFD) feeding in C57BL/6J mice induced HO-1 expression in the visceral adipose tissue, particularly the stromal vascular fraction. When the irradiated C57BL/6J mice reconstituted with wild-type or HO-1(+/-) bone marrow were fed with HFD for over 24 weeks, the HO-1(+/-) chimeras were protected from HFD-induced insulin resistance and this was associated with reduced adipose macrophage infiltration and angiogenesis, suggesting that HO-1 affects myeloid cell migration toward adipose tissue during obesity. In vivo and in vitro migration assays revealed that HO-1(+/-) macrophages exhibited an impaired migration response. Chemoattractant-induced phosphorylation of p38 and focal adhesion kinase (FAK) declined faster in HO-1(+/-) macrophages. Further experiments demonstrated that carbon monoxide and bilirubin, the byproducts derived from heme degradation by HO-1, enhanced macrophage migration by increasing phosphorylation of p38 and FAK, respectively. These data disclose a novel role of hematopoietic cell HO-1 in promoting adipose macrophage infiltration and the development of insulin resistance during obesity.