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Aims: The aim of this project was to explore the different CTX-M expression levels occurring from a single conserved promoter with different spacer sequences, the variation of which is hypothesized to be a key factor in fluctuating levels of CTX-M., Methods: The blaCTX-M promoter fragments with five different spacer sequences were amplified, sequenced and cloned into the pUA66 expression vector carrying the green fluorescent protein (GFP) gene. The expression of blaCTX-M in the transconjugants was analyzed using fluorescence microscopy, flow cytometry and qRT-PCR., Results: The promoters of all the blaCTX-M genes were provided by ISEcp1 and were extremely conserved. The promoter-associated spacer sequences varied from 42 to 127 bp and variations in GFP expression in the five transconjugants were observed. A nucleic acid deletion and point mutation were detected in the spacer sequences by variations in which the expression of blaCTX-M was influenced., Conclusion: The different spacer sequences have a significant impact on the activity of the conserved promoter. The shorter spacer sequence between the conserved promoter and the blaCTX-M gene does not specifically enhance the expression of blaCTX-M, contrary to previous reports. The expression of blaCTX-M may be regulated by changes in promoter activity caused by diverse spacer sequences., (© 2016 S. Karger AG, Basel.)

As a part of breast-conserving therapy (BCT), postoperative radiotherapy is one of the main means to improve the clinical efficacy of breast cancer (BCa). However, ionizing radiation (IR) may induce BCa cells to develop radioresistance, which causes tumor recurrence and metastasis after treatment. Recently, DOC-2/DAB2 interactive protein (DAB2IP) has been reported often down-regulated in a variety of cancers and is related to tumor tolerance to radiotherapy. In this study, BCa cell lines were introduced to study how DAB2IP deficient influenced BCa cell radiosensitivity in vitro and in vivo and discuss the possible mechanism. Small RNA interference system (siRNA) was employed to decrease DAB2IP expression in two BCa cell lines, MDA-MB-231 and 4T1. Cells in response to IR or antineoplastics were detected by clone formation assay or MTT method, respectively. For in vivo studies, siDAB2IP or siControl cells were subcutaneously injected into the right flank of each female mouse. Sphere formation assay, soft agar colony anchoring assay and in vivo tumorigenesis assay were implemented to examine the stem cell-like features of BCa cells. Tube formation assay as well as immunofluorescence assay (IFA) were respectively applied to determine the angiogenesis of tumor cells in vitro and in vivo. The expression of a series of angiogenesis-related molecules was analyzed by qRT-PCR, western blot and IFA. It was observed that the downregulation of DAB2IP could significantly improve the clone formation ability of BCa cells, reduce their sensitivity to radiation and chemotherapy drugs, enhance their migration and invasion abilities and increase their stemness characteristics. It was also noted that either DAB2IP-knocking down or treated with the conditioned medium from DAB2IP-deficient BCa cells could promote the tube-forming ability of the endothelial cell. Similarly, in vivo studies showed that tumors developed from siDAB2IP BCa cells had higher tumor microvascular density (MVD) and more severe oxygen deficiency than that in DAB2IP- sufficient tumors. Meanwhile, Knock-down of DAB2IP inhibited vascular maturation and promoted the formation of vasculogenic mimicry (VM) in BCa tissues. Down-regulation of STAT3 could enhance siDAB2IP cells sensitivity to IR, accompanied by the decrease of VEGF expression. Our data support that loss of DAB2IP confers radio-resistance of BCa could be due to increased hypoxia, inhibited vascular maturation and promoted VM formation. STAT3 inhibition could be a potential way to overcome such DAB2IP-deficient induced tolerance in BCT. [ABSTRACT FROM AUTHOR]

Background : A large proportion of pulmonary embolism (PE) heritability remains unexplained, particularly among the East Asian (EAS) population. Our study aims to expand the genetic architecture of PE and reveal more genetic determinants in Han Chinese. Methods: We conducted the first genome-wide association study (GWAS) of PE in Han Chinese, then performed the GWAS meta-analysis based on the discovery and replication stages. To validate the effect of the risk allele, qPCR and Western blotting experiments were used to investigate possible changes in gene expression. Mendelian randomization (MR) analysis was employed to implicate pathogenic mechanisms, and a polygenic risk score (PRS) for PE risk prediction was generated. Results: After meta-analysis of the discovery dataset (622 cases, 8853 controls) and replication dataset (646 cases, 8810 controls), GWAS identified 3 independent loci associated with PE, including the reported loci FGG rs2066865 (p-value = 3.81 × 10−14), ABO rs582094 (p-value = 1.16 × 10−10) and newly reported locus FABP2 rs1799883 (p-value = 7.59 × 10−17). Previously reported 10 variants were successfully replicated in our cohort. Functional experiments confirmed that FABP2-A163G(rs1799883) promoted the transcription and protein expression of FABP2. Meanwhile, MR analysis revealed that high LDL-C and TC levels were associated with an increased risk of PE. Individuals with the top 10% of PRS had over a fivefold increased risk for PE compared to the general population. Conclusions: We identified FABP2, related to the transport of long-chain fatty acids, contributing to the risk of PE and provided more evidence for the essential role of metabolic pathways in PE development. [ABSTRACT FROM AUTHOR]

Simple Summary: Forensic entomology plays a major role in postmortem interval (PMI) estimation, mainly based on morphological characteristics of larvae colonized on decomposed corpses. However, weight and length cannot be used as relevant parameters to calculate the age of wandering at the larval and pupal stages. In order to identify a specific indicator that may serve as a potential target, studies on the mechanism during postembryonic development may provide a foundation to establish a molecular age determination method. Sarcophaga peregrina (Robineau-Desvoidy, 1830) is a species of medical and forensic importance. In this study, transcriptome analysis was performed to investigate gene expression profiles from the larval to pupal stages of S. peregrina, which were used to identify differentially expressed genetic markers as candidates for molecular age estimation. This study explores molecular mechanisms during postembryonic development of S. peregrina. The developmental, stage-specific gene profiles suggest genetic markers for age prediction of forensically important flies. Sarcophaga peregrina (Robineau-Desvoidy, 1830) is a species of medical and forensic importance. In order to investigate the molecular mechanism during postembryonic development and identify specific genes that may serve as potential targets, transcriptome analysis was used to investigate its gene expression dynamics from the larval to pupal stages, based on our previous de novo-assembled genome of S. peregrina. Totals of 2457, 3656, 3764, and 2554 differentially expressed genes were identified. The specific genes encoding the structural constituent of cuticle were significantly differentially expressed, suggesting that degradation and synthesis of cuticle-related proteins might actively occur during metamorphosis. Molting (20-hydroxyecdysone, 20E) and juvenile (JH) hormone pathways were significantly enriched, and gene expression levels changed in a dynamic pattern during the developmental stages. In addition, the genes in the oxidative phosphorylation pathway were significantly expressed at a high level during the larval stage, and down-regulated from the wandering to pupal stages. Weighted gene co-expression correlation network analysis (WGCNA) further demonstrated the potential regulation mechanism of tyrosine metabolism in the process of puparium tanning. Moreover, 10 consistently up-regulated genes were further validated by qRT-PCR. The utility of the models was then examined in a blind study, indicating the ability to predict larval development. The developmental, stage-specific gene profiles suggest novel molecular markers for age prediction of forensically important flies. [ABSTRACT FROM AUTHOR]

Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae), is a blowfly species widely studied in medical, veterinary, and entomological research. Our study examined the impact of constant (15, 20, 25, 30, and 35 °C) and variable (ranging from 21.0 to 25.4 °C, with an average of 23.31 °C) temperatures on the development and larval body length of C. megacephala. Additionally, we analyzed the age of the adult C. megacephala through pteridine content and related metabolic genes analysis. Our findings revealed three distinct growth patterns: isomorphen diagram, isomegalen diagram, and thermal accumulated models. At constant temperatures of 15, 20, 25, 30, and 35 °C, egg-hatching times were 44.5 ± 8.9, 26.7 ± 4.6, 12.6 ± 1.1, 11.0 ± 1.0, and 9.9 ± 1.9 h, respectively, while it was 15.3 ± 5.9 h at variable temperatures. The total development times from oviposition to adult eclosion in C. megacephala required 858.1 ± 69.2, 362.3 ± 5.9, 289.6 ± 17.8, 207.3 ± 9.3, and 184.7 ± 12.1 h at constant temperatures of 15, 20, 25, 30, and 35 °C, respectively. This duration was extended to 282.0 ± 64.1 h under variable temperatures. However, no significant differences were found in hatching times and the total developmental durations between 25 °C and variable temperatures. A developmental threshold temperature (D 0) of 9.90 ± 0.77 °C and a thermal summation constant (K) of 4244.0 ± 347.0° hours were ascertained. Pteridine content patterns varied significantly across constant temperatures, but not between 25 °C and variable temperatures. Sex and temperature were identified as the primary factors influencing pteridine levels in the head of C. megacephala. Gene expression associated with pteridine metabolism decreased following adult eclosion, matching with increased pteridine concentration. Further investigations are needed to explore the use of pteridine cofactors for age-grading adult necrophagous flies. These findings provide valuable insights into the lifespan of C. megacephala , thereby offering valuable groundwork for forthcoming investigations and PMImin determination. • No significant differences in development times were found between 25 °C and VT. • Pteridine concentration increased following adult eclosion in all temperatures. • Dhpr , PTS , and TH decreased following adult eclosion in all temperatures. [ABSTRACT FROM AUTHOR]

Sarcophaga dux (Diptera: Sarcophagidae) is a necrophagous flesh fly species with potential forensic value for estimating minimum postmortem interval (PMI min). The basic developmental data and precise age estimates of the pupae are significant for PMI min estimation in forensic investigations. In the present study, we investigated the development data of that species at seven constant temperatures varying from 16 °C to 34 °C, including body length changes of the larve, developmental duration and accumulated degree hours of the preadults. Several reference genes for relative quantification of the differentially expressed genes (DEGs) were firstly selected and evaluated in the pupae of different ages under different temperatures. The DEGs of the insects during the pupal period at different constant temperatures (34, 25 and 16 °C) were further analyzed for more precise age estimation. The results showed that the developmental durations of the preadults at 16, 19, 22, 25, 28, 31 and 34 °C were 1478.6 ± 18.3 h, 726.1 ± 15.8 h, 538.5 ± 0.9 h, 394.1 ± 9.5 h, 375.6 ± 10.8 h, 284.1 ± 7.3 h, and 252.5 ± 6.1 h, respectively. The developmental threshold temperature the flies was 12.27 ± 0.35 °C, and the thermal summation constant was 5341.71 ± 249.29° hours. The most reliable reference genes during the pupal period at different temperatures were found: GST1 and 18S rRNA for the 34 °C group, GST1 and RPL49 for 25 °C, and 18S rRNA and 28S rRNA for 16 °C. The four differential expression genes (Hsp60 , A-alpha , ARP , and RPL8) have the potential to be used for more precise age estimation of pupal S. dux. This work provides important basic developmental data and a more precise age estimation method for pupal S. dux , and improves the value of this species for PMI min estimation in forensic investigations. Image 1 • The basic developmental data of S. dux during different temperatures were collected. • Four appropriate reference genes at intrapuparial stage of S. dux were selected. • The expression patterns of the same DEGs at the three temperature regimes (34°C, 25°C and 16°C) were similar. [ABSTRACT FROM AUTHOR]