Rehmanniae Radix (Di Huang) is one of the most important traditional Chinese medicines (TCM), and is used for multiple therapeutic purposes. In our investigation of the chemical constituents of ...Rehmanniae Radix, steamed roots were prepared by the classical processing method. Reversed-phase HPLC of the 50% MeOH extract of steamed Rehmanniae Radix yielded three 5-hydroxymethylfurfural derivatives. The new furfural disaccharide 5-(α-
d
-glucopyranosyl-(1→6)-α-
d
-glucopyranosyloxymethyl)-2-furancarboxaldehyde (
1
) was isolated and characterized, together with its known aglycone 5-hydroxymethyl-2-furfural (
3
), which is currently in sickle cell anemia Phase I clinical trials, and its corresponding monosaccharide 5-(α-
d
-glucopyranosyloxymethyl)-2-furancarboxaldehyde (
2
), which was isolated as a natural product for the first time. The presence of these three compounds, particularly
3
, which were not found in the unprocessed extract of Rehmanniae Radix, could substantiate the traditional medicinal use of steamed Rehmanniae Radix.
We previously reported a gradual increase of relative mitochondrial DNA (mtDNA) copy number during the progression of esophageal squamous cell carcinoma (ESCC). Because mitochondria are the ...intracellular organelles responsible for ATP production, we investigated the associations among mtDNA copy number, mitochondrial bioenergetic function, tumor invasion and the expression levels of epithelial mesenchymal transition (EMT) markers in a series of seven ESCC cell lines, including 48T, 81T, 146T, TE1, TE2, TE6 and TE9. Among them, TE1 had the highest relative mtDNA copy number of 240.7%. The mRNA of mtDNA-encoded ND1 gene (2.80), succinate-supported oxygen consumption rate (11.21 nmol/min/10(6) cells), ATP content (10.7 fmol/cell), and the protein level of mitochondrial transcription factor A (TFAM) were the highest and the lactate concentration in the culture medium (3.34 mM) was the lowest in TE1. These findings indicate that TE1 exhibited the highest bioenergetic function of mitochondria. Furthermore, TE1 showed the highest trans-well migration activity of 223.0 cells/field, the highest vimentin but the lowest E-cadherin protein expression levels, which suggest that TE1 had the highest invasion capability. We then conducted a knockdown study using pLKO.1-based lentiviral particles to infect TE1 cells to suppress the expression of TFAM. Molecular analyses of the parental TE1, control TE1-NT and TFAM knockdown TE1-sh-TFAM(97) cells were performed. Interestingly, as compared to the control TE1-NT, TE1-sh-TFAM(97) exhibited lower levels of the relative mtDNA copy number (p = 0.001), mRNA of mtDNA-encoded ND1 gene (p = 0.050), succinate-supported oxygen consumption rate (p = 0.065), and ATP content (p = 0.007), but had a higher lactate concentration in the culture medium (p = 0.010) and higher protein level of lactate dehydrogenase. A decline in mitochondrial bioenergetic function was observed in TE1-sh-TFAM(97). Significantly, compared to the control TE1-NT, TE1-sh-TFAM(97) had a lower trans-well migration activity (p < 0.001), a higher E-cadherin level but a lower vimentin protein level, which indicates a decrease of invasiveness. Taken together, we suggest that high relative mtDNA copy number and bioenergetic function of mitochondria may confer an advantage for tumor invasion of ESCC.
Urinary extracellular vesicles (uEVs) are rich in valuable biomolecule information which are increasingly recognized as potential biomarkers for various diseases. uEV long RNAs are among the critical ...cargos capable of providing unique transcriptome information of the source cells. However, consensus regarding ideal reference genes for relative long RNAs quantification in uEVs is not available as of date. Here we explored stable reference genes through profiling the long RNA expression by RNA‐seq following unsupervised analysis and validation studies. Candidate reference genes were identified using four algorithms: NormFinder, GeNorm, BestKeeper and the Delta Ct method, followed by validation. RNA profile showed uEVs contained abundant long RNAs information and the core transcriptome was related to cellular structures, especially ribosome which functions mainly as translation, protein and RNA binding molecules. Analysis of RNA‐seq data identified RPL18A, RPL11, RPL27, RACK1, RPSA, RPL41, H1‐2, RPL4, GAPDH, RPS27A as candidate reference genes. RT‐qPCR validation revealed that RPL41, RPSA and RPL18A were reliable reference genes for long RNA quantification in uEVs from patients with diabetes mellitus (DM), diabetic nephropathy (DN), IgA nephropathy (IgAN) and prostate cancer (PCA). Interestingly, RPL41 also outperformed traditional reference genes in renal tissues of DN and IgAN, as well as in plasma EVs of several types of cancers. The stable reference genes identified in this study may facilitate development of uEVs as novel biomarkers and increase the accuracy and comparability of biomarker studies.
Four new bromophycolides, R−U (1−4), were isolated from the Fijian red alga Callophycus serratus and were identified by 1D and 2D NMR and mass spectroscopic analyses. These compounds expand the known ...structural variety of diterpene-benzoate macrolides and exhibited modest cytotoxicity toward selected human cancer cell lines. Bromophycolide S (2) also showed submicromolar activity against the human malaria parasite Plasmodium falciparum.
A new series of pregnenolone derivatives was synthesized by parallel synthesis. Their anticancer activity against cancer cell lines was evaluated and SAR established.
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► The synthesis ...of a series of pregnenolone derivatives
2–
107 has been described which were evaluated for their cytotoxicity against HepG2 and MDA-MB-231 cell lines. ► Among all derivatives, the heterocyclic enone
8 (IC
50
=
0.74
μM/mL against HepG2), its pyrazoline derivative
48 (IC
50
=
0.91
μM/mL against MDA-MB-230 cancer cell lines) were identified as the most active compounds. ► All (
O-carboxymethyl) oxime, hydazones and pyrazoles derivatives of pregnenolone were found to be inactive against both the cancer cell lines.
Pregnenolone (
1) was used as a template to develop new anticancer compounds. Ring-D modification of
1 resulted in the synthesis of benzylidenes
2–
17, pyrazolines
18–
76, pyrazoles
85–
91, hydrazones
77–
84, and oximes
92–
107 derivatives. The structure of compound
107 was also deduced through single crystal X-ray diffraction studies. The inclusion of furanyl and pyridyl rings to pregnenolone skeleton increases the cytotoxicity of all compounds significantly. Among benzylidene derivatives, only heterocyclic enone
8 (IC
50
=
0.74
μM/mL against HepG2), and
17 (IC
50
=
4.49
μM/mL against HepG2, IC
50
=
5.01
μM/mL against MDA-MB-230 cancer cell line) exhibited a significant activity. The cytotoxicity data of pyrazoline derivatives
18–
76 revealed that only furanyl bearing pyrazolines
40,
42–
44,
48, and
49 exhibited significant activities. While all (
O-carboxymethyl) oximes, hydazones, and pyrazoles derivatives of pregnenolone did not show any significant activity against both the cell lines. Thus the furanyl bearing enone
8 (IC
50
=
0.74
μM/mL against HepG2), and its pyrazoline derivative
48 (IC
50
=
0.91
μM/mL against MDA-MB-230 cancer cell lines) were identified as the most active compounds in all derivatives of pregnenolone.
Summary Objectives Toll-like receptor 2 (TLR2) is a major mediator of innate immunity against tuberculosis (TB). This study aimed to determine if TLR2 promoter DNA methylation is associated with ...pulmonary TB. Methods The DNA methylation levels of 20 CpG sites over the TLR2 promoter region and TLR2 gene/protein expressions of immune cells of the blood were examined in 99 sputum culture-positive pulmonary TB patients and 77 healthy subjects (HS). Results TB patients had higher methylation levels over five CpG sites (3, 7, 9, 13, and 18), lower TLR2 gene expression, lower TLR2 expression on monocyte, higher TLR2 expression on NK cell, and higher serum TNF-α/IFN-γ levels than HS after adjusting for confounding factors. Patients with a high bacillary load had lower methylation levels at CpG-15, -17, and -20. Patients with drug-resistant TB had higher CpG-18 methylation levels and lower TLR2 expression on NK cell. Patients with far advanced lesion on chest radiograph had higher serum TNF-α level and higher TLR2 expression on NK cell. Patients with a high TLR2 expression on NK cell had lower one-year survival. CpG-18 methylation level, TLR2 expressions on monocyte/NK cell, and TNF-α/IFN-γ levels were all reversed to normal after 6-month anti-TB treatment. Conclusions Aberrant methylation of certain CpG sites over TLR2 promoter region is associated with active pulmonary TB or its phenotypes, probably through the down-regulation of TLR2 expression.
Microfluidic chip-based high-performance-liquid-chromatography coupled to mass spectrometry (chip-HPLC-MS) has been widely used in proteomic research due to its enhanced sensitivity. We employed a ...chip-HPLC-MS system for determining small molecules such as drug metabolites in biological fluids. This chip-HPLC-MS system integrates a microfluidic switch, a 2-dimensional column design including an enrichment column (160 nL) for sample pre-concentration and an analytical column for chromatographic separation, as well as a nanospray emitter on a single polyimide chip. In this study, a relatively large sample volume (500 nL) was injected into the enrichment column for pre-concentration and an additional 4 μL of the initial mobile phase was applied to remove un-retained components from the sample matrix prior to chromatographic separation. The 2-dimensional column design provides the advantages of online sample concentration and reducing matrix influence on MS detection. 7-Aminoflunitrazepam (7-aminoFM2), a major metabolite of flunitrazepam (FM2), was determined in urine samples using the integrated chip-HPLC-MS system. The linear range was 0.1-10 ng mL
−1
and the method detection limit (signal-to-noise ratio of 3) was 0.05 ng mL
−1
for 7-aminoFM2. After consecutive liquid-liquid extraction (LLE) and solid-phase extraction (SPE), the chip-HPLC-MS exhibited high correlation between 7-aminoFM2 spiked Milli-Q water and 7-aminoFM2 spiked urine samples. This system also showed good precision (
n
= 5) and recovery for spiked urine samples at the levels of 0.1, 1.0, and 10 ng mL
−1
. Intra-day and inter-day precision were 2.0-7.1% and 4.3-6.0%, respectively. Clinical urine samples were also analyzed by this chip-HPLC-MS system and acceptable relative differences (−1.3 to −13.0%) compared with the results using a GC-MC method were determined. Due to its high sensitivity and ease of operation, the chip-HPLC-MS system can be utilized for the determination of small molecules such as drug metabolites and neurotransmitters in biological fluids for clinical diagnosis.
Two-dimensional chip-HPLC-MS/MS has been illustrated as a sensitive analytical method for quantifying drug metabolites such as 7-aminoflunitrazepam in human urine.
This study investigated the effects of solid-state aging on Sn–3Ag–0.5Cu (SAC305) and Sn–9Zn (SZ) solder joints with two Cu electroplated layers: PC-Cu and PCS-Cu. Distinct surface features and ...impurity levels were evident in PC-Cu and PCS-Cu owing to the use of different additives in the electroplating solutions. The SAC305 solder joints exhibited notable voids and cracks at the joint interface during aging, which were attributed to the high impurity content of PC-Cu. In contrast, low-impurity PCS-Cu did not exhibit void formation. Regardless of whether PC-Cu or PCS-Cu was used, no interfacial voids were observed in the SZ solder joints, implying that the SZ solder effectively suppressed voids. Mechanisms related to void suppression were also proposed. The presence of Zn in the solder changed the interfacial reactions, forming a Cu
5
Zn
8
intermetallic compound (IMC) in addition to the Cu–Sn IMCs in the SAC305 solder joints. Inhibited grain-boundary diffusion in high-impurity scenarios hinders voids owing to the effective substitution of Cu vacancies by Zn. With Zn consumption near the soldering interface, the Cu
5
Zn
8
IMC detached and a Cu
6
Sn
5
phase was formed between the Cu–Zn IMC and Cu. Meanwhile, PCS-Cu exhibited localized Cu
6
Sn
5
growth. In addition, SZ showed less influence of Cu impurities on the interfacial IMC thickness compared to SAC/PC-Cu with rapid IMC growth. The findings revealed the void formation mechanisms and highlighted the void-suppressing features of SZ in solder joints.
Temperature gradient is inevitable in solder joints in advanced electronic packaging; it induces thermomigration (TM), critically affecting the reliability of micro solder joints. Although a FeCoNiMn ...alloy (FCNM) can resist thermal aging in solder joints, its TM resistance has seldom been examined. Thus, this study investigated the TM in Cu/Sn3.5Ag (SA3.5)/FCNM solder joints between a cold end (50 °C) and a hot end (200 °C) at different times. A multielement intermetallic compound (MEIMC), (Cu, Mn, Fe, Co, Ni)6Sn5, is formed at the SA3.5/FCNM interface in the reflowed and aged solder joints. A (Fe, Co, Ni, Mn)Sn2 MEIMC also formed, but it was very thin. The (Cu, Mn, Fe, Co, Ni)6Sn5 was completely decomposed by TM from the hot-end FCNM. In contrast, the (Cu, Mn, Fe, Co, Ni)6Sn5 MEIMC thickened as the rapid TM flux of Cu from the hot-end Cu cap; however, the low chemical flux from FCNM hindered its formation, which was replaced by Cu6Sn5 formation near the Cu/SA3.5 interface. The findings demonstrate that FCNM providing a very low chemical flux is a remarkably promising diffusion barrier for TM in micro solder joints.
•Excellent suppression of thermomigration in Cu/Sn3.5Ag/FeCoNiMn high-entropy alloy.•Very low chemical flux provided by FeCoNiMn to resist thermomigration.•Demonstrating a novel diffusion barrier of FeCoNiMn against thermomigration.
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