[Changes of HER2 low expression status in primary and recurrent/metastatic breast cancer].

Objective: To investigate the evolution and clinical significance of HER2 low expression status in HER2 negative patients in primary and recurrent/metastatic breast cancers. Methods: The data and archived sections of 259 breast cancer patients with recurrence/metastasis and HER2-negative primary foci were collected from January 2015 to January 2022 at the Fourth Hospital of Hebei Medical University, and the HER2 status of primary and recurrence/metastasis foci was determined by immunohistochemistry (IHC), among which IHC 2+patients were subject to fluorescence in situ hybridization (FISH). The HER2 status was classified as HER2-0 group; patients with IHC 1+, IHC 2+and no FISH amplification were classified as HER2 low expression group; and patients with IHC 3+, IHC 2+and FISH amplified were classified as HER2-positive group. The changes of HER2 status in patients with HER2 low expression in primary versus recurrent/metastatic breast cancer foci were compared, and their clinicopathologic characteristics and prognosis were analyzed. Results: The overall concordance rate between primary and recurrent/metastatic HER2 status in breast cancer was 60.6% (157/259, κ=0.178). A total of 102 patients (102/259, 39.4%) had inconsistent primary and recurrent/metastatic HER2 status; 37 patients (37/259, 14.3%) had HER2-0 at the primary foci and HER2-low expression at the recurrent/metastatic; and 56 patients (56/259, 21.6%) had HER2-low expression in the primary foci and HER2-0 in the recurrent/metastatic. The recurrent/metastatic foci became low-expressing compared with the recurrent/metastatic foci which remained HER2-0 patients, with longer overall survival time, higher ER and PR positivity, lower Ki-67 positivity index, and lower tumor histological grade; all with statistically significant differences (all P<0.05). In the primary HER2-low group, patients with recurrent/metastatic foci became HER2-0 while those with recurrent/metastatic foci remained low expression; there were no statistically significant differences in clinicopathological features and overall survival time (all P>0.05). Conclusions: Unstable HER2 status in patients with HER2-0 and low expression in primary versus recurrent/metastatic breast cancer foci, and HER2-0 in the primary foci but low HER2 expression status in recurrence/metastasis is associated with favourable prognosis, and testing HER2 status in recurrence/metastasis can provide more treatment options for such patients.

Identification of ELAVL1 gene and miRNA-139-3p involved in the aggressiveness of NSCLC.

OBJECTIVE: Tumor metastasis remains the main cause for the cancer-associated death of human non-small-cell lung carcinoma (NSCLC). Many studies have verified that microRNAs (miRNAs) exert crucial functions in the development of NSCLC. Nevertheless, the functions of miR-139-3p in NSCLC remain unexplored. PATIENTS AND METHODS: The quantitative Real Time-PCR (qRT-PCR) assay was applied to assess the level of miR-139-3p and ELAV like RNA binding protein 1 (ELAVL1) in NSCLC tissues and cell lines. The growth of NSCLC cell was analyzed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and colony formation assay. The migration ability and invasiveness of NSCLC cells were analyzed using wound healing and transwell invasion analysis. The expression of ELAVL1 was determined by immunoblotting assay. The growth of NSCLC cell in vivo was assessed using xenograft model. RESULTS: We uncovered that miR-139-3p was down expressed in NSCLC. MiR-139-3p repressed NSCLC cell growth, migration as well as invasion in vitro, and suppressed the progression of NSCLC cell in vivo. Mechanistically, ELAVL1 was proved as a downstream target of miR-139-3p. The level of ELAVL1 was upregulated in NSCLC and inversely associated with miR-139-3p level. Immunoblotting assay suggested that ELAVL1 was negatively modulated by miR-139-3p in NSCLC cell. In vivo, miR-139-3p repressed NSCLC cell growth and metastasis. Several recuse assays revealed that ELAVL1 mediated the inhibitory actions of miR-139-3p on the growth and metastatic-related traits of NSCLC cell. CONCLUSIONS: Our results indicate that miR-139-3p acts as a suppressor in modulating the aggressiveness of NSCLC via regulating ELAVL1.

Nanoemulsion formulation of florfenicol improves bioavailability in pigs.

Nanotechnology applications in medicine have seen a tremendous growth in the past decade and are being employed to enhance the stability and bioavailability of lipophilic substances, such as florfenicol. This study aimed to examine the pharmacokinetic properties of the formulated oil-in-water florfenicol-loaded nanoemulsion (FF-NE). FF-NE and florfenicol control (Nuflor) were administered to the pigs at a dose of 20 mg/kg. Nanoemulsion formulation of florfenicol was highly influenced in vivo plasma profile. The in vivo absorption study in pigs indicated that Cmax (14.54 µg/mL) was significantly higher in FF-NE, 3.42 times higher than the marketed formulation. In comparison with the control group, the relative bioavailability of formulated nanoemulsion was up to 134.5%. Assessment of bioequivalence using log-transformed data showed that the 90% confidence intervals (90% CI) of Cmax and AUC0-∞ were 2.48-4.60 and 1.21-1.72, respectively.

Residue depletion of colistin in swine after intramuscular administration.

A newly formulated colistin sulphate solution was prepared in a previous study as a potential agent for intramuscular injection and its effectiveness, toxicity and pharmacokinetics were investigated. In order to provide more information to establish scientific guidance for safe use of this preparation, its residue depletion in swine tissues following intramuscular administration was investigated in this experiment. Fifty healthy cross-bred piglets (13.3 +/- 0.9 kg) were used in this study. Five animals were kept as untreated controls and the other 45 animals were intramuscularly injected with the colistin preparation at a dose of 2.5 mg/kg of body weight. From the treated piglets, 5 animals were randomly selected and sacrificed at different withdrawal times. Liver, kidney and muscle tissues were sampled to examine the colistin residue levels by microbiological assay. The results showed that the colistin residue in liver and muscle decreased quickly and could not be detected at 1 day after the final dosing. However, the residue depletion in the kidneys was much slower than that in other tissues and even a small quantity of drug could be detected at 14 days after withdrawal. Using the method recommended by the Committee for Veterinary Medical Products (CVMP), a withdrawal time of 10 days was established for the safe use of the newly formulated colistin sulphate solution.

Mechanical properties and in-vivo performance of calcium phosphate cement-chitosan fibre composite.

Self-hardened calcium phosphate cement (CPC) sets to form hydroxyapatite and possesses excellent osteoconductivity. However, lack of macroporosity and low strength constrain its application in bone tissue engineering. Recent studies have incorporated various fibres into CPC to improve its mechanical strength. The present approach focused on the reinforcement of CPC with chitosan fibres and then the effects of the fibre structure on the mechanical properties and macrochannels formation characteristics of CPC-fibre composite were investigated. Chitosan fibres of diameter 200 microm were used to fabricate two types of three-dimensional structure, which were then coated with collagen and incorporated into CPC to fabricate CPC-fibre implants with a fibre volume content of 5 per cent. The compressive strength of the CPC-fibre implant was 33 MPa when the strain was 2.4 per cent, which is fourfold higher than that of the CPC control. Nine cylindrical implants including six CPC-fibre implants were implanted in the bone defects of nine dogs and were then post-operatively observed. After 20 weeks in vivo, new callus from the healthy tissue of the defect entirely integrated with the CPC-fibre implant and new bone was formed as the implant degraded. Scanning electronic microscopy images indicated that macrochannels were formed in the CPC-fibre implants with the degradation of fibres, but only micropores with a scale of less than 50 microm could be observed in the CPC control. Briefly, the incorporation of a suitable chitosan-fibre structure into a CPC implant not only could improve its mechanical properties but also facilitated the bone repair process in vivo.

Glycogen debranching enzyme: purification, antibody characterization, and immunoblot analyses of type III glycogen storage disease.

Type III glycogen storage disease is caused by a deficiency of glycogen debranching-enzyme activity. Many patients with this disease have both liver and muscle involvement, whereas others have only liver involvement without clinical or laboratory evidence of myopathy. To improve our understanding of the molecular basis of the disease, debranching enzyme was purified 238-fold from porcine skeletal muscle. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified enzyme gave a single band with a relative molecular weight of 160,000 that migrated to the same position as purified rabbit-muscle debranching enzyme. Antiserum against porcine debranching enzyme was prepared in rabbit. The antiserum reacted against porcine debranching enzyme with a single precipitin line and demonstrated a reaction having complete identity to those of both the enzyme present in crude muscle and the enzyme present in liver extracts. Incubation of antiserum with purified porcine debranching enzyme inhibited almost all enzyme activity, whereas such treatment with preimmune serum had little effect. The antiserum also inhibited debranching-enzyme activity in crude liver extracts from both pigs and humans to the same extent as was observed in muscle. Immunoblot analysis probed with anti-porcine-muscle debranching-enzyme antiserum showed that the antiserum can detect debranching enzyme in both human muscle and human liver. The bands detected in human samples by the antiserum were the same size as the one detected in porcine muscle. Five patients with Type III and six patients with other types of glycogen storage disease were subjected to immunoblot analysis. Although anti-porcine antiserum detected specific bands in all liver and muscle samples from patients with other types of glycogen storage disease (Types I, II, and IX), the antiserum detected no cross-reactive material in any of the liver or muscle samples from patients with Type III glycogen storage disease. These data indicate (1) immunochemical similarity of debranching enzyme in liver and muscle and (2) that deficiency of debranching-enzyme activity in Type III glycogen storage disease is due to absence of debrancher protein in the patients that we studied.

Expression of gene(s) in restricted fragment of lambda DNA in E. coli.

Restriction endonucleases EcoR1 and BamH1 are used to produce fragments pBR322C (375 bp) and pBR322B (3987 bp) from pBR322, and to produce lambda F2A (65.6--71.3% of lambda DNA, 2679 bp) and lambda F2B (71.3--81% of lambda DNA, 4559 bp) from EcoR1 restriction fragment lambda F2 (65.6--81% of lambda DNA) of lambda cI857S7 DNA. By recombining pBR322B and lambda F2B in vitro, a new plasmid called pCB2 carrying promoters and structural genes cI and cro is constructed. The desired strain with pCB2 is selected from 338 transformants for its AprTcs and for its immunity to lambda infection. The length of the pCB2 DNA molecule is 2.66 +/- 0.33 micrometers and its MW is 5.51 +/- 0.68 x 10(6)d, as determined by electron microscope and agarose gel electrophoresis. The lengths of single strands and the double strands of the heteroduplex formed between lambda F2 and linear pCB2 (EcoR1 digested) agree well with the original design for its construction, From the above data, we come to the conclusion that pCB2 we constructed is a new plasmid with cI and/or cro gene expressed in E. coli.