Comparative application of biological and ninhydrin- derivatized spectrophotometric assays in the evaluation and validation of amikacin sulfate injection

Abstract Instrumental techniques are preferred over bioassay methods for antibiotic quantification mainly due to speed and ability to quantify metabolites in biological samples; however, the potency and biological activity of these drugs cannot be assessed. Two methods - agar well diffusion (bio-assay) and spectrophotometric methods were used to evaluate amikacin sulfate injection. Agar plates were inoculated with S. aureus inoculum; zones of inhibition from its susceptibility to amikacin were obtained, while spectrophotometric absorption at 650 nm of ninhydrin- derivatized amikacin in phosphate buffer (pH 8) was measured. Methods performance showed linearity from 1 - 16 µgmL-1 (bioassay, r = 0.9994) and 10-50 µgmL-1 (spectrophotometric, r = 0.9998). Molar absorptivity was 2.595 x 104 Lmol-1cm-1. Limits of detection and quantification were 1.07 and 3.24 µgmL-1 respectively for bioassay method, while corresponding values for spectrophotometric method were 0.98 and 2.97 µg mL-1. Relative standard deviations were ≤ 2.0% for both methods, with recoveries from 95.93 - 100.25%. Amikacin in brands ranged from 97.53 ± 2.68 to 100.84 ± 1.82%, student's t-test was ≤ 2.78 (n = 4) with respect to label claim for both methods. Experimental paired t-test (t = 2.07; n = 4) and F-test (F = 3.94; n = 4) values indicated no significant difference between both methods, hence comparable and can jointly be used in quality control assessment of antibiotics

Protective effects of Baicalin injection on severe acute pancreatitis through regulating follistatin-like-1 signaling pathway by down-regulating miR-429 expression in mice

Abstract In China, Scutellaria is used for treating inflammatory-related diseases. Baicalin is the main active component of Scutellaria and has protective effects on acute pancreatitis. However, the mechanism of Baicalin is still unclear. In this study, the protective effects of baicalin on acute pancreatitis induced by taurocholate and its mechanism are investigated. In this study, mice were randomly divided into three groups: sham operation, model, and treatment groups. Acute pancreatitis in mice was induced by intraperitoneal injection of taurocholate (35 mg/kg). The treatment group was given baicalin (100 mg/kg) 2 h before acute pancreatitis induction. The mRNA expression levels of miR-429, nuclear factor kappa B65(NF-kB65), toll-like receptor 4(TLR4), TNF receptor associated factor6 (TRAF6), NF-kappa-B inhibitor(IkB), Follistatin-like 1 (FSTL1), and interleukin-1 receptor-associated kinase (IRAK) in the liver tissues 24 h after intraperitoneal injection were detected by RT-PCR. Then, the expression levels of NF-kB65, p-NF-κB65, TLR4, TRAF6, IkB, FSTL1, IRAK, p- IRAK, and p- IkB-а proteins were detected by Western blot. IL-6, TNF-α and IL-1 ß in plasma were measured by ELISA, and histopathological changes in the pancreases of the mice were observed. The results showed that after baicalin treatment, miR-429 expression in the pancreatic tissues and the expression levels of NF-kB65, TLR4, TRAF6, p-IkB-а, FSTL1, and p-IRAK decreased. Similarly, pancreatic myeloperoxidase (MPO) activity and the plasma levels of IL-6, TNF-а, IL-12, IL-1ß1, endotoxin, serum amylase, and lipase were reduced. Thus, the pancreatic injury induced by taurocholate was alleviated. The present study indicates that pretreatment with Baicalin can alleviate acute pancreatic injury induced by taurocholate in mice. The mechanism may be associated with the decreased miR-429 expression, reduced FSTL1 signaling pathway activity, TLR4 and TLR4/MyD88 signaling pathway inhibition, and reduced pancreatic inflammation. FSTL1 is the regulatory target for miR-429

AAV9 transduction mediated by systemic delivery of vector via retro-orbital injection in newborn, neonatal and juvenile mice.

Adeno-associated virus (AAV)-based gene therapy is gaining popularity owing to its excellent safety profile and effective therapeutic outcomes in a number of diseases. Intravenous (IV) injection of AAV into the tail vein, facial vein and retro-orbital (RO) venous sinus have all been useful strategies to infuse the viral vector systemically. However, tail vein injection is technically challenging in juvenile mice, and injection at young ages (≤ postnatal day-(P)21) is essentially impossible. The temporal or facial vein is localized anterior to the ear bud and is markedly visible in the first couple of days postnatally. However, this method is age-dependent and requires a dissecting microscope. Retro-orbital injection (ROI), on the other hand, is suitable for all murine ages, including newborn and older mice, and is relatively less stressful to animals compared to tail vein injection. Although many reports have shown ROI as an effective route of AAV delivery, herein we aim to highlight and summarize the methods and benefits of ROI. To capture the full spectrum of transduction efficiency mediated by ROI, we transduced the editing-dependent reporter mice (Ai9 Cre reporter mice) with the AAV9 vector, which targets a wide range of peripheral tissues with exceptional brain tropism. We also provide a comprehensive description of the ROI technique to facilitate viral vector administration without complications.

Local anesthetic effects on gene transcription in human skeletal muscle biopsies.

INTRODUCTION: We examined if epinephrine in the local anesthetic to help control incision-related bleeding interferes with molecular measurements obtained with the Duchenne-Bergström percutaneous needle biopsy technique for sampling human skeletal muscle. METHODS: Three groups received 2.5-3.0 ml of 1% lidocaine in 2 injections: (1) 0.5-1.0 ml superficially, which varied among the groups according to (i) -Epi; intra- and subcutaneous without epinephrine, (ii) +Epi -Fascia; intra- and subcutaneous with epinephrine, avoiding the fascia, and (iii) +Epi +Fascia; intra- and subcutaneous with epinephrine, directing a small amount (∽0.2 ml) into the fascia area; and (2) ∽2.0 ml without epinephrine into the fascia area for all subjects. A muscle biopsy was obtained 5-10 min later for IL-6 and MuRF-1 mRNA levels. RESULTS: IL-6 mRNA levels were low in -Epi and +Epi -Fascia, but ∽300-fold higher in +Epi +Fascia. MuRF-1 mRNA levels were similar among the groups. CONCLUSIONS: Lidocaine with epinephrine can confound intramuscular measurements from needle biopsies, but this can be avoided with a careful injection approach.

Macrophage subtyping in the determination of age of injection sites.

Determination of the age of injection marks in skin may be of particular interest in the investigation of drug abuse-related fatalities. The aim of our study was to assess the value of macrophage subtyping by antibody-based markers in the determination of the age of injection marks. Immunohistochemical investigations were performed with the antibodies Ki-M1P, 27E10, MRP14, MRP8 and 25F9. Monocytes/macrophages in acute lesions (several hours to 2 days old) expressed proteins detectable with the antibodies 27E10 and MRP14 and showed acute erythrophagia. An additional reaction with the antibody MRP8 was seen in lesions a few days old. An antigen recognized by the antibody 25F9 was found in tissue macrophages, multinucleated giant cells of active granulomas and siderophages. The expression of the 25F9 detectable antigen was absent in inactive granulomas and siderophages, whereas the macrophages were always detectable with the pan-macrophage marker Ki-M1P.