Reverse micelle-based microencapsulation of oxytetracycline hydrochloride into poly-d,l-lactide-co-glycolide microspheres

The objectives of this study were to solubilize oxytetracycline hydrochloride (HCl) in reverse micelles to prepare poly-d,l-lactide-co-glycolide (PLGA) microspheres and to explore parameters affecting its encapsulation efficiency. Oxytetracycline HCl was dissolved in the reverse micelles consisting of cetyltrimethylammonium bromide, water, and ethyl formate. A PLGA polymer was then dissolved in the reverse micellar solution, and a modified solvent quenching procedure was carried out to prepare PLGA microspheres. Encapsulation efficiencies of oxytetracycline HCl ranged from 2.3 ± 0.2 to 24.9 ± 4.6%, depending on experimental conditions. Important parameters affecting its encapsulation efficiency included the amounts of water used to prepare the reverse micelles and PLGA polymer. With regard to microsphere morphology, the reverse micellar process produced the microspheres with smooth and pore-free surfaces. In particular, their internal matrices did not possess hollow cavities that were frequently observed when a typical double emulsion technique was used to make microspheres. In summary, it was possible to encapsulate oxytetracycline HCl into PLGA microspheres via the ethyl formate-based reverse micellar technique. We also anticipate that the use of ethyl formate could avoid environmental and human toxicity issues associated with methylene chloride.     

'Twist theory' for the roles of estrogen and testosterone in the process of spermatogenesis

Estrogen and testosterone have different effects on different steps of spermatogenesis and for each step optimal levels of estrogen and testosterone are different. The effects of these hormones behave as if 'twisted' from one end to the other end of the spectrum of steps of spermatogenesis. A graphic model is presented to explain this 'Twist Theory'.     

Piriformis syndrome in a patient after cesarean section under spinal anesthesia

OBJECTIVES: The diagnosis, pathogenesis, and treatment of piriformis syndrome as a cause of persistent buttock and hip pain after spinal anesthesia are presented in this case report. CASE REPORT: A 29-year-old woman presented 5 days after cesarean delivery with sudden onset of pain in the left buttock and left hip radiating to the posterior knee with fever. She was subsequently diagnosed with piriformis syndrome. CONCLUSIONS: Back pain with radiation into the buttocks after spinal anesthesia is frequently attributed to the spinal procedure itself. However, prolonged sitting and weight bearing in the upright position after cesarean delivery can cause sciatic nerve compression at the sacroiliac joint with concomitant irritation, inflammation, and spasm of the piriformis muscle. Piriformis syndrome is frequently underdiagnosed in the obstetric population. Because the diagnosis of piriformis syndrome is based mainly on clinical signs and symptoms, the clinician must have a high index of suspicion.     

Profiling Cancer Gene Mutations in Clinical Formalin‐Fixed,Paraffin‐Embedded Colorectal Tumor Specimens Using Targeted Next‐Generation Sequencing

Purpose.

The success of precision oncology relies on accurate and sensitive molecular profiling. The Ion AmpliSeq Cancer Panel, a targeted enrichment method for next-generation sequencing (NGS) using the Ion Torrent platform, provides a fast, easy, and cost-effective sequencing workflow for detecting genomic “hotspot” regions that are frequently mutated in human cancer genes. Most recently, the U.K. has launched the AmpliSeq sequencing test in its National Health Service. This study aimed to evaluate the clinical application of the AmpliSeq methodology.

Methods.

We used 10 ng of genomic DNA from formalin-fixed, paraffin-embedded human colorectal cancer (CRC) tumor specimens to sequence 46 cancer genes using the AmpliSeq platform. In a validation study, we developed an orthogonal NGS-based resequencing approach (SimpliSeq) to assess the AmpliSeq variant calls.

Results.

Validated mutational analyses revealed that AmpliSeq was effective in profiling gene mutations, and that the method correctly pinpointed “true-positive” gene mutations with variant frequency >5% and demonstrated high-level molecular heterogeneity in CRC. However, AmpliSeq enrichment and NGS also produced several recurrent “false-positive” calls in clinically druggable oncogenes such as PIK3CA.

Conclusion.

AmpliSeq provided highly sensitive and quantitative mutation detection for most of the genes on its cancer panel using limited DNA quantities from formalin-fixed, paraffin-embedded samples. For those genes with recurrent “false-positive” variant calls, caution should be used in data interpretation, and orthogonal verification of mutations is recommended for clinical decision making.     

siRNA knock-down of mutant torsinA restores processing through secretory pathway in DYT1 dystonia cells

Most cases of the dominantly inherited movement disorder, earlyonset torsion dystonia (DYT1) are caused by a mutant form oftorsinA lacking a glutamic acid residue in the C-terminal region(torsinAE). TorsinA is an AAA+ protein located predominantlyin the lumen of the endoplasmic reticulum (ER) and nuclear envelopeapparently involved in membrane structure/movement and processingof proteins through the secretory pathway. A reporter proteinGaussia luciferase (Gluc) shows a reduced rate of secretionin primary fibroblasts from DYT1 patients expressing endogenouslevels of torsinA and torsinAE when compared with control fibroblastsexpressing only torsinA. In this study, small interfering RNA(siRNA) oligonucleotides were identified, which downregulatethe levels of torsinA or torsinAE mRNA and protein by over 65%following transfection. Transfection of siRNA for torsinA messagein control fibroblasts expressing Gluc reduced levels of luciferasesecretion compared with the same cells non-transfected or transfectedwith a non-specific siRNA. Transfection of siRNA selectivelyinhibiting torsinAE message in DYT fibroblasts increased luciferasesecretion when compared with cells non-transfected or transfectedwith a non-specific siRNA. Further, transduction of DYT1 cellswith a lentivirus vector expressing torsinA, but not torsinB,also increased secretion. These studies are consistent witha role for torsinA as an ER chaperone affecting processing ofproteins through the secretory pathway and indicate that torsinAEacts to inhibit this torsinA activity. The ability of allele-specificsiRNA for torsinAE to normalize secretory function in DYT1 patientcells supports its potential role as a therapeutic agent inearly onset torsion dystonia.     

The noncoding RNA, miR-126, suppresses the growth of neoplastic cells by targeting phosphatidylinositol 3-kinase signaling and is frequently lost in colon cancers

MicroRNAs (miRNA/miR) are a class of small noncoding RNAs implicated in the pathogenesis of various malignancies. In the current study, using micro(RNA) arrays, we found a ubiquitous loss of miR-126 expression in colon cancer lines when compared to normal human colon epithelia. Reconstitution of miR-126 in colon cancer cells resulted in a significant growth reduction as evidenced in clonogenic assays. A search for miR-126 gene targets revealed p85beta, a regulatory subunit involved in stabilizing and propagating the phosphatidylinositol 3-kinase (PI3K) signal, as one of the potential substrates. Restoration of miR-126 in cancer cells induced a > or =3-fold reduction in p85beta protein levels, with no concomitant change in p85alpha, a gene that is functionally related to p85beta but not a supposed target of miR-126. Additionally, using reporter constructs, we show that the p85beta-3' untranslated region is directly targeted by miR-126. Furthermore, this miR-126 mediated reduction of p85beta was accompanied by a substantial reduction in phosphorylated AKT levels in the cancer cells, suggesting an impairment in PI3K signaling. Finally, in a panel of matched normal colon and primary colon tumors, each of the tumors demonstrated miR-126 down-regulation together with an increase in the p85beta protein level. Taken together, we propose that miR-126 regulates PI3K signaling partly by targeting p85beta, and that the loss of miR-126 may provide a selective growth advantage during colon carcinogenesis.     

Compaction enhances extracellular matrix content and mechanical properties of tissue-engineered cartilaginous constructs

Many cell-based tissue-engineered cartilaginous constructs are mechanically softer than native tissue and have low content and abnormal proportions of extracellular matrix (ECM) constituents. We hypothesized that the load-bearing mechanical properties of cartilaginous constructs improve with the inclusion of collagen (COL) and proteoglycan (PG) during assembly. The objectives of this work were to determine (1) the effect of addition of PG, COL, or COL+PG on compressive properties of 2% agarose constructs and (2) the ability of mechanical compaction to concentrate matrix content and improve the compressive properties of such constructs. The inclusion of COL+PG improved the compressive properties of hydrogel constructs compared with PG or COL alone. Mechanical compaction increased the PG and COL concentrations in and compressive stiffness of the constructs. Chondrocytes included in the constructs maintained high viability after compaction. These results support the concepts that the assembly of cartilaginous constructs with COL+PG and application of mechanical compaction enhance the ECM content and compressive properties of engineered cartilaginous constructs.     

Efficacy of rBm86 against Rhipicephalus (Boophilus) microplus (IVRI-I line) and Hyalomma anatolicum anatolicum (IVRI-II line) infestations on bovine calves

With an aim to evaluate the protective potentiality of rBm86 against Rhipicephalus (Boophilus) microplus Indian Veterinary Research Institute (IVRI)-I line and Hyalomma anatolicum anatolicum IVRI-II line infestations on crossbred (Bos indicus?×?Bos taurus) calves, 20 animals of 3 months of age were randomly divided in to four equal groups and maintained in tick-proof conditions. Animals of groups 1 and 2 were immunized with 2 ml of rBm86 (100 μg)-based vaccine (procured from Revetmex S.A. de C.V, Mexico City, Mexico) thrice at 30 days interval. Animals of groups 3 and 4 were kept as negative control and inoculated with PBS only. Each animal of group 1 and 3 was challenged with 7-day-old 50 unfed adults of H. anatolicum anatolicum (1:1, male and female), and each animal of groups 2 and 4 was challenged with 6-8-day-old R. (B.) microplus larvae obtained from 50 mg of eggs, on 17th day of the last immunization. The efficacy of rBm86 against tick infestations was determined as percentage reduction in number of adults dropped (DT%), engorged body weight (DR%), egg masses (DO%), and immunogen efficacy (E%). The calculated data were 11.8, 10.8, 15.0, and 25.1 %, respectively, for DT, DR, DO, and E% against H. anatolicum anatolicum infestation, while in the case of R. (B.) microplus infestation, the corresponding data were 6.4, 11.24, 40.7, and 44.5 %, respectively. The results indicated partial effectiveness of rBm86 antigen(s) in imparting protection against homologous and heterologous challenge infestations of Indian ticks. The results indicated identification of more effective antigen(s) for the development of vaccine against economically important tick species in India.     

胰腺部位的Castleman病1例

1临床资料患者,男性,49岁。因车祸后行CT检查发现胰腺体尾部占位入院。既往无腹痛,否认胰腺炎病史。体检：皮肤巩膜无黄染,全身浅表淋巴结未见肿大,全腹无压痛,未扪及包块。