Introduction of Foreign Genes into the Tissue of Live Fish by Direct Injection and Particle Bombardment

We compared 2 methods of direct gene delivery into live rainbow trout Oncorhynchus mykiss tissue, with the final goal of developing DNA-based vaccines for bacterial diseases in salmonids. The introduction of plasmid constructs containing the luciferase and β-galactosidase reporter genes was achieved either by direct injection or by particle bombardment with DNA-coated gold microparticles. Luciferase expression was observed in homogenates of trout flank muscle and skin 2 d after injection of 10 to 100 pg of DNA per fish or bombardment of 1 pm gold particles coated with 5 to 25 pg DNA per fish at helium pressures ranging from 2750 to 12 400 kPa. Expression levels increased over 10 d and persisted for at least 60 d after injection. For particle bombardment, the most reproducible levels of luciferase expression were obtained with the eye as a target (83% of fish positive versus 59% of fish positive when the flank was the target). The levels of luciferase expression observed after particle bombardment were significantly lower (t-test, p \u3c 0.02) than the levels measured after direct injection. Immunohistochemical analysis indicated β-galactosidase gene expression in muscle cells at the site of injection and in the dermis, epidermis and muscle after bombardment. These 2 methods may prove valuable for the development of a new generation of DNA-based vaccines for fish

Aberrant Methylation Inactivates Transforming Growth Factor β Receptor I in Head and Neck Squamous Cell Carcinoma

Background. Alterations in TGF-β signaling are common in head and neck cancer (HNSCC). Mutations in TGF-β type II receptor (TβR-II) occur frequently in HNSCC while TGF-β type I receptor (TβR-I) mutations are rare, suggesting that other molecular alterations in the TGF-β pathway are likely. To identify abnormalities in TβR-I expression we analyzed 50 HNSCCs and correlated the results with clinical-pathologic features. Methods. Hypermethylation of TβR-I was evaluated via methylation-specific PCR (MSP) and restriction enzyme-mediated PCR (MSRE). Mutations in exons 1 and 7, mRNA and protein expression were analyzed by direct sequencing, semiquantitative RT—PCR and immunohistochemistry, respectively. Results. TβR-I expression was lost in 83% HNSCCs and was linked to DNA hypermethylation of the CpG-rich promoter region in 62% of the tumors. The variants 9A/6A and Int7G24A were found in two patients. Conclusions. This study shows that suppression of TβR-I expression in HNSCC is associated with DNA hypermethylation

Aberrant methylation inactivates transforming growth factor beta receptor I in head and neck squamous cell carcinoma

Background. Alterations in TGF-β signaling are common in head and neck cancer (HNSCC). Mutations in TGF-β type II receptor (TβR-II) occur frequently in HNSCC while TGF-β type I receptor (TβR-I) mutations are rare, suggesting that other molecular alterations in the TGF-β pathway are likely. To identify abnormalities in TβR-I expression we analyzed 50 HNSCCs and correlated the results with clinical-pathologic features. Methods. Hypermethylation of TβR-I was evaluated via methylation-specific PCR (MSP) and restriction enzyme-mediated PCR (MSRE). Mutations in exons 1 and 7, mRNA and protein expression were analyzed by direct sequencing, semiquantitative RT-PCR and immunohistochemistry, respectively. Results. TβR-I expression was lost in 83% HNSCCs and was linked to DNA hypermethylation of the CpG-rich promoter region in 62% of the tumors. The variants 9A/6A and Int7G24A were found in two patients. Conclusions. This study shows that suppression of TβR-I expression in HNSCC is associated with DNA hypermethylation

Tetrahydropyrazolo[1,5-a]Pyrimidine-3-Carboxamide and N-Benzyl-6′,7′-Dihydrospiro[Piperidine-4,4′-Thieno[3,2-c]Pyran] analogues with bactericidal efficacy against Mycobacterium tuberculosis targeting MmpL3

Mycobacterium tuberculosis is a major human pathogen and the causative agent for the pulmonary disease, tuberculosis (TB). Current treatment programs to combat TB are under threat due to the emergence of multi-drug and extensively-drug resistant TB. As part of our efforts towards the discovery of new anti-tubercular leads, a number of potent tetrahydropyrazolo[1,5-a]pyrimidine-3-ca​rboxamide(THPP) and N-benzyl-6′,7′-dihydrospiro[piperidine-4,​4′-thieno[3,2-c]pyran](Spiro) analogues were recently identified against Mycobacterium tuberculosis and Mycobacterium bovis BCG through a high-throughput whole-cell screening campaign. Herein, we describe the attractive in vitro and in vivo anti-tubercular profiles of both lead series. The generation of M. tuberculosis spontaneous mutants and subsequent whole genome sequencing of several resistant mutants identified single mutations in the essential mmpL3 gene. This ‘genetic phenotype’ was further confirmed by a ‘chemical phenotype’, whereby M. bovis BCG treated with both the THPP and Spiro series resulted in the accumulation of trehalose monomycolate. In vivo efficacy evaluation of two optimized THPP and Spiro leads showed how the compounds were able to reduce >2 logs bacterial cfu counts in the lungs of infected mice

Aggressive proximal tibio-fibular joint ganglion cyst after a total knee arthroplasty--a case report

Although the knee is a common location, a ganglion cyst arising from the proximal tibio-fibular joint (PTFJ) is relatively rarer. Unique diagnostic and management difficulties are encountered as these can extend into subcutaneous tissue, or spread along peroneal muscles and nerve, or erode adjacent bone. We report here a PTFJ ganglion cyst which was indolent for at least 12 years and then caused destructive and expansile changes in the fibula over a period of 6 years after a total knee arthroplasty (TKA), simulating a malignancy. Although no wear or foreign body reaction could be shown, this alarming progression of a PTFJ ganglion cyst in the fibula after the TKA is a noteworthy association, and has not been recognized and reported before. Awareness of such a lesion can aid in the diagnosis and prevent unnecessary aggressive management

Adenoid Cystic Carcinoma of the Base of the Tongue Metastasizing to the Scalp

Adenoid cystic carcinoma is a rare neoplasm that originates from secretory glands, most commonly from the salivary glands. We present a 76 year-old white man with a history of adenoid cystic carcinoma from the base of the tongue diagnosed 15 years prior to the development of the metastatic lesion on his mid-posterior scalp. The present case represents the second reported instance of an extracutaneous adenoid cystic carcinoma metastasizing to the scalp. Differentiating between a primary cutaneous adenoid cystic carcinoma and an extracutaneous adenoid cystic carcinoma metastasizing to cutaneous structures is crucial in determining prognosis and management