Negative/low HER2 expression alone or combined with E-cadherin positivity is predictive of better prognosis in patients with breast carcinoma

The loss of E-cadherin expression leads to absence of tissue integrity, an essential step in tumor progression. Methylation of CpG islands in the promoter region of the CDH1 gene coding E-cadherin might be an alternative for gene silencing. In the present study, we investigate the expression of E-cadherin and hormone receptors in invasive ductal breast carcinoma (IDCs). Protein expression was analysed immunohistochemically in 87 cases, including 26 familial tumors. The most interesting results revealed a significantly reduced E-cadherin expression in cases with familial history compared to sporadic tumors (p=0.009), as well as with tumors ≤5 cm (p=0.022). Moreover, HER2 over-expression was associated with distant metastasis (p=0.011) and overall survival (p log rank=0.028). Tumors displaying negative/low HER2 expression combined with E-cadherin positivity confer better patient survival (p=0.052). Triple Negative tumors (TN) were more frequently found in patients with advanced grade (GIII) (p=0.001) and TNM (III+IV) (p=0.018) which supports the aggressive behavior of TN tumors. On the other hand, hypermethylation of CDH1 gene promoter was observed in 46% of hereditary cases and strongly associated with loss of E-cadherin expression (p=0.002). Furthermore, patients with unmethylated CDH1 pattern have a better 5-year disease free survival (p=0.021). In conclusion, in patients with hereditary breast cancer, the CpG methylation event contributes to the loss of E-cadherin expression. On the other hand, HER2 over-expression is predictive of worse prognosis, either alone or combined with loss of E-cadherin expression in Tunisian patients with breast cancer.     

Prevalence of coagulation factor II G20210A and factor V G1691A Leiden polymorphisms in Chechans, a genetically isolated population in Jordan

Background Coagulation factor II G20210A and coagulation factor V (Leiden) G1691A single nucleotide polymorphisms (SNPs) are major inherited risk factors of venous thromboembolism. In view of the heterogeneity in their world distribution and lack of sufficient information about their distribution among Chechans, we addressed the prevalence of these SNPs in the Chechan population in Jordan, a genetically isolated population. Methods and Results factor II G20210A and factor V Leiden SNPs were analysed by polymerase chain reaction and restriction fragment length polymorphism (PCR?CRFLP) method and Amplification refractory mutation detection system (ARMS) respectively in 120 random unrelated subjects from the Chechan population in Jordan. Among the subjects studied for factor II G20210A mutation there were three individuals carrying this mutation as heterozygous (one female and two male), giving a prevalence of 2.5?% and an allele frequency of 1.25?%. No homozygous factor II allele was found. Factor V Leiden G1691A mutation was detected as heterozygous in 22 of 120 of individuals (17 female and five male) indicating a prevalence of 18.3?% and allele frequency of 9.2?%. No homozygous allele was found. Conclusion Our results indicated that prevalence of factor II G20210A mutation in the Chechan population is similar to prevalence in Jordan and Caucasian populations (1?C6?%) while the prevalence of factor V Leiden was higher in the Chechan population compared to Jordan and Caucasian populations (2?C15?%).     

Directed, strong, and reversible immobilization of proteins tagged with a β-trefoil lectin domain: a simple method to immobilize biomolecules on plain agarose matrixes

A highly stable lipase from Geobacillus thermocatenolatus (BTL2) and the enhanced green fluorescent protein from Aquorea victoria (EGFP) were recombinantly produced N-terminally tagged to the lectin domain of the hemolytic pore-forming toxin LSLa from the mushroom Laetiporus sulphureus . Such a domain (LSL(150)), recently described as a novel fusion tag, is based on a β-trefoil scaffold with two operative binding sites for galactose or galactose-containing derivatives. The fusion proteins herein analyzed have enabled us to characterize the binding mode of LSL(150) to polymeric and solid substrates such as agarose beads. The lectin-fusion proteins are able to be quantitatively bound to both cross-linked and non-cross-linked agarose matrixes in a very rapid manner, resulting in a surprisingly dynamic protein distribution inside the porous beads that evolves from heterogeneous to homogeneous along the postimmobilization time. Such dynamic distribution can be related to the reversible nature of the LSL(150)-agarose interaction. Furthermore, this latter interaction is temperature dependent since it is 4-fold stronger when the immobilization takes place at 25 °C than when it does at 4 °C. The strongest lectin-agarose interaction is also quite stable under a survey of different conditions such as high temperatures (up to 60 °C) or high organic solvent concentrations (up to 60% of acetonitrile). Notably, the use of cross-linked agarose would endow the system with more robustness due to its better mechanical properties compared to the noncross-linked one. The stability of the LSL(150)-agarose interaction would prevent protein leaching during the operation process unless high pH media are used. In summary, we believe that the LSL(150) lectin domain exhibits interesting structural features as an immobilization domain that makes it suitable to reversibly immobilize industrially relevant enzymes in very simple carriers as agarose.     

Activation of fast skeletal muscle troponin as a potential therapeutic approach for treating neuromuscular diseases

Limited neural input results in muscle weakness in neuromuscular disease because of a reduction in the density of muscle innervation, the rate of neuromuscular junction activation or the efficiency of synaptic transmission. We developed a small-molecule fast-skeletal-troponin activator, CK-2017357, as a means to increase muscle strength by amplifying the response of muscle when neural input is otherwise diminished secondary to neuromuscular disease. Binding selectively to the fast-skeletal-troponin complex, CK-2017357 slows the rate of calcium release from troponin C and sensitizes muscle to calcium. As a consequence, the force-calcium relationship of muscle fibers shifts leftwards, as does the force-frequency relationship of a nerve-muscle pair, so that CK-2017357 increases the production of muscle force in situ at sub-maximal nerve stimulation rates. Notably, we show that sensitization of the fast-skeletal-troponin complex to calcium improves muscle force and grip strength immediately after administration of single doses of CK-2017357 in a model of the neuromuscular disease myasthenia gravis. Troponin activation may provide a new therapeutic approach to improve physical activity in diseases where neuromuscular function is compromised.     

Efficacy of tetracycline encapsulated O-carboxymethyl chitosan nanoparticles against intracellular infections of Staphylococcus aureus

Intracellular bacterial infections are recurrent, persistent and are difficult to treat because of poor penetration and limited availability of antibiotics within macrophages and epithelial cells. We developed biocompatible, 200nm sized tetracycline encapsulated O-carboxymethyl chitosan nanoparticles (Tet-O-CMC Nps) via ionic gelation for its sustained delivery of Tet into cells. S. aureus binds and aggregates with Tet-O-CMC Nps increasing drug concentrations at the infection site. Tet-O-CMC Nps were sixfold more effective in killing intracellular S. aureus compared to Tet alone in HEK-293 and differentiated THP1 macrophage cells proving it to be an efficient nanomedicine to treat intracellular S. aureus infections.     

Transferring knowledge towards understanding the pore stabilizing variations in K+ channels

Recent advances in structural biology underlying mechanisms of channel gating have strengthened our knowledge about how K⁺ channels can be inter-convertible between conductive and non-conductive states. We have reviewed and combined mutagenesis with biochemical, biophysical and structural information in order to understand the critical roles of the pore residues in stabilizing the pore structure and channel open state. We also discuss how the latest knowledge on the K⁺ channel KcsA may provide a step towards better understanding of distinct pore stabilizing differences among diversified K⁺ channels.     

A vaccinia virus-driven interplay between the MKK4/7-JNK1/2 pathway and cytoskeleton reorganization

Viral manipulation of transduction pathways associated with key cellular functions such as survival, response to microbial infection, and cytoskeleton reorganization can provide the supportive milieu for a productive infection. Here, we demonstrate that vaccinia virus (VACV) infection leads to activation of the stress-activated protein kinase (SAPK)/extracellular signal-regulated kinase (ERK) 4/7 (MKK4/7)-c-Jun N-terminal protein kinase 1/2 (JNK1/2) pathway; further, the stimulation of this pathway requires postpenetration, prereplicative events in the viral replication cycle. Although the formation of intracellular mature virus (IMV) was not affected in MKK4/7- or JNK1/2-knockout (KO) cells, we did note an accentuated deregulation of microtubule and actin network organization in infected JNK1/2-KO cells. This was followed by deregulated viral trafficking to the periphery and enhanced enveloped particle release. Furthermore, VACV infection induced alterations in the cell contractility and morphology, and cell migration was reduced in the JNK-KO cells. In addition, phosphorylation of proteins implicated with early cell contractility and cell migration, such as microtubule-associated protein 1B and paxillin, respectively, was not detected in the VACV-infected KO cells. In sum, our findings uncover a regulatory role played by the MKK4/7-JNK1/2 pathway in cytoskeleton reorganization during VACV infection.     

A tetravalent dengue nanoparticle stimulates antibody production in mice

Background

Dengue is a major public health problem worldwide, especially in the tropical and subtropical regions of the world. Infection with a single Dengue virus (DENV) serotype causes a mild, self-limiting febrile illness called dengue fever. However, a subset of patients experiencing secondary infection with a different serotype progresses to the severe form of the disease, dengue hemorrhagic fever/dengue shock syndrome. Currently, there are no licensed vaccines or antiviral drugs to prevent or treat dengue infections. Biodegradable nanoparticles coated with proteins represent a promising method for in vivo delivery of vaccines.

Findings

Here, we used a murine model to evaluate the IgG production after administration of inactivated DENV corresponding to all four serotypes adsorbed to bovine serum albumin nanoparticles. This formulation induced a production of anti-DENV IgG antibodies (p < 0.001). However, plaque reduction neutralization assays with the four DENV serotypes revealed that these antibodies have no neutralizing activity in the dilutions tested.

Conclusions

Our results show that while the nanoparticle system induces humoral responses against DENV, further investigation with different DENV antigens will be required to improve immunogenicity, epitope specicity, and functional activity to make this platform a viable option for DENV vaccines.     

Microgenomics: Identification of new expression profiles via small and single-cell sample analyses.

BACKGROUND: Since the sequencing of the human genome has been finished, microgenomics has been booming, employing highly sophisticated, high-throughput platforms. But these mainly chip-based methods can only generate biologically relevant data if the samples investigated consist of homogeneous cell populations, in which no unwanted cells of different specificity and/or developmental stage obscure the results. METHODS: Different sampling methods have been routinely applied to overcome the problem presented by heterogeneous samples, e.g., global surveys, cell cultures, and microdissection. Various methods of laser-assisted microdissection, employing either positive or negative selection of tissue areas or even single cells, are available. RESULTS: These laser-assisted microdissection methods allow for fast and precise procurement of extremely small samples. Through subsequent application of recently developed methods of linear mRNA amplification in a pool of isolated total RNA, it has now become possible to perform complex high-throughput RNA expression profiling by microdissecting and processing even single-cell samples. CONCLUSIONS: Studies using the tools and methods of microgenomics have shed light on how those new approaches will eventually aid in the development of a new generation of diagnostics, e.g., leading to new patient-specific drugs tailored to the requirements assessed by assaying only a few biopsy cells.