Expression of the Androgen Receptor,pAkt, and pPTEN in Breast Cancer and Their Potential in Prognostication

BACKGROUNDImportance of androgen receptor (AR) as an independent prognostic marker in Pakistani women with breast cancer (BCa) remains unexplored. Our aim was to identify the expression and potential prognostic value of AR, its upstream regulator (pAkt) and target gene (pPTEN) in invasive BCa.METHODSThis study used a cohort of 200 Pakistani women with invasive BCa diagnosed during 2002-2011. Expression of AR, pAkt and pPTEN was determined on formalin fixed paraffin embedded tissue sections by immunohistochemistry. The association of AR, pAkt and pPTEN with clinicopathological parameters was determined. Survival analyses were undertaken on patients with ≥ 5 years of follow-up (n = 82).RESULTSExpression of AR, pAkt and pPTEN was observed in 47.5%, 81.3% and 50.6% of patients, respectively. AR-expressing tumors were low or intermediate in grade (P < .001) and expressed ER (P = .002) and PR (P = .001). Patients with AR⁺ tumors had significantly higher OS (Mean OS = 10.2 ± 0.465 years) compared to patients with AR^? tumors (Mean OS = 5.8 ± 0.348 years) (P = .047). Furthermore, AR-positivity was associated with improved OS in patients receiving endocrine therapy (P = .020). Patients with AR⁺ /pAkt⁺ /pPTEN^? tumors, had increased OS (Mean OS = 7.1 ± 0.535 years) compared to patients with AR^?/pAkt⁺/pPTEN^? tumors (Mean OS = 5.1 ± 0.738 years).CONCLUSIONAR-expressing tumors are frequently characterized by low or intermediate grade tumors, expressing ER and PR. In addition, expression of AR, pAkt and pPTEN, could be considered in prognostication of patients with invasive BCa.     

Phagocytosis-coupled flow cytometry for detection and size discrimination of anionic polystyrene particles

Flow cytometry was evaluated for its capacity to detect and distinguish a wide size range (20–2000 nm) of fluorescent polystyrene particles (PSPs). Side scatter and fluorescence parameters could predict dispersed PSP sizes down to 200 nm, but the forward scatter parameter was not discriminatory. Confocal microscopy of flow-sorted fractions confirmed that dispersed PSPs appeared as a single sharp peak on fluorescence histograms, whereas agglomerated PSPs were detected as smaller adjacent peaks. Particles as small as 200 nm could also be detected by flow cytometry after they were first phagocytized by J774A.1 murine macrophages. Confocal microscopy demonstrated that these PSPs were internalized within the cytoplasm. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and calcein–AM (acetoxymethyl ester) assays showed that they were not cytotoxic. Internalized PSP size correlated to both cellular side scatter (R² = 0.9821) and fluorescence intensity (R² = 0.9993). Furthermore, PSPs of various sizes could be distinguished when J774A.1 cells were loaded with a single size of PSP and mixed with cells containing other sizes. However, spectra of cells loaded with a mixture of PSP sizes resembled those containing only the largest PSP. These data demonstrate the capacity and limitations of phagocytosis-coupled flow cytometry to distinguish between dispersed and agglomerated states and detect a wide size range of particles.     

Induced fit docking,free energy calculation and molecular dynamics studies on Mycobacterium tuberculosis alanine racemase inhibitor

Alar, a Pyridoxal 5′-phosphate (PLP)-dependent bacterial enzyme is responsible for the racemisation of L-alanine into D-alanine which is essential for the peptidoglycan biosynthesis in both Gram-positive and Gram-negative bacteria. In the present study, we performed induced fit docking, binding free energy calculation and molecular dynamics simulation to elucidate the Alar inhibition potential of 1,2,4-thiadiazolidine-3,5-dione-based inhibitor 1. The inhibitor binds to the hydrophobic groove of Alar and the binding was found to be stable throughout 20-ns MD simulation. Induced fit docking result showed that Lys42, Tyr46, Tyr175 and Tyr364 residues are primarily responsible for the stabilisation of inhibitor–protein complex. Further, high negative van der Waals binding free energy value of –38.88 kcal/mol, indicated it as the main driving force for the inhibitor binding. Based on the information obtained from this study, we designed few molecules as potent Alar inhibitor. In order to gain structural insight and to validate the stability of complex, we performed 20-ns MD simulation of the designed molecule D1. Results obtained from this study can be used for the design of M. tuberculosis Alar potent inhibitors lacking affinity for the co-factor PLP.     

Antibacterial and Cytotoxic Efficacy of Extracellular Silver Nanoparticles Biofabricated from Chromium Reducing Novel OS4 Strain of Stenotrophomonas maltophilia

Biofabricated metal nanoparticles are generally biocompatible, inexpensive, and ecofriendly, therefore, are used preferably in industries, medical and material science research. Considering the importance of biofabricated materials, we isolated, characterized and identified a novel bacterial strain OS4 of Stenotrophomonas maltophilia (GenBank: {"type":"entrez-nucleotide","attrs":{"text":"JN247637.1","term_id":"341941875","term_text":"JN247637.1"}}JN247637.1). At neutral pH, this Gram negative bacterial strain significantly reduced hexavalent chromium, an important heavy metal contaminant found in the tannery effluents and minings. Subsequently, even at room temperature the supernatant of log phase grown culture of strain OS4 also reduced silver nitrate (AgNO₃) to generate nanoparticles (AgNPs). These AgNPs were further characterized by UV–visible, Nanophox particle size analyzer, XRD, SEM and FTIR. As evident from the FTIR data, plausibly the protein components of supernatant caused the reduction of AgNO₃. The cuboid and homogenous AgNPs showed a characteristic UV-visible peak at 428 nm with average size of ∼93 nm. The XRD spectra exhibited the characteristic Bragg peaks of 111, 200, 220 and 311 facets of the face centred cubic symmetry of nanoparticles suggesting that these nanoparticles were crystalline in nature. From the nanoparticle release kinetics data, the rapid release of AgNPs was correlated with the particle size and increasing surface area of the nanoparticles. A highly significant antimicrobial activity against medically important bacteria by the biofabricated AgNPs was also revealed as decline in growth of Staphylococcus aureus (91%), Escherichia coli (69%) and Serratia marcescens (66%) substantially. Additionally, different cytotoxic assays showed no toxicity of AgNPs to liver function, RBCs, splenocytes and HeLa cells, hence these particles were safe to use. Therefore, this novel bacterial strain OS4 is likely to provide broad spectrum benefits for curing chromium polluted sites, for biofabrication of AgNPs and ultimately in the nanoparticle based drug formulation for the treatment of infectious diseases.     

Synthesis, spectral studies and in vitro assessment for antiamoebic activity of new cyclooctadiene ruthenium(II) complexes with 5-nitrothiophene-2-carboxaldehyde thiosemicarbazones

We report here the synthesis, characterization and in vitro antiamoebic activity of 5-nitrothiophene-2-carboxaldehyde thiosemicarbazones (TSC), 1–5, and their bidentate complexes [Ru(η⁴-C₈H₁₂)(TSC)Cl₂] 1a–5a. The biological studies of these compounds were investigated against HK-9 strain of Entamoeba histolytica and the concentration causing 50% cell growth inhibition (IC₅₀) was calculated in the micromolar range. The ligands exhibited antiamoebic activity in the range (2.05–5.29 μM). Screening results indicated that the potencies of the compounds increased by the incorporation of ruthenium(II) in the thiosemicarbazones. The complexes 1a–5a showed antiamoebic activity with an IC₅₀ of 0.61–1.43 μM and were better inhibitors of growth of E. histolytica, based on IC₅₀ values. The most promising among them is Ru(II) complex 2a having 1,2,3,4-tetrahydroquinoline as N⁴ substitution.     

Signaling Crosstalk of FHIT,p53, and p38 in etoposide-induced apoptosis in MCF-7 cells

Fragile histidine trail (FHIT) is a tumor suppressor in response to DNA damage which has been deleted in various tumors. However, the signaling mechanisms and interactions of FHIT with regard to apoptotic proteins including p53 and p38 in the DNA damage-induced apoptosis are not well described. In the present study, we used etoposide-induced DNA damage in MCF-7 as a model to address these crosstalks. The time course study showed that the expression of FHIT, p53, and p38MAPK started after 1 hour following etoposide treatment. FHIT overexpression led to increase p53 expression, p38 activation, and augmented apoptosis following etoposide-induced DNA damage compared to wild-type cells. However, FHIT knockdown blocked p53 expression, delayed p38 activation, and completely inhibited etoposide-induced apoptosis. Inhibition of p38 activity prevented induction of p53, FHIT, and apoptosis in this model. Thus, activation of p38 upon etoposide treatment leads to increase in FHIT and p53 expression. In p53 knockdown MCF-7, the FHIT induction was hampered but p38 activation was induced in lower doses of etoposide. In p53 knockdown cells, inhibition of p38 induced FHIT expression and apoptosis. Our data demonstrated that the exposure of MCF-7 cells to etoposide increases apoptosis through a mechanism involving the activation of the p38-FHIT-p53 pathway. Moreover, our findings suggest signaling interaction for these pathways may represent a promising therapy for breast cancer.     

Corals shed bacteria as a potential mechanism of resilience to organic matter enrichment

Understanding the mechanisms of resilience of coral reefs to anthropogenic stressors is a critical step toward mitigating their current global decline. Coral–bacteria associations are fundamental to reef health and disease, but direct observations of these interactions remain largely unexplored. Here, we use novel technology, high-speed laser scanning confocal microscopy on live coral (Pocillopora damicornis), to test the hypothesis that corals exert control over the abundance of their associated bacterial communities by releasing (‘shedding'') bacteria from their surface, and that this mechanism can counteract bacterial growth stimulated by organic inputs. We also test the hypothesis that the coral pathogen Vibrio coralliilyticus can evade such a defense mechanism. This first report of direct observation with high-speed confocal microscopy of living coral and its associated bacterial community revealed a layer (3.3–146.8 μm thick) on the coral surface where bacteria were concentrated. The results of two independent experiments showed that the bacterial abundance in this layer was not sensitive to enrichment (5 mg l⁻¹ peptone), and that coral fragments exposed to enrichment released significantly more bacteria from their surfaces than control corals (P<0.01; 35.9±1.4 × 10⁵ cells cm⁻² coral versus 1.3±0.5 × 10⁵ cells cm⁻² coral). Our results provide direct support to the hypothesis that shedding bacteria may be an important mechanism by which coral-associated bacterial abundances are regulated under organic matter stress. Additionally, the novel ability to watch this ecological behavior in real-time at the microscale opens an unexplored avenue for mechanistic studies of coral–microbe interactions.     

Changes in photosynthetic pigments and chlorophyll-<Emphasis Type="Italic">a</Emphasis> fluorescence attributes of sweet-forage and grain sorghum cultivars under salt stress

Water shortage leads to a low quality of water, especially saline water in most parts of agricultural regions. This experiment was designed to determine the effects of saline irrigation on sorghum as a moderately salt-tolerant crop. To study salinity effects on photosynthetic pigment attributes including the chlorophyll content and chlorophyll fluorescence, an experiment was performed in a climate-controlled greenhouse at two vegetative and reproductive stages. The experimental design was factorial based on a completely randomized design with five NaCl concentrations (control, 50, 100, 150, and 200 mM), two grain and sweet-forage sorghum cultivars (Kimia and Pegah, respectively) and four replications. According to the experimental data, there were no significant differences between two grain and sweet-forage cultivars. Except for 100 and 150 mM NaCl, salinity significantly decreased the chlorophyll index and pigment contents of the leaf, while it increased the chlorophyll-a fluorescence characteristics. Although salinity reduced photosynthetic pigments and the crop yield, either grain or sweet-forage cultivars could significantly control the effect of salinity between 100 and 150 mM NaCl at both developmental stages, showing the possibility of using saline water in sorghum cultivation up to 150 mM NaCl.     

Detection and phylogenetic profiling of nodavirus associated with white tail disease in Malaysian <Emphasis Type="Italic">Macrobrachium rosenbergii</Emphasis> de Man

White tail disease (WTD) is a serious viral disease in the hatcheries and nursery ponds of Macrobrachium rosenbergii in many parts of the world. A new disease similar to WTD was observed in larvae and post larvae of M. rosenbergii cultured in Malaysia. In the present study, RT-PCR assay was used to detect the causative agents of WTD, M. rosenbergii nodavirus (MrNV) and extra small virus (XSV) using specific primers for MrNV RNA2 and XSV. The results showed the presence of MrNV in the samples with or without signs of WTD. However, XSV was only detected in some of the MrNV-positive samples. Phylogenetic analysis showed that the RNA2 of our Malaysian isolates were significantly different from the other isolates. Histopathological studies revealed myofiber degeneration of the tail muscles and liquefactive myopathy in the infected prawns. This was the first report on the occurrence of MrNV in the Malaysian freshwater prawn.     

Affinity maturation and characterization of the ofatumumab monoclonal antibody

CD20 molecule, a phosphoprotein with 297 amino acids and four transmembrane domains, is a member of MS4A protein family. Anti-CD20 antibodies such as ofatumumab, which have been developed for cancer treatment and has demonstrated efficacy in relapsed/refractory chronic lymphocytic leukemia, are among the most successful therapies to date. Rational engineering methods can be applied with reasonable success to improve functional characteristics of antibodies. Considering the importance of this issue, we have used in silico modeling approach for the improvement of ofatumumab monoclonal antibody. Four mutated variants of ofatumumab were developed and expressed in Chinese hamster ovary (CHO) cells along with the unmodified antibody. Analysis of affinity of the purified antibodies with CD20 showed significant improvement in antigen-binding characteristics of one of the variants compared with the control antibody. This study represents the first step toward development of the second generation ofatumumab antibody with improved affinity.