Further evidence for role of leukotrienes as mediators of decidualization in the rat

Inhibitors of leukotrieens were utilized to investigate the role of leukoteines (LTs) in the induction of decidualization in the rat. Alzet osmotic minipumps, filled with either FPL 55712 (FPL, a specific antagonist of peptidoleukotrienes), nordihydroguaiaretic acid (NDGA, an inhibitor of LT synthesis) or in combination with leukotriene C₄ (LTC₄) and/or prostaglandin E2 (PGE₂), were instilled at the ovarian end of uterine horns of day 5 pseudopregnant rats. Intraluminal infusion of FPL or DNGA, for 4 days, induced a dose dependent decrease in the uterine wet weights when compared to that induced by the infusion of their corresponding vehicles (1 μl/h). Furthermore, simultaneous infusion of LTC₄ (10 ng/h) with different doses of FPL (1, 0.5, or 0.25 μg/h) produced an increase in uterine weights as compared to that produced by FPL alone. Maximum response, however, was noted when LTC₄ (n0 ng/h) was infused with FPL at a rate of 0.5 μg/h. The infusion of LTC₄ (10 ng/h) or PGE₂ (1 μg/h) with NDGA, at 1 and 5 μg/h, could not overcome its inhibitory effect on decidualization. On the contrary, a combination of LTC₄ (10 ng/h) and PGE₂ (1 μg/h) was comparable to that induced by the infusion of the vehicle. To determine if the synthesis of PGs and LTs was inhibited by NDGA, one uterine horn was infused with NDGA (5 μg/h) and the other horn with the vehicle. The intrauterine infusion of NDGA for 24 h inhibited the release of PGE₂, PGF_2α, LTC₄ and LTB₄ as compared to those released by the vehicle-infused horns. These data suggest that both PGs and LTs are required for the induction and progression of decidualization.     

Aquatic fungi of Iraq: Species of Saprolegnia

Five species of Saprolegnia have been isolated from the water samples obtained monthly from Shatt Al-Arab estuary from September, 1976 to August, 1977. These are S. ferax, S. anisospora, S. diclina, S. terrestris and S. parasitica. Out of 81 isolates 43 reached sexual maturity while 38 remained sterile. Maximum abundance was found during winter months while minimum in summer months.     

PKCλ/ι signaling promotes triple-negative breast cancer growth and metastasis

Triple-negative breast cancer (TNBC) is a distinct breast cancer subtype defined by the absence of estrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor 2 (HER2/neu), and the patients with TNBC are often diagnosed with higher rates of recurrence and metastasis. Because of the absence of ER, PR and HER2/neu expressions, TNBC patients are insensitive to HER2-directed and endocrine therapies available for breast cancer treatment. Here, we report that expression of atypical protein kinase C isoform, PKCλ/ι, significantly increased and activated in all invasive breast cancer (invasive ductal carcinoma or IDC) subtypes including the TNBC subtype. Because of the lack of targeted therapies for TNBC, we choose to study PKCλ/ι signaling as a potential therapeutic target for TNBC. Our observations indicated that PKCλ/ι signaling is highly active during breast cancer invasive progression, and metastatic breast cancers, the advanced stages of breast cancer disease that developed more frequently in TNBC patients, are also characterized with high levels of PKCλ/ι expression and activation. Functional analysis in experimental mouse models revealed that depletion of PKCλ/ι significantly reduces TNBC growth as well as lung metastatic colonization. Furthermore, we have identified a PKCλ/ι-regulated gene signature consisting of 110 genes, which are significantly associated with indolent to invasive progression of human breast cancer and poor prognosis. Mechanistically, cytokines such as TGFβ and IL1β could activate PKCλ/ι signaling in TNBC cells and depletion of PKCλ/ι impairs NF-κB p65 (RelA) nuclear localization. We observed that cytokine-PKCλ/ι-RelA signaling axis, at least in part, involved in modulating gene expression to regulate invasion of TNBC cells. Overall, our results indicate that induction and activation of PKCλ/ι promote TNBC growth, invasion and metastasis. Thus, targeting PKCλ/ι signaling could be a therapeutic option for breast cancer, including the TNBC subtype.Breast cancer is a clinically heterogeneous disease and both intra and inter-tumor heterogeneities provide great challenges for developing successful therapies. Expressions (or absence thereof) of estrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor 2 (HER2)/neu are widely used to clinically classify breast tumors into multiple therapeutic groups.¹ The ER/PR-positive and the HER2-positive breast cancer patients could be benefited from endocrine and HER2-targeted therapies.¹ However, triple-negative breast cancers (TNBCs), which represent ∼12–17% of all breast cancer,² lack ER, PR and HER2/neu expressions² and are not responsive to therapies targeting these receptors. Therefore, the only systemic therapy available for TNBC is chemotherapy.³ Furthermore, TNBC is associated with aggressive pathologic features like higher histology grade and mitotic index⁴ and often found to be associated with higher rate of metastasis and recurrence leading to limited clinical outcome.^{5, 6, 7, 8} Recurrence of TNBC tends to recur within a few years after successful initial treatment^{6, 9} and often develops metastasis to the bone, brain and lungs with poor prognosis.^{2, 6} Thus, identification of signaling pathways that regulate malignant progression of breast cancer subtypes, especially TNBCs, would be therapeutically important.In recent years, PKC signaling has been implicated in modulating invasion and metastasis of multiple tumors.^{10, 11} The PKC family consists of multiple serine/threonine kinases and the relative contribution of individual PKC isoforms during cancer progression varies due to pleiotropism.¹² PKC isoforms regulate diverse cellular functions such as cell-cycle regulation, cellular survival, cell–cell communications and apoptosis.¹³ In particular, atypical PKC isoforms, PKCζ and atypical protein kinase C lamda/iota (PKCλ/ι), are known to be important for chemotaxis, cell polarity, migration and wound healing processes.^{14, 15} Aberrations in all these processes are manifested in tumor progression and metastasis.¹⁴ Consistent with these notions, recent studies indicated that atypical PKCs are associated with various human cancers.^{10, 11} Importantly, the PKCλ/ι gene is located at the 3q26.2 genomic region, which is most frequently amplified in human cancer^{16, 17}, and overexpression of PKCλ/ι has been implicated in cancer development in multiple tissues including the lung,^{18, 19} pancreas,²⁰ stomach,²¹ colon,²² esophagus,²³ liver,²⁴ bile duct,²⁵ ovary,¹⁷ prostate²⁶ and brain.²⁷ Recently, few studies have been reported higher expression of PKCλ/ι in ER/PR- and HER-positive breast cancer and also in lymph node metastases.^{28, 29} Kojima et. al.²⁸ showed that PKCλ/ι expression is highly induced in the ER/PR- and HER2-positive IDCs compared with ductal carcinoma in situ (DCIS) and normal breast. PKCλ/ι forms apical-junctional complexes (AJCs) with other polarity proteins such as partitioning defective 3 homolog (PAR3) and partitioning defective 6 homolog (PAR6),^{30, 31, 32, 33} and invasiveness of breast tumor cells was shown to be associated with loss of PKCλ/ι localization from their apical domains.²⁸ In addition, predominant nuclear localization of PKCλ/ι in both normal and atypical ductal hyperplasia (ADH) lesions prompted the concept that PKCλ/ι might be in an inactive state in these lesions.²⁸ However, expression and activation of PKCλ/ι in TNBCs and the functional importance of PKCλ/ι signaling in relation to invasive breast cancer progression and metastasis are very poorly understood.^{10, 11}Here, we studied PKCλ/ι signaling during invasive progression of TNBC. We utilized expression evaluations in triple-negative IDCs as well as metastatic breast cancers of human patients, in vitro and in vivo functional assays, and global gene expression analysis of human patient samples. We concluded that PKCλ/ι signaling is an important regulator for invasion and metastatic progression of human breast cancers including triple-negative subtypes.     

Evolutionary Optimization of Computationally Designed Enzymes: Kemp Eliminases of the KE07 Series

Understanding enzyme catalysis through the analysis of natural enzymes is a daunting challenge—their active sites are complex and combine numerous interactions and catalytic forces that are finely coordinated. Study of more rudimentary (wo)man-made enzymes provides a unique opportunity for better understanding of enzymatic catalysis. KE07, a computationally designed Kemp eliminase that employs a glutamate side chain as the catalytic base for the critical proton abstraction step and an apolar binding site to guide substrate binding, was optimized by seven rounds of random mutagenesis and selection, resulting in a > 200-fold increase in catalytic efficiency. Here, we describe the directed evolution process in detail and the biophysical and crystallographic studies of the designed KE07 and its evolved variants. The optimization of KE07's activity to give a k_cat/K_M value of ∼ 2600 s^− 1 M^− 1 and an ∼ 10⁶-fold rate acceleration (k_cat/k_uncat) involved the incorporation of up to eight mutations. These mutations led to a marked decrease in the overall thermodynamic stability of the evolved KE07s and in the configurational stability of their active sites. We identified two primary contributions of the mutations to KE07's improved activity: (i) the introduction of new salt bridges to correct a mistake in the original design that placed a lysine for leaving-group protonation without consideration of its “quenching” interactions with the catalytic glutamate, and (ii) the tuning of the environment, the pK_a of the catalytic base, and its interactions with the substrate through the evolution of a network of hydrogen bonds consisting of several charged residues surrounding the active site.     

Dehydration-induced alterations in growth and osmotic potential of callus from six tepary bean lines varying in drought resistance

Growth and osmotic potential of calli induced from leaf- and root-derived tissues of six tepary bean lines (Phaseolus acutifolius) varying in drought resistance were assessed in vitro after polyethylene glycol-induced (10%, PEG-10,000) dehydration. Calli of resistant teparies were characterized by low initial osmotic potential (ψ_s) and relative growth rate (RGR) on medium lacking PEG (−0.30 MPa). However, calli of both resistant and sensitive lines were similar in dry matter percent (DM). Presence of PEG in the medium (−0.58 MPa) elevated DM in all teparies except one resistant line. Both leaf- and root-derived calli of sensitive teparies exhibited osmotic adjustment (OA) but reduced RGR that remained after rehydration in one line. We concluded that preexisting force of low cellular ψ_s rather than induced OA plays an important role in buffering adverse effects of dehydration and conditioning drought resistance of tepary beans. This information may aid Phaseolus breeders in screening for drought resistance among large number of accessions.     

Suppression of leopard moth (Lepidoptera: Cossidae) populations in olive trees in Egypt through mating disruption

The leopard moth, Zeuzera pyrina (L.) (Lepidoptera: Cossidae), is a damaging pest for many fruit trees (e.g., apple [Malus spp.], pear [Pyrus spp.] peach [Prunus spp.], and olive [Olea]). Recently, it caused serious yield losses in newly established olive orchards in Egypt, including the death of young trees. Chemical and biological control have shown limited efficiency against this pest. Field tests were conducted in 2005 and 2006 to evaluate mating disruption (MD) for the control of the leopard moth, on heavily infested, densely planted olive plots (336 trees per ha). The binary blend of the pheromone components (E,Z)-2,13-octadecenyl acetate and (E,Z)-3,13-octadecenyl acetate (95:5) was dispensed from polyethylene vials. Efficacy was measured considering reduction of catches in pheromone traps, reduction of active galleries of leopard moth per tree and fruit yield in the pheromone-treated plots (MD) compared with control plots (CO). Male captures in MD plots were reduced by 89.3% in 2005 and 82.9% in 2006, during a trapping period of 14 and 13 wk, respectively. Application of MD over two consecutive years progressively reduced the number of active galleries per tree in the third year where no sex pheromone was applied. In all years, larval galleries outnumbered moth captures. Fruit yield from trees where sex pheromone had been applied in 2005 and 2006 increased significantly in 2006 (98.8 +/- 2.9 kg per tree) and 2007 (23 +/- 1.3 kg per tree) compared with control ones (61.0 +/- 3.9 and 10.0 +/- 0.6 kg per tree, respectively). Mating disruption shows promising for suppressing leopard moth infestation in olives.     

Evaluation of a new method for antifungal susceptibility testing for azoles

The interpretation of the end points in azole antifungal drug susceptibility testing is challenging, in part due to incomplete growth inhibition of Candida species. Since the reference Clinical and Laboratory Standards Institute (CLSI) broth microdilution method have limitation with azoles, a new modification of the CLSI microdilution protocol was evaluated. We measure the decrease in growth rate (μ) of exponentially growing cultures in accordance to different azole concentrations at time intervals up to 10 h. Using 15 different Candida strains, an overall agreement within ± 2 dilutions by the CLSI method at 24 h in RPMI and the μ-dependent method for three antifungal agents (fluconazole- itraconazole and voriconazole) was achieved. MIC measurement by the new method was less sensitive to the medium used or the inoculum size applied. The presented data suggested that, measuring the in vitro inhibition kinetics at the logarithmic phase could have advantages for addressing susceptibility testing toward azoles.     

Inflammatory markers in end-stage renal disease patients on haemodialysis

BackgroundCXC chemokine ligand 16 (CXCL16) is an inflammatory chemokine that mediates renal infiltration of macrophages and activated T cells. Aim: To investigate serum levels of CXCL16 in patients undergoing hemodialysis and their correlation with other inflammatory markers such as C-reactive protein (CRP) and intact parathyroid hormone (iPTH).MethodsThe study included 40 hemodialysis patients (22 males) and 40 age and gender-matched controls (24 males). Fasting blood sugar (FBS), urea, creatinine, calcium and inorganic phosphorous were assayed in participants using routine methods, glycosylated hemoglobin (HbA1c) by quantitative chromatographic spectrophotometry, iPTH by chemiluminescent microparticle immunoassay, CRP by nephelometry and CXCL16 by ELISA technique.ResultsSerum CXCL16, CRP, PTH, FBS, HbA1c, phosphorus, urea, and creatinine levels were significantly higher in hemodialysis patients compared to controls (p<0.00001). No statistically significant differences were observed between patients and controls for calcium. Serum CXCL16 levels correlated positively with CRP (r=0.956, p<0.00001) and iPTH (r=-0.403, p<0.001). Hemodialysis patients (diabetics or hypertensives) had significantly higher CXCL16 levels compared to non-diabetics or non-hypertensives.ConclusionsHigh levels of serum CXCL16, CRP and iPTH reflect the inflammatory status of hemodialysis patients and help avoid complications. Serum CXCL16 could be used as a biomarker together with CRP in these patients.