Alcohol dehydrogenase expression as a biomarker of denitrification activity in activated sludge using methanol and glycerol as electron donors

Carbon sources such as methanol and glycerol are used for enhancing denitrification at wastewater treatment plants, which are required to meet increasingly stringent effluent nitrogen limits. Consequently, dosing strategies for these compounds could benefit from the development and application of molecular activity biomarkers to infer and distinguish between methanol- or glycerol-based denitrification in activated sludge. In this study, the applicability of genes coding for methanol dehydrogenase (mdh2 and mxaF) and glycerol dehydrogenase (dhaD) as potential biomarkers of denitrification activity using these specific substrates was explored and confirmed using a two-pronged approach. First, during short-term spikes of activated sludge biomass with glycerol, the ability of dhaD mRNA concentrations to closely track nitrate depletion profiles was demonstrated. Second, a high-degree of correlation of the mRNA concentrations of mdh2, mxaF and dhaD with methanol- and glycerol-based denitrification kinetics during long-term bioreactor operation using these substrates was also shown. Based on these results, expression of mdh2, mxaF and dhaD genes are promising biomarkers of in situ denitrification activity on methanol and glycerol, respectively, in mixed-culture engineered wastewater treatment processes.     

Mitochondrial dysfunction links ceramide activated HRK expression and cell death

Purpose

Cell death is an essential process in normal development and homeostasis. In eyes, corneal epithelial injury leads to the death of cells in underlying stroma, an event believed to initiate corneal wound healing. The molecular basis of wound induced corneal stromal cell death is not understood in detail. Studies of others have indicated that ceramide may play significant role in stromal cell death following LASIK surgery. We have undertaken the present study to investigate the mechanism of death induced by C6 ceramide in cultures of human corneal stromal (HCSF) fibroblasts.

Methods

Cultures of HCSF were established from freshly excised corneas. Cell death was induced in low passage (p<4) cultures of HCSF by treating the cells with C6 ceramide or C6 dihydroceramide as a control. Cell death was assessed by Live/Dead cell staining with calcein AM and ethidium homodimer-1 as well as Annexin V staining, caspase activation and TUNEL staining Mitochondrial dysfunction was assessed by Mito Sox Red, JC-1 and cytochrome C release Gene expression was examined by qPCR and western blotting.

Results

Our data demonstrate ceramide caused mitochondrial dysfunction as evident from reduced MTT staining, cyto c release from mitochondria, enhanced generation of ROS, and loss in mitochondrial membrane potential (ΔΨ_m). Cell death was evident from Live -Dead Cell staining and the inability to reestablish cultures from detached cells. Ceramide induced the expression of the harikari gene(HRK) and up-regulated JNK phosphorylation. In ceramide treated cells HRK was translocated to mitochondria, where it was found to interact with mitochondrial protein p32. The data also demonstrated HRK, p32 and BAD interaction. Ceramide-induced expression of HRK, mitochondrial dysfunction and cell death were reduced by HRK knockdown with HRK siRNA.

Conclusion

Our data document that ceramide is capable of inducing death of corneal stromal fibroblasts through the induction of HRK mediated mitochondria dysfunction.     

Candidate nsSNPs that can affect the functions and interactions of cell cycle proteins

Nonsynonymous single nucleotide polymorphisms (nsSNPs) alter the encoded amino acid sequence, and are thus likely to affect the function of the proteins, and represent potential disease-modifiers. There is an enormous number of nsSNPs in the human population, and the major challenge lies in distinguishing the functionally significant and potentially disease-related ones from the rest. In this study, we analyzed the genetic variations that can alter the functions and the interactions of a group of cell cycle proteins (n = 60) and the proteins interacting with them (n = 26) using computational tools. As a result, we extracted 249 nsSNPs from 77 cell cycle proteins and their interaction partners from public SNP databases. Only 31 (12.4%) of the nsSNPs were validated. The majority (64.5%) of the validated SNPs were rare (minor allele frequencies < 5%). Evolutionary conservation analysis using the SIFT tool suggested that 16.1% of the validated nsSNPs may disrupt the protein function. In addition, 58% of the validated nsSNPs were located in functional protein domains/motifs, which together with the evolutionary conservation analysis enabled us to infer possible biological consequences of the nsSNPs in our set. Our study strongly suggests the presence of naturally occurring genetic variations in the cell cycle proteins that may affect their interactions and functions with possible roles in complex human diseases, such as cancer.     

Conservation of cysteine residues in fungal histidine acid phytases

Amino acid sequence analysis of fungal histidine acid phosphatases displaying phytase activity has revealed a conserved eight-cysteine motif. These conserved amino acids are not directly associated with catalytic function; rather they appear to be essential in the formation of disulfide bridges. Their role is seen as being similar to another eight-cysteine motif recently reported in the amino acid sequence of nearly 500 plant polypeptides. An additional disulfide bridge formed by two cysteines at the N-terminus of all the filamentous ascomycete phytases was also observed. Disulfide bridges are known to increase both stability and heat tolerance in proteins. It is therefore plausible that this extra disulfide bridge contributes to the higher stability found in phytase from some Aspergillus species. To engineer an enhanced phytase for the feed industry, it is imperative that the role of disulfide bridges be taken into cognizance and possibly be increased in number to further elevate stability in this enzyme.     

Mechanisms of sex steroid effects on bone

Sex steroids play a major role in the regulation of bone turnover. Thus, gonadectomy in either sex is associated with an increase in bone remodeling, increased bone resorption, and a relative deficit in bone formation, resulting in accelerated bone loss. Recent physiological studies have established an important role for estrogen in regulating bone turnover not only in females, but also in males. Studies in mice with knock out of the estrogen receptor, aromatase, or androgen receptor have provided important insights into the in vivo mechanisms of sex steroid action on bone. The cellular and molecular mediators of sex steroid effects on the bone-forming osteoblasts and bone-resorbing osteoclasts are also being increasingly better defined. Estrogen inhibits bone remodeling by concurrently suppressing osteoblastogenesis and osteoclastogenesis from marrow precursors. Both estrogen and androgens inhibit bone resorption via effects on the receptor activator of NF-kappaB ligand (RANKL)/RANK/osteoprotegerin system, as well as by reducing the production of a number of pro-resorptive cytokines, along with direct effects on osteoclast activity and lifespan. Sex steroid effects on bone formation are also likely mediated by multiple mechanisms, including a prolongation of osteoblast lifespan via non-genotropic mechanisms, as well as effects on osteoblast differentiation and function. These pleiotropic actions of sex steroids on virtually all aspects of bone metabolism belie the importance of the skeleton not only in providing structural support for the body and in locomotion, but also as a dynamic tissue responsive, among other things, to the reproductive needs of the organism for calcium.     

MAPK signaling to the early secretory pathway revealed by kinase/phosphatase functional screening

To what extent the secretory pathway is regulated by cellular signaling is unknown. In this study, we used RNA interference to explore the function of human kinases and phosphatases in controlling the organization of and trafficking within the secretory pathway. We identified 122 kinases/phosphatases that affect endoplasmic reticulum (ER) export, ER exit sites (ERESs), and/or the Golgi apparatus. Numerous kinases/phosphatases regulate the number of ERESs and ER to Golgi protein trafficking. Among the pathways identified, the Raf–MEK (MAPK/ERK [extracellular signal-regulated kinase] kinase)–ERK cascade, including its regulatory proteins CNK1 (connector enhancer of the kinase suppressor of Ras-1) and neurofibromin, controls the number of ERESs via ERK2, which targets Sec16, a key regulator of ERESs and COPII (coat protein II) vesicle biogenesis. Our analysis reveals an unanticipated complexity of kinase/phosphatase-mediated regulation of the secretory pathway, uncovering a link between growth factor signaling and ER export.     

Denitrification and nitrous oxide emissions from riparian forests soils exposed to prolonged nitrogen runoff

Compared to upland forests, riparian forest soils have greater potential to remove nitrate (NO₃) from agricultural runoff through denitrification. It is unclear, however, whether prolonged exposure of riparian soils to nitrogen (N) loading will affect the rate of denitrification and its end products. This research assesses the rate of denitrification and nitrous oxide (N₂O) emissions from riparian forest soils exposed to prolonged nutrient runoff from plant nurseries and compares these to similar forest soils not exposed to nutrient runoff. Nursery runoff also contains high levels of phosphate (PO₄). Since there are conflicting reports on the impact of PO₄ on the activity of denitrifying microbes, the impact of PO₄ on such activity was also investigated. Bulk and intact soil cores were collected from N-exposed and non-exposed forests to determine denitrification and N₂O emission rates, whereas denitrification potential was determined using soil slurries. Compared to the non-amended treatment, denitrification rate increased 2.7- and 3.4-fold when soil cores collected from both N-exposed and non-exposed sites were amended with 30 and 60 μg NO₃-N g⁻¹ soil, respectively. Net N₂O emissions were 1.5 and 1.7 times higher from the N-exposed sites compared to the non-exposed sites at 30 and 60 μg NO₃-N g⁻¹ soil amendment rates, respectively. Similarly, denitrification potential increased 17 times in response to addition of 15 μg NO₃-N g⁻¹ in soil slurries. The addition of PO₄ (5 μg PO₄-P g⁻¹) to soil slurries and intact cores did not affect denitrification rates. These observations suggest that prolonged N loading did not affect the denitrification potential of the riparian forest soils; however, it did result in higher N₂O emissions compared to emission rates from non-exposed forest soils.     

Temperature-dependent development and reproductive traits of <Emphasis Type="Italic">Tetranychus macfarlanei</Emphasis> (Acari: Tetranychidae)

Development and reproductive traits of Tetranychus macfarlanei Baker & Pritchard (Acari: Tetranychidae) were investigated on kidney bean, Phaseolus vulgaris L., at eleven constant temperatures. Tetranychus macfarlanei was able to develop and complete its life cycle at temperatures ranging from 17.5 to 37.5°C. At 15 and 40°C, a few eggs (2–4%) hatched but further development was arrested. Development from egg to adult was slowest at 17.5°C and fastest at 35°C for both females and males. Using Ikemoto and Takai’s linear model, the estimated lower developmental thresholds for egg-to-female adult, egg-to-male adult and egg-to-egg development were 12.9–13.0°C. The thermal constants for the respective stages were 110.85, 115.99 and 125.32 degree-days (DD). The intrinsic optimum temperatures (T _Φ) calculated by non-linear SSI model were determined as 24.4, 24.4 and 24.2°C for egg-to-female adult, egg-to-male adult and egg-to-egg development, respectively. The net reproductive rate (R ₀) was highest at 25°C (167.4 females per female) and lowest at 17.5°C (42.6 females per female). The intrinsic rate of natural increase, r _m, increased linearly with the rising of temperature from 0.102 at 17.5°C to 0.441 day⁻¹ at 35°C. These values suggested that T. macfarlanei could be growing quickly in response to increasing temperatures from 17.5 to 35°C and provide a basis for predicting its potential geographical range.     

Age-dependent renal cortical microvascular loss in female mice

Renal function and blood flow decline during aging in association with a decrease in the number of intrarenal vessels, but if loss of estrogen contributes to this microvascular, rarefaction remains unclear. We tested the hypothesis that the decreased renal microvascular density with age is aggravated by loss of estrogen. Six-month-old female C57/BL6 mice underwent ovariectomy (Ovx) or sham operation and then were allowed to age to 18-22 mo. Another comparable group was replenished with estrogen after Ovx (Ovx+E), while a 6-mo-old group served as young controls. Kidneys were then dissected for evaluation of microvascular density (by micro-computed tomography) and angiogenic and fibrogenic factors. Cortical density of small microvessels (20-200 μm) was decreased in all aged groups compared with young controls (30.3 ± 5.8 vessels/mm2, P < 0.05), but tended to be lower in sham compared with Ovx and Ovx+E (9.9 ± 1.7 vs. 17.2 ± 4.2 and 18 ± 3.0 vessels/mm2, P = 0.08 and P = 0.02, respectively). Cortical density of larger microvessels (200-500 μm) decreased only in aged sham (P = 0.04 vs. young control), and proangiogenic signaling was attenuated. On the other hand, renal fibrogenic mechanisms were aggravated in aged Ovx compared with aged sham, but blunted in Ovx+E, in association with downregulated transforming growth factor-β signaling and decreased oxidative stress in the kidney. Therefore, aging induced in female mice renal cortical microvascular loss, which was likely not mediated by loss of endogenous estrogen. However, estrogen may play a role in protecting the kidney by decreasing oxidative stress and attenuating mechanisms linked to renal interstitial fibrosis.