The ecological integrity of the lower Olifants River,Limpopo province,South Africa: 2009–2015 – Part B: Tributaries of the Olifants River

Monitoring on the Lowveld reaches of the Olifants River, Limpopo River System, and its Steelpoort, Blyde, Klaserie and Selati tributaries was initiated in 2009. Analysis of the 2009–2015 data from four Olifants River sites showed deterioration in the river’s ecological condition between where it enters the Lowveld and where it enters the Kruger National Park, with a slight recovery within the Kruger National Park. Physico-chemical, aquatic macroinvertebrate and fish data collected in 2009–2015 at six sites on the Steelpoort, Blyde, Klaserie and Selati tributaries of the Olifants River corroborated the ecological condition of these tributaries. The Selati was the most polluted and was in a critically modified condition, whereas the Klaserie and Steelpoort were in fair condition and the Blyde was in good condition. The Selati appeared to have a significant negative impact on the water quality, macroinvertebrates and fish of the Olifants River within the Kruger National Park.     

Human health risks of metals and metalloids in muscle tissue of Synodontis zambezensis Peters, 1852 from Flag Boshielo Dam,South Africa

Muscle tissue from 63 Synodontis zambezensis collected bimonthly in 2013 at Flag Boshielo Dam were analysed for metals and metalloids in a desktop human health risk assessment. The Hazard Quotient, based on a weekly meal of 67 g of fish muscle, exceeded the maximum acceptable level of one for lead, cobalt, cadmium, mercury, arsenic and selenium. The concentrations of these elements were higher in 2013 than those recorded in 2009 and 2012 in other fish species from Flag Boshielo Dam and these may pose a long-term health risk if consumed regularly by impoverished rural communities reliant on fish as a source of protein.     

Do roe deer react to wildlife warning reflectors? A test combining a controlled experiment with field observations

Millions of animals are killed by vehicle collisions each year. As mitigation measures, wildlife warning reflectors have become increasingly popular, although clear evidence for their effectiveness is lacking. A reason for inconclusive results in the literature may be that most previous studies on the effectiveness of wildlife warning reflectors compare animal-vehicle collision rates with and without reflectors, a setting characterised by low event rates and weak experimental control. Animal behaviour can be expected to provide a more direct evidence for a possible effect of reflectors. In this study, we analyse roe deer behaviour in the presence of a blue semicircle reflector, one of the most frequently applied wildlife warning reflectors in Germany and other parts of Europe. Behavioural response to these reflectors (classified as no reaction, vigilance, short-distance flight and long-distance flight) was recorded both under controlled experimental conditions with captive roe deer and for free-ranging roe deer at road sections with traffic occurrence. We used generalised linear mixed models (GLMMs) to test if reflector presence induced threat-related behaviour (vigilance, flight) and movement away from the reflectors. We found no significant evidence that the light stimulus emitted by reflectors was perceived as a threat or induced evasive movement. We conclude that our study provides no evidence that blue semicircle reflectors induce behaviour in roe deer that seems suitable to reduce roe deer-vehicle collisions.     

Chitosan-based hybrid immobilization in bienzymatic reactions and its application to the production of laminaribiose

A hybrid-immobilization method was developed to improve the long-term stability of laminaribiose phosphorylase immobilized on epoxy supports Sepabeads EC-EP/S. Entrapment in chitosan retained all of the enzyme activity depending on the amount of entrapped solid materials and increased half-life by a factor of 10–94.4 h. No enzyme activity loss was determined during 12 times reuse. The immobilization method is also applicable to sucrose phosphorylase immobilized on Sepabeads EC-EP/S. Up to 31.9 g/L laminaribiose were produced in bienzymatic batch experiments with reaction-integrated product separation by adsorption on zeolites.

     

The interaction of beta-amyloid protein with cellular membranes stimulates its own production

Gradual changes in steady-state levels of beta amyloid peptides (Aβ) in brain are considered an initial step in the amyloid cascade hypothesis of Alzheimer's disease. Aβ is a product of the secretase cleavage of amyloid precursor protein (APP). There is evidence that the membrane lipid environment may modulate secretase activity and alters its function. Cleavage of APP strongly depends on membrane properties. Since Aβ perturbs cell membrane fluidity, the cell membrane may be the location where the neurotoxic cascade of Aβ is initiated. Therefore, we tested effects of oligomeric Aβ on membrane fluidity of whole living cells, the impact of exogenous and cellular Aβ on the processing of APP and the role of GM-1 ganglioside. We present evidence that oligoAβ_(1-40) stimulates the amyloidogenic processing of APP by reducing membrane fluidity and complexing with GM-1 ganglioside. This dynamic action of Aβ may start a vicious circle, where endogenous Aβ stimulates its own production. Based on our novel findings, we propose that oligoAβ_(1-40) accelerates the proteolytic cleavage of APP by decreasing membrane fluidity.     

Molecular mechanisms that funnel RNA precursors into endogenous small-interfering RNA and microRNA biogenesis pathways in Drosophila

In Drosophila, three types of endogenous small RNAs—microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs), and endogenous small-interfering RNAs (endo-siRNAs or esiRNAs)—function as triggers in RNA silencing. Although piRNAs are produced independently of Dicer, miRNA and esiRNA biogenesis pathways require Dicer1 and Dicer2, respectively. Recent studies have shown that among the four isoforms of Loquacious (Loqs), Loqs-PB and Loqs-PD are involved in miRNA and esiRNA processing pathways, respectively. However, how these Loqs isoforms function in their respective small RNA biogenesis pathways remains elusive. Here, we show that Loqs-PD associates specifically with Dicer2 through its C-terminal domain. The Dicer2–Loqs-PD complex contains R2D2, another known Dicer2 partner, and excises both exogenous siRNAs and esiRNAs from their corresponding precursors in vitro. However, Loqs-PD, but not R2D2, enhanced Dicer2 activity. The Dicer2–Loqs-PD complex processes esiRNA precursor hairpins with long stems, which results in the production of AGO2-associated small RNAs. Interestingly, however, small RNAs derived from terminal hairpins of esiRNA precursors are loaded onto AGO1; thus, they are classified as a new subset of miRNAs. These results suggest that the precursor RNA structure determines the biogenesis mechanism of esiRNAs and miRNAs, thereby implicating hairpin structures with long stems as intermediates in the evolution of Drosophila miRNA.     

Imidazo[4,5-c]pyridines as corticotropin releasing factor receptor ligands

A series of high affinity CRF receptor ligands with an imidazo[4,5-c]pyridine core is described. Individual analogues were synthesized and tested in vitro in rat brain receptors to determine binding affinity. The best compound was further tested in the dog N-in-1 pharmacokinetic model to assess oral bioavailability at 1 mg/kg po.     

Assessment of pharmacological activities of two medicinal plant of Bangladesh: Launaea sarmentosa and Aegialitis rotundifolia roxb in the management of pain,pyrexia and inflammation

Background

The current study aims at evaluating the analgesic, anti-pyretic and anti-inflammatory properties of methanolic extract of the stem, bark and leaves of Launaea sarmentosa and Aegialitis rotundifolia roxb.

Results

The AELS and AEAR extract presented a significant (***p < 0.001) dose dependent increase in reaction time in writhing method and showed inhibition of 63.1% and 57.1% respectively at the doses of 400 mg/kg body weight while standard drug showed (P < 0.001) inhibition of 69.23%. In tail immersion method, AELS and AEAR showed maximum time of tail retention at 30 min in hot water i.e. 6.93 sec and 6.54 sec respectively at highest doses of 400 mg/kg body weight than lower dose while standard pentazocine showed reaction time of 7.62 sec. The AELS and AEAR extract also exhibited promising anti-inflammatory effect as demonstrated by statistically significant inhibition of paw volume by 32.48% and 26.75% respectively at the dose of 400 mg/kg body weight while the value at the dose of 200 mg/kg body weight were linear to higher dose at the 3^rd hour of study. On the other hand, Standard indomethacin inhibited 40.13% of inflammation (***P < 0.001). In Cotton-pellet granuloma method, AELS and AEAR extract at the dose of 400 mg/kg body weight exhibited inhibition of inflammation of 34.7% and 29.1% respectively while standard drug showed (P < 0.001) inhibition of 63.22%. Intraperitoneal administration of AELS and AEAR showed dose dependent decrease in body temperature in brewer’s yeast induced hyperthermia in rats at both doses. However, AELS significantly decreased body temperature (***p < 0.001) at 400 mg/kg compared to control.

Conclusions

Present work propose that the methanolic extract of Launaea sarmentosa and Aegialitis rotundifolia roxb possesses dose dependent pharmacological action which supports its therapeutic use in folk medicine possibly mediated through the inhibition or blocking of release of prostaglandin and/or actions of vasoactive substances such as histamine, serotonin and kinins.     

Effects of double ligation of Stensen's duct on the rabbit parotid gland

Salivary gland duct ligation is an alternative to gland excision for treating sialorrhea or reducing salivary gland size prior to tumor excision. Duct ligation also is used as an approach to study salivary gland aging, regeneration, radiotherapy, sialolithiasis and sialadenitis. Reports conflict about the contribution of each salivary cell population to gland size reduction after ductal ligation. Certain cell populations, especially acini, reportedly undergo atrophy, apoptosis and proliferation during reduction of gland size. Acini also have been reported to de-differentiate into ducts. These contradictory results have been attributed to different animal or salivary gland models, or to methods of ligation. We report here a bilateral double ligature technique for rabbit parotid glands with histologic observations at 1, 7, 14, 30, 60 days after ligation. A large battery of special stains and immunohistochemical procedures was employed to define the cell populations. Four stages with overlapping features were observed that led to progressive shutdown of gland activities: 1) marked atrophy of the acinar cells occurred by 14 days, 2) response to and removal of the secretory material trapped in the acinar and ductal lumens mainly between 30 and 60 days, 3) reduction in the number of parenchymal (mostly acinar) cells by apoptosis that occurred mainly between 14–30 days, and 4) maintenance of steady-state at 60 days with a low rate of fluid, protein, and glycoprotein secretion, which greatly decreased the number of leukocytes engaged in the removal of the luminal contents. The main post- ligation characteristics were dilation of ductal and acinar lumens, massive transient infiltration of mostly heterophils (rabbit polymorphonuclear leukocytes), acinar atrophy, and apoptosis of both acinar and ductal cells. Proliferation was uncommon except in the larger ducts. By 30 days, the distribution of myoepithelial cells had spread from exclusively investing the intercalated ducts pre-ligation to surrounding a majority of the residual duct-like structures, many of which clearly were atrophic acini. Thus, both atrophy and apoptosis made major contributions to the post-ligation reduction in gland size. Structures also occurred with both ductal and acinar markers that suggested acini differentiating into ducts. Overall, the reaction to duct ligation proceeded at a considerably slower pace in the rabbit parotid glands than has been reported for the salivary glands of the rat.     

Nitric oxide is a positive regulator of the Warburg effect in ovarian cancer cells

Ovarian cancer (OVCA) is among the most lethal gynecological cancers leading to high mortality rates among women. Increasing evidence indicate that cancer cells undergo metabolic transformation during tumorigenesis and growth through nutrients and growth factors available in tumor microenvironment. This altered metabolic rewiring further enhances tumor progression. Recent studies have begun to unravel the role of amino acids in the tumor microenvironment on the proliferation of cancer cells. One critically important, yet often overlooked, component to tumor growth is the metabolic reprogramming of nitric oxide (NO) pathways in cancer cells. Multiple lines of evidence support the link between NO and tumor growth in some cancers, including pancreas, breast and ovarian. However, the multifaceted role of NO in the metabolism of OVCA is unclear and direct demonstration of NO''s role in modulating OVCA cells'' metabolism is lacking. This study aims at indentifying the mechanistic links between NO and OVCA metabolism. We uncover a role of NO in modulating OVCA metabolism: NO positively regulates the Warburg effect, which postulates increased glycolysis along with reduced mitochondrial activity under aerobic conditions in cancer cells. Through both NO synthesis inhibition (using L-arginine deprivation, arginine is a substrate for NO synthase (NOS), which catalyzes NO synthesis; using L-Name, a NOS inhibitor) and NO donor (using DETA-NONOate) analysis, we show that NO not only positively regulates tumor growth but also inhibits mitochondrial respiration in OVCA cells, shifting these cells towards glycolysis to maintain their ATP production. Additionally, NO led to an increase in TCA cycle flux and glutaminolysis, suggesting that NO decreases ROS levels by increasing NADPH and glutathione levels. Our results place NO as a central player in the metabolism of OVCA cells. Understanding the effects of NO on cancer cell metabolism can lead to the development of NO targeting drugs for OVCAs.Despite recent medical and pharmaceutical advances in cancer research, ovarian cancer (OVCA) remains one of the most deadly gynecological malignancies, with most of the cancer first detected in late stages when metastasis has already occurred.¹ Only 20% of OVCA patients are diagnosed when cancer has not spread past the ovaries; in the other 80% of cases, the cancer has metastasized, most frequently to the peritoneum.² Platinum-based preoperative chemotherapy is the standard of care of early stage disease, and surgical resection along with platinum-based postoperative chemotherapy is the standard of care for late stage disease.¹ However, many platinum-based chemotherapy drugs come with unwanted side effects. Therefore, an alternative therapy for OVCA is needed.Nitric oxide (NO) shows promise either as a cancer therapeutic agent by itself or as a target of cancer therapies.³ This may be because NO can act as a signaling molecule or as a source of oxidative and nitrosative stress.⁴ NO can stimulate mitochondrial biogenesis through PGC-1-related coactivator⁵ and increase mitochondrial function.^{6, 7} In follicular thyroid carcinoma cells, S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a NO donor, was shown to increase the expression of genes involved in mitochondrial biogenesis.^{8, 9} A 14-day treatment of lung carcinoma cells with dipropylenetriamine NONOate (DETA-NONOate), another NO donor, increased cell migration compared with the absence of treatment.¹⁰ In breast cancer cells, exogenous NO increased cell proliferation, as well as cyclin-D1 and ornithine decarboxylase expression.¹¹ In prostate cancer cells, NO was shown to inhibit androgen receptor-dependent promoter activity and proliferation of androgen-dependent cells, indicating that NO would select for the development of prostate cancer cells that are androgen-independent.¹² NO has even been shown to inhibit mitochondrial ATP production, and therefore inhibit apoptosis, as ATP is necessary for the apoptotic process.¹³ Moreover, inducible nitric oxide synthase (iNOS) knockout mice had less tumor formation than wild-type mice, indicating that NO promotes lung tumorigenesis.¹⁴ On the other hand, NO production, as induced by proinflammatory cytokines, induced apoptosis in OVCA cells.³ NOS overexpression by transfection of a plasmid containing NOS-3 DNA resulted in increased cell death in HepG2 cells.¹⁵ In another study, NO was implicated in N-(4-hydroxyphenyl) retinamide-mediated apoptosis.¹⁶ Finally, iNOS expression in p53-depleted mice increased apoptosis of lymphoma cells compared with p53-deficient mice without iNOS expression.¹⁷ Therefore, NO has been seen to have both an anti-tumorigenic as well as a pro-tumorigenic effect.Arginine, a conditionally essential amino acid used to produce NO, is also a potential target for cancer therapy. L-arginine is normally produced by the body; however, in some diseased states, more arginine than what the body normally produces is required.¹⁸ Arginine sources include protein breakdown or directly from the diet, in addition to de novo synthesis.¹⁹ In the de novo production of L-arginine, citrulline and aspartate are first converted to argininosuccinate by arginase, which is then split into arginine and fumarate by argininosuccinate lyase.²⁰ L-arginine can also be converted to citrulline and NO through NO synthase (NOS).¹⁹ Some cancer cells, including melanoma and hepatocellular carcinoma, do not express argininosuccinate synthase (ASS), an enzyme involved in arginine production and thus rely on exogenous arginine.¹⁹ For these cancers, arginine-deprivation therapy is being heavily explored as a treatment.^{21, 22} OVCA cells have been shown to express ASS.²³ In fact, OVCA cells were shown to have increased expression of ASS compared with normal ovarian surface epithelium.²⁴ As OVCA can synthesize arginine de novo, strategies which target arginine''s conversion into citrulline are needed for regulating OVCA tumor growth.Recent studies suggest that cancer cells undergo metabolic reprogramming, which drives cancer cells'' growth and progression.^{25, 26, 27, 28, 29, 30, 31, 32, 33} One critically important, yet often overlooked, component to tumor growth is the metabolic rewiring of NO pathways in OVCA cells. Despite considerable investigation on NO''s regulation of cancer cell proliferation and growth, mechanistic details regarding the effect of NO on cancer cell metabolism is still lacking: specifically, how NO affects glycolysis, TCA cycle flux, and ROS production. Studies on the effects of NO on cancer cell metabolism have mainly focused on the effect of NO on mitochondrial respiration.^{34, 35, 36, 37} NO has been shown to inhibit cytochrome c oxidase (COX) in the mitochondria of breast cancer cells, as well as decrease oxygen consumption rate.^{37, 38, 39} Moncada and colleagues studied the effect of NO on the metabolism of rat cortical astrocytes and neurons, two cells with different glycolytic capacities. They showed that NO decreased ATP concentration, which led to an increase in glycolysis in astrocytes, but not in neurons, indicating that glycolytic capacity affects the metabolic response of these cells to NO.⁴⁰ NO was shown to reduce ATP production via OXPHOS in rat reticulocytes, cells that produce 90% of their ATP from OXPHOS.⁴¹ Endothelial NOS (eNOS) was shown to have a role in the upregulation of GLUT4 transporters by AMPK and AICAR in the heart muscle.⁴² Additionally, NO can serve to stabilize HIF-1α in hypoxic conditions through S-nitrosylation of PHD2,⁴ and as HIF-1α upregulates GLUT transporters and glycolysis,⁴³ NO may affect the metabolism of cancer cells.Although NO is found to affect glycolysis of normal cells, how NO modulates glycolysis of OVCA cells is less understood. The multifaceted role of NO in the metabolism of OVCA is unclear, and direct demonstration of NO''s role in modulating the metabolism of OVCA cells is lacking. This study aims at understanding the mechanistic links between NO and the overall cancer metabolism – specifically, its effects on glycolysis, TCA cycle, OXPHOS, and ROS production – of OVCA cells. Our results show that NO decreases mitochondrial respiration, forcing OVCA cells to undergo higher glycolytic rates to maintain ATP production levels. Our work is the first to illustrate the central role of NO on OVCA metabolism – specifically, showing how NO (i) positively regulates the Warburg effect in OVCA cell, (ii) maintains low ROS levels by upregulating NADPH generation, and (ii) negatively alters mitochondrial respiration, thus promoting cancer growth and proliferation. Our work is also unique in that it is the first to explore the effects of NO on TCA cycle flux and glutaminolysis, potentially also affecting ROS levels by affecting antioxidant levels. In conclusion, by elucidating the effects of NO on cancer metabolism and ROS levels, we have a better understanding of the different mechanisms by which NO affects cancer cell growth. This understanding may lead to potentially useful therapies to halt cancer progression.