However, the fat-reducing activity of GE in a glucose-loaded medium was mediated by a significant upregulation of the daf-16 and daf-2 genes.DAF-2 and DAF-16 are the most critical components involved in the IIS pathway of C. elegans, and constitute one of the major nutrient-sensing pathways that act as regulators of fat metabolism . To validate our results, we determined the effect of GE on lipid accumulation using both skn-1 and daf-16 mutant strains. As can be observed in Figure 4E, treatment with our GE from L1 to L4 with the skn-1 mutant induced a significant reduction in the lipid content in comparison with the untreated mutant, demonstrating that this gene would not be crucial for the antiobesity activity of our extract, which would support our previous findings. However, treatment with the GE extract did not have the effect of lipid accumulation on the daf-16 mutant when grown on NGM plates,nor in glucose-loaded plates , suggesting that the activity of this transcription factor is crucial for the lipid-reducing activity of GE. Thus, our findings demonstrate that the fat-reducing activity of our GE extract is dependent on the daf-16/FOXO mediator of the IIS signalling pathway.

An imbalance in the lipid metabolism can lead to an increase in inflammation, which in turn promotes the generation of intracellular reactive oxygen species , causing oxidative stress. Excessive ROS accumulation can alter proteins and other molecules, such as lipids and DNA, contributing to cellular damage and, in turn, the development of aging-related diseases, including diabetes . Previous studies have suggested the in vivo antioxidant activity of G. frondosa, attributed to its content in polysaccharides . Thus, Kou and colleagues suggested that the hypoglycaemic activity of G. frondosa polysaccharides seen in diet–streptozotocin-induced diabetic rats were mediated by a reduction in oxidative stress through the NF-kB signalling pathway . Furthermore, other mushrooms have been demonstrated to exert antioxidant activities, some of them evaluated using the C. elegans model . As we have previously described, our GE exhibited high in vitro antioxidant activity, probably due to its high content in polysaccharides, but also to the presence of phenolic compounds, PUFAs, and MUFAs with antioxidant activities . Moreover, as demonstrated before, treatment with GE induced a significant overexpression of skn-1/Nrf, which was not related to the reduced fat content of the worm. This transcription factor plays a critical role in the regulation of the C. elegans response to oxidative stress , together with additional functions including proteostasis and aging .

The overexpression of daf-16 induced by GE could also result in increased resistance to oxidative stress, in addition to a prolonged lifespan.For this reason, we also evaluated the in vivo antioxidant activity of GE in C. elegans when treated from L1 to L4.Thus, treatment with GE induced a significant reduction in ROS production , quantified by DHE , suggesting GE’s potential role in reducing oxidative stress in vivo. One of the methods to determine the oxidative status of the nematodes is to evaluate their tolerance to heat stress. No differences were observed in the percentage of survival between GE-treated and untreated worms exposed to a temperature of 35 °C , suggesting that the antioxidant activity of GE is independent to any effect on heat stress resistance. However, SKN-1 activity has recently been demonstrated to be suppressed upon heat stress . For this reason, we aimed to further investigate if the ROS-reducing activity of our GE is dependent on skn-1 overexpression. As can be observed, both skn-1 and daf-16 mutants lacked ROS-reducing activity, as no differences were observed between GE-treated and untreated control worms . Our results demonstrate that GE treatment induces a significant reduction in ROS in C. elegans grown under normal conditions, and that this activity is mediated by the skn-1/Nrf-2 and daf-16/FOXO transcription factors.Watershed degradation in the developing countries due to anthropogenic activities is increasingly becoming a threat to the natural water resources . As a result, most rural areas are undergoing rapid and far reaching land use changes.

Many researchers including Olson and Matina concur that most of the changes are mainly associated with intensification of agriculture and expansion of mixed-crop livestock systems into former grazing land and other natural areas. In addition there is increased deforestation and encroachment of the forests hence reduced forest cover.This growth rate translates to an increased pressure and demand for land resources especially in the watershed regions such as Mau Forest Complex. As a result, there is continuing watershed degradation reflected through diminishing forest cover, reduced water quality, soil productivity, loss of riparian vegetation and wetland areas leading to a decline in the ecological stability of these systems . SWMFC is considered the most important of the five main watershed areas in Kenya because of its immense economic, social and environmental contributions to the country . It is the main source of Chemosit and Kipsonoi rivers among other major rivers that drain into the Lake Victoria Basin. Despite the critical role in supporting environmental, socio-economic and biological processes, South West Mau Forest Complex has been greatly deforested through excision and encroachment for settlement and farmland . This high rate of vegetation loss has led to decline in the ecological and hydrological changes that may threaten the sustainable future of areas downstream, biodiversity conservation and livelihood support systems.Riparian areas are essential for diminishing negative impacts of land use activities on rivers.However, the riparian areas in SWMFC are experiencing diverse development initiatives likely to have also considerably reduced the biodiversity and increased threats to these river systems.

This study set out to determine how land use activities have affected riparian structure, water and soil qualities along Chemosit and Kipsonoi rivers in South West Mau Forest Complex.This study was carried out along Chemosit and Kipsonoi rivers in South West Mau Forest Complex.It lies at 0.14˚ – 0.78˚ South and 35.28˚ – 35.70˚ West at an altitude ranging from 1600 m – 3000 m above sea level.Kipsonoi river extends from the South Western Mau main escarpment, flows through Sotik and finally drains to Sondu river.On the other hand,Chemosit river, flows through Kimulot, Itare, and Nyakach before entering Lake Victoria .The area receives conventional type of rainfall generally exhibiting bimodal pattern which is well distributed throughout the year with an average rainfall of 1000 mm to 2000 mm∙yr−1 with the highland areas receiving cumulative annual rainfall of approximately 1835 mm which decreases to about 1500 mm∙yr−1 towards the lowlands. There is a short dry spell in January and February while April and May are the wettest months and short rains in November and December. The temperatures in the area range from 18˚C to 22˚C with the lowland having an annual temperature of 26˚C. July is the coldest month with an average of 16˚C while February is hottest with average temperatures of 20˚C .Sampling sites were restricted to a 200 m long upstream section of the river on either side of the bank and near bridges to enhance accessibility. In addition, sampling was started 30 m away from the bridge and forest margins to avoid edge effects. In each of the sampling site, 3 water sampling points were identified at an interval of 100m. For soil sampling 3 belt transects were laid systematically on both sides of the river bank at an interval of 70 m apart. The transects were laid perpendicular to the river flow to encompass the most heterogeneity . In each transect, plots were created according to a modified procedure developed by Hitimana et al. . The belts were subdivided into 5 contiguously units of 10 m × 20 m sampling plots. 2 m × 10 m nested sampling units were established within each 0.02 ha plots.

The same sampling design was adopted in all sampling sites along the two rivers.Water sampling was done from the middle and the two edges of the river bank. This was replicated 3 times at every 100 m section of the river. On the other hand, soil samples were collected diagonally in 0.02 ha plots transects at 10 cm depth and at the 1st,3rd and 5th 0.02 ha plots created in each transect from the edge of river.Soil was scooped, packed in well labeled transparent polythene bags. A total of 108 samples were collected for analysis. Adjacent land use activities were obtained through direct observation and photographs. Information on major crops was collected using a structured questionnaire administered to the sampled households living in the immediate vicinity and within 1 km on either side of the river bank. Local land use in every sampling site was recorded. Physical parameters , chemical parameters and heavy metals was done according to the standard procedures as described in APHA.

These are as follows: Total suspended solids were estimated gravimetrically, water pH using a pH meter, Total Nitrogen determined by Kjeldahl digestion. Total phosphorous was determined using the ascorbic acid method while analysis for potassium, copper, cadmium and lead was carried out using Atomic Absorption Spectrophotometer . Before analysis, calibration standards of different concentrations were prepared. In this particular analysis, each replica representing samples from the middle and the two edges were mixed separately to form a composite sample. A total of 18 composite samples were obtained for the entire water samples collected. Soil samples were separately air-dried at room temperature, crushed, homogenized and passed through a 2 mm sieve. Soil organic carbon content was determined by the WalkeyBlack titration method . Total Nitrogen was determined using the Kjeldahl distillation method . Potassium, Sulphur, Phosphorous, Cadmium, Copper, and Lead were analyzed using inductivity couple plasma spectrophotometer .