Role of KLHL3 and dietary K⁺ in regulating KS-WNK1 expression

This is the author accepted manuscript. The final version is available from the American Physiological Society via the DOI in this recordThe physiological role of the shorter isoform of WNK1 that is exclusively expressed in the kidney (KS-WNK1), with particular abundance in the distal convoluted tubule, remains elusive. KS-WNK1 despite lacking the kinase domain, is nevertheless capable of stimulating the NaCl cotransporter (NCC), apparently through activation of WNK4. It has recently been shown that a less severe form of the Familial Hyperkalemic Hypertension featuring only hyperkalemia is caused by missense mutations in the WNK1 acidic domain that preferentially affect CUL3-KLHL3 E3-induced degradation of KS-WNK1, rather than that of the full-length WNK1 (L-WNK1). Here we show that L-WNK1 is indeed less impacted by the CUL3-KLHL3 E3 ligase complex compared to KS-WNK1. We demonstrate that the unique 30 amino acid amino N-terminal fragment of KS-WNK1 is essential for its activating effect on NCC and recognition by KLHL3. We identify specific amino acid residues in this region critical for the functional effect of KS-WNK1 and KLHL3 sensitivity. To further explore this, we generated KLHL3-R528H knock-in mice that mimic human mutations causing Familial Hyperkalemic Hypertension. These mice revealed that the KLHL3 mutation specifically increased expression of KS-WNK1 in the kidney. We also observed that in wild type mice, expression of KS-WNK1 is only detectable after exposure to low potassium diet. These findings provide new insights into the regulation and function of KS-WNK1 by the CUL3-KLHL3 complex in DCT and indicate that this pathway is regulated by dietary K+ levels.National Institutes of Health (NIH)Conacyt MexicoPAPIIT UNAML'OréalMedical Research Council (MRC

Differential Response of High-Elevation Planktonic Bacterial Community Structure and Metabolism to Experimental Nutrient Enrichment

Nutrient enrichment of high-elevation freshwater ecosystems by atmospheric deposition is increasing worldwide, and bacteria are a key conduit for the metabolism of organic matter in these oligotrophic environments. We conducted two distinct in situ microcosm experiments in a high-elevation lake (Emerald Lake, Sierra Nevada, California, USA) to evaluate responses in bacterioplankton growth, carbon utilization, and community structure to short-term enrichment by nitrate and phosphate. The first experiment, conducted just following ice-off, employed dark dilution culture to directly assess the impact of nutrients on bacterioplankton growth and consumption of terrigenous dissolved organic matter during snowmelt. The second experiment, conducted in transparent microcosms during autumn overturn, examined how bacterioplankton in unmanipulated microbial communities responded to nutrients concomitant with increasing phytoplankton-derived organic matter. In both experiments, phosphate enrichment (but not nitrate) caused significant increases in bacterioplankton growth, changed particulate organic stoichiometry, and induced shifts in bacterial community composition, including consistent declines in the relative abundance of Actinobacteria. The dark dilution culture showed a significant increase in dissolved organic carbon removal in response to phosphate enrichment. In transparent microcosms nutrient enrichment had no effect on concentrations of chlorophyll, carbon, or the fluorescence characteristics of dissolved organic matter, suggesting that bacterioplankton responses were independent of phytoplankton responses. These results demonstrate that bacterioplankton communities in unproductive high-elevation habitats can rapidly alter their taxonomic composition and metabolism in response to short-term phosphate enrichment. Our results reinforce the key role that phosphorus plays in oligotrophic lake ecosystems, clarify the nature of bacterioplankton nutrient limitation, and emphasize that evaluation of eutrophication in these habitats should incorporate heterotrophic microbial communities and processes

Predominant Non-additive Effects of Multiple Stressors on Autotroph C:N:P Ratios Propagate in Freshwater and Marine Food Webs

A continuing challenge for scientists is to understand how multiple interactive stressor factors affect biological interactions, and subsequently, ecosystems–in ways not easily predicted by single factor studies. In this review, we have compiled and analyzed available research on how multiple stressor pairs composed of temperature (T), light (L), ultraviolet radiation (UVR), nutrients (Nut), carbon dioxide (CO2), dissolved organic carbon (DOC), and salinity (S) impact the stoichiometry of autotrophs which in turn shapes the nature of their ecological interactions within lower trophic levels in streams, lakes and oceans. Our analysis from 66 studies with 320 observations of 11 stressor pairs, demonstrated that non-additive responses predominate across aquatic ecosystems and their net interactive effect depends on the stressor pair at play. Across systems, there was a prevalence of antagonism in freshwater (60–67% vs. 47% in marine systems) compared to marine systems where synergism was more common (49% vs. 33–40% in freshwaters). While the lack of data impeded comparisons among all of the paired stressors, we found pronounced system differences for the L × Nut interactions. For this interaction, our data for C:P and N:P is consistent with the initial hypothesis that the interaction was primarily synergistic in the oceans, but not for C:N. Our study found a wide range of variability in the net effects of the interactions in freshwater systems, with some observations supporting antagonism, and others synergism. Our results suggest that the nature of the stressor pairs interactions on C:N:P ratios regulates the “continuum” commensalistic-competitive-predatory relationship between algae and bacteria and the food chain efficiency at the algae-herbivore interface. Overall, the scarce number of studies with even more fewer replications in each study that are available for freshwater systems have prevented a more detailed, insightful analysis. Our findings highlighting the preponderance of antagonistic and synergistic effects of stressor interactions in aquatic ecosystems—effects that play key roles in the functioning of feedback loops in the biosphere—also stress the need for further studies evaluating the interactive effects of multiple stressors in a rapidly changing world facing a confluence of tipping points

Comparison of viable cell counts and fluorescent in situ hybridization using specific rRNA-based probes for the quantification of human fecal bacteria

Conventional cultivation and fluorescence in situ hybridization (FISH) using 16S rRNA-based probes were compared for the enumeration of human colonic bacteria. Groups of common intestinal anaerobic bacteria were enumerated in slurries prepared from fecal samples of three healthy volunteers. To introduce variation between the samples, they were incubated for 48 h in batch culture (anaerobic) fermenters at 37 degrees C, and pure cultures of Bifidobacterium infantis, Clostridium perfringens, or Lactobacillus acidophilus were added. Samples were taken from the fermenters at different times. Total anaerobes, bifidobacteria, bacteroides, clostridia, and lactobacilli were enumerated by both plating and FISH. The results showed that plate counts of total anaerobes, bifidobacteria, lactobacilli and bacteroides were approximately ten-fold lower than the corresponding FISH counts. Numbers of clostridia were higher using the plating method, probably because the clostridia probe used in FISH analyses was designed to only detect part of the genus Clostridium. The introduced variation in the methods could be detected by both methods and was comparable

Reduced Efficacy of Commercial Acaricides against Populations of Resistant Cattle Tick Rhipicephalus microplus from Two Municipalities of Antioquia, Colombia

Two distant Antioquian cattle farms where systemic and topical acaricides had previously failed to control infestations by Rhipicephalus ( Boophilus) microplus were studied. An initial in vivo study was conducted using single subcutaneous injections with a long-acting formulation of ivermectin (630 μg/kg). Injections were made at 3-month intervals on animals at each farm to evaluate the therapeutic and persistent efficacy of ivermectin against R. microplus. Body tick counts and reproductive parameters of semi- or fully engorged females ≥5 mm) were assessed at 10-day intervals, and since no negative control group could be included, values were compared against those for day 0. Although there was an overall reduction of 50%-75% in tick numbers that persisted for 30-40 days, it was not significantly different at one of the farms and not enough to afford protection from severe infestations. The engorgement weight and egg mass weight of ticks from treated animals were significantly lower throughout the 50-day posttreatment period. Egg hatch was not significantly reduced posttreatment and remained at levels of 80%-90%. A random selection of 9 out of 28 commercial formulations of ivermectin sold in Colombia were analyzed by High Performance Liquid Chromatography (HPLC). All were within the expected labeled concentration (∓15% deviation) of 1% and 3.15% ivermectin except for one. A popular unregistered injectable widely used in both farms and labeled as “natural pyrethrin”, was found to contain 10.5% ivermectin. An adult immersion test was conducted to evaluate the efficacy of topical acaricides to recommended concentrations of five commercial products and/or their combinations. Efficacy was determined by comparing the reproductive index of each treated group to that of the control group. Cypermethrin (150 ppm) was completely ineffective at both farms. Amitraz (208 ppm) exhibited low and intermediate efficacies of 14% and 56%. The combination of amitraz (100 ppm) and cypermethrin (150 ppm) was less efficacious than the amitraz alone. A generic product based on amitraz + citronella (208 ppm + 10 ppm, respectively) was shown to be less efficacious than the name-brand amitraz product. Products containing the organophosphate chlorpyrifos or trichlorfon exhibited intermediate efficacies of approximately 60% at the Tarso farm. We conclude that at these two locations, there is a high degree of resistance to many of the acaricides available in Colombia and confirm suspicions that ivermectin is no longer able to eliminate tick infestations

Comparison of viable cell counts and fluorescence in situ hybridization using specific rRNA-based probes for the quantification of human fecal bacteria.

Conventional cultivation and fluorescence in situ hybridization (FISH) using 16S rRNA-based probes were compared for the enumeration of human colonic bacteria. Groups of common intestinal anaerobic bacteria were enumerated in slurries prepared from fecal samples of three healthy volunteers. To introduce variation between the samples, they were incubated for 48 h in batch culture (anaerobic) fermenters at 37 degrees C, and pure cultures of Bifidobacterium infantis, Clostridium perfringens, or Lactobacillus acidophilus were added. Samples were taken from the fermenters at different times. Total anaerobes, bifidobacteria, bacteroides, clostridia, and lactobacilli were enumerated by both plating and FISH. The results showed that plate counts of total anaerobes, bifidobacteria, lactobacilli and bacteroides were approximately ten-fold lower than the corresponding FISH counts. Numbers of clostridia were higher using the plating method, probably because the clostridia probe used in FISH analyses was designed to only detect part of the genus Clostridium. The introduced variation in the methods could be detected by both methods and was comparable

The human connection: First evidence of microplastics in remote high mountain lakes of Sierra Nevada, Spain.

Microplastics have become one of the most serious global threats to animal and human health. While their presence has been documented in all Earth water ecosystems, including remote mountain lakes, the observation that the abundance of microplastics is largely different across nearby lakes has rarely been examined. As part of a citizen science initiative, this study analyzed for the first time the abundance of microplastics in the surface of 35 glacial lakes of Sierra Nevada National Park in Southern Spain with the objective of determining the local factors that control their abundance. First, we described the shape, size, color and nature of microplastics. Second, we tested whether the number of microplastics differed between basins and analyzed environmental and morphometrical features of lakes affecting their abundance. We found that microplastics were common in most lakes, with a maximum abundance of 21.3 particles per liter that akin to some of the most microplastic polluted lakes worldwide. Fragments were the predominant shape (59.7%) followed by fibers (38.8%) and very scarce spheres (1.5%). Microplastics were observed for all size-fractions, but the abundance of particle

Does the nutrient stoichiometry of primary producers affect the secondary consumer Pleurobrachia pileus?

We investigated whether phosphorus limitations of primary producers propagate upwards through the food web, not only to the primary consumer level but also onto the secondary consumers' level. A tri-trophic food chain was used to assess the effects of phosphorus-limited phytoplankton (the cryptophyte Rhodomonas salina) on herbivorous zooplankters (the copepod Acartia tonsa) and finally on zooplanktivores (the ctenophore Pleurobrachia pileus). The algae were cultured in phosphorus-replete and phosphorus-limited media before being fed to two groups of copepods. The copepods in turn were fed to the top predator, P. pileus, in a mixture resulting in a phosphorus-gradient, ranging from copepods having received only phosphorus-replete algae to copepods reared solely on phosphorus-limited algae. The C:P ratio of the algae varied significantly between the two treatments, resulting in higher C:P ratios for those copepods feeding on phosphorus-limited algae, albeit with a significance of 0.07. The differences in the feeding environment of the copepods could be followed to Pleurobrachia pileus. Contrary to our expectations, we found that phosphorus-limited copepods represented a higher quality food source for P. pileus, as shown by the better condition (expressed as nucleic acid content) of the ctenophore. This could possibly be explained by the rather high C:P ratios of ctenophores, their resulting low phosphorus demand and relative insensitivity to P deficiency. This might potentially be an additional explanation for the observed increasing abundances of gelatinous zooplankton in our increasingly phosphorus-limited coastal seas