Vacuolar Na+/H+ antiporter cation selectivity is regulated by calmodulin from within the vacuole in a Ca2+- and pH-dependent manner

The selective movement of ions between intracellular compartments is fundamental for eukaryotes. Arabidopsis thaliana Na(+)/H(+) exchanger 1 (AtNHX1), the most abundant vacuolar Na(+)/H(+) antiporter in A. thaliana, has important roles affecting the maintenance of cellular pH, ion homeostasis, and the regulation of protein trafficking. Previously, we have shown that the AtNHX1 C-terminal hydrophilic region localized in the vacuolar lumen plays an important role in regulating the antiporter's activity. Here, we have identified A. thaliana calmodulin-like protein 15 (AtCaM15), which interacts with the AtNHX1 C terminus. When expressed in yeast, AtCaM15 is localized in the vacuolar lumen. The transient expression of AtCaM15 in Arabidopsis leaf protoplasts showed that AtCaM15 is present in the central vacuole. The binding of AtCaM15 to AtNHX1 was Ca(2+)- and pH-dependent and decreased with increasing pH values. Our results also show that the binding of AtCaM15 to AtNHX1 modified the Na(+)/K(+) selectivity of the antiporter, decreasing its Na(+)/H(+) exchange activity. Taken together, the presence of a vacuolar calmodulin-like protein acting on the vacuolar-localized AtNHX1 C terminus in a Ca(2+)- pH-dependent manner suggests the presence of signaling entities acting within the vacuole.     

Iron oxides stimulate sulfate-driven anaerobic methane oxidation in seeps

Seep sediments are dominated by intensive microbial sulfate reduction coupled to the anaerobic oxidation of methane (AOM). Through geochemical measurements of incubation experiments with methane seep sediments collected from Hydrate Ridge, we provide insight into the role of iron oxides in sulfate-driven AOM. Seep sediments incubated with ¹³C-labeled methane showed co-occurring sulfate reduction, AOM, and methanogenesis. The isotope fractionation factors for sulfur and oxygen isotopes in sulfate were about 40‰ and 22‰, respectively, reinforcing the difference between microbial sulfate reduction in methane seeps versus other sedimentary environments (for example, sulfur isotope fractionation above 60‰ in sulfate reduction coupled to organic carbon oxidation or in diffusive sedimentary sulfate–methane transition zone). The addition of hematite to these microcosm experiments resulted in significant microbial iron reduction as well as enhancing sulfate-driven AOM. The magnitude of the isotope fractionation of sulfur and oxygen isotopes in sulfate from these incubations was lowered by about 50%, indicating the involvement of iron oxides during sulfate reduction in methane seeps. The similar relative change between the oxygen versus sulfur isotopes of sulfate in all experiments (with and without hematite addition) suggests that oxidized forms of iron, naturally present in the sediment incubations, were involved in sulfate reduction, with hematite addition increasing the sulfate recycling or the activity of sulfur-cycling microorganisms by about 40%. These results highlight a role for natural iron oxides during bacterial sulfate reduction in methane seeps not only as nutrient but also as stimulator of sulfur recycling.Microbial dissimilatory processes generate energy through the decomposition of substrates, whereas assimilatory processes use substrates for intracellular biosynthesis of macromolecules. The most known and energetically favorable dissimilatory process is the oxidation of organic carbon coupled to oxygen as terminal electron acceptor (Eq. 1). In sediments with a high supply of organic carbon, oxygen can be depleted within the upper few millimeters, leading to anoxic conditions deeper in the sediment column. Under these conditions, microbial dissimilatory processes are coupled to the reduction of a series of other terminal electron acceptors besides oxygen (1). The largest free-energy yields are associated with nitrate reduction (denitrification), followed by manganese and iron oxide reduction, and then sulfate reduction. Due to the high concentration of sulfate in the ocean, dissimilatory bacterial sulfate reduction (Eq. 2) is responsible for the majority of organic matter oxidation in marine sediments (2). Below the depth of sulfate depletion, traditionally the only presumed process is methanogenesis (methane production), where its main pathways are fermentation of organic matter, mainly acetate (Eq. 3), or the reduction of carbon dioxide with hydrogen as substrate (Eq. 4) (3):O₂ + CH₂O → H₂O + CO₂[1]SO42−+2CH2O→H2S+2HCO3−[2]CH₃COOH→CH₄ + CO₂[3]CO₂ + 4H₂→CH₄ + 2H₂O[4]When methane that has been produced deep in sediments diffuses into contact with an available electron acceptor, it can be oxidized (methanotrophy). Methanotrophy is the main process that prevents the escape of methane produced within marine and fresh water sediments into the atmosphere. In fresh water systems, methanotrophic bacteria are responsible for oxidizing methane to dissolved inorganic carbon (DIC) typically using oxygen as an electron acceptor (4, 5). In marine sediments, however, where oxygen diffusion is limited, anaerobic oxidation of methane (AOM) coupled to sulfate reduction [e.g., refs. 6 and 7 (Eq. 5)] has been shown to consume up to 90% of the methane produced within the subseafloor environment (8). Often, when methane is present, the majority of sulfate available in marine pore fluids is reduced through sulfate-driven AOM (9–13):CH4+SO42−→HS−+HCO3−+H2O.[5]Other electron acceptors such as nitrate and oxides of iron and manganese, could also oxidize methane anaerobically and provide a greater free-energy yield than sulfate-coupled methane oxidation (14). Indeed, Beal et al. (15) showed the potential for iron- and manganese-driven AOM in microcosm experiments with methane seep sediments from Eel River Basin and Hydrate Ridge, and iron-driven AOM has been interpreted from modeling geochemical profiles in deep-sea sediments (13, 16). AOM has been shown to occur in nonmarine sediments via denitrification (17–21) and iron reduction (22, 23). However, all geochemical and microbiological studies point to sulfate-driven AOM as the dominant sink for methane in marine sediments.Sulfate-driven AOM is understood to involve microbial consortia of archaea and bacteria affiliated with archaeal methanotrophs (“methane oxidizers”) and sulfate-reducing bacteria (11, 24). A common view is that anaerobic methanotrophic archaea (ANME) oxidize methane, while the sulfate-reducing syntrophic partner scavenges the resulting reducing equivalents to reduce sulfate to sulfide (7, 25, 26). Recently, however, cultured AOM enrichments from seeps were reported to be capable of direct coupling of methane oxidation and sulfate reduction by the ANME-2 archaea, with the passage of zero valent sulfur to a disproportionating bacterial partner, capable of simultaneously oxidizing and reducing this substrate to sulfate and sulfide in a ratio of 1:7, respectively (27). Whether this “single organism mechanism” for sulfate-driven AOM is widespread in the natural environment, or whether there is a diversity of mechanisms for sulfate-driven AOM, remains enigmatic.Carbon isotopes provide a good constraint on the depth distribution and location of methanogenesis and methanotrophy because of the carbon isotope fractionation associated with these processes (e.g., refs. 28 and 29). During methanogenesis, ¹²C is strongly partitioned into methane; the δ¹³C of the methane produced can be between −50‰ to −110‰. In parallel, the residual DIC pool in methanogenic zones becomes highly enriched in ¹³C, occasionally by as much as 50‰ to 70‰ (e.g., ref. 28). Oxidizing this methane on the other hand, results in ¹³C-depleted DIC and slightly heavier δ¹³C values of the residual methane, caused by a fractionation of 0‰ to 10‰ during methane oxidation and the initial negative δ¹³C value of the methane itself (30, 31).The sulfur and oxygen isotopes in dissolved sulfate (δ³⁴S_SO4 and δ¹⁸O_SO4) may also be a diagnostic tool for tracking the pathways of sulfate reduction by methane or other organic compounds. Sulfur isotope fractionation during dissimilatory bacterial sulfate reduction, which partitions ³²S into the sulfide, leaving ³⁴S behind in the residual sulfate, can be as high as 72‰ (32–35). As sulfate is reduced to sulfide via intracellular intermediates (34, 36–40), the magnitude of this sulfur isotope fractionation depends upon the isotope partitioning at each of the intercellular steps and on the ratio between the backward and forward sulfur fluxes within the bacterial cells (34, 36).Oxygen isotopes in sulfate, however, have been shown to be strongly influenced by the oxygen isotope composition of water in which the bacteria are grown (41–45). The consensus is that, within the cell, sulfur compounds, such as sulfite, and water exchange oxygen atoms; some of these isotopically equilibrated molecules return to the extracellular sulfate pool. As all of the intercellular steps are considered to be reversible (e.g., refs. 34, 36, 46, and 47), water–oxygen is also incorporated during the oxidation of these sulfur intermediates back to sulfate (41–43, 48–51).Therefore, both oxygen and sulfur isotopes in the residual sulfate during dissimilatory sulfate reduction are affected by the changes in the intracellular fluxes of sulfur species. However, these isotopes in the residual sulfate are affected in different ways, and thus the change of one isotope vs. the other helps uniquely solve for the relative change in the flux of each intracellular step as sulfate is being reduced (42, 43, 50). The sulfur and oxygen isotope composition of residual sulfate has been used to explore the mechanism of traditional (organoclastic) sulfate reduction both in pure culture (e.g., refs. 44, 45, and 52) and in the natural environment (e.g., refs. 12, 49, 50, and 53–55). The coupled isotope approach has been used specifically to study sulfate-driven AOM recently in estuaries (56). In the work of Antler et al. (56), it was shown that the oxygen and sulfur isotopes in the residual sulfate in the pore fluids are linearly correlated during sulfate-driven AOM, whereas during organoclastic bacterial sulfate reduction, the isotopes exhibit a concaved curve relationship.Although iron and manganese oxides should be reduced before the onset of dissimilatory bacterial sulfate reduction in the natural environment from thermodynamic considerations, due to their low solubility, they may not be completely reduced through dissimilatory respiration when sulfate reduction starts (e.g., ref. 22). These lower reactivity manganese and iron oxides therefore may still be present during the lower-energy yielding anaerobic processes such as sulfate reduction, methanotrophy, and methanogenesis. Indeed, iron oxides have been shown to serve as electron acceptors for methane oxidation even in the sulfate “zone” (15, 16, 22, 57), although the mechanism of this coupling remains enigmatic. In the context of deep-sea methane seep ecosystems, earlier work by Beal et al. (15) demonstrated stimulation of AOM by the addition of iron and manganese oxides in sediment incubation experiments. In that work, however, the nature of the coupling between methane oxidation and metal oxides was not ascertained, and the multiple links between the sediment sulfur, iron, and methane cycles are equivocal.Here, we conducted microcosm experiments with sediments collected from Hydrate Ridge South (Fig. S1) and used synergistic combinations of isotope analyses (δ³⁴S_SO4, δ¹⁸O_SO4, and δ¹³C_DIC) to aid in assessing whether methane oxidation is directly coupled to the respiration of iron oxides or whether stimulation in methanotrophy is a result of the coupling between iron and sulfate. We provide compelling evidence for the stimulation of AOM in seep sediments through the coupling between iron and sulfate, and propose a mechanism for iron involvement in sulfate-driven AOM. Using microcosm experiments with seep sediments dominated by sulfate-driven AOM and amended with hematite and ¹³C-labeled methane and glucose, we are able to demonstrate the role of iron in sulfate-driven AOM. Hematite is a less reactive form of iron oxide than, for example, amorphous iron (58), and it was used to prevent the microbial populations from “switching” completely to the more energetically favorable process of iron reduction.     

Regulatory element copy number differences shape primate expression profiles

Gene expression differences are shaped by selective pressures and contribute to phenotypic differences between species. We identified 964 copy number differences (CNDs) of conserved sequences across three primate species and examined their potential effects on gene expression profiles. Samples with copy number different genes had significantly different expression than samples with neutral copy number. Genes encoding regulatory molecules differed in copy number and were associated with significant expression differences. Additionally, we identified 127 CNDs that were processed pseudogenes and some of which were expressed. Furthermore, there were copy number-different regulatory regions such as ultraconserved elements and long intergenic noncoding RNAs with the potential to affect expression. We postulate that CNDs of these conserved sequences fine-tune developmental pathways by altering the levels of RNA.     

Enhanced cellulose degradation by targeted integration of a cohesin-fused β-glucosidase into the Clostridium thermocellum cellulosome

The conversion of recalcitrant plant-derived cellulosic biomass into biofuels is dependent on highly efficient cellulase systems that produce near-quantitative levels of soluble saccharides. Similar to other fungal and bacterial cellulase systems, the multienzyme cellulosome system of the anaerobic, cellulolytic bacterium Clostridium thermocellum is strongly inhibited by the major end product cellobiose. Cellobiose-induced inhibition can be relieved via its cleavage to noninhibitory glucose by the addition of exogenous noncellulosomal enzyme β-glucosidase; however, because the cellulosome is adsorbed to the insoluble substrate only a fraction of β-glucosidase would be available to the cellulosome. Towards this end, we designed a chimeric cohesin-fused β-glucosidase (BglA-CohII) that binds directly to the cellulosome through an unoccupied dockerin module of its major scaffoldin subunit. The β-glucosidase activity is thus focused at the immediate site of cellobiose production by the cellulosomal enzymes. BglA-CohII was shown to retain cellobiase activity and was readily incorporated into the native cellulosome complex. Surprisingly, it was found that the native C. thermocellum cellulosome exists as a homooligomer and the high-affinity interaction of BglA-CohII with the scaffoldin moiety appears to dissociate the oligomeric state of the cellulosome. Complexation of the cellulosome and BglA-CohII resulted in higher overall degradation of microcrystalline cellulose and pretreated switchgrass compared to the native cellulosome alone or in combination with wild-type BglA in solution. These results demonstrate the effect of enzyme targeting and its potential for enhanced degradation of cellulosic biomass.     

LASIK versus PRK for high astigmatism

Purpose

To compare the refractive outcomes of laser in situ keratomileusis (LASIK) and photorefractive keratectomy (PRK) for myopic astigmatism of 3 diopters (D) or more.

Study design

Retrospective matched comparative study.

Methods

This study include consecutive myopic patients (SE 0 to –10 D) undergoing LASIK or PRK between 2007 and 2016 with astigmatism of 3 to 6 D, and postoperative follow-up of at least 30 days for LASIK and 60 days for PRK, compared outcomes of LASIK and PRK eyes.

Results

The LASIK and PRK groups comprised 175 eyes of 175 patients each, with median follow-up of 39 and 139 days, respectively (P?<?0.001). Mean preoperative manifest astigmatism was –3.35?±?0.46 and –3.42?±?0.51 D (P?=?0.92), postoperative SE was –0.43?±?0.55 and –0.16?±?0.64 D (P?<?0.001), and arithmetic astigmatism was –0.59?±?0.46 and –0.88?±?0.60 D (P?<?0.001), for the LASIK and PRK groups, respectively. Fifty-seven and 64.0% eyes had postoperative SE within?±?0.5 D of emmetropia (P?=?0.19), and 57.7 and 38.8% eyes were within 0.5 D of attempted astigmatic correction (P?<?0.001) for the LASIK and PRK groups, respectively. More PRK eyes were overtreated regarding both SE and astigmatism than LASIK eyes (P?<?0.001). The efficacy and safety indices were close to 1.0 in both groups. The surgically induced astigmatism, magnitude of error, index of success, correction index and flattening index were all better in the LASIK group.

Conclusion

Both LASIK and PRK achieve good outcomes in high astigmatism. LASIK achieved mild superiority over PRK.

     

Evaluation of hip flexion strength following lateral lumbar interbody fusion

Background context

Lateral interbody fusion (LIF) is a minimally invasive procedure that is designed to achieve a solid interbody fusion while minimizing the damage to the surrounding soft tissue. Although short-term results have been promising, few data have been published to date regarding its risks and complication rate.

Purpose

The aim was to evaluate the extent of injury to the psoas muscle after the LIF procedure by measuring hip flexion strength.

Study design

A prospective case series was used in the study.

Method

Hip flexion strength was measured using a handheld digital dynamometer while the patient was seated on a chair; the examiner held the device against the patient’s attempt to flex the hip. Both sides were measured to compare the operated and nonoperated psoas muscles. Each side was measured three times and the average amount (in pounds) was recorded. Measurements were done before and after surgery on Day 2-3, at 2 weeks, 6 weeks, and at 3 and 6 months.

Results

Thirty-three patients were recruited for this study. Mean preoperative hip flexion strength values were 20.7±3.47 lb and 21.3±4.31 lb for operated and nonoperated legs, respectively, with no significant difference (p=.85). With a mean of 11.2±2.24 lb postoperative measurements on Day 2, the operated side showed statistically significant reduction of strength (p=.0001). The nonoperated side was also weaker postoperatively, but not significantly (mean=19.12±1.74 lb; p=.097). From the first follow-up visit at 2 weeks, the values on the operated leg had returned to baseline values (20.6, p=.97) and were not significantly different from preoperative values on either side.

Discussion

Hip flexion was weakened immediately after the LIF procedure, which may be attributed to psoas muscle injury during the procedure. However, this damage was temporary, with almost complete return to baseline values by 2 weeks.     

Frequency of hand movements as a possible diagnostic tool for parkinsonian bradykinesia. Proposal of a simple bedside test

Neurological Sciences - Bradykinesia, dysrhythmia, and decrement in hand movements (HM) are core symptoms of Parkinson’s disease (PD). The maximal rate of repetitive rhythm-preserving HM can...