Circularly permuted monomeric red fluorescent proteins with new termini in the ��-sheet

Circularly permuted fluorescent proteins (FPs) have a growing number of uses in live cell fluorescence biosensing applications. Most notably, they enable the construction of single fluorescent protein‐based biosensors for Ca²⁺ and other analytes of interest. Circularly permuted FPs are also of great utility in the optimization of fluorescence resonance energy transfer (FRET)‐based biosensors by providing a means for varying the critical dipole–dipole orientation. We have previously reported on our efforts to create circularly permuted variants of a monomeric red FP (RFP) known as mCherry. In our previous work, we had identified six distinct locations within mCherry that tolerated the insertion of a short peptide sequence. Creation of circularly permuted variants with new termini at the locations corresponding to the sites of insertion led to the discovery of three permuted variants that retained no more than 18% of the brightness of mCherry. We now report the extensive directed evolution of the variant with new termini at position 193 of the protein sequence for improved fluorescent brightness. The resulting variant, known as cp193g7, has 61% of the intrinsic brightness of mCherry and was found to be highly tolerant of circular permutation at other locations within the sequence. We have exploited this property to engineer an expanded series of circularly permuted variants with new termini located along the length of the 10th β‐strand of mCherry. These new variants may ultimately prove useful for the creation of single FP‐based Ca²⁺ biosensors.     

Identification of the N-linked glycosylation sites of vitamin K-dependent carboxylase and effect of glycosylation on carboxylase function

The vitamin K-dependent carboxylase is an integral membrane protein which is required for the post-translational modification of a variety of vitamin K-dependent proteins. Previous studies have suggested carboxylase is a glycoprotein with N-linked glycosylation sites. In this study, we identify the N-glycosylation sites of carboxylase by mass spectrometric peptide mapping analyses combined with site-directed mutagenesis. Our mass spectrometric results show that the N-linked glycosylation in carboxylase occurs at positions N459, N550, N605, and N627. Eliminating these glycosylation sites by changing asparagine to glutamine caused the mutant carboxylase to migrate faster on SDS-PAGE gels, adding further evidence that these sites are glycosylated. In addition, the mutation studies identified N525, a site that cannot be recovered by mass spectroscopy analysis, as a glycosylation site. Furthermore, the potential glycosylation site at N570 is glycosylated only if all five natural glycosylation sites are simultaneously mutated. Removal of the oligosaccharides by glycosidase from wild-type carboxylase or by elimination of the functional glycosylation sites by site-directed mutagenesis did not affect either the carboxylation or epoxidation activity when the small FLEEL pentapeptide was used as a substrate, suggesting that N-linked glycosylation is not required for the enzymatic function of carboxylase. In contrast, when site N570 and the five natural glycosylation sites were mutated simultaneously, the resulting carboxylase protein was degraded. Our results suggest that N-linked glycosylation is not essential for carboxylase enzymatic activity but is important for protein folding and stability.     

When vegetation change alters ecosystem water availability

The combined effects of vegetation and climate change on biosphere–atmosphere water vapor (H₂O) and carbon dioxide (CO₂) exchanges are expected to vary depending, in part, on how biotic activity is controlled by and alters water availability. This is particularly important when a change in ecosystem composition alters the fractional covers of bare soil, grass, and woody plants so as to influence the accessibility of shallower vs. deeper soil water pools. To study this, we compared 5 years of eddy covariance measurements of H₂O and CO₂ fluxes over a riparian grassland, shrubland, and woodland. In comparison with the surrounding upland region, groundwater access at the riparian sites increased net carbon uptake (NEP) and evapotranspiration (ET), which were sustained over more of the year. Among the sites, the grassland used less of the stable groundwater resource, and increasing woody plant density decoupled NEP and ET from incident precipitation (P), resulting in greater exchange rates that were less variable year to year. Despite similar gross patterns, how groundwater accessibility affected NEP was more complex than ET. The grassland had higher respiration (R_eco) costs. Thus, while it had similar ET and gross carbon uptake (GEP) to the shrubland, grassland NEP was substantially less. Also, grassland carbon fluxes were more variable due to occasional flooding at the site, which both stimulated and inhibited NEP depending upon phenology. Woodland NEP was large, but surprisingly similar to the less mature, sparse shrubland, even while having much greater GEP. Woodland R_eco was greater than the shrubland and responded strongly and positively to P, which resulted in a surprising negative NEP response to P. This is likely due to the large accumulation of carbon aboveground and in the surface soil. These long‐term observations support the strong role that water accessibility can play when determining the consequences of ecosystem vegetation change.     

Moving Towards a New Urban Systems Science

Ecosystems - Research on urban ecosystems rapidly expanded in the 1990s and is now a central topic in ecosystem science. In this paper, we argue that there are two critical challenges for ecosystem...     

An Estimate of the Average Number of Recessive Lethal Mutations Carried by Humans

The effects of inbreeding on human health depend critically on the number and severity of recessive, deleterious mutations carried by individuals. In humans, existing estimates of these quantities are based on comparisons between consanguineous and nonconsanguineous couples, an approach that confounds socioeconomic and genetic effects of inbreeding. To overcome this limitation, we focused on a founder population that practices a communal lifestyle, for which there is almost complete Mendelian disease ascertainment and a known pedigree. Focusing on recessive lethal diseases and simulating allele transmissions, we estimated that each haploid set of human autosomes carries on average 0.29 (95% credible interval [0.10, 0.84]) recessive alleles that lead to complete sterility or death by reproductive age when homozygous. Comparison to existing estimates in humans suggests that a substantial fraction of the total burden imposed by recessive deleterious variants is due to single mutations that lead to sterility or death between birth and reproductive age. In turn, comparison to estimates from other eukaryotes points to a surprising constancy of the average number of recessive lethal mutations across organisms with markedly different genome sizes.     

FEBio finite element models of the human lumbar spine

Finite element analysis has proven to be a viable method for assessing many structure-function relationships in the human lumbar spine. Several validated models of the spine have been published, but they typically rely on commercial packages and are difficult to share between labs. The goal of this study is to present the development of the first open-access models of the human lumbar spine in FEBio. This modeling framework currently targets three deficient areas in the field of lumbar spine modeling: 1) open-access models, 2) accessibility for multiple meshing schemes, and 3) options to include advanced hyperelastic and biphasic constitutive models.     

Wetting-induced soil CO2 emission pulses are driven by interactions among soil temperature,carbon, and nitrogen limitation in the Colorado Desert

Warming-induced changes in precipitation regimes, coupled with anthropogenically enhanced nitrogen (N) deposition, are likely to increase the prevalence, duration, and magnitude of soil respiration pulses following wetting via interactions among temperature and carbon (C) and N availability. Quantifying the importance of these interactive controls on soil respiration is a key challenge as pulses can be large terrestrial sources of atmospheric carbon dioxide (CO₂) over comparatively short timescales. Using an automated sensor system, we measured soil CO₂ flux dynamics in the Colorado Desert—a system characterized by pronounced transitions from dry-to-wet soil conditions—through a multi-year series of experimental wetting campaigns. Experimental manipulations included combinations of C and N additions across a range of ambient temperatures and across five sites varying in atmospheric N deposition. We found soil CO₂ pulses following wetting were highly predictable from peak instantaneous CO₂ flux measurements. CO₂ pulses consistently increased with temperature, and temperature at time of wetting positively correlated to CO₂ pulse magnitude. Experimentally adding N along the N deposition gradient generated contrasting pulse responses: adding N increased CO₂ pulses in low N deposition sites, whereas adding N decreased CO₂ pulses in high N deposition sites. At a low N deposition site, simultaneous additions of C and N during wetting led to the highest observed soil CO₂ fluxes reported globally at 299.5 μmol CO₂ m⁻² s⁻¹. Our results suggest that soils have the capacity to emit high amounts of CO₂ within small timeframes following infrequent wetting, and pulse sizes reflect a non-linear combination of soil resource and temperature interactions. Importantly, the largest soil CO₂ emissions occurred when multiple resources were amended simultaneously in historically resource-limited desert soils, pointing to regions experiencing simultaneous effects of desertification and urbanization as key locations in future global C balance.