Genome-Wide Screens Reveal that Resveratrol Induces Replicative Stress in Human Cells.

Resveratrol is a natural product associated with wide-ranging effects in animal and cellular models, including lifespan extension. To identify the genetic target of resveratrol in human cells, we conducted genome-wide CRISPR-Cas9 screens to pinpoint genes that confer sensitivity or resistance to resveratrol. An extensive network of DNA damage response and replicative stress genes exhibited genetic interactions with resveratrol and its analog pterostilbene. These genetic profiles showed similarity to the response to hydroxyurea, an inhibitor of ribonucleotide reductase that causes replicative stress. Resveratrol, pterostilbene, and hydroxyurea caused similar depletion of nucleotide pools, inhibition of replication fork progression, and induction of replicative stress. The ability of resveratrol to inhibit cell proliferation and S phase transit was independent of the histone deacetylase sirtuin 1, which has been implicated in lifespan extension by resveratrol. These results establish that a primary impact of resveratrol on human cell proliferation is the induction of low-level replicative stress.

Microbes vary strategically in their metalation of mononuclear enzymes.

Studies have determined that nonredox enzymes that are cofactored with Fe(II) are the most oxidant-sensitive targets inside Escherichia coli. These enzymes use Fe(II) cofactors to bind and activate substrates. Because of their solvent exposure, the metal can be accessed and oxidized by reactive oxygen species, thereby inactivating the enzyme. Because these enzymes participate in key physiological processes, the consequences of stress can be severe. Accordingly, when E. coli senses elevated levels of H2O2, it induces both a miniferritin and a manganese importer, enabling the replacement of the iron atom in these enzymes with manganese. Manganese does not react with H2O2 and thereby preserves enzyme activity. In this study, we examined several diverse microbes to identify the metal that they customarily integrate into ribulose-5-phosphate 3-epimerase, a representative of this enzyme family. The anaerobe Bacteroides thetaiotaomicron, like E. coli, uses iron. In contrast, Bacillus subtilis and Lactococcus lactis use manganese, and Saccharomyces cerevisiae uses zinc. The latter organisms are therefore well suited to the oxidizing environments in which they dwell. Similar results were obtained with peptide deformylase, another essential enzyme of the mononuclear class. Strikingly, heterologous expression experiments show that it is the metal pool within the organism, rather than features of the protein itself, that determine which metal is incorporated. Further, regardless of the source organism, each enzyme exhibits highest turnover with iron and lowest turnover with zinc. We infer that the intrinsic catalytic properties of the metal cannot easily be retuned by evolution of the polypeptide.

Reconciling carbon quality with availability predicts temperature sensitivity of global soil carbon mineralization.

Soil organic carbon (SOC) mineralization is a key component of the global carbon cycle. Its temperature sensitivity Q10 (which is defined as the factor of change in mineralization with a 10 °C temperature increase) is crucial for understanding the carbon cycle-climate change feedback but remains uncertain. Here, we demonstrate the universal control of carbon quality-availability tradeoffs on Q10. When carbon availability is not limited, Q10 is controlled by carbon quality; otherwise, substrate availability controls Q10. A model driven by such quality-availability tradeoffs explains 97% of the spatiotemporal variability of Q10 in incubations of soils across the globe and predicts a global Q10 of 2.1 ± 0.4 (mean ± one SD) with higher Q10 in northern high-latitude regions. We further reveal that global Q10 is predominantly governed by the mineralization of high-quality carbon. The work provides a foundation for predicting SOC dynamics under climate and land use changes which may alter soil carbon quality and availability.

Checkpoint Kinase Rad53 Couples Leading- and Lagging-Strand DNA Synthesis under Replication Stress.

The checkpoint kinase Rad53 is activated during replication stress to prevent fork collapse, an essential but poorly understood process. Here we show that Rad53 couples leading- and lagging-strand synthesis under replication stress. In rad53-1 cells stressed by dNTP depletion, the replicative DNA helicase, MCM, and the leading-strand DNA polymerase, Pol ε, move beyond the site of DNA synthesis, likely unwinding template DNA. Remarkably, DNA synthesis progresses further along the lagging strand than the leading strand, resulting in the exposure of long stretches of single-stranded leading-strand template. The asymmetric DNA synthesis in rad53-1 cells is suppressed by elevated levels of dNTPs in vivo, and the activity of Pol ε is compromised more than lagging-strand polymerase Pol δ at low dNTP concentrations in vitro. Therefore, we propose that Rad53 prevents the generation of excessive ssDNA under replication stress by coordinating DNA unwinding with synthesis of both strands.

Role of adaptor protein complexes in generating functionally distinct synaptic vesicle pools.

The synaptic vesicle (SV) cycle ensures the release of neurotransmitters and the replenishment of SVs to sustain neuronal activity. Multiple endocytosis and sorting pathways contribute to the recapture of the SV membrane and proteins after fusion. Adaptor protein (AP) complexes are among the critical components of the SV retrieval machinery. The canonical clathrin adaptor AP2 ensures the replenishment of most SVs across many neuronal populations. An alternative AP1/AP3-dependent process mediates the formation of a subset of SVs that differ from AP2 vesicles in molecular composition and respond preferentially during higher frequency firing. Furthermore, recent studies show that vesicular transporters for different neurotransmitters depend to a different extent on the AP3 pathway and this affects the release properties of the respective neurotransmitters. This review focuses on the current understanding of the AP-dependent molecular and functional diversity among SVs. We also discuss the contribution of these pathways to the regulation of neurotransmitter release across neuronal populations.

A complex array of factors regulate the activity of Arabidopsis thaliana δ1 -pyrroline-5-carboxylate synthetase isoenzymes to ensure their specific role in plant cell metabolism.

The first and committed step in proline synthesis from glutamate is catalyzed by δ1 -pyrroline-5-carboxylate synthetase (P5CS). Two P5CS genes have been found in most angiosperms, one constitutively expressed to satisfy proline demand for protein synthesis, the other stress-induced. Despite the number of papers to investigate regulation at the transcriptional level, to date, the properties of the enzymes have been subjected to limited study. The isolation of Arabidopsis thaliana P5CS isoenzymes was achieved through heterologous expression and affinity purification. The two proteins were characterized with respect to kinetic and biochemical properties. AtP5CS2 showed KM values in the micro- to millimolar range, and its activity was inhibited by NADP+ , ADP and proline, and by glutamine and arginine at high levels. Mg2+ ions were required for activity, which was further stimulated by K+ and other cations. AtP5CS1 displayed positive cooperativity with glutamate and was almost insensitive to inhibition by proline. In the presence of physiological, nonsaturating concentrations of glutamate, proline was slightly stimulatory, and glutamine strongly increased the catalytic rate. Data suggest that the activity of AtP5CS isoenzymes is differentially regulated by a complex array of factors including the concentrations of proline, glutamate, glutamine, monovalent cations and pyridine dinucleotides.

Not all soil carbon is created equal: Labile and stable pools under nitrogen input.

Anthropogenic activities have raised nitrogen (N) input worldwide with profound implications for soil carbon (C) cycling in ecosystems. The specific impacts of N input on soil organic matter (SOM) pools differing in microbial availability remain debatable. For the first time, we used a much-improved approach by effectively combining the 13C natural abundance in SOM with 21 years of C3-C4 vegetation conversion and long-term incubation. This allows to distinguish the impact of N input on SOM pools with various turnover times. We found that N input reduced the mineralization of all SOM pools, with labile pools having greater sensitivity to N than stable ones. The suppression in SOM mineralization was notably higher in the very labile pool (18%-52%) than the labile and stable (11%-47%) and the very stable pool (3%-21%) compared to that in the unfertilized control soil. The very labile C pool made a strong contribution (up to 60%) to total CO2 release and also contributed to 74%-96% of suppressed CO2 with N input. This suppression of SOM mineralization by N was initially attributed to the decreased microbial biomass and soil functions. Over the long-term, the shift in bacterial community toward Proteobacteria and reduction in functional genes for labile C degradation were the primary drivers. In conclusion, the higher the availability of the SOM pools, the stronger the suppression of their mineralization by N input. Labile SOM pools are highly sensitive to N availability and may hold a greater potential for C sequestration under N input at global scale.

Global pattern of organic carbon pools in forest soils.

Understanding the mechanisms of soil organic carbon (SOC) sequestration in forests is vital to ecosystem carbon budgeting and helps gain insight in the functioning and sustainable management of world forests. An explicit knowledge of the mechanisms driving global SOC sequestration in forests is still lacking because of the complex interplays between climate, soil, and forest type in influencing SOC pool size and stability. Based on a synthesis of 1179 observations from 292 studies across global forests, we quantified the relative importance of climate, soil property, and forest type on total SOC content and the specific contents of physical (particulate vs. mineral-associated SOC) and chemical (labile vs. recalcitrant SOC) pools in upper 10 cm mineral soils, as well as SOC stock in the O horizons. The variability in the total SOC content of the mineral soils was better explained by climate (47%-60%) and soil factors (26%-50%) than by NPP (10%-20%). The total SOC content and contents of particulate (POC) and recalcitrant SOC (ROC) of the mineral soils all decreased with increasing mean annual temperature because SOC decomposition overrides the C replenishment under warmer climate. The content of mineral-associated organic carbon (MAOC) was influenced by temperature, which directly affected microbial activity. Additionally, the presence of clay and iron oxides physically protected SOC by forming MAOC. The SOC stock in the O horizons was larger in the temperate zone and Mediterranean regions than in the boreal and sub/tropical zones. Mixed forests had 64% larger SOC pools than either broadleaf or coniferous forests, because of (i) higher productivity and (ii) litter input from different tree species resulting in diversification of molecular composition of SOC and microbial community. While climate, soil, and forest type jointly determine the formation and stability of SOC, climate predominantly controls the global patterns of SOC pools in forest ecosystems.

Significant accrual of soil organic carbon through long-term rice cultivation in paddy fields in China.

Paddy fields serve as significant reservoirs of soil organic carbon (SOC) and their potential for terrestrial carbon (C) sequestration is closely associated with changes in SOC pools. However, there has been a dearth of comprehensive studies quantifying changes in SOC pools following extended periods of rice cultivation across a broad geographical scale. Using 104 rice paddy sampling sites that have been in continuous cultivation since the 1980s across China, we studied the changes in topsoil (0-20 cm) labile organic C (LOC I), semi-labile organic C (LOC II), recalcitrant organic C (ROC), and total SOC. We found a substantial increase in both the content (48%) and density (39%) of total SOC within China's paddy fields between the 1980s to the 2010s. Intriguingly, the rate of increase in content and density of ROC exceeded that of LOC (I and II). Using a structural equation model, we revealed that changes in the content and density of total SOC were mainly driven by corresponding shifts in ROC, which are influenced both directly and indirectly by climatic and soil physicochemical factors; in particular temperature, precipitation, phosphorous (P) and clay content. We also showed that the δ13 CLOC were greater than δ13 CROC , independent of the rice cropping region, and that there was a significant positive correlation between δ13 CSOC and δ13 Cstraw . The δ13 CLOC and δ13 CSOC showed significantly negative correlation with soil total Si, suggesting that soil Si plays a part in the allocation of C into different SOC pools, and its turnover or stabilization. Our study underscores that the global C sequestration of the paddy fields mainly stems from the substantial increase in ROC pool.

Peatland pools are tightly coupled to the contemporary carbon cycle.

Peatlands are globally important stores of soil carbon (C) formed over millennial timescales but are at risk of destabilization by human and climate disturbance. Pools are ubiquitous features of many peatlands and can contain very high concentrations of C mobilized in dissolved and particulate organic form and as the greenhouses gases carbon dioxide (CO2 ) and methane (CH4 ). The radiocarbon content (14 C) of these aquatic C forms tells us whether pool C is generated by contemporary primary production or from destabilized C released from deep peat layers where it was previously stored for millennia. We present novel 14 C and stable C (δ13 C) isotope data from 97 aquatic samples across six peatland pool locations in the United Kingdom with a focus on dissolved and particulate organic C and dissolved CO2 . Our observations cover two distinct pool types: natural peatland pools and those formed by ditch blocking efforts to rewet peatlands (restoration pools). The pools were dominated by contemporary C, with the majority of C (~50%-75%) in all forms being younger than 300 years old. Both pool types readily transform and decompose organic C in the water column and emit CO2 to the atmosphere, though mixing with the atmosphere and subsequent CO2 emissions was more evident in natural pools. Our results show little evidence of destabilization of deep, old C in natural or restoration pools, despite the presence of substantial millennial-aged C in the surrounding peat. One possible exception is CH4 ebullition (bubbling), with our observations showing that millennial-aged C can be emitted from peatland pools via this pathway. Our results suggest that restoration pools formed by ditch blocking are effective at preventing the release of deep, old C from rewetted peatlands via aquatic export.

Standardizing cassette-based deep mutagenesis by Golden Gate assembly.

Protocols for the construction of large, deeply mutagenized protein encoding libraries via Golden Gate assembly of synthetic DNA cassettes employ disparate, system-specific methodology. Here we present a standardized Golden Gate method for building user-defined libraries. We demonstrate that a 25 µL reaction, using 40 fmol of input DNA, can generate a library on the order of 1 × 106 members and that reaction volume or input DNA concentration can be scaled up with no losses in transformation efficiency. Such libraries can be constructed from dsDNA cassettes generated either by degenerate oligonucleotides or oligo pools. We demonstrate its real-world effectiveness by building custom, user-defined libraries on the order of 104 -107 unique protein encoding variants for two orthogonal protein engineering systems. We include a detailed protocol and provide several general-use destination vectors.

Fast and robust recombinant protein production utilizing episomal stable pools in WAVE bioreactors.

Protein reagents are essential resources for several stages of drug discovery projects from structural biology and assay development through lead optimization. Depending on the aim of the project different amounts of pure protein are required. Small-scale expressions are initially used to determine the reachable levels of production and quality before scaling up protein reagent supply. Commonly, amounts of several hundreds of milligrams to grams are needed for different experiments, including structural investigations and activity evaluations, which require rather large cultivation volumes. This implies that cultivation of large volumes of either transiently transfected cells or stable pools/stable cell lines is needed. Hence, a production process that is scalable, speeds up the development projects, and increases the robustness of protein reagent quality throughout scales. Here we present a protein production pipeline with high scalability. We show that our protocols for protein production in Chinese hamster ovary cells allow for a seamless and efficient scale-up with robust product quality and high performance. The flexible scale of the production process, as shown here, allows for shorter lead times in drug discovery projects where there is a reagent demand for a specific protein or a set of target proteins.

Impact of Climate on Soil Organic Matter Composition in Soils of Tropical Volcanic Regions Revealed by EGA-MS and Py-GC/MS.

Soil organic matter (SOM) crucially influences the global carbon cycle, yet its molecular composition and determinants are understudied, especially for tropical volcanic regions. We investigated how SOM compounds change in response to climate, vegetation, soil horizon, and soil properties and the relationship between SOM composition and microbial decomposability in Tanzanian and Indonesian volcanic regions. We collected topsoil (0-15 cm) and subsoil (20-40 cm) horizons (n = 22; pH: 4.6-7.6; SOC: 10-152 g kg-1) with undisturbed vegetation and wide mean annual temperature and moisture ranges (14-26 °C; 800-3300 mm) across four elevational transects (340-2210 m asl.). Evolved gas analysis-mass spectrometry (EGA-MS) documented a simultaneous release of SOM compounds and clay mineral dehydroxylation. Subsequently applying double-shot pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) at 200 and 550 °C, we detailed the molecular composition of topsoil and subsoil SOM. A minor portion (2.7 ± 1.9%) of compounds desorbed at 200 °C, limiting its efficacy for investigating overall SOM characteristics. Pyrolyzed SOM closely aligns with the intermediate decomposable SOM pool, with most pyrolysates (550 °C) originating from this pool. Pyrolysates composition suggests tropical SOM is mainly microbial-derived and subsoil contains more degraded compounds. Higher litter inputs and attenuated SOM decomposition due to cooler temperatures and lower soil pH (<5.5) produce less-degraded SOM at higher elevations. Redundancy analyses revealed the crucial role of active Al/Fe (oxalate-extractable Al/Fe), abundant in low-temperature/high-moisture conditions, in stabilizing these less-degraded components. Our findings provide new insights into SOM molecular composition and its determinants, critical for understanding the carbon cycle in tropical ecosystems.

Investigating the interactive effects of habitat type and light intensity on rocky shores.

Light availability and habitat complexity are two key drivers of community assembly. Urbanisation has been shown to affect both, with important consequences to ecological communities. On the intertidal, for instance, studies have shown that light intensity is greater on natural rocky shores than on less complex artificial habitats (seawalls), though different habitats can also experience similar light intensities, for example when shaded by urban structures. Understanding therefore how these factors individually, and combined, affect communities is important to understand the mechanisms driving changes in community structure, and consequently provide solutions to tackle the increasing homogenisation of habitats and lightscapes in urbanised spaces through smart infrastructure designs. Here, we assessed how different light levels affect the recruitment of communities in rock pools and on emergent rock on an intertidal rocky shore. We cleared 30 patches of emergent rock and 30 rock pools and manipulated light using shades with different light transmissions (full light, procedural control, 75%, 35%, and 15% light transmission, full shade) and assessed mobile and sessile communities monthly for 6 months. Effects of reducing light levels were generally stronger on rock than in pools. Fully shaded plots supported double the amount of mobile organisms than plots in full sunlight, in both habitats. Algal cover was higher in pools compared to rock, and at intermediate light levels, but effects varied with site. This study highlights the importance of variable light conditions and different habitats for rocky shore communities, which should be considered in future coastal developments to retain natural biodiversity.

Increased number of cryptosporidiosis cases with travel history to Croatia might be related to swimming pools, Germany, 2023.

In August and September 2023, an unusually high number of cryptosporidiosis cases identified by routine German surveillance had travelled to Croatia (n = 23). Nine cases had stayed in the same camping resort and seven further cases had stayed at other camping sites within 15 km. Based on our standardised questionnaires, the most likely source of infection was swimming pools (93%). Further environmental investigations on site might reveal potential common sources of contamination that could be targeted by control measures.

A Reduction in the Readily Releasable Vesicle Pool Impairs GABAergic Inhibition in the Hippocampus after Blood-Brain Barrier Dysfunction.

Major burdens for patients suffering from stroke are cognitive co-morbidities and epileptogenesis. Neural network disinhibition and deficient inhibitive pulses for fast network activities may result from impaired presynaptic release of the inhibitory neurotransmitter GABA. To test this hypothesis, a cortical photothrombotic stroke was induced in Sprague Dawley rats, and inhibitory currents were recorded seven days later in the peri-infarct blood-brain barrier disrupted (BBBd) hippocampus via patch-clamp electrophysiology in CA1 pyramidal cells (PC). Miniature inhibitory postsynaptic current (mIPSC) frequency was reduced to about half, and mIPSCs decayed faster in the BBBd hippocampus. Furthermore, the paired-pulse ratio of evoked GABA release was increased at 100 Hz, and train stimulations with 100 Hz revealed that the readily releasable pool (RRP), usually assumed to correspond to the number of tightly docked presynaptic vesicles, is reduced by about half in the BBBd hippocampus. These pathophysiologic changes are likely to contribute significantly to disturbed fast oscillatory activity, like cognition-associated gamma oscillations or sharp wave ripples and epileptogenesis in the BBBd hippocampus.

Stabilization of carbon through co-addition of water treatment residuals with anaerobic digested sludge in a coarse textured soil.

Coarse textured soils have low potential to store carbon (C) due to lack of mineral oxides and have low clay content to protect C from biodegradation and leaching. This study evaluated the potential of stabilizing C by adding metal oxyhydroxide-rich water treatment residuals (WTRs) to an aeolian pure sand (<5% clay) topsoil amended with anaerobic digestate (AD) sludge. The AD sludge was applied at 5% (w/w) with aluminum based WTR (Al-WTR) and iron based WTR (Fe-WTR) co-applied at 1:1 and 2:1 WTR:AD (w/w) ratios and incubated at room temperature for 132 days. The cumulative mineralized C was normalized to the total organic C of the treatments. Co-addition with Al-WTR showed to be more effective in stabilizing C through decreased cumulative mineralized C by 48% and 57% in 1Al-WTR:1AD and 2Al-WTR:1AD, respectively, compared to AD sludge sole amendment. Co-application with Al-WTR also decreased permanganate oxidizable C by 37% and dissolved organic C by 51%. Co-application with Fe-WTR did not decrease the concentration of these labile C pools to the same extent, possibly due to the selective use of Fe-WTRs to treat organic-rich raw water. This makes it less effective in stabilizing C in a pure sand relative to Al-WTR due to chemical instability of the Fe-organic complexes. The Al-WTR provides a promising co-amendment to increase C sequestration in pure sands when co-applied with biosolids. The co-amendment approach will not only facilitate C sequestration but also contributes to waste management, aligning to the objectives of a circular economy.

Quantitative macroinvertebrate bioassessment in seasonally astatic aquatic habitats, Part I. Quantitative method.

Seasonally astatic aquatic habitats are important ecologically, municipally, and agriculturally. Regulatory agencies and conservation organizations have developed various plans for protecting or constructing temporary wetlands, resulting in habitat monitoring requirements, particularly as relates to restoration and constructed habitats. Unfortunately, there has been no effort to develop a unified, consistent method for wetland biological monitoring. This is particularly true for habitats important in a regulatory sense. We conducted macroinvertebrate bioassessment in constructed vernal pools in California, USA, to assess habitat functionality. This tool is modified from aquatic bioassessment; a primary tool of regulatory agencies in measuring habitat health and water quality and should be equally applicable to seasonally astatic wetlands globally.

Quantitative macroinvertebrate bioassessment in seasonally astatic aquatic habitats, Part II. Applying the method.

Seasonally astatic aquatic habitats are important ecologically, municipally, and agriculturally. Regulatory agencies and conservation organizations have developed various plans for protecting or constructing temporary wetlands, resulting in habitat monitoring requirements, particularly as relates to restoration and constructed habitats. Unfortunately, there has been no effort to develop a unified, consistent method for wetland biological monitoring. In Part I, we presented a quantifiable, replicable method for assessing seasonally astatic wetlands, which would allow for direct comparison between individual wetlands, wetland sites, and wetland types. Here in Part II, we apply the method and present the results from more than a decade of a data on two disparate sites that support California vernal pool habitats. These habitats include natural, restored, and constructed vernal pools. Our results demonstrate that the method we present yields reliable, statistically useful, and actionable data and provides a better method for assessing astatic wetland ecological health and the persistence of federally listed vernal pool crustaceans than other methods so far employed.

Physicochemical origins of prokaryotic and eukaryotic organisms.

Origins research currently rests on a vitalistic foundation and requires reconceptualization. From a cellular perspective, prokaryotic cells grow and divide in stable, colloidal processes, throughout which the cytoplasm remains crowded (concentrated) with closely interacting proteins and nucleic acids. Their functional stability is ensured by repulsive and attractive non-covalent forces, especially van der Waals forces, screened electrostatic forces, and hydrogen bonding (hydration and the hydrophobic effect). On average, biomacromolecules are crowded at above 15% volume fraction, surrounded by up to 3 nm layer of aqueous electrolyte at ionic strength above 0.01 molar; they are energized by biochemical reactions coupled to nutrient environments. During cellular growth, non-covalent molecular forces and biochemical reactions stabilize the cytoplasm as a two-phase, colloidal system comprising vectorially structured cytogel and dilute cytosol. From a geochemical perspective, Earth's rotation kept prebiotic molecules in continuous cyclic disequilibria in Usiglio-type intertidal pools, rich in potassium and magnesium ions, the last cations to precipitate from evaporatig seawater. These ions impart biochemical functionality to extant proteins and RNAs. The prebiotic molecules were repeatedly purified by phase separation in response to tidal drying and rewetting; they were chemically evolving as briny, carbonaceous inclusions in tidal sediments until the crowding transition allowed chemical evolution to proceeed toward Woesian progenotes, the Last Universal Common Ancestors (LUCAs) and the first prokaryotes. These cellular and geochemical processes are summarized as a jigsaw puzzle of the emerging and evolving prokaryotes. Their unavoidable cyclic fusions and rehydrations along Archaean coastlines initiated the emergence of complex Precambrian eukaryotes.