Slow Turnover Research Articles

Long-term biochar and soil organic carbon stability – Evidence from field experiments in Germany

Organic soil amendments (OSA) with long residence times, such as biochar, have a high potential for soil organic carbon (SOC) sequestration. The highly aromatic structure of biochar reduces microbial decomposition and explains the slow turnover of biochar, indicating long persistence in soils and thus potential SOC sequestration. However, there is a lack of data on biochar-induced SOC sequestration in the long-term and under field conditions. We sampled two long-term field experiments in Germany, where biochar was applied 12 and 14 years ago. Both locations differ in soil characteristics and in the types and amounts of biochar and other OSA. Amendments containing compost and 31.5 Mg ha−1 of biochar on a loamy soil led to a SOC stock increase of 38 Mg ha−1 after OSA addition. The additional increase is due to non-biochar co-amendments such as compost or biogas digestate. After eleven years, this SOC stock increase was still stable. High biochar amount additions of 40 Mg ha−1 combined with biogas digestate, compost or synthetic fertilizer on a sandy soil led to an increase of SOC stocks of 61 Mg ha−1; 38 Mg ha−1 dissipated in the following four years most likely due to lacking physical protection of the coarse soil material, and after nine years the biochar-amended soils showed only slightly higher SOC stocks (+7 Mg ha−1) than the control. Black carbon stocks on the same soil increased in the short- and mid-term and decreased almost to the original stock levels after nine years. Our results indicate that in most cases the long-term effect on SOC and black carbon stocks is controlled by biochar quality and amount, while non-biochar co-amendments can be neglected. This study proves that SOC sequestration through the use of biochar is possible, especially in loamy soils, while non-biochar OSA cannot sequester SOC in the long term.

Sequential recruitment of body fluid spaces for increasing volumes of crystalloid fluid.

The interstitial space harbours two fluid compartments linked serially to the plasma. This study explores conditions that lead to fluid accumulation in the most secluded compartment, termed the "third space". Retrospective data was collected from 326 experiments in which intravenous crystalloid fluid was administered to conscious volunteers as well as a small group of anaesthetized patients. The urinary excretion and plasma dilution derived from haemoglobin served as input variables in nine population volume kinetic analyses representing subtly different settings. An infusion of 250-500mL of Ringer's solution expanded only the central fluid space (plasma), whereas the infusion of 500-1,000mL extended into a rapidly exchanging interstitial fluid space. When more than 1L was infused over 30min, it was distributed across plasma and both interstitial fluid compartments. The remote space, characterized by slow turnover, abruptly accommodated fluid upon accumulation of 700-800mL in the rapidly exchanging space, equivalent to an 11%-13% volume increase. However, larger expansion was necessary to trigger this event in a perioperative setting. The plasma half-life of crystalloid fluid was 25 times longer when 2,000-2,700mL expanded all three fluid compartments compared to when only 250-500mL expanded the central space (14h versus 30min). As the volume of crystalloid fluid increases, it apparently occupies a larger proportion of the interstitial space. When more than 1L is administered at a high rate, there is expansion of a remote "third space", which considerably extends the intravascular half-life.

Mannosidase-inhibiting iminosugar production by recombinant Corynebacterium glutamicum harboring the 1-deoxynojirimycin biosynthetic gene cluster

The iminosugar class of carbohydrate-active enzyme inhibitors has therapeutic applications in metabolic syndrome conditions, viral infections and cancer. Compared to chemical synthesis, microbial iminosugar production has benefits of cost, sustainability and optimization. In this study, the 1-deoxynojirimycin (DNJ) biosynthetic gene cluster from Bacillus velezensis MBLB0692, and its individual genes, were cloned into Corynebacterium glutamicum (Cg). Characterizations of the encoded aminotransferase GabT1, phosphatase Yktc1, and dehydrogenase GutB1, were performed with purified enzymes and whole cell biocatalysts bearing individual and clustered (TYB) genes. GabT1 showed a variable pattern in its half-reaction with a slow turnover. GutB1 was an alkaline dehydrogenase with a broad substrate specificity and no divalent ion dependency while the zinc-dependent phosphatase Yktc1 had substrate specificity that was both pH- and ion-dependent. The CgYktc1 and CgGutB1 whole cells were viable biocatalysts with wider ranges of substrates than their enzyme counterparts. The CgTYB cells produced mannosidase-inhibiting iminosugars corresponding to mannojirimycin dehydrate (162 m/z) and deoxymannojirimycin (164 m/z). Mannosidase inhibitors have been found to be effective in treating orphan diseases, cancer and viral infections, and their biosynthesis by recombinant C. glutamicum can be optimized for industrial production and novel drug development.

How Does Escherichia coli Allocate Proteome?

Microorganisms are shown to actively partition their intracellular resources, such as proteins, for growth optimization. Recent experiments have begun to reveal molecular components unpinning the partition; however, quantitatively, it remains unclear how individual parts orchestrate to yield precise resource allocation that is both robust and dynamic. Here, we developed a coarse-grained mathematical framework that centers on guanosine pentaphosphate (ppGpp)-mediated regulation and used it to systematically uncover the design principles of proteome allocation in Escherichia coli. Our results showed that the cellular ability of resource partition lies in an ultrasensitive, negative feedback-controlling topology with the ultrasensitivity arising from zero-order amino acid kinetics and the negative feedback from ppGpp-controlled ribosome synthesis. In addition, together with the time-scale separation between slow ribosome kinetics and fast turnovers of ppGpp and amino acids, the network topology confers the organism an optimization mechanism that mimics sliding mode control, a nonlinear optimization strategy that is widely used in man-made systems. We further showed that such a controlling mechanism is robust against parameter variations and molecular fluctuations and is also efficient for biomass production over time. This work elucidates the fundamental controlling mechanism of E. coli proteome allocation, thereby providing insights into quantitative microbial physiology as well as the design of synthetic gene networks.

A viral effector blocks the turnover of a plant NLR receptor to trigger a robust immune response

Plant intracellular nucleotide-binding and leucine-rich repeat immune receptors (NLRs) play a key role in activating a strong pathogen defense response. Plant NLR proteins are tightly regulated and accumulate at very low levels in the absence of pathogen effectors. However, little is known about how this low level of NLR proteins is able to induce robust immune responses upon recognition of pathogen effectors. Here, we report that, in the absence of effector, the inactive form of the tomato NLR Sw-5b is targeted for ubiquitination by the E3 ligase SBP1. Interaction of SBP1 with Sw-5b via only its N-terminal domain leads to slow turnover. In contrast, in its auto-active state, Sw-5b is rapidly turned over as SBP1 is upregulated and interacts with both its N-terminal and NB-LRR domains. During infection with the tomato spotted wilt virus, the viral effector NSm interacts with Sw-5b and disrupts the interaction of Sw-5b with SBP1, thereby stabilizing the active Sw-5b and allowing it to induce a robust immune response.

Keratin 5 basal cells are temporally regulated developmental and tissue repair progenitors in bladder urothelium.

Urothelium forms a distensible yet impermeable barrier, senses and transduces stimuli, and defends the urinary tract from mechanical, chemical, and bacterial injuries. Biochemical and genetic labeling studies support the existence of one or more progenitor populations with the capacity to rapidly regenerate the urothelium following injury, but slow turnover, a low mitotic index, and inconsistent methodologies obscure progenitor identity. The progenitor properties of basal keratin 5 urothelial cells (K5-UCs) have been previously investigated, but those studies focused on embryonic or adult bladder urothelium. Urothelium undergoes desquamation and apoptosis after birth, which requires postnatal proliferation and restoration. Therefore, we mapped the fate of bladder K5-UCs across postnatal development/maturation and following administration of cyclophosphamide to measure homeostatic and reparative progenitor capacities, respectively. In vivo studies demonstrate that basal K5-UCs are age-restricted progenitors in neonates and juveniles, but not in adult mice. Neonatal K5-UCs retain a superior progenitor capacity in vitro, forming larger and more differentiated urothelial organoids than adult K5-UCs. Accordingly, K5-UC transcriptomes are temporally distinct, with enrichment of transcripts associated with cell proliferation and differentiation in neonates. Induction of urothelial proliferation is sufficient to restore adult K5-UC progenitor capacity. Our findings advance the understanding of urothelial progenitors and support a linear model of urothelial formation and regeneration, which may have significant impact on therapeutic development or tissue engineering strategies.NEW & NOTEWORTHY Fate mapping reveals an important linear relationship, whereby bladder basal urothelial cells give rise to intermediate and superficial cells in an age-restricted manner and contribute to tissue repair. Neonatal basal cells reprise their role as superior progenitors in vitro and display distinct transcriptional signatures, which suggest progenitor function is at least partially cell intrinsic. However, the urothelium progenitor niche cannot be overlooked, since FGF7 rescues adult basal cell progenitor function.

Limitations and consequences of public health models centred on hospitals and lacking connections with territorial and home-based social and health services

BackgroundDelayed discharge from hospital to home or other care institutions is a significant problem and has been investigated in the international scientific literature for many years.Behind this condition is a health care system based on a hospital-centered concept characterized by a lack of territorial health and social welfare services.This phenomenon causes two different problems: an excessive length of hospital stay, resulting in slow turnover of bed utilization; and overcrowding in emergency rooms (ERs).The phenomenon of frequent users assumes particular importance in this context. These patients repeatedly visit the emergency department (ED) in the same year because care needs are not met by primary care services.The authors in this study tried to describe the Frequent users (FUs) population and the variables associated with this condition.Materials and methodsA retrospective "single-arm" descriptive study was conducted by analysing all accesses made to the ED of Policlinico Tor Vergata (PTV) from January 1, 2022, to December 31, 2022.FUs were defined as patients who had 4 or more accesses to PTV ER during the year.ResultsA total of 37,800 accesses occurred during the study period. A total of 31,691 users accessed the PS, with a mean age of 55.8 ± 22.2 years.There were 359 FU patients (approximately 1%) who had a total of 1984 accesses, corresponding to 5.2% of the total accesses.The triage codes for the FU patients were red, 2%; orange, 21%; blue, 45%; green, 26%; white, 5%; and not performed, 1%.Considering the 1984 FU accesses, the most frequently attributed "main problems" in the ED were "other symptoms or disorders" (54%), "psychomotor agitation" (12%), "trauma or burn" (8%), "abdominal pain" (6%), "chest pain" (4%), "dyspnea" (4%) and "urological symptoms or disorders" (4%). Multivariate analysis revealed that the main determinants of FUs were psychomotor agitation (HR = 7,23; CL95%:6,194–8,443), urological disorders (HR = 2,16; CL95%:1,68–2,76) and poor socioeconomic status (HR = 2,40; CL95%:2,213–2,663).ConclusionsThe FUs phenomenon expresses an area of health and social distress where poverty and lack of territorial services oblige people to refer to the ED. Primary care interventions integrated with social support are crucial for managing access to the ED.

Are vehicle lifespan caps an effective and efficient method for reducing US light-duty vehicle fleet GHG emissions?

With light duty vehicles (LDVs) responsible for 17% of annual US greenhouse gas (GHG) emissions, integrating emerging GHG-reducing technologies into the fleet is essential. However, the slow rate of vehicle turnover presents a significant barrier to the market penetration of new technologies, with adoption delayed by the low number of vehicles needing replacement each year. A strategy of accelerated vehicle turnover through a vehicle lifespan cap could potentially mitigate this limit. While older studies reach differing conclusions on their effectiveness, two newer studies that incorporate life cycle assessment find that accelerated turnover strategies can be effective if coupled with high levels of electric vehicle deployment. We seek to determine whether a vehicle lifespan cap strategy can be an effective and efficient (cost-effective) method for reducing US LDV fleet GHG emissions. We augment the capabilities of the Fleet Life Cycle Assessment and Material Flow Estimation (FLAME) fleet life cycle assessment model, integrating vehicle lifespan caps and comprehensive calculations of cost along with sensitivity analysis for electric vehicle survival curves and battery degradation. The augmented FLAME model is used to analyse the impact of vehicle lifespan caps of varying lengths on a suite of scenarios, including a business as usual (BAU) scenario and eight scenarios modelling different technology improvement assumptions. This work confirms that vehicle lifespan caps have limited effectiveness in reducing GHG emissions under a BAU scenario but show potential to meaningfully reduce GHG emissions in a scenario with accelerated deployment of electric vehicles. However, abatement costs are high, exceeding 2020 USD 1000/tCO2eq under baseline assumptions, but falling within the range of current estimates of the social cost of carbon under more optimistic assumptions. Overall, vehicle lifespan caps must be carefully considered as they accelerate both the benefits and costs of new vehicle technologies, and are best positioned as part of a larger integrated strategy for tackling transportation GHG emissions.

Mitochondrial defects in sporadic inclusion body myositis-causes and consequences.

Sporadic inclusion body myositis (sIBM) is a distinct subcategory of Idiopathic Inflammatory Myopathies (IIM), characterized by unique pathological features such as muscle inflammation, rimmed vacuoles, and protein aggregation within the myofibers. Although hyperactivation of the immune system is widely believed as the primary cause of IIM, it is debated whether non-immune tissue dysfunction might contribute to the disease's onset as patients with sIBM are refractory to conventional immunosuppressant treatment. Moreover, the findings that mitochondrial dysfunction can elicit non-apoptotic programmed cell death and the subsequent immune response further support this hypothesis. Notably, abnormal mitochondrial structure and activities are more prominent in the muscle of sIBM than in other types of IIM, suggesting the presence of defective mitochondria might represent an overlooked contributor to the disease onset. The large-scale mitochondrial DNA deletion, aberrant protein aggregation, and slowed organelle turnover have provided mechanistic insights into the genesis of impaired mitochondria in sIBM. This article reviews the disease hallmarks of sIBM, the plausible contributors of mitochondrial damage in the sIBM muscle, and the immunological responses associated with mitochondrial perturbations. Additionally, the potential application of mitochondrial-targeted chemicals as a new treatment strategy to sIBM is explored and discussed.

OPN, BSP, and Bone Quality-Structural, Biochemical, and Biomechanical Assessment in OPN-/-, BSP-/-, and DKO Mice.

Osteopontin (OPN) and Bone Sialoprotein (BSP), abundantly expressed by osteoblasts and osteoclasts, appear to have important, partly overlapping functions in bone. In gene-knockout (KO, -/-) models of either protein and their double (D)KO in the same CD1/129sv genetic background, we analyzed the morphology, matrix characteristics, and biomechanical properties of femur bone in 2 and 4month old, male and female mice. OPN-/- mice display inconsistent, perhaps localized hypermineralization, while the BSP-/- are hypomineralized throughout ages and sexes, and the low mineralization of young DKO mice recovers with age. The higher contribution of primary bone remnants in OPN-/- shafts suggests a slow turnover, while their lower percentage in BSP-/- indicates rapid remodeling, despite FTIR-based evidence in this genotype of a high maturity of the mineralized matrix. In 3-point bending assays, OPN-/- bones consistently display higher Maximal Load, Work to Max. Load and in young mice Ultimate Stress, an intrinsic characteristic of the matrix. Young male and old female BSP-/- also display high Work to Max. Load along with low Ultimate Stress. Principal Component Analysis confirms the major role of morphological traits in mechanical competence, and evidences a grouping of the WT phenotype with the OPN-/- and of BSP-/- with DKO, driven by both structural and matrix parameters, suggesting that the presence or absence of BSP has the most profound effects on skeletal properties. Single or double gene KO of OPN and BSP thus have multiple distinct effects on skeletal phenotypes, confirming their importance in bone biology and their interplay in its regulation.

Tumor Protein D52 Regulates Paligenosis in Exocrine Pancreatic Acinar Cells

Background: Paligenosis is a conserved cellular plasticity program in which fully differentiated cells reenter the cell cycle in order to regenerate lost tissue during injury. This process is utilized by cells that have a slow turnover rate and lack dedicated stem cells, as is the case with exocrine pancreatic acinar cells. Acinar cells, which synthesize and secrete digestive enzymes necessary for macronutrient digestion, undergo paligenosis in response to damage induced by the inflammatory disease pancreatitis. During paligenosis, acinar cells first downscale their secretory apparatus by upregulating autophagy, and then take on a mucinous-ductal-progenitor-like identity in a process called acinar to ductal metaplasia (ADM) prior to proliferation. This phenomenon can be recapitulated in rodent models in vivo utilizing repeated episodes of cerulein-induced pancreatitis or in 18-hour suspension culture during which primary isolated acinar cells dedifferentiate to a progenitor-like state. We have previously reported that markers of the endolysosomal secretory pathway in acinar cells are rapidly lost during models of experimental pancreatitis and suspension culture. Tumor Protein D52, which regulates endolysosomal traffcking and secretion in acinar cells, undergoes lysosomal degradation at the onset of experimental pancreatitis; furthermore, restoring D52 expression by adenovirus in isolated acinar cells maintains secretion during experimental pancreatitis as well as maintains acinar differentiation in suspension culture. Hypothesis: Degradation of D52, and loss of the endolysosomal secretory pathway, are pivotal events during the initial stages of paligenosis. Methods: To study the role of D52 and the regulation of paligenosis, we generated tamoxifen-inducible, acinar specific D52 knockout mice (D52KOac), and markers of paligenosis (ADM, proliferation) were assessed by immunoblot, qPCR, and immunostaining. Mice were given tamoxifen at 2-3 months of age to ensure normal pancreatic development, and tissues were harvested 21 days after tamoxifen. Results: Compared to controls, D52KOac exhibited 2.5-fold upregulation of the ductal markers Sox9 and CK19, 50% reduction of Ptf1α, and a 5-fold increase in β-catenin; these markers signify the presence of ADM and cells with progenitor-like characteristics. Cell cycle regulation and proliferation were also observed in D52KOac by a nearly 3-fold increase in cyclin D and increased Ki67+ nuclei. Conclusions: These data support that loss of D52 in pancreatic acinar cells induces spontaneous paligenosis in the absence of cellular injury and inflammation. These findings point to a previously unrecognized role for D52 in the regulation of acinar cell differentiation and plasticity. Given that paligenosis is a key pathway involved in the development of pancreatic neoplasms and adenocarcinoma, studying D52-regulated paligenosis will reveal additional insights into this critical protective mechanism. American Pancreatic Association Foundation Young Investigator Award. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Ligand Field Sensitive Spin Acceleration in the Iron-Catalyzed [2 + 2] Cycloaddition of Unactivated Alkenes and Dienes.

Redox-active pyridine(diimine) (PDI) iron catalysts promote the reversible [2 + 2] cycloaddition of alkenes and dienes to cyclobutane derivatives that have applications ranging from fuels to chemically recyclable polymers. Metallacycles were identified as key intermediates, and spin crossover from the singlet to the triplet surface was calculated to facilitate the reductive coupling step responsible for the formation of the four-membered ring. In this work, a series of sterically and electronically differentiated PDI ligands was studied for the [2 + 2] cycloaddition of ethylene and butadiene to vinylcyclobutane. Kinetic studies revealed that the fastest and slowest turnover were observed with equally electron-deficient supporting ligands that either feature phenyl-substituted imine carbon atoms (MeBPDI) or a pyrazine core (MePZDI). While the oxidative cyclization was comparatively slow for both catalysts, the rate of reductive coupling─determined by stoichiometric 13C2H4 labeling studies─correlated with the turnover frequencies. Two-state density functional theory studies and the distinct electronic structures of related (iPrBPDI) and (iPrPZDI) iron methyl complexes revealed significantly different ligand field strengths due to either diminished ligand σ-donation (MeBPDI) or promoted metal π-backbonding (MePZDI). Spin acceleration, leading to fast reductive coupling and catalytic turnover, was promoted in the case of the weaker ligand field and depends on both the nature and position of the electron-withdrawing group. This study provides strong evidence for the role of two-state reactivity in C(sp3)-C(sp3) bond formation and insights on how ligand design either promotes or inhibits spin acceleration in earth-abundant metal catalysis.

Measuring bioavailability, utilization, and excretion of rumen-protected lysine in lactating cows using an isotope technique

Supplementing a diet with rumen-protected amino acids (AAs) is a common feeding strategy for efficient production. For a cost-effective use of rumen-protected AA, the accurate bioavailability of rumen-protected amino acids should be known and their metabolism after absorption needs to be well understood. The current study determined the bioavailability, absorption, utilization, and excretion of rumen-protected Lys (RP-Lys). Four ruminally cannulated cows in a 4 × 4 Latin square design (12 d for diet adaptation; 5 or 6 d for total collections) received the following treatments: L0, a basal diet; L25, the basal diet and L-Lys infused into the abomasum to provide 25.9 g/d L-Lys; L50, the basal diet and L-Lys infused into the abomasum to provide 51.8 g/d L-Lys; and RPL, the basal diet supplemented with 105 g/d (as-is) of RP-Lys to provide 26.7 g of digestible Lys. During the last 5 or 6 d in each period, 15N-Lys (0.38 g/d) was infused into the abomasum for all cows to label the pool of AA, and the total collection of milk, urine, and feces were conducted. 15N enrichment of samples on d 4 and 5 were used to calculate the bioavailability and Lys metabolism. We used a model containing a fast AA turnover (≤ 5 d) and slow AA turnover pool (> 5 d) to calculate fluxes of Lys. The Lys flux to the fast AA turnover pool (absorbed Lys + Lys from the slow AA turnover pool to fast AA turnover pool) was calculated using 15N enrichment of milk Lys. The flux of Lys from the fast AA turnover pool to milk and urine was calculated using 15N transfer into milk and urine. Then, absorbed Lys was estimated by the sum of Lys flux to milk and urine assuming no net utilization of Lys by body tissues. Duodenal Lys flow was estimated by 15N enrichment of fecal Lys. The bioavailability of RP-Lys was calculated from duodenal Lys flows and Lys absorption for RPL. Increasing Lys supply from L25 to L50 increased Lys utilization for milk by 9 g/d but also increased urinary excretion by 10 g/d. For RPL, absorbed Lys was estimated to be 136 g/d where 28 g of absorbed Lys originated from RP-Lys. In conclusion, 68% of bioavailability was obtained for RP-Lys. The Lys provided from RP-Lys was not only utilized for milk protein (48%) but also excreted in urine (20%) after oxidation.

Nitrate-induced hydroxyl radical releases deep soil organic carbon by opening the ‘enzyme latch’ under micro-aerobic conditions

Deep soils (defined here as soil layers >1 m beneath the ground) contain a significant amount of soil organic carbon (SOC) with a relatively slow turnover rate. In oxygen (O2) limit conditions, terms here as “micro-aerobic environment”, a high concentration of phenol usually suppresses SOC decomposition by inhibiting hydrolase activities, a mechanism known as the ‘enzyme latch’. Here, we showed that the addition of nitrate (NO3−) into deep soil significantly decreased the soil phenol content. This reduction alleviated the inhibitory effect of phenol on hydrolase activities, resulting in an increased rate of deep soil SOC decomposition. NO3− addition enhanced deep soil SOC decomposition by opening the ‘enzyme latch’ under micro-aerobic conditions. Under micro-aerobic conditions, NO3− addition increased the concentration of hydroxyl radicals (•OH), with the extent of increase negatively correlated with the soil phenol content. Quenching •OH using terephthalic acid removed the suppressing effect of NO3− on phenol content, indicating that NO3− addition opened the ‘enzyme latch’ by promoting •OH generation, which oxidized phenol. Additionally, the addition of NO3− promoted the production of nicotinamide adenine dinucleotide (NADH) and hydrogen peroxide (H2O2) in deep soil. The H2O2 was significantly positively correlated with •OH, suggesting that the enhanced •OH generation under NO3− addition was caused by the Fenton reaction. Overall, our results shed light on a new pathway through which NO3− leaching affected SOC stability in micro-aerobic deep soils. Reducing nitrate leaching is beneficial for the conservation of deep soil SOC.

Maize seed aid and seed systems development: Opportunities for synergies in Uganda

In the name of food security, governments and NGOs purchase large volumes of maize seed in non-relief situations to provide at reduced or no cost to producers. At the same time, efforts to build formal maize seed systems have been frustrated by slow turnover rates – the dominance of older seed products in the market over newer, higher performing ones. Under certain conditions, governments and NGO seed aid purchases can support formal seed systems development in three ways: i) support increased producer awareness of new products, ii) support local private seed industry development, and iii) advance equity goals by targeting aid to the most vulnerable of producers who lack the capacity to purchase seeds. This study explores the objectives and activities of seed aid programmes in Uganda and their interactions with the maize seed sector. We draw insights from interviews with representatives of seed companies, NGOs and government agencies, as well as focus group discussions with producers. The findings indicated that seed aid programme objectives are largely disconnected from broader seed systems development goals. There is little evidence of public-private collaboration in design of these programmes. Better designed programs have the potential to align with varietal turnover objectives, commercial sector development and targeting of underserved markets could promote equity and ‘crowd in’ demand.

Probing the role of protein conformational changes in the mechanism of prenylated-FMN-dependent phenazine-1-carboxylic acid decarboxylase

Phenazine-1-carboxylic acid decarboxylase (PhdA) is a prenylated-FMN-dependent (prFMN) enzyme belonging to the UbiD family of decarboxylases. Many UbiD-like enzymes catalyze (de)carboxylation reactions on aromatic rings and conjugated double bonds and are potentially valuable industrial catalysts. We have investigated the mechanism of PhdA using a slow turnover substrate, 2,3-dimethylquinoxaline-5-carboxylic acid (DQCA). Detailed analysis of the pH dependence and solvent deuterium isotope effects associated with the reaction uncovered unusual kinetic behavior. At low substrate concentrations, a substantial inverse solvent isotope effect (SIE) is observed on Vmax/KM of ∼ 0.5 when reaction rates of DQCA in H2O and D2O are compared. Under the same conditions, a normal SIE of 4.15 is measured by internal competition for proton transfer to the product. These apparently contradictory results indicate that the SIE values report on different steps in the mechanism. A proton inventory analysis of the reaction under Vmax/KM and Vmax conditions points to a "medium effect" as the source of the inverse SIE. Molecular dynamics simulations of the effect of D2O on PhdA structure support that D2O reduces the conformational lability of the enzyme and results in a more compact structure, akin to the active, "closed" conformer observed in crystal structures of some UbiD-like enzymes. Consistent with the simulations, PhdA was found to be more stable in D2O and to bind DQCA more tightly, leading to the observed rate enhancement under Vmax/KM conditions.

A Bispecific Modeling Framework Enables the Prediction of Efficacy, Toxicity, and Optimal Molecular Design of Bispecific Antibodies Targeting MerTK

Inhibiting MerTK on macrophages is a promising therapeutic strategy for augmenting anti-tumor immunity. However, blocking MerTK on retinal pigment epithelial cells (RPEs) results in retinal toxicity. Bispecific antibodies (bsAbs) containing an anti-MerTK therapeutic and anti-PD-L1 targeting arm were developed to reduce drug binding to MerTK on RPEs, since PD-L1 is overexpressed on macrophages but not RPEs. In this study, we present a modeling framework using in vitro receptor occupancy (RO) and pharmacokinetics (PK) data to predict efficacy, toxicity, and therapeutic index (TI) of anti-MerTK bsAbs. We first used simulations and in vitro RO data of anti-MerTK monospecific antibody (msAb) to estimate the required MerTK RO for in vivo efficacy and toxicity. Using these estimated RO thresholds, we employed our model to predict the efficacious and toxic doses for anti-MerTK bsAbs with varying affinities for MerTK. Our model predicted the highest TI for the anti-MerTK/PD-L1 bsAb with an attenuated MerTK binding arm, which was consistent with in vivo efficacy and toxicity observations. Subsequently, we used the model, in combination with sensitivity analysis and parameter scans, to suggest an optimal molecular design of anti-MerTK bsAb with the highest predicted TI in humans. Our prediction revealed that this optimized anti-MerTK bsAb should contain a MerTK therapeutic arm with relatively low affinity, along with a high affinity targeting arm that can bind to a low abundance target with slow turnover rate. Overall, these results demonstrated that our modeling framework can guide the rational design of bsAbs.Graphical

HGRAD: A versatile "one-fits-all" system to acutely deplete RNA binding proteins from condensates.

Nuclear RNA binding proteins (RBPs) are difficult to study because they often belong to large protein families and form extensive networks of auto- and crossregulation. They are highly abundant and many localize to condensates with a slow turnover, requiring long depletion times or knockouts that cannot distinguish between direct and indirect or compensatory effects. Here, we developed a system that is optimized for the rapid degradation of nuclear RBPs, called hGRAD. It comes as a "one-fits-all" plasmid, and integration into any cell line with endogenously GFP-tagged proteins allows for an inducible, rapid, and complete knockdown. We show that the nuclear RBPs SRSF3, SRSF5, SRRM2, and NONO are completely cleared from nuclear speckles and paraspeckles within 2 h. hGRAD works in various cell types, is more efficient than previous methods, and does not require the expression of exogenous ubiquitin ligases. Combining SRSF5 hGRAD degradation with Nascent-seq uncovered transient transcript changes, compensatory mechanisms, and an effect of SRSF5 on transcript stability.

A Systematic Review of the Causes, Consequences, and Solutions of Emergency Department Overcrowding in Saudi Arabia.

This study aims to investigate and address the issue of emergency department (ED) overcrowding, a significant problem worldwide. The study seeks to understand the impacts of ED overcrowding on emergency medical healthcare services and patient outcomes. This systematic review follows the PRISMA flow diagram and the guidelines of the Cochrane Handbook. We systematically reviewed the causes and solutions of emergency department overcrowding. We went through Google Scholar, the National Center for Biotechnology Information, the British Medical Journal, Science Direct, Ovid, Cochrane, the Saudi Journal of Emergency Medicine, Medline, and PubMed as databases. Our criteria were articles done in Saudi Arabia from 2012 to 2022. One hundred and ninety-six (196) research papers were extracted; only 28 articles met our paper inclusion-exclusion criteria. The result of these papers regarding causes, consequences, and solutions was that non-urgent and returned visits lacked knowledge of PHC, triad, and telemedicine services. Prolonged LOS is due to slow bed turnover, laboratory and consultation time, and physical response to the final decision resulting in burnout staff, wrong diagnoses, and management plans. The crowding issues can be resolved by awareness, PHC access, triad systems, and technological and telemedicine services. High demand for emergency treatment should not be a hindrance to quality treatment. Physical, technological, and strategic measures should be put in place to fight the crowding problem in EDs in Saudi Arabia, as it may cause adverse effects such as transmission of diseases and death of patients.

Groundwater Circulation Mechanism of the Upstream Area of Beiniuchuan River Using Isotope–Hydrochemical Tracer

In order to achieve the rational development and utilization of underground water resources in the Dongsheng mining area under coal mining conditions, we selected the upstream area of Beiniuchuan River as a typical region. Through field investigations, sampling tests, and the application of hydrochemical and isotope techniques, we traced the groundwater circulation mechanism in the Dongsheng mining area. The results indicate that the majority of the Quaternary alluvial and Salawusu Formation groundwater is of the HCO3-Ca type, with a TDS content below 300 mg/L. However, in some areas, the hydrochemical type becomes complex due to anthropogenic contamination. The shallow-buried Yan’an Formation groundwater is either of the HCO3-Ca·Mg type or the HCO3·SO4-Ca·Mg type, with TDS content ranging from 200 to 750 mg/L. The Yan’an Formation at depths greater than 40 m exhibits complex water chemistry, with a TDS content higher than 500 mg/L, and it belongs to the Cl-Na type, with TDS around 700 mg/L. The hydrogen and oxygen isotope results indicate that the local groundwater is primarily recharged via atmospheric precipitation. The 3H and 14C results show that the Quaternary alluvial and shallow-buried Yan’an Formation groundwater has a fast turnover rate, while the deep-buried Yan’an Formation and Yan’chang Formation groundwater have a slower turnover rate. The regional groundwater circulation can be generalized into three flow systems: shallow, intermediate, and deep. Under the influence of coal mining activities, the water circulation conditions in the study area have undergone significant changes. The sealing integrity of the Yan’an Formation has been compromised, and precipitation and shallow groundwater have enhanced the vertical infiltration capacity of the formation, increasing the proportion of groundwater participating in the intermediate flow system. As a result, the river runoff mainly dependent on the discharge from the shallow flow system has drastically decreased.