An assembly chaperone collaborates with the SMN complex to generate spliceosomal SnRNPs.

Spliceosomal small nuclear ribonucleoproteins (snRNPs) are essential components of the nuclear pre-mRNA processing machinery. A hallmark of these particles is a ring-shaped core domain generated by the binding of Sm proteins onto snRNA. PRMT5 and SMN complexes mediate the formation of the core domain in vivo. Here, we have elucidated the mechanism of this reaction by both biochemical and structural studies. We show that pICln, a component of the PRMT5 complex, induces the formation of an otherwise unstable higher-order Sm protein unit. In this state, the Sm proteins are kinetically trapped, preventing their association with snRNA. The SMN complex subsequently binds to these Sm protein units, dissociates pICln, and catalyzes ring closure on snRNA. Our data identify pICln as an assembly chaperone and the SMN complex as a catalyst of spliceosomal snRNP formation. The mode of action of this combined chaperone/catalyst system is reminiscent of the mechanism employed by DNA clamp loaders.

Evolutionary patterns of chromosomal instability and mismatch repair deficiency in proximal and distal colorectal cancer.

AIM: Colorectal carcinomas (CRCs) progress through heterogeneous pathways. The aim of this study was to analyse whether or not the cytogenetic evolution of CRC is linked to tumour site, level of chromosomal imbalance and metastasis. METHOD: A set of therapy-naïve pT3 CRCs comprising 26 proximal and 49 distal pT3 CRCs was studied by combining immunohistochemistry of mismatch repair (MMR) proteins, microsatellite analyses and molecular karyotyping as well as clinical parameters. RESULTS: A MMR deficient/microsatellite-unstable (dMMR/MSI-H) status was associated with location of the primary tumour proximal to the splenic flexure, and dMMR/MSI-H tumours presented with significantly lower levels of chromosomal imbalances compared with MMR proficient/microsatellite-stable (pMMR/MSS) tumours. Oncogenetic tree modelling suggested two evolutionary clusters characterized by dMMR/MSI-H and chromosomal instability (CIN), respectively, for both proximal and distal CRCs. In CIN cases, +13q, -18q and +20q were predicted as preferentially early events, and -1p, -4 -and -5q as late events. Separate oncogenetic tree models of proximal and distal cases indicated similar early events independent of tumour site. However, in cases with high CIN defined by more than 10 copy number aberrations, loss of 17p occurred earlier in cytogenetic evolution than in cases showing low to moderate CIN. Differences in the oncogenetic trees were observed for CRCs with lymph node and distant metastasis. Loss of 8p was modelled as an early event in node-positive CRC, while +7p and +8q comprised early events in CRC with distant metastasis. CONCLUSION: CRCs characterized by CIN follow multiple, interconnected genetic pathways in line with the basic 'Vogelgram' concept proposed for the progression of CRC that places the accumulation of genetic changes at centre of tumour evolution. However, the timing of specific genetic events may favour metastatic potential.

Mitigating greenhouse gas emissions from irrigated rice cultivation through improved fertilizer and water management.

Greenhouse gas (GHG) emissions from agriculture sector play an important role for global warming and climate change. Thus, it is necessary to find out GHG emissions mitigation strategies from rice cultivation. The efficient management of nitrogen fertilizer using urea deep placement (UDP) and the use of the water-saving alternate wetting and drying (AWD) irrigation could mitigate greenhouse gas (GHG) emissions and reduce environmental pollution. However, there is a dearth of studies on the impacts of UDP and the integrated plant nutrient system (IPNS) which combines poultry manure and prilled urea (PU) with different irrigation regimes on GHG emissions, nitrogen use efficiency (NUE) and rice yields. We conducted field experiments during the dry seasons of 2018, 2019, and 2020 to compare the effects of four fertilizer treatments including control (no N), PU, UDP, and IPNS in combination with two irrigation systems- (AWD and continuous flooding, CF) on GHG emissions, NUE and rice yield. Fertilizer treatments had significant (p < 0.05) interaction effects with irrigation regimes on methane (CH4) and nitrous oxide (N2O) emissions. PU reduced CH4 and N2O emissions by 6% and 20% compared to IPNS treatment, respectively under AWD irrigation, but produced similar emissions under CF irrigation. Similarly, UDP reduced cumulative CH4 emissions by 9% and 15% under AWD irrigation, and 9% and 11% under CF condition compared to PU and IPNS treatments, respectively. Across the year and fertilizer treatments, AWD irrigation significantly (p < 0.05) reduced cumulative CH4 emissions and GHG intensity by 28%, and 26%, respectively without significant yield loss compared to CF condition. Although AWD irrigation increased cumulative N2O emissions by 73%, it reduced the total global warming potential by 27% compared to CF irrigation. The CH4 emission factor for AWD was lower (1.67 kg ha-1 day-1) compared to CF (2.33 kg ha-1 day-1). Across the irrigation regimes, UDP increased rice yield by 21% and N recovery efficiency by 58% compared to PU. These results suggest that both UDP and AWD irrigation might be considered as a carbon-friendly technology.

E-cadherin to P-cadherin switching in lobular breast cancer with tubular elements.

Loss of E-cadherin expression due to mutation of the CDH1 gene is a characteristic feature of invasive lobular breast cancer (ILBC). Beta-catenin, which binds to the cytoplasmic domain of E-cadherin, is simultaneously downregulated, reflecting disassembly of adherens junctions (AJs) and loss of cell adhesion. E-cadherin to P-cadherin expression switching can rescue AJs and cell adhesion. However, P-cadherin has not been implicated in ILBC, so far. We aimed to characterize 13 ILBCs with exceptional histomorphology, which we termed ILBCs with tubular elements. The CDH1 mutational status was determined by next generation sequencing and whole-genome copy number (CN) profiling. Expression of cadherins was assessed by immunohistochemistry. ILBCs with tubular elements were ER-positive (13/13) and HER2-negative (13/13) and harbored deleterious CDH1 mutations (11/13) accompanied by loss of heterozygosity due to deletion of chromosome 16q22.1 (9/11). E-cadherin expression was lost or reduced in noncohesive tumor cells and in admixed tubular elements (13/13). Beta-catenin expression was lost in noncohesive tumor cells, but was retained in tubular elements (11/13), indicating focal rescue of AJ formation. N-cadherin and R-cadherin were always negative (0/13). Strikingly, P-cadherin was commonly positive (12/13) and immunoreactivity was accentuated in tubular elements. Adjacent lobular carcinoma in situ (LCIS) was always P-cadherin-negative (0/7). In a reference cohort of LCIS specimens, P-cadherin was constantly not expressed (0/25). In a reference cohort of invasive mammary carcinomas, P-cadherin-positive cases (36/268, 13%) were associated with triple-negative nonlobular breast cancer (P < 0.001). Compared with ILBCs from the reference cohort, P-cadherin expression was more common in ILBCs with tubular elements (12/13 versus 7/84, P < 0.001). In summary, E-cadherin to P-cadherin switching occurs in a subset of ILBCs. P-cadherin is the molecular determinant of a mixed-appearing histomorphology in ILBCs with tubular elements.

Structure and activation of C1, the complex initiating the classical pathway of the complement cascade.

The complement system is an important antimicrobial and inflammation-generating component of the innate immune system. The classical pathway of complement is activated upon binding of the 774-kDa C1 complex, consisting of the recognition molecule C1q and the tetrameric protease complex C1r2s2, to a variety of activators presenting specific molecular patterns such as IgG- and IgM-containing immune complexes. A canonical model entails a C1r2s2 with its serine protease domains tightly packed together in the center of C1 and an intricate intramolecular reaction mechanism for activation of C1r and C1s, induced upon C1 binding to the activator. Here, we show that the serine protease domains of C1r and C1s are located at the periphery of the C1r2s2 tetramer both when alone or within the nonactivated C1 complex. Our structural studies indicate that the C1 complex adopts a conformation incompatible with intramolecular activation of C1, suggesting instead that intermolecular proteolytic activation between neighboring C1 complexes bound to a complement activating surface occurs. Our results rationalize how a multitude of structurally unrelated molecular patterns can activate C1 and suggests a conserved mechanism for complement activation through the classical and the related lectin pathway.

The FlpTRAP system for purification of specific, endogenous chromatin regions.

The repressor element 1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) binds to repressor element 1/neuron-restrictive silencer element (RE1/NRSE) sites in the genome and recruits effector proteins to repress its target genes. Here, we developed the FlpTRAP system to isolate endogenously assembled DNA-protein complexes such as the REST/NRSF complex. In the FlpTRAP system, we take advantage of the step-arrest variant of the Flp recombinase, FlpH305L, which, in the presence of Flp recognition target (FRT) DNA, accumulates as FRT DNA-protein adduct. The FlpTRAP system consists of three elements: (i) FlpH305L-containing cell extracts or isolates, (ii) a cell line engineered to harbor the DNA motif of interest flanked by FRT sites, and (iii) affinity selection steps to isolate the target chromatin. Specifically, 3×FLAG-tagged FlpH305L was expressed in insect cell cultures infected with baculovirus, and cell lysates were prepared. The lysate was used to capture the FRT-SNAP25 RE1/NRSE-FRT chromatin from a human medulloblastoma cell line, and the target RE1/NRSE chromatin was isolated by anti-FLAG immunoaffinity chromatography. Using electrophoretic mobility shift assays (EMSAs) and chromatin immunopurification (ChIP), we show that FlpH305L recognized and bound to the FRT sites. Overall, we suggest the FlpTRAP system as a tool to purify endogenous, specific chromatin loci from eukaryotic cells.

The ATPase BRG1/SMARCA4 is a protein interaction platform that recruits BAF subunits and the transcriptional repressor REST/NRSF in neural progenitor cells.

The BAF complex (SWI/SNF) is an ATP-dependent chromatin remodeler that adapts the structural organization of the chromatin. Despite a growing understanding of the composition of BAF in different cell types, the interaction network within the BAF complex is poorly understood. Here, we characterized an isoform of the BRG1/SMARCA4 ATPase expressed in human neural progenitor cells. By electron microscopy and image processing, the neural BRG1/SMARCA4 shows an elongated globular structure, which provides a considerably larger surface than anticipated. We show that neural BRG1/SMARCA4 binds to BAF57/SMARCE1 and BAF60A/SMARCD1, two further components of BAF. Moreover, we demonstrate an interaction between the neural BRG1/SMARCA4 isoform and the central neurodevelopmental transcriptional repressor REST/NRSF. Our results provide insights into the assembly of a central transcriptional repressor complex, link the structure of the neural BRG1/SMARCA4 to its role as a protein-protein interaction platform and suggest BRG1/SMARCA4 as a key determinant that directs the BAF complex to specific DNA sites by interacting with transcription factors and regulators.

Folding and assembly defects of pyruvate dehydrogenase deficiency-related variants in the E1α subunit of the pyruvate dehydrogenase complex.

The pyruvate dehydrogenase complex (PDC) bridges glycolysis and the citric acid cycle. In human, PDC deficiency leads to severe neurodevelopmental delay and progressive neurodegeneration. The majority of cases are caused by variants in the gene encoding the PDC subunit E1α. The molecular effects of the variants, however, remain poorly understood. Using yeast as a eukaryotic model system, we have studied the substitutions A189V, M230V, and R322C in yeast E1α (corresponding to the pathogenic variants A169V, M210V, and R302C in human E1α) and evaluated how substitutions of single amino acid residues within different functional E1α regions affect PDC structure and activity. The E1α A189V substitution located in the heterodimer interface showed a more compact conformation with significant underrepresentation of E1 in PDC and impaired overall PDC activity. The E1α M230V substitution located in the tetramer and heterodimer interface showed a relatively more open conformation and was particularly affected by low thiamin pyrophosphate concentrations. The E1α R322C substitution located in the phosphorylation loop of E1α resulted in PDC lacking E3 subunits and abolished overall functional activity. Furthermore, we show for the E1α variant A189V that variant E1α accumulates in the Hsp60 chaperonin, but can be released upon ATP supplementation. Our studies suggest that pathogenic E1α variants may be associated with structural changes of PDC and impaired folding of E1α.

Hemoglobin polymerization via disulfide bond formation in the hypoxia-tolerant turtle Trachemys scripta: implications for antioxidant defense and O2 transport.

The ability of many reptilian hemoglobins (Hbs) to form high-molecular weight polymers, albeit known for decades, has not been investigated in detail. Given that turtle Hbs often contain a high number of cysteine (Cys), potentially contributing to the red blood cell defense against reactive oxygen species, we have examined whether polymerization of Hb could occur via intermolecular disulfide bonds in red blood cells of freshwater turtle Trachemys scripta, a species that is highly tolerant of hypoxia and oxidative stress. We find that one of the two Hb isoforms of the hemolysate HbA is prone to polymerization in vitro into linear flexible chains of different size that are visible by electron microscopy but not the HbD isoform. Polymerization of purified HbA is favored by hydrogen peroxide, a main cellular reactive oxygen species and a thiol oxidant, and inhibited by thiol reduction and alkylation, indicating that HbA polymerization is due to disulfide bonds. By using mass spectrometry, we identify Cys5 of the αA-subunit of HbA as specifically responsible for forming disulfide bonds between adjacent HbA tetramers. Polymerization of HbA does not affect oxygen affinity, cooperativity, and sensitivity to the allosteric cofactor ATP, indicating that HbA is still fully functional. Polymers also form in T. scripta blood after exposure to anoxia but not normoxia, indicating that they are of physiological relevance. Taken together, these results show that HbA polymers may form during oxidative stress and that Cys5αA of HbA is a key element of the antioxidant capacity of turtle red blood cells.

3D cryo-EM structure of an active step I spliceosome and localization of its catalytic core.

The spliceosome excises introns from pre-mRNA in a two-step splicing reaction. So far, the three-dimensional (3D) structure of a spliceosome with preserved catalytic activity has remained elusive. Here, we determined the 3D structure of the human, catalytically active step I spliceosome (C complex) by cryo-electron microscopy (cryo-EM) in vitrified ice. Via immunolabeling we mapped the position of the 5' exon. The C complex contains an unusually salt-stable ribonucleoprotein (RNP) core that harbors its catalytic center. We determined the 3D structure of this RNP core and also that of a post-step II particle, the 35S U5 snRNP, which contains most of the C complex core proteins. As C complex domains could be recognized in these structures, their position in the C complex could be determined, thereby allowing the region harboring the spliceosome's catalytic core to be localized.

Paddy soil drainage influences residue carbon contribution to methane emissions.

Water drainage is an important mitigation option for reducing CH4 (methane) emissions from residue-amended paddy soils. Several studies have indicated a long-term reduction in CH4 emissions, even after re-flooding, suggesting that the mechanism goes beyond creating temporary oxidized conditions in the soil. In this pot trial, the effects of different drainage patterns on straw-derived CH4 and CO2 (carbon dioxide) emissions were compared to identify the balance between straw-carbon CH4 and CO2 emissions influenced by soil aeration over different periods, including effects of drainage on emissions during re-flooding. The water treatments included were: continuous flooding [C] as the control and five drainage patterns (pre-planting drainage [P], early-season drainage [E], midseason drainage [M], pre-planting plus midseason drainage [PM], early-season-plus-midseason drainage [EM]). An equal amount of 13C-enriched rice straw was applied to all treatments to identify straw-derived 13C-gas emissions from soil carbon derived emissions. The highest fluxes of CH4 and δ13C-CH4 were recorded from the control treatment in the first week after straw application. The CH4 flux and δ13C-CH4 were reduced the most (0.1-0.8 µg CH4 g-1 soil day-1 and -13 to -34‰) in the pre-planting and pre-planting plus midseason drainage treatments at day one after transplanting. Total and straw-derived CH4 emissions were reduced by 69% and 78% in pre-planting drainage and 77% and 87% in pre-planting plus midseason drainage respectively, compared to control. The early-season, midseason, pre-planting plus midseason and early-season-plus-midseason drainage treatments resulted in higher total and straw-derived CO2 emissions compared to the control and pre-planting drainage treatments. The pre-planting and pre-planting plus midseason drainage treatments lowered the global warming potential by 47-53%, and early-season and early-season-plus-midseason drainage treatments reduced it by 24-31% compared to control. By using labelled crop residues, this experiment demonstrates a direct link between early drainage and reduced CH4 emissions from incorporated crop residues, eventually leading to a reduction in total global warming potential. It is suggested that accelerated decomposition of the residues during early season drainage prolonged the reduction in CH4 emissions. Therefore, it is important to introduce the early drainage as an effective measure to mitigate CH4 emissions from crop residues.

Dual tagging as an approach to isolate endogenous chromatin remodeling complexes from Saccharomyces cerevisiae.

Affinity isolation has been an essential technique for molecular studies of cellular assemblies, such as the switch/sucrose non-fermentable (SWI/SNF) family of ATP-dependent chromatin remodeling complexes. However, even biochemically pure isolates can contain heterogeneous mixtures of complexes and their components. In particular, purification strategies that rely on affinity tags fused to only one component of a complex may be susceptible to this phenomenon. This study demonstrates that fusing purification tags to two different proteins enables the isolation of intact complexes of remodels the structure of chromatin (RSC). A Protein A tag was fused to one of the RSC proteins and a Twin-Strep tag to another protein of the complex. By mass spectrometry, we demonstrate the enrichment of the RSC complexes. The complexes had an apparent Svedberg value of about 20S, as shown by glycerol gradient ultracentrifugation. Additionally, purified complexes were demonstrated to be functional. Electron microscopy and single-particle analyses revealed a conformational rearrangement of RSC upon interaction with acetylated histone H3 peptides. This purification method is useful to purify functionally active, structurally well-defined macromolecular assemblies.

Optimized preoperative fasting times decrease ketone body concentration and stabilize mean arterial blood pressure during induction of anesthesia in children younger than 36 months: a prospective observational cohort study.

BACKGROUND: In pediatric anesthesia, preoperative fasting guidelines are still often exceeded. OBJECTIVE: The objective of this noninterventional clinical observational cohort study was to evaluate the effect of an optimized preoperative fasting management (OPT) on glucose concentration, ketone bodies, acid-base balance, and change in mean arterial blood pressure (MAP) during induction of anesthesia in children. METHODS: Children aged 0-36 months scheduled for elective surgery with OPT (n = 50) were compared with peers studied before optimizing preoperative fasting time (OLD) (n = 50) who were matched for weight, age, and height. RESULTS: In children with OPT (n = 50), mean fasting time (6.0 ± 1.9 h vs 8.5 ± 3.5 h, P < 0.001), deviation from guideline (ΔGL) (1.2 ± 1.4 h vs 3.7 ± 3.1 h, P < 0.001, ΔGL>2 h 8% vs 70%), ketone bodies (0.2 ± 0.2 mmol·l(-1) vs 0.6 ± 0.6 mmol·l(-1) , P < 0.001), and incidence of hypotension (MAP <40 mmHg, 0 vs 5, P = 0.022) were statistically significantly lower and MAP after induction was statistically significantly higher (55.2 ± 9.5 mmHg vs 50.3 ± 9.8 mmHg, P = 0.015) as compared to children in the OLD (n = 50) group. Glucose, lactate, bicarbonate, base excess, and anion gap did not significantly differ. CONCLUSION: Optimized fasting times improve the metabolic and hemodynamic condition during induction of anesthesia in children younger than 36 months of age.

Self-assembly of a nanoscale DNA box with a controllable lid.

The unique structural motifs and self-recognition properties of DNA can be exploited to generate self-assembling DNA nanostructures of specific shapes using a 'bottom-up' approach. Several assembly strategies have been developed for building complex three-dimensional (3D) DNA nanostructures. Recently, the DNA 'origami' method was used to build two-dimensional addressable DNA structures of arbitrary shape that can be used as platforms to arrange nanomaterials with high precision and specificity. A long-term goal of this field has been to construct fully addressable 3D DNA nanostructures. Here we extend the DNA origami method into three dimensions by creating an addressable DNA box 42 x 36 x 36 nm(3) in size that can be opened in the presence of externally supplied DNA 'keys'. We thoroughly characterize the structure of this DNA box using cryogenic transmission electron microscopy, small-angle X-ray scattering and atomic force microscopy, and use fluorescence resonance energy transfer to optically monitor the opening of the lid. Controlled access to the interior compartment of this DNA nanocontainer could yield several interesting applications, for example as a logic sensor for multiple-sequence signals or for the controlled release of nanocargos.

Snapshots of the RNA editing machine in trypanosomes captured at different assembly stages in vivo.

Mitochondrial pre-messenger RNAs in kinetoplastid protozoa are substrates of uridylate-specific RNA editing. RNA editing converts non-functional pre-mRNAs into translatable molecules and can generate protein diversity by alternative editing. Although several editing complexes have been described, their structure and relationship is unknown. Here, we report the isolation of functionally active RNA editing complexes by a multistep purification procedure. We show that the endogenous isolates contain two subpopulations of approximately 20S and approximately 35-40S and present the three-dimensional structures of both complexes by electron microscopy. The approximately 35-40S complexes consist of a platform density packed against a semispherical element. The approximately 20S complexes are composed of two subdomains connected by an interface. The two particles are structurally related, and we show that RNA binding is a main determinant for the interconversion of the two complexes. The approximately 20S editosomes contain an RNA-binding site, which binds gRNA, pre-mRNA and gRNA/pre-mRNA hybrid molecules with nanomolar affinity. Variability analysis indicates that subsets of complexes lack or possess additional domains, suggesting binding sites for components. Together, a picture of the RNA editing machinery is provided.

Drug loading of polymeric micelles.

PURPOSE: To gain mechanistic insights into drug loading and lyophilization of polymeric micelles. METHODS: PEGylated poly-4-(vinylpyridine) micelles were loaded with dexamethasone. Three different methods were applied and compared: O/W emulsion, direct dialysis, cosolvent evaporation. Micellar dispersions with the highest drug load were lyophilized with varying lyoprotectors: sucrose, trehalose, maltose, a polyvinylpyrrolidine derivative, and ß-cyclodextrin derivatives. For comparison, other PEGylated block copolymer micelles (PEGylated polylactic acid, polylactic acid-co-glycolic acid, polycaprolactone) were freeze-dried. RESULTS: Drug loading via direct dialysis from acetone was a less effective loading method which led to dexamethasone loads <2% w/w. O/W emulsion technique from dichlormethane increased drug load up to ~13% w/w; optimized cosolvent evaporation increased load up to ~19% w/w. An important step for cosolvent evaporation was solubility screen of the drug prior to preparation. Loading was maintained upon lyophilization with ß-cyclodextrins which proved to be versatile stabilizers for other block copolymer micelles. CONCLUSION: Careful solvent selection prior to cosolvent evaporation was a beneficial approach to load hydrophobic drugs into polymeric micelles. Moreover, ß-cyclodextrins could be used as versatile lyoprotectors for these micelles.

Genomic Complexity as a Biomarker to De-Escalate Adjuvant Imatinib Treatment in High-Risk Gastrointestinal Stromal Tumor.

PURPOSE: Adjuvant imatinib treatment is recommended for patients with localized gastrointestinal stromal tumor (GIST) at high risk of recurrence. Almost half of high-risk patients are cured by surgery alone, indicating a need for improved selection of patients for adjuvant therapy. The aim of this study was to investigate if genomic tumor complexity could be used as a prognostic biomarker. METHODS: The discovery cohort consisted of patients who underwent resection of primary GIST at Oslo University Hospital between 1998 and 2020. Karyotypes were categorized as simple if they had ≤ 5 chromosomal changes and complex if there were > 5 chromosomal aberrations. Validation was performed in an independent patient cohort where chromosomal imbalances were mapped using comparative genomic hybridization. RESULTS: Chromosomal aberrations were detected in 206 tumors, of which 76 had a complex karyotype. The most frequently observed changes were losses at 14q, 22q, 1p, and 15q. The 5-year recurrence-free survival (RFS) in patients classified as very low, low, or intermediate risk was 99%. High-risk patients with a simple tumor karyotype had an estimated 5-year RFS of 94%, and patients with a complex karyotype had an estimated 5-year RFS of 51%. A complex karyotype was associated with poor RFS in patients with and without adjuvant imatinib treatment and in multivariable analysis adjusted for tumor site, size, mitotic count, and rupture. The prognostic impact of genomic complexity was confirmed in the validation cohort. In both cohorts, the 5-year disease-specific survival was > 90% for high-risk patients with genomically simple tumors. CONCLUSION: Genomic tumor complexity is an independent prognostic biomarker in localized, high-risk GIST. Recurrences were infrequent for tumors with simple karyotypes. De-escalation of adjuvant imatinib treatment should be explored in patients with cytogenetically simple GISTs.

Effects of integrated plant nutrition systems with fertilizer deep placement on rice yields and nitrogen use efficiency under different irrigation regimes.

Improved fertilizer management, with a combination of organic and inorganic inputs, has the potential to enhance rice yield while maintaining soil health. However, studies on the effects of broadcast prilled urea (PU) and urea deep placement (UDP) applied in combination with organic inputs (poultry litter [PL] and vermicompost [VC]), as integrated plant nutrition systems (IPNSs), on rice yields and nitrogen use efficiency (NUE) under alternate wetting and drying (AWD) irrigation are limited. We conducted field experiments during the dry and wet seasons of 2018, 2019, and 2020 to investigate the effects of fertilizer treatments, including control (no nitrogen), UDP, PU, and IPNSs (PU + VC, PU + PL, and UDP + PL) on rice yield and NUE under two irrigation regimes - AWD and continuous flooding (CF). The results revealed that fertilizer treatment and irrigation regime had significant (p < 0.05) interaction effects on rice yield and the agronomic efficiency of N (AEN) during the dry season. UDP significantly (p < 0.05) boosted rice yield, total dry matter (TDM), and NUE as compared to broadcast PU in both wet and dry seasons. Similarly, the IPNS treatment of UDP with PL significantly (p < 0.05) boosted rice yield, TDM, and NUE in comparison to broadcast PU. Under AWD irrigation, UDP alone produced higher rice yields than other treatments, while UDP, and UDP with PL produced similar yields under CF irrigation. During the dry season, AWD irrigation significantly (p < 0.05) increased rice yield, TDM, and AEN when compared to CF conditions, but during the wet season, AWD irrigation demonstrated a rice yield and NUE equivalent to CF. This research implies that using a UDP alone or in combination with PL as an IPNS could be a good way to boost crop productivity while also maintaining soil fertility.

Characterization of purified human Bact spliceosomal complexes reveals compositional and morphological changes during spliceosome activation and first step catalysis.

To better understand the compositional and structural dynamics of the human spliceosome during its activation, we set out to isolate spliceosomal complexes formed after precatalytic B but prior to catalytically active C complexes. By shortening the polypyrimidine tract of the PM5 pre-mRNA, which lacks a 3' splice site and 3' exon, we stalled spliceosome assembly at the activation stage. We subsequently affinity purified human B(act) complexes under the same conditions previously used to isolate B and C complexes, and analyzed their protein composition by mass spectrometry. A comparison of the protein composition of these complexes allowed a fine dissection of compositional changes during the B to B(act) and B(act) to C transitions, and comparisons with the Saccharomyces cerevisiae B(act) complex revealed that the compositional dynamics of the spliceosome during activation are largely conserved between lower and higher eukaryotes. Human SF3b155 and CDC5L were shown to be phosphorylated specifically during the B to B(act) and B(act) to C transition, respectively, suggesting these modifications function at these stages of splicing. The two-dimensional structure of the human B(act) complex was determined by electron microscopy, and a comparison with the B complex revealed that the morphology of the human spliceosome changes significantly during its activation. The overall architecture of the human and S. cerevisiae B(act) complex is similar, suggesting that many of the higher order interactions among spliceosomal components, as well as their dynamics, are also largely conserved.