Distinct Linkage Research Articles

Click‐iG: Simultaneous Enrichment and Profiling of Intact N‐linked, O‐GalNAc, and O‐GlcNAcylated Glycopeptides

AbstractProteins are ubiquitously modified with glycans of varied chemical structures through distinct glycosidic linkages, making the landscape of protein glycosylation challenging to map. Profiling of intact glycopeptides with mass spectrometry (MS) has recently emerged as a powerful tool for revealing matched information of the glycosylation sites and attached glycans (i.e., intact glycosites), but is largely limited to individual glycosylation types. Herein, we describe Click‐iG, which integrates metabolic labeling of glycans with clickable unnatural sugars, an optimized MS method, and a tailored version of pGlyco3 software to enable simultaneous enrichment and profiling of three types of intact glycopeptides: N‐linked, mucin‐type O‐linked, and O‐GlcNAcylated glycopeptides. We demonstrate the utility of Click‐iG by the identification of thousands of intact glycosites in cell lines and living mice. From the mouse lung, heart, and spleen, a total of 2053 intact N‐glycosites, 262 intact O‐GalNAc glycosites, and 1947 O‐GlcNAcylation sites were identified. Click‐iG‐enabled comprehensive coverage of the protein glycosylation landscape lays the foundation for interrogating crosstalk between different glycosylation pathways.

Click-iG: Simultaneous Enrichment and Profiling of Intact N-linked, O-GalNAc, and O-GlcNAcylated Glycopeptides.

Proteins are ubiquitously modified with glycans of varied chemical structures through distinct glycosidic linkages, making the landscape of protein glycosylation challenging to map. Profiling of intact glycopeptides with mass spectrometry (MS) has recently emerged as a powerful tool for revealing matched information of the glycosylation sites and attached glycans (i.e., intact glycosites), but is largely limited to individual glycosylation types. Herein, we describe Click-iG, which integrates metabolic labeling of glycans with clickable unnatural sugars, an optimized MS method, and a tailored version of pGlyco3 software to enable simultaneous enrichment and profiling of three types of intact glycopeptides: N-linked, mucin-type O-linked, and O-GlcNAcylated glycopeptides. We demonstrate the utility of Click-iG by the identification of thousands of intact glycosites in cell lines and living mice. From the mouse lung, heart, and spleen, a total of 2053 intact N-glycosites, 262 intact O-GalNAc glycosites, and 1947 O-GlcNAcylation sites were identified. Click-iG-enabled comprehensive coverage of the protein glycosylation landscape lays the foundation for interrogating crosstalk between different glycosylation pathways.

Variability of volume transport in the Taiwan Strait and its response to tropical MJO convection: A numerical approach

Taiwan Strait (TWS) plays a crucial role in material exchange and nutrient budget between the South China Sea and the East China Sea. In this study, we investigate the variability of volume transport in the TWS and its response to tropical Madden–Julian Oscillation (MJO) convection based on the simulated results from a three-dimensional operational numerical model. Validated by the observational data, the model generally reproduced the physical field well. The volume transport in the TWS has strong seasonal cycles as well as higher-frequency variations. Intra-seasonal fluctuations dominate the along-strait currents variability, while secondary and the last signal are seasonal one and inter-annual variability, respectively. Overall, the along-strait wind stress plays a more important role in controlling the variability of volume transport in the TWS than the pressure gradient induced by north-to-south sea level slope. At intra-seasonal time scales, the volume transport in the TWS varies with the movement of the tropical MJO convection from Indian Ocean eastward to the western Pacific Ocean. These oceanic anomalies are related to atmospheric anomalies, with a distinct physical linkage from the tropical atmosphere to the mid-latitude ocean. The tropical MJO deep convection can modify the upper tropospheric heights and generate a wave train pattern that propagates to mid-latitudes. In addition, these anomalous upper tropospheric heights modulate the surface pressure, resulting in a cyclonic anomaly. The upper tropospheric heights anomaly and its corresponding mean sea level pressure anomaly move eastward in the MJO cycle following the migrating MJO heat source in the tropics from phase 2 to 5. Consequently, surface winds change as the cyclonic anomaly moves from central China to the east of Japan, resulting in a northeast volume transport anomaly during MJO phase 2 and 3 and a southwest volume transport anomaly during MJO phase 4 and 5.

Discovery and Biosynthesis of Cihunamides, Macrocyclic Antibacterial RiPPs with a Unique C-N Linkage Formed by CYP450 Catalysis.

Cihunamides A-D (1-4), novel antibacterial RiPPs, were isolated from volcanic-island-derived Streptomyces sp. The structures of 1-4 were elucidated by 1 H, 13 C, and 15 N NMR, MS, and chemical derivatization; they contain a tetrapeptide core composed of WNIW, cyclized by a unique C-N linkage between two Trp units. Genome mining of the producer strain revealed two biosynthetic genes encoding a cytochrome P450 enzyme and a precursor peptide. Heterologous co-expression of the core genes demonstrated the biosynthesis of cihunamides through P450-mediated oxidative Trp-Trp cross-linking. Further bioinformatic analysis uncovered 252 homologous gene clusters, including that of tryptorubins, which possess a distinct Trp-Trp linkage. Cihunamides do not display the non-canonical atropisomerism shown in tryptorubins, which are the founding members of the "atropitide" family. Therefore, we propose to use a new RiPP family name, "bitryptides", for cihunamides, tryptorubins, and their congeners, wherein the Trp-Trp linkages define the structural class rather than non-canonical atropisomerism.

Bifidobacterial GH146 β-l-Arabinofuranosidase (Bll4HypBA1) as the Last Enzyme for the Complete Removal of Oligoarabinofuranosides from Hydroxyproline-Rich Glycoproteins.

In plant cell walls, the hydroxyproline-rich glycoproteins (HRGPs) such as extensin contain oligoarabinofuranoside linked to a hydroxyproline (Hyp) residue. The mature arabinooligosaccharide was revealed to be a tetrasaccharide (α-l-Araf-(1→3)-β-l-Araf-(1→2)-β-l-Araf-(1→2)-β-l-Araf, l-Araf4 ), whose linkages are targets of the bifidobacterial and Xanthomonas arabinooligosaccharide-degrading enzymes. The l-Araf4 motif was cleaved by GH43 α-l-arabinofuranosidase (Arafase) and converted to an l-Araf3 -linked structure. The latter is then cleaved by GH121 β-l-arabinobiosidase (HypBA2), producing β-l-Araf-(1→2)-l-Ara (β-l-arabinobiose) and mono-β-l-Araf linked to the HRGP backbone. In bifidobacteria, the β-l-arabinobiose is then hydrolyzed by GH127 β-l-Arafase (Bll1HypBA1), a mechanistically unique cysteine glycosidase. We recently identified the distantly related homologue from Xanthomonas euvesicatoria as GH146 β-l-Arafase along with paralogues from Bifidobacterium longum, one of which, Bll4HypBA1 (BLLJ_0089), can degrade l-Araf1 -Hyp in a similar way to that of GH146. As the chemical synthesis of the extensin hydrophilic motif 1 a, which possesses three distinct linkages that connect four oligoAraf residues [Hyp(l-Arafn ) (n=4, 3, 1)], was achieved previously, we precisely monitored the step-wise enzymatic cleavage of 1 a in addition to that of potato lectin. The results unequivocally revealed that this enzyme specifically degrades the Hyp(l-Araf1 ) motif.

The unifying catalytic mechanism of the RING-between-RING E3 ubiquitin ligase family

The RING-between-RING (RBR) E3 ubiquitin ligase family in humans comprises 14 members and is defined by a two-step catalytic mechanism in which ubiquitin is first transferred from an E2 ubiquitin-conjugating enzyme to the RBR active site and then to the substrate. To define the core features of this catalytic mechanism, we here structurally and biochemically characterise the two RBRs HOIL-1 and RNF216. Crystal structures of both enzymes in their RBR/E2-Ub/Ub transthiolation complexes capturing the first catalytic step, together with complementary functional experiments, reveal the defining features of the RBR catalytic mechanism. RBRs catalyse ubiquitination via a conserved transthiolation complex structure that enables efficient E2-to-RBR ubiquitin transfer. Our data also highlight a conserved RBR allosteric activation mechanism by distinct ubiquitin linkages that suggests RBRs employ a feed-forward mechanism. We finally identify that the HOIL-1 RING2 domain contains an unusual Zn2/Cys6 binuclear cluster that is required for catalytic activity and substrate ubiquitination.

Population genetic analysis of 12 X-STR markers in Slovakia

Background: During the last 20 years, X-chromosomal STR markers have become widely used in forensic genetics and paternity testing. Nevertheless, to exploit their full potential in any given population, a reliable reference dataset needs to be established. Since no relevant studies concerning these markers have been performed on the Slovak population so far, we decided to analyse several commonly used markers in this population. Aim: To create an informative set of Slovak population data concerning X-STR markers. Subjects and methods: We genotyped 378 individuals and analysed 12 loci (DXS10148, DX10135, DXS8378, DXS7132, DXS10079, DXS10074, DXS10103, HPRTB, DXS10101, DXS10146, DXS10134 and DXS742) localised in four distinct linkage groups. Results: Our analysis showed that the most informative marker is DXS10135 (PIC = 0,927) and the most informative linkage group (LG) is LG1 with 149 different haplotypes. This analysis also confirmed linkage disequilibrium for two pairs of markers (DX10101-DX10103 and DX10101-HPRTB) within LG3 in female samples. No statistically significant departure from HWE was observed for any locus. Moreover, the interpopulation comparison of 8 European populations based on haplotype frequencies showed no statistically significant FST values in any LG, except for LG2 in comparison with the German population. Conclusion: We created a haplotype database for forensic analyses and kinship testing in Slovakia, as well as the CE dataset which can be used to further increase the decision power in similar analyses in the future.

Water-Harvesting Metal-Organic Frameworks with Gigantic Al24 Units and their Deconstruction into Molecular Clusters.

In contrast to the vast Al-oxo molecular cluster chemistry, Al-based building units for metal-organic framework (MOF) construction are limited in structural diversity and complexity. Synthesis of single crystalline MOFs based on this "hard" metal is further complicated by the poor reversibility of the Al-organic coordination linkages. Here, a strategy to employ two kinds of linkages with distinct strength-strong Al-carboxylate linkage and weak Cu-pyrazol N linkage-gives FDM-91 (FDM=Fudan Materials) with gigantic Al24 -based units. After replacing the weak moieties with organic linkers post-synthetically, two new stable MOFs with exceptional water harvesting capacity (up to 0.53 g g-1 ) and outstanding cycling performance are developed. Linkage-selective dissociation of FDM-91 further leads to the isolation of the Al24 molecular clusters. The versatile chemistry performed here to reinforce or deconstruct MOFs provides a new way to make important extended and discrete structures.

Vegetation Pattern and Regeneration Dynamics of the Progressively Declining Monotheca buxifolia Forests in Pakistan: Implications for Conservation

Monotheca buxifolia (Falc.) A. DC., a wild edible fruit-yielding tree species, has economic and ecological importance, yet there is a lack of studies concerning its distribution pattern and regeneration dynamics at a larger-scale. This study aims to produce the first country-level classification of Monotheca forests based on their unique floristic composition and influential abiotic factors, besides their natural regeneration dynamics, across the environmentally diverse landscapes in Pakistan. For this purpose, floristic inventory was carried out in 440 plots where environmental variables, stand dendrometric characteristics and woody-species regeneration were recorded. During this survey, 3789 individuals of 27 woody tree species belonging to 25 genera and 22 families were sampled. These native and exotic tree species were mostly dicot (73%) with predominately mega-phanerophytic (88%) lifeforms, which largely reflect strong chorological differentiation and distinct linkage (55%) to the Sino-Japanese phytogeographical region. M. buxifolia and co-occurring species exhibiting similar environmental affinities were grouped into four ecologically distinct communities by Ward’s cluster analysis. Ordinations further highlight the special effects of topographic and edaphic factors besides anthropogenic interference on the sampled plots. Generally, Monotheca stands were moderately dense, with the average density varying considerably, ranging from 296 to 325 individuals/ha, with basal area ranging from 41.26 to 93.35 m2 ha−1. In the understory stratum, natural regeneration of Monotheca was mostly scant and mainly covered by Dodonaea shrubs. Size class structure of the dominant species shows pronounced effect of anthropogenic intervention as reflected by the presence of fewer individuals of juveniles and larger trees. Overall, the cut stump frequency was higher at juvenile and mature stages, which may be attributed to over-harvesting and extraction of fuelwood apart from the adverse effect of climate change in the region. We concluded that both topographic and edaphic factors coupled with biotic interventions are more influential in the distribution and persistence of M. buxifolia and co-occurring woody species and might be considered in its restoration and conservation. Thus, we recommend an urgent management plan to favor Monotheca regeneration for allowing the renewal of these rapidly declining remnant stands in Pakistan.

Genetic analysis of CNS autoimmunity using the diversity of the Collaborative Cross reveals unique phenotypes and mechanisms

Abstract Multiple Sclerosis (MS) is a complex disease with remarkable heterogeneity in disease course and progression, the genetic basis of which remains obscure. Here we leveraged the Collaborative Cross (CC) - a highly genetically diverse mouse strain panel - and myelin oligodendrocyte glycoprotein peptide 35–55 (MOG35–55) induced experimental autoimmune encephalomyelitis (EAE), to model genetics of MS disease course. 33 CC strains were selected based on compatible MHC haplotypes (H2b and H2g7), which captured a wide spectrum of distinct EAE phenotypes, compared with typical chronic EAE in C57BL/6 mice. CC028 mice exhibited severe and rapidly progressing disease. In contrast, several strains, including CC011 and CC040, were highly resistant to EAE. Sex differences in EAE course were observed in 4 strains, including CC042. Remitting-relapsing EAE was observed in 4 strains, including CC002. In addition to classical EAE clinical signs (ascending paralysis), we identified two strains, CC004 and CC083 that exhibited high incidence of axial rotary (AR)-EAE, including profound ataxia, head tilt, and axial rotation. Preliminary quantitative trait locus (QTL) analysis revealed a distinct linkage pattern in each sex for classical EAE severity, with several emerging peaks on chromosome (Chr) 2, 8, 12, 18, and X in females, Chr10 in males, and Chr4 in both sexes. QTL analysis of AR-EAE severity revealed a narrow interval on Chr18 (39.2–41.0Mb) passing suggestive linkage significance at the 90% confidence threshold. Experiments are ongoing to determine the immunopathologic basis of distinct genetically controlled EAE phenotypes in CC strains of interest. Supported by grants from NIH (R21 AI145306-01) and the National MS Society (RG-1901-33309)

K27‐linked ubiquitylation promotes p97 substrate processing and is essential for cell proliferation

Conjugation of ubiquitin (Ub) to numerous substrate proteins regulates virtually all cellular processes. Eight distinct ubiquitin polymer linkages specifying different functional outcomes are generated in cells. However, the roles of some atypical poly‐ubiquitin topologies, in particular linkages via lysine 27 (K27), remain poorly understood due to a lack of tools for their specific detection and manipulation. Here, we adapted a cell‐based ubiquitin replacement strategy to enable selective and conditional abrogation of K27‐linked ubiquitylation, revealing that this ubiquitin linkage type is essential for proliferation of human cells. We demonstrate that K27‐linked ubiquitylation is predominantly a nuclear modification whose ablation deregulates nuclear ubiquitylation dynamics and impairs cell cycle progression in an epistatic manner with inactivation of the ATPase p97/VCP. Moreover, we show that a p97‐proteasome pathway model substrate (Ub(G76V)‐GFP) is directly modified by K27‐linked ubiquitylation, and that disabling the formation of K27‐linked ubiquitin signals or blocking their decoding via overexpression of the K27 linkage‐specific binder UCHL3 impedes Ub(G76V)‐GFP turnover at the level of p97 function. Our findings suggest a critical role of K27‐linked ubiquitylation in supporting cell fitness by facilitating p97‐dependent processing of ubiquitylated nuclear proteins.

Genomic regions associated with virulence in Setosphaeria turcica identified by linkage mapping in a biparental population

Northern corn leaf blight (NCLB) and sorghum leaf blight (SLB) are significant diseases of maize and sorghum, respectively, caused by the filamentous fungus Setosphaeria turcica. Strains of S. turcica are typically host-specific and infect either maize or sorghum. Host specificity in this pathogen is attributed to a single locus for maize and a second distinct locus for sorghum. To identify the genetic basis of host specificity in S. turcica, we generated a biparental population of S. turcica by crossing strains specific to maize and sorghum, phenotyped the population for leaf blight on sorghum and maize, genotyped the population to create a linkage map of S. turcica, and located candidate virulence regions. A total of 190 ascospores from 35 pseudothecia were isolated from the cross of maize and sorghum-specific strains. Greenhouse phenotyping of the biparental population (n = 144) showed independent inheritance of virulence, as indicated by a 1:1:1:1 segregation for virulence to maize, sorghum, both maize and sorghum, and avirulence to both crops. The population and host-specific parent strains were genotyped using genome skim sequencing on an Illumina NovaSeq 6000 platform resulting in over 780 million reads. A total of 32,635 variants including single nucleotide polymorphisms and indels were scored. There was evidence for a large deletion in the sorghum-specific strain of S. turcica. A genetic map consisting of 17 linkage groups spanning 3,069 centimorgans was constructed. Virulence to sorghum and maize mapped on distinct linkage groups with a significant QTL detected for virulence to maize. Furthermore, a single locus each for the in vitro traits hyphal growth rate and conidiation were identified and mapped onto two other linkage groups. In vitro traits did not correlate with in planta virulence complexity, suggesting that virulence on both hosts does not incur a fitness cost. Hyphal growth rate and conidiation were negatively correlated, indicating differences in hyphal growth versus dispersal ability for this pathogen. Identification of genetic regions underlying virulence specificity and saprotrophic growth traits in S. turcica provides a better understanding of the S. turcica- Andropogoneae pathosystem.

Genome-wide association findings from the brains for dementia research cohort

The Brains for Dementia Research (BDR) cohort (~3200) is a longitudinal clinicopathological programme, complimented with genetic analysis for the purposes of aetiological investigation into dementia. Here the data from genetic association analyses are presented from the initial collection of DNA from the BDR cohort. The aim of this study was to investigate the preliminary association signals for pathologically confirmed Alzheimer's disease samples compared to controls with no other pathology (n = 520). Genome-wide genotyping was carried out using the NeuroChip platform. Analysis utilised the standard PLINK software for association studies. Genome-wide Bonferroni significant association were observed on chr19 around the APOE/TOMM40 locus across 2 distinct linkage disequilibrium blocks. Eleven of the top 35 association signals have been identified in previous studies, in addition to an intriguing SNP association within the FPR1 gene locus. This study suggests the BDR is genetically comparable to other Alzheimer's disease cohorts and offers an independent resource to verify findings, and additional genetic data for meta-analyses.

Gathered into One

Abstract This article reconsiders the two enigmatic scenes of Mary at Jesus’ feet in John 11 and 12. Previous scholarship has recognized ordered connections between John 11–12, and between John 12–13. Examination of John’s distinctive linkage between Mary and Judas uncovers an artistic network of figural cantilevers and triads that connect, or gather into one, scenes from all three chapters: Jesus’ Raising of Lazarus, Mary’s Anointing of Jesus, and the Foot Washing. This network architecture enables information transfer and implicit commentary between the three contexts, which in turn illuminates Jesus’ emotional anagnorisis with Mary on his way to Lazarus’ tomb, as well as Mary’s unconventional wiping of the ointment from his feet with her hair. Her opposition to Judas points up her alignment with Jesus, wherein he shares her tears and she shares his anointing. Finally, this network model provides an intelligible platform for the migration of key Synoptic elements away from John’s Anointing into his Lazarus story.

Network diversity, distance and economic impact in a cluster: visualising linkages and assessing network capital

Purpose Effective policy to support business ecosystems should build on evidence-based analyses of firm-level activities and outcomes. This paper aims to contribute to this requirement and makes three contributions. The first contribution is to extend the application of the network capital concept to a variety of eight distinct linkage categories (e.g. suppliers, customers and business support agencies) that support networking and clustering, in both activity and impact terms. The second contribution is outlining a novel method of network visualisation (V-LINC) based on the collection of primary and qualitative data. The third contribution is in applying the method to one cluster, information and communications technologies. Design/methodology/approach Qualitative research on the nature and extent of organisational network linkages was undertaken. Structured interviews with a set of focal firms followed a tailored design approach. The concept of network capital was extended and applied to the cluster context by measuring network inputs and output (i.e. investments and impact). The approach was operationalised via a novel impact measurement approach, denoted as V-LINC, an acronym for visualising linkages in networks and clusters. Findings The authors develop a business impact framework exploiting novel linkage visualisations and qualitative data from firms in a cluster in one city region across eight linkage types to capture distinct network capital elements. Organisational inputs into network development, measured as investment and involvement indicators and organisational outcomes from those networks, measured as importance and intensity indicators, are used to assess network performance. A comprehensive, systematic and robust analysis of network elements and performance is possible. Distance is found to interact differently across linkage types. Targeted recommendations may be made from the analysis of local or regional business ecosystems in light of measured business impacts of linkages. Research limitations/implications Due to the resource-intensive nature of data collection, the current study engages a limited sample of firms and interviewees. Applications of this approach in other contexts will permit further research into its usefulness in evaluating business impacts generated through networking activities. Originality/value The method introduced here (V-LINC) offers a novel means to include both geography network theory into an understanding of knowledge relationships and networks within clusters. Accounting for both distance and linkage type reveals which categories of intra-regional and extra-regional linkages generate the greatest impact, given their frequency. The approach adds to available cluster visualisation and analysis approaches through identifying patterns of disaggregated knowledge flows and their impacts, with application to evaluation demands of policy.

Tourism and Renewable Energy in South Asia: A Panel Study

Purpose - The distinct linkage between tourism and energy has received significant attention due to the recent momentum towards green tourism and environmental sustainability. Given the present growth trajectory of the tourism industry, we examine the selected South Asian countries' tourismrenewable energy nexus. Design - We obtain data (1995 to 2018) from the World Bank (2018) and the BP Statistical Review of World Energy (2018). Methodology - Second generation cointegration tests were conducted to verify the cointegrating relationship. DCCE and ARDL-PMG methods were used for long-run estimation. DumitrescuHurlin panel causality test was conducted to check long-run causalities. Finally, Variance Decomposition analysis was used to assess the effect of tourism on REs in the future Findings - Tourism and renewable energy consumption are positively associated in the long-run. Unidirectional causalities are observed from tourism to renewable energy uses at both the aggregate and disaggregate levels. Furthermore, tourism can significantly explain the predicted variation in the use of different renewable energies for the next 15 years. Originality - To our knowledge, this is the first study conducted to analyse the relationship between tourism and renewable energy consumption at both aggregate and disaggregated levels in South Asia. We also propose tourism-friendly renewable energy policies that may be key for continuing sustainable tourism development in the selected South Asian countries.

Building a Data Linkage Foundation for Mother-Child Pharmacoepidemiology Research.

Expanding our understanding of the effects of maternal medication exposure through research is a public health priority and will help inform both clinical and policy decision making, ultimately improving outcomes for pregnant women and their children. Our objective was to describe a linked-data research platform that facilitates studies of pregnancy medication exposures and policy changes on maternal and child health outcomes. Mothers receiving Medicaid benefits were probabilistically linked with newborns in the Tennessee Medicaid program (TennCare) through three distinct linkage processes. Medicaid claims data and state birth and fetal death certificate records (vital records) were used to identify and link potential mothers, deliveries, and newborn children. The linkage process started with the creation of a merged pool of potential mothers and eligible deliveries, which was linked to vital records and to children's records. In the last step, linked records from the preceding steps were combined into the final Mother-child linked records. For each data linkage step, rubrics and scoring systems for exact and partial matches and mismatches among key linkage fields were applied and used to examine the strength of the probabilistic linkages. Summary linkage yields for year 2013 are reported for illustration purposes. Among the 84,253 potential deliveries, 1,761,557 eligible potential mothers, and 51,400 eligible children identified in Tennessee Medicaid records in 2013, a total of 60,265 of these records were uniquely linked to vital records, including 46,172 (77%) with linked mother-child-vital records. Among the 51,400 eligible children records identified in Tennessee Medicaid for that year, 97% (50,053) had at least one link to vital records or a mother-delivery record. In linked records, the median maternal age was 24years, and the median gestational age was 39weeks. About 33% of pregnant women underwent cesarean birth, and 1% of births were classified as complicated deliveries. Supplementing existing Medicaid claims data with birth certificate records complements administrative claims information and allows for detailed assessments of pregnancy exposures and policy changes on mother and child outcomes.

Solid-state additive manufacturing of aluminum and copper using additive friction stir deposition: Process-microstructure linkages

Among metal additive manufacturing technologies, additive friction stir deposition stands out for its ability to create freeform and fully-dense structures without melting and solidification. Here, we employ a comparative approach to investigate the process-microstructure linkages in additive friction stir deposition, utilizing two materials with distinct thermomechanical behavior—an Al-Mg-Si alloy and Cu—both of which are challenging to print using beam-based additive processes. The deposited Al-Mg-Si is shown to exhibit a relatively homogeneous microstructure with extensive subgrain formation and a strong shear texture, whereas the deposited Cu is characterized by a wide distribution of grain sizes and a weaker shear texture. We show evidence that the microstructure in Al-Mg-Si primarily evolves by continuous dynamic recrystallization, including geometric dynamic recrystallization and progressive lattice rotation, while the heterogeneous microstructure of Cu results from discontinuous recrystallization during both deposition and cooling. In Al-Mg-Si, the continuous recrystallization progresses with an increase of the applied strain, which correlates with the ratio between the tool rotation rate Ω and travel velocityV. Conversely, the microstructure evolution in Cu is found to be less dependent on Ω, instead varying more with changes to V. This difference originates from the absence of Cu rotation in the deposition zone, which reduces the influence of tool rotation on strain development. We attribute the distinct process-microstructure linkages and the underlying mechanisms between Al-Mg-Si and Cu to their differences in intrinsic thermomechanical properties and interactions with the tool head.

Identification of ubiquitin Ser57 kinases regulating the oxidative stress response in yeast.

Ubiquitination regulates many different cellular processes, including protein quality control, membrane trafficking, and stress responses. The diversity of ubiquitin functions in the cell is partly due to its ability to form chains with distinct linkages that can alter the fate of substrate proteins in unique ways. The complexity of the ubiquitin code is further enhanced by post-translational modifications on ubiquitin itself, the biological functions of which are not well understood. Here, we present genetic and biochemical evidence that serine 57 (Ser57) phosphorylation of ubiquitin functions in stress responses in Saccharomyces cerevisiae, including the oxidative stress response. We also identify and characterize the first known Ser57 ubiquitin kinases in yeast and human cells, and we report that two Ser57 ubiquitin kinases regulate the oxidative stress response in yeast. These studies implicate ubiquitin phosphorylation at the Ser57 position as an important modifier of ubiquitin function, particularly in response to proteotoxic stress.

Heterogeneity and Actin Cytoskeleton in Osteoclast and Macrophage Multinucleation.

Osteoclast signatures are determined by two transcriptional programs, the lineage-determining transcription pathway and the receptor activator of nuclear factor kappa-B ligand (RANKL)-dependent differentiation pathways. During differentiation, mononuclear precursors become multinucleated by cell fusion. Recently, live-cell imaging has revealed a high level of heterogeneity in osteoclast multinucleation. This heterogeneity includes the difference in the differentiation states and the mobility of the fusion precursors, as well as the mode of fusion among the fusion precursors with different numbers of nuclei. In particular, fusion partners often form morphologically distinct actin-based linkages that allow two cells to exchange lipids and proteins before membrane fusion. However, the origin of this heterogeneity remains elusive. On the other hand, osteoclast multinucleation is sensitive to the environmental cues. Such cues promote the reorganization of the actin cytoskeleton, especially the formation and transformation of the podosome, an actin-rich punctate adhesion. This review covers the heterogeneity of osteoclast multinucleation at the pre-fusion stage with reference to the environment-dependent signaling pathway responsible for reorganizing the actin cytoskeleton. Furthermore, we compare osteoclast multinucleation with macrophage fusion, which results in multinucleated giant macrophages.