Decoding Arabidopsis thaliana CPK/SnRK Superfamily Kinase Client Signaling Networks Using Peptide Library and Mass Spectrometry.

Members of the calcium-dependent protein kinase (CDPK/CPK) and SNF-related protein kinase (SnRK) superfamilies are commonly found in plants and some protists. Our knowledge of client specificity of the members of this superfamily is fragmentary. As this family is represented by over 30 members in Arabidopsis thaliana, the identification of kinase-specific and overlapping client relationships is crucial to our understanding the nuances of this large family of kinases as directed towards signal transduction pathways. Herein, we used the kinase client (KiC) assay-a relative, quantitative, high-throughput mass spectrometry-based in vitro phosphorylation assay-to identify and characterize potential CPK/SnRK targets of Arabidopsis. Eight CPKs (1, 3, 6, 8, 17, 24, 28, and 32), four SnRKs (subclass 1 and 2), and PPCK1 and PPCK2 were screened against a synthetic peptide library that contains 2095 peptides and 2661 known phosphorylation sites. A total of 625 in vitro phosphorylation sites corresponding to 203 non-redundant proteins were identified. The most promiscuous kinase, CPK17, had 105 candidate target proteins, many of which had already been discovered. Sequence analysis of the identified phosphopeptides revealed four motifs: LxRxxS, RxxSxxR, RxxS, and LxxxxS, that were significantly enriched among CPK/SnRK clients. The results provide insight into both CPK- and SnRK-specific and overlapping signaling network architectures and recapitulate many known in vivo relationships validating this large-scale approach towards discovering kinase targets.

Insights into the ISG15 transfer cascade by the UBE1L activating enzyme.

The attachment of the ubiquitin-like protein ISG15 to substrates by specific E1-E2-E3 enzymes is a well-established signalling mechanism of the innate immune response. Here, we present a 3.45 Å cryo-EM structure of a chemically trapped UBE1L-UBE2L6 complex bound to activated ISG15. This structure reveals the details of the first steps of ISG15 recognition and UBE2L6 recruitment by UBE1L (also known as UBA7). Taking advantage of viral effector proteins from severe acute respiratory coronavirus 2 (SARS-CoV-2) and influenza B virus (IBV), we validate the structure and confirm the importance of the ISG15 C-terminal ubiquitin-like domain in the adenylation reaction. Moreover, biochemical characterization of the UBE1L-ISG15 and UBE1L-UBE2L6 interactions enables the design of ISG15 and UBE2L6 mutants with altered selectively for the ISG15 and ubiquitin conjugation pathways. Together, our study helps to define the molecular basis of these interactions and the specificity determinants that ensure the fidelity of ISG15 signalling during the antiviral response.

ISGylation-independent protection of cell growth by USP18 following interferon stimulation.

Type 1 interferon stimulation highly up-regulates all elements of a ubiquitin-like conjugation system that leads to ISGylation of target proteins. An ISG15-specific member of the deubiquitylase family, USP18, is up-regulated in a co-ordinated manner. USP18 can also provide a negative feedback by inhibiting JAK-STAT signalling through protein interactions independently of DUB activity. Here, we provide an acute example of this phenomenon, whereby the early expression of USP18, post-interferon treatment of HCT116 colon cancer cells is sufficient to fully suppress the expression of the ISG15 E1 enzyme, UBA7. Stimulation of lung adenocarcinoma A549 cells with interferon reduces their growth rate but they remain viable. In contrast, A549 USP18 knock-out cells show similar growth characteristics under basal conditions, but upon interferon stimulation, a profound inhibition of cell growth is observed. We show that this contingency on USP18 is independent of ISGylation, suggesting non-catalytic functions are required for viability. We also demonstrate that global deISGylation kinetics are very slow compared with deubiquitylation. This is not influenced by USP18 expression, suggesting that enhanced ISGylation in USP18 KO cells reflects increased conjugating activity.

Self-esteem's impacts on intimacy-building: Pathways through self-disclosure and responsiveness.

Building intimate relationships is rewarding but entails risking rejection. Trait self-esteem-a person's overall self-evaluation-has important implications for how people behave in socially risky situations. Integrating established models of responsiveness and intimacy with theory and research on self-esteem, we present a model that highlights the ways in which self-esteem impacts intimacy-building. A review of relevant research reveals that compared to people with high self-esteem, people with low self-esteem exhibit interpersonal perceptions and behaviors that can hinder intimacy development-for example, disclosing less openly, and eliciting and perceiving less responsiveness from others. We identify important directions for future research and consider methods for encouraging intimacy-promoting processes among people with low self-esteem.

Pregnancy and neonatal outcomes of COVID-19: The PAN-COVID study.

OBJECTIVE: To assess perinatal outcomes for pregnancies affected by suspected or confirmed SARS-CoV-2 infection. METHODS: Prospective, web-based registry. Pregnant women were invited to participate if they had suspected or confirmed SARS-CoV-2 infection between 1st January 2020 and 31st March 2021 to assess the impact of infection on maternal and perinatal outcomes including miscarriage, stillbirth, fetal growth restriction, pre-term birth and transmission to the infant. RESULTS: Between April 2020 and March 2021, the study recruited 8239 participants who had suspected or confirmed SARs-CoV-2 infection episodes in pregnancy between January 2020 and March 2021. Maternal death affected 14/8197 (0.2%) participants, 176/8187 (2.2%) of participants required ventilatory support. Pre-eclampsia affected 389/8189 (4.8%) participants, eclampsia was reported in 40/ 8024 (0.5%) of all participants. Stillbirth affected 35/8187 (0.4 %) participants. In participants delivering within 2 weeks of delivery 21/2686 (0.8 %) were affected by stillbirth compared with 8/4596 (0.2 %) delivering ≥ 2 weeks after infection (95 % CI 0.3-1.0). SGA affected 744/7696 (9.3 %) of livebirths, FGR affected 360/8175 (4.4 %) of all pregnancies. Pre-term birth occurred in 922/8066 (11.5%), the majority of these were indicated pre-term births, 220/7987 (2.8%) participants experienced spontaneous pre-term births. Early neonatal deaths affected 11/8050 livebirths. Of all neonates, 80/7993 (1.0%) tested positive for SARS-CoV-2. CONCLUSIONS: Infection was associated with indicated pre-term birth, most commonly for fetal compromise. The overall proportions of women affected by SGA and FGR were not higher than expected, however there was the proportion affected by stillbirth in participants delivering within 2 weeks of infection was significantly higher than those delivering ≥ 2 weeks after infection. We suggest that clinicians' threshold for delivery should be low if there are concerns with fetal movements or fetal heart rate monitoring in the time around infection. The proportion affected by pre-eclampsia amongst participants was not higher than would be expected, although we report a higher than expected proportion affected by eclampsia. There appears to be no effect on birthweight or congenital malformations in women affected by SARS-CoV-2 infection in pregnancy and neonatal infection is uncommon. This study reflects a population with a range of infection severity for SARS-COV-2 in pregnancy, generalisable to whole obstetric populations.

Contribution of cerebral microvascular mechanisms to age-related cognitive impairment and dementia.

Cognitive impairment and dementia are significant health burdens worldwide. Aging, hypertension, and diabetes are the primary risk factors for Alzheimer's disease and Alzheimer's disease and related dementias (AD/ADRD). There are no effective treatments for AD/ADRD to date. An emerging body of evidence indicates that cerebral vascular dysfunction and hypoperfusion precedes the development of other AD pathological phenotypes and cognitive impairment. However, vascular contribution to dementia is not currently well understood. This commentary highlights the emerging concepts and mechanisms underlying the microvascular contribution to AD/ADRD, including hypotheses targeting the anterograde and retrograde cerebral vascular pathways, as well as the cerebral capillaries and the venous system. We also briefly discuss vascular endothelial dysfunction, oxidative stress, inflammation, and cellular senescence that may contribute to impaired cerebral blood flow autoregulation, neurovascular uncoupling, and dysfunction of cerebral capillaries and the venous system.

The adducin saga: pleiotropic genomic targets for precision medicine in human hypertension-vascular, renal, and cognitive diseases.

Hypertension is a leading risk factor for stroke, heart disease, chronic kidney disease, vascular cognitive impairment, and Alzheimer's disease. Previous genetic studies have nominated hundreds of genes linked to hypertension, and renal and cognitive diseases. Some have been advanced as candidate genes by showing that they can alter blood pressure or renal and cerebral vascular function in knockout animals; however, final validation of the causal variants and underlying mechanisms has remained elusive. This review chronicles 40 years of work, from the initial identification of adducin (ADD) as an ACTIN-binding protein suggested to increase blood pressure in Milan hypertensive rats, to the discovery of a mutation in ADD1 as a candidate gene for hypertension in rats that were subsequently linked to hypertension in man. More recently, a recessive K572Q mutation in ADD3 was identified in Fawn-Hooded Hypertensive (FHH) and Milan Normotensive (MNS) rats that develop renal disease, which is absent in resistant strains. ADD3 dimerizes with ADD1 to form functional ADD protein. The mutation in ADD3 disrupts a critical ACTIN-binding site necessary for its interactions with actin and spectrin to regulate the cytoskeleton. Studies using Add3 KO and transgenic strains, as well as a genetic complementation study in FHH and MNS rats, confirmed that the K572Q mutation in ADD3 plays a causal role in altering the myogenic response and autoregulation of renal and cerebral blood flow, resulting in increased susceptibility to hypertension-induced renal disease and cerebral vascular and cognitive dysfunction.

Observational study to estimate the proportion of surgical site infection following excision of ulcerated skin tumours (OASIS study).

BACKGROUND: Ulceration is a recognized risk factor for surgical site infection (SSI); however, the proportion of patients developing SSI after excision of an ulcerated skin cancer is unknown. AIM: To determine the proportion of participants with SSI after surgical excision of an ulcerated skin cancer. A secondary aim was to assess feasibility outcomes to inform the design of a randomized controlled trial to investigate the benefits and harms of perioperative antibiotics following excision of ulcerated tumours. METHODS: This was a multicentre, prospective, observational study of patients undergoing excision of an ulcerated skin cancer between March 2019 and March 2020. Prior to surgical excision, surface swabs of the ulcerated tumours of participants recruited from one centre were undertaken to determine organism growth. At 4 weeks after surgery, all participants were e-mailed or posted the Wound Healing Questionnaire (WHQ) to determine whether they had developed SSI. RESULTS: In total, 148 participants were recruited 105 (70.9%) males; mean ± SD age 77.1 ± 12.3 years. Primary outcome data were available for 116 (78.4%) participants, of whom 35 (30.2%) were identified as having an SSI using the WHQ with a cutoff score of 8, and 47 (40.5%) were identified with a cutoff score of 6. Using the modified WHQ in participants with wounds left to heal by secondary intention, 33 (28.4%) and 43 (37.1%) were identified to have SSI respectively. CONCLUSION: This prospective evaluation of SSI identified with the WHQ following excision of ulcerated skin cancers demonstrated a high proportion with SSI. The WHQ was acceptable to patients; however, further evaluation is required to ensure validity in assessing skin wounds.

Luseogliflozin, a sodium-glucose cotransporter-2 inhibitor, reverses cerebrovascular dysfunction and cognitive impairments in 18-mo-old diabetic animals.

Diabetes mellitus (DM) is a leading risk factor for age-related dementia, but the mechanisms involved are not well understood. We previously discovered that hyperglycemia induced impaired myogenic response (MR) and cerebral blood flow (CBF) autoregulation in 18-mo-old DM rats associated with blood-brain barrier (BBB) leakage, impaired neurovascular coupling, and cognitive impairment. In the present study, we examined whether reducing plasma glucose with a sodium-glucose cotransporter-2 inhibitor (SGLT2i) luseogliflozin can ameliorate cerebral vascular and cognitive function in diabetic rats. Plasma glucose and HbA1c levels of 18-mo-old DM rats were reduced, and blood pressure was not altered after treatment with luseogliflozin. SGLT2i treatment restored the impaired MR of middle cerebral arteries (MCAs) and parenchymal arterioles and surface and deep cortical CBF autoregulation in DM rats. Luseogliflozin treatment also rescued neurovascular uncoupling, reduced BBB leakage and cognitive deficits in DM rats. However, SGLT2i did not have direct constrictive effects on vascular smooth muscle cells and MCAs isolated from normal rats, although it decreased reactive oxygen species production in cerebral vessels of DM rats. These results provide evidence that normalization of hyperglycemia with an SGLT2i can reverse cerebrovascular dysfunction and cognitive impairments in rats with long-standing hyperglycemia, possibly by ameliorating oxidative stress-caused vascular damage.NEW & NOTEWORTHY This study demonstrates that luseogliflozin, a sodium-glucose cotransporter-2 inhibitor, improved CBF autoregulation in association with reduced vascular oxidative stress and AGEs production in the cerebrovasculature of 18-mo-old DM rats. SGLT2i also prevented BBB leakage, impaired functional hyperemia, neurodegeneration, and cognitive impairment seen in DM rats. Luseogliflozin did not have direct constrictive effects on VSMCs and MCAs isolated from normal rats. These results provide evidence that normalization of hyperglycemia with an SGLT2i can reverse cerebrovascular dysfunction and cognitive impairments in rats with long-standing hyperglycemia, possibly by ameliorating oxidative stress-caused vascular damage.

Oral Anticoagulation Discontinuation Following Catheter Ablation of Typical Atrial Flutter.

Catheter ablation (CA) of typical atrial flutter (AFL) is the preferred treatment for typical AFL due to its excellent long-term success rate. However, current guidelines recommend pursuing oral anticoagulation (OAC) based on established indices of stroke regardless of the perceived success of ablation. We conducted a retrospective study of all patients who underwent typical AFL ablation at our institute from 2011 to 2017. All patients continued OAC for at least six weeks post-CA and underwent 24-hour Holter monitoring. OAC was discontinued if there was no evidence of recurrence at six weeks. In patients with low left ventricular ejection fraction or prior atrial fibrillation episodes, OAC was continued for six months with repeat Holter monitoring at six months. A total of 106 patients were included in our analysis, with a mean age of 64 ± 14 years and 78.3% of whom were male. The mean CHA2DS2-VASc score was 3 ± 1 points. OAC was discontinued by six weeks in 17% and at one year in 55.7% of patients, respectively, but was continued indefinitely in 44.3%. Over a mean follow-up period of 28.6 ± 27.3 months, there was one ischemic stroke in the OAC discontinuation group and no ischemic events in the continued OAC group. There were a total of three major bleeding events, all in the OAC group. In patients undergoing successful AFL ablation, a strategy of OAC discontinuation with close rhythm monitoring appears feasible. The benefit of continued OAC in this cohort may be outweighed by an adverse risk of bleeding. Further studies examining rhythm-guided OAC can minimize unnecessary exposure to long-term anticoagulation.

DNA dosimeter measurements of beam profile using a novel simultaneous processing technique.

A DNA dosimeter (DNAd) was previously developed that uses double-strand breaks (DSB) to measure dose. This dosimeter has been tested to measure dose in scenarios where transient-charged particle equilibrium (TCPE) has been established. The probability of double strand break (PDSBo), which is the ratio of broken double-stranded DNA (dsDNA) to the initial unbroken dsDNA in the dosimeter, was used to quantify DSBs and related to dose. The goal of this work is to produce a new technique to process and analyze the DNAd and quantify DNA-DSBs. This technique included simultaneously processing multiple DNAds and also establishing a new form to the probability of double strand break (PDSBn), which was then used to test the DNAd in a non-TCPE condition by taking beam penumbra measurements. The technique utilized a 384-well plate, and the measurements were made at the edge of a 10 × 10 cm field and compared to film measurements. During these penumbra measurements, while observing the positional differences in the higher gradient region at 4.1 and 4.55 cm from the center of the radiation field, the distance to agreement of PDSBo to film were 0.38 cm and 0.26 cm while the distance to agreement of PDSBn to film were 0.11 cm and 0.06 cm, respectively. Finally, the developed new separation technique reduced the time needed for the analysis of 25 samples from 200 min to 30 min.

Dissecting distinct proteolytic activities of FMDV Lpro implicates cleavage and degradation of RLR signaling proteins, not its deISGylase/DUB activity, in type I interferon suppression.

The type I interferon response is an important innate antiviral pathway. Recognition of viral RNA by RIG-I-like receptors (RLRs) activates a signaling cascade that leads to type I interferon (IFN-α/ß) gene transcription. Multiple proteins in this signaling pathway (e.g. RIG-I, MDA5, MAVS, TBK1, IRF3) are regulated by (de)ubiquitination events. Most viruses have evolved mechanisms to counter this antiviral response. The leader protease (Lpro) of foot-and-mouth-disease virus (FMDV) has been recognized to reduce IFN-α/ß gene transcription; however, the exact mechanism is unknown. The proteolytic activity of Lpro is vital for releasing itself from the viral polyprotein and for cleaving and degrading specific host cell proteins, such as eIF4G and NF-κB. In addition, Lpro has been demonstrated to have deubiquitination/deISGylation activity. Lpro's deubiquitination/deISGylation activity and the cleavage/degradation of signaling proteins have both been postulated to be important for reduced IFN-α/ß gene transcription. Here, we demonstrate that TBK1, the kinase that phosphorylates and activates the transcription factor IRF3, is cleaved by Lpro in FMDV-infected cells as well as in cells infected with a recombinant EMCV expressing Lpro. In vitro cleavage experiments revealed that Lpro cleaves TBK1 at residues 692-694. We also observed cleavage of MAVS in HeLa cells infected with EMCV-Lpro, but only observed decreasing levels of MAVS in FMDV-infected porcine LFPK αVß6 cells. We set out to dissect Lpro's ability to cleave RLR signaling proteins from its deubiquitination/deISGylation activity to determine their relative contributions to the reduction of IFN-α/ß gene transcription. The introduction of specific mutations, of which several were based on the recently published structure of Lpro in complex with ISG15, allowed us to identify specific amino acid substitutions that separate the different proteolytic activities of Lpro. Characterization of the effects of these mutations revealed that Lpro's ability to cleave RLR signaling proteins but not its deubiquitination/deISGylation activity correlates with the reduced IFN-ß gene transcription.

The Tumour Suppressor TMEM127 Is a Nedd4-Family E3 Ligase Adaptor Required by Salmonella SteD to Ubiquitinate and Degrade MHC Class II Molecules.

The Salmonella enterica effector SteD depletes mature MHC class II (mMHCII) molecules from the surface of infected antigen-presenting cells through ubiquitination of the cytoplasmic tail of the mMHCII ß chain. Here, through a genome-wide mutant screen of human antigen-presenting cells, we show that the NEDD4 family HECT E3 ubiquitin ligase WWP2 and a tumor-suppressing transmembrane protein of unknown biochemical function, TMEM127, are required for SteD-dependent ubiquitination of mMHCII. Although evidently not involved in normal regulation of mMHCII, TMEM127 was essential for SteD to suppress both mMHCII antigen presentation in mouse dendritic cells and MHCII-dependent CD4+ T cell activation. We found that TMEM127 contains a canonical PPxY motif, which was required for binding to WWP2. SteD bound to TMEM127 and enabled TMEM127 to interact with and induce ubiquitination of mature MHCII. Furthermore, SteD also underwent TMEM127- and WWP2-dependent ubiquitination, which both contributed to its degradation and augmented its activity on mMHCII.

Global Landscape and Dynamics of Parkin and USP30-Dependent Ubiquitylomes in iNeurons during Mitophagic Signaling.

The ubiquitin ligase Parkin, protein kinase PINK1, USP30 deubiquitylase, and p97 segregase function together to regulate turnover of damaged mitochondria via mitophagy, but our mechanistic understanding in neurons is limited. Here, we combine induced neurons (iNeurons) derived from embryonic stem cells with quantitative proteomics to reveal the dynamics and specificity of Parkin-dependent ubiquitylation under endogenous expression conditions. Targets showing elevated ubiquitylation in USP30-/- iNeurons are concentrated in components of the mitochondrial translocon, and the ubiquitylation kinetics of the vast majority of Parkin targets are unaffected, correlating with a modest kinetic acceleration in accumulation of pS65-Ub and mitophagic flux upon mitochondrial depolarization without USP30. Basally, ubiquitylated translocon import substrates accumulate, suggesting a quality control function for USP30. p97 was dispensable for Parkin ligase activity in iNeurons. This work provides an unprecedented quantitative landscape of the Parkin-modified ubiquitylome in iNeurons and reveals the underlying specificity of central regulatory elements in the pathway.

Insights into ubiquitin chain architecture using Ub-clipping.

Protein ubiquitination is a multi-functional post-translational modification that affects all cellular processes. Its versatility arises from architecturally complex polyubiquitin chains, in which individual ubiquitin moieties may be ubiquitinated on one or multiple residues, and/or modified by phosphorylation and acetylation1-3. Advances in mass spectrometry have enabled the mapping of individual ubiquitin modifications that generate the ubiquitin code; however, the architecture of polyubiquitin signals has remained largely inaccessible. Here we introduce Ub-clipping as a methodology by which to understand polyubiquitin signals and architectures. Ub-clipping uses an engineered viral protease, Lbpro∗, to incompletely remove ubiquitin from substrates and leave the signature C-terminal GlyGly dipeptide attached to the modified residue; this simplifies the direct assessment of protein ubiquitination on substrates and within polyubiquitin. Monoubiquitin generated by Lbpro∗ retains GlyGly-modified residues, enabling the quantification of multiply GlyGly-modified branch-point ubiquitin. Notably, we find that a large amount (10-20%) of ubiquitin in polymers seems to exist as branched chains. Moreover, Ub-clipping enables the assessment of co-existing ubiquitin modifications. The analysis of depolarized mitochondria reveals that PINK1/parkin-mediated mitophagy predominantly exploits mono- and short-chain polyubiquitin, in which phosphorylated ubiquitin moieties are not further modified. Ub-clipping can therefore provide insight into the combinatorial complexity and architecture of the ubiquitin code.

Optimizing the response, precision, and cost of a DNA double-strand break dosimeter.

We developed a dosimeter that measures biological damage following delivery of therapeutic beams in the form of double-strand breaks (DSBs) to DNA. The dosimeter contains DNA strands that are labeled on one end with biotin and on the other with fluorescein and attached to magnetic microbeads. Following irradiation, a magnet is used to separate broken from unbroken DNA strands. Then, fluorescence is utilized to measure the relative amount of broken DNA and determine the probability for DSB. The long-term goal for this research is to evaluate whether this type of biologically based dosimeter holds any advantages over the conventional techniques. The purpose of this work was to optimize the dosimeter fabrication and usage to enable higher precision for the long-term research goal. More specifically, the goal was to optimize the DNA dosimeter using three metrics: the response, precision, and cost per dosimeter. Six aspects of the dosimeter fabrication and usage were varied and evaluated for their effect on the metrics: (1) the type of magnetic microbeads, (2) the microbead to DNA mass ratio at attachment, (3) the type of suspension buffer used during irradiation, (4) the concentration of the DNA dosimeter during irradiation, (5) the time waited between fabrication and irradiation of the dosimeter, and (6) the time waited between irradiation and read out of the response. In brief, the best results were achieved with the dosimeter when attaching 4.2 µg of DNA with 1 mg of MyOne T1 microbeads and by suspending the microbead-connected DNA strands with 200 µl of phosphate-buffered saline for irradiation. Also, better results were achieved when waiting a day after fabrication before irradiating the dosimeter and also waiting an hour after irradiation to measure the response. This manuscript is meant to serve as guide for others who would like to replicate this DNA dose measurement technique.

Etched ion tracks in amorphous SiO2 characterized by small angle x-ray scattering: influence of ion energy and etching conditions.

Small angle x-ray scattering was used to study the morphology of conical structures formed in thin films of amorphous SiO2. Samples were irradiated with 1.1 GeV Au ions at the GSI UNILAC in Darmstadt, Germany, and with 185, 89 and 54 MeV Au ions at the Heavy Ion Accelerator Facility at ANU in Canberra, Australia. The irradiated material was subsequently etched in HF using two different etchant concentrations over a series of etch times to reveal conically shaped etched channels of various sizes. Synchrotron based SAXS measurements were used to characterize both the radial and axial ion track etch rates with unprecedented precision. The results show that the ion energy has a significant effect on the morphology of the etched channels, and that at short etch times resulting in very small cones, the increased etching rate of the damaged region in the radial direction with respect to the ion trajectory is significant.

OTULIN deficiency in ORAS causes cell type-specific LUBAC degradation, dysregulated TNF signalling and cell death.

The deubiquitinase OTULIN removes methionine-1 (M1)-linked polyubiquitin signals conjugated by the linear ubiquitin chain assembly complex (LUBAC) and is critical for preventing TNF-driven inflammation in OTULIN-related autoinflammatory syndrome (ORAS). Five ORAS patients have been reported, but how dysregulated M1-linked polyubiquitin signalling causes their symptoms is unclear. Here, we report a new case of ORAS in which an OTULIN-Gly281Arg mutation leads to reduced activity and stability in vitro and in cells. In contrast to OTULIN-deficient monocytes, in which TNF signalling and NF-κB activation are increased, loss of OTULIN in patient-derived fibroblasts leads to a reduction in LUBAC levels and an impaired response to TNF Interestingly, both patient-derived fibroblasts and OTULIN-deficient monocytes are sensitised to certain types of TNF-induced death, and apoptotic cells are evident in ORAS patient skin lesions. Remarkably, haematopoietic stem cell transplantation leads to complete resolution of inflammatory symptoms, including fevers, panniculitis and diarrhoea. Therefore, haematopoietic cells are necessary for clinical manifestation of ORAS Together, our data suggest that ORAS pathogenesis involves hyper-inflammatory immune cells and TNF-induced death of both leukocytes and non-haematopoietic cells.

A Chlamydia effector combining deubiquitination and acetylation activities induces Golgi fragmentation.

Pathogenic bacteria are armed with potent effector proteins that subvert host signalling processes during infection1. The activities of bacterial effectors and their associated roles within the host cell are often poorly understood, particularly for Chlamydia trachomatis2, a World Health Organization designated neglected disease pathogen. We identify and explain remarkable dual Lys63-deubiquitinase (DUB) and Lys-acetyltransferase activities in the Chlamydia effector ChlaDUB1. Crystal structures capturing intermediate stages of each reaction reveal how the same catalytic centre of ChlaDUB1 can facilitate such distinct processes, and enable the generation of mutations that uncouple the two activities. Targeted Chlamydia mutant strains allow us to link the DUB activity of ChlaDUB1 and the related, dedicated DUB ChlaDUB2 to fragmentation of the host Golgi apparatus, a key process in Chlamydia infection for which effectors have remained elusive. Our work illustrates the incredible versatility of bacterial effector proteins, and provides important insights towards understanding Chlamydia pathogenesis.

A structural comparison of casein micelles in cow, goat and sheep milk using X-ray scattering.

The casein micelle is a flexible construct, with its key structural components being casein proteins and colloidal calcium phosphate nanoclusters. According to literature, milk from different species exhibits differences in composition and physicochemical properties. X-ray scattering techniques were used to investigate and compare the nanoscale structure of casein micelles present in cow, goat and sheep milk. Although there were differences in the size and density of larger scale protein structures, at an atomic level the protein structures were similar. There were also strong similarities in the structure of the calcium-containing nanoclusters, namely that they had similar sizes and separations within the casein micelle for all three species.