Next Generation SICLOPPS Screening for the Identification of Inhibitors of the HIF-1α/HIF-1ß Protein-Protein Interaction.

Split-intein circular ligation of proteins and peptides (SICLOPPS) is a method for generating intracellular libraries of cyclic peptides that has yielded several first-in-class inhibitors. Here, we detail a revised high-content, high-throughput SICLOPPS screening protocol that utilizes next-generation sequencing, biopanning, and computational tools to identify hits against a given protein-protein interaction. We used this platform for the identification of inhibitors of the HIF-1α/HIF-1ß protein-protein interaction. The revised platform resulted in a significantly higher positive hit rate than that previously reported for SICLOPPS screens, and the identified cyclic peptides were more active in vitro and in cells than our previously reported inhibitors. The platform detailed here may be used for the identification of inhibitors of a wide range of other targets.

Effect of Temperature-Induced Aging on the Gas Permeation Behavior of Thin Film Composite Membranes of PIM-1 and Carboxylated PIM-1.

Polymers of intrinsic microporosity (PIMs) are a class of promising gas separation materials due to their high membrane permeabilities and reasonable selectivities. When processed into thin film composite (TFC) membranes, their high gas throughput aligns closely with industrial requirements, but they are prone to physical aging and plasticization effects. TFC membranes based on the prototypical PIM-1 and its carboxylated derivative cPIM-1 exhibit temperature-dependent gas permeation behavior, which has not been extensively studied before. In single CO2 permeation tests, measurable physical aging occurred when the temperature was raised to 55 °C within a period of 90 min, and the aging rate accelerated as temperature was raised further. TFC membranes prepared from cPIM-1 exhibited a faster aging rate compared to PIM-1 at the same temperature. The decreased permeance could be at least partially recovered through a 5 day methanol vapor treatment. In mixed gas experiments, all membranes showed decreased permselectivities at elevated temperatures. The plasticization pressure of TFC membranes occurred at around 1 bar of CO2 partial pressure, independent of temperature. Significant plasticization was particularly evident for cPIM-1 TFC membranes under CO2/CH4 conditions with increasing temperature, which resulted in increased gas permeance for both components.

Intergenerational Power Shift and the Rise of Nonarranged Marriages Among Refugees.

The experiences of war and refugee status can alter intrafamily dynamics, with implications for family formation, including marriage. We use the nationally representative Syrian Migrant Sample of the 2018 Turkey Demographic Health Survey (TDHS-S) to conduct a duration analysis of marriage outcomes among Syrian refugees in Turkey, tracking women throughout their residence in prewar Syria (before the conflict began in 2011), postwar Syria (after the conflict began but before arrival in Turkey), and Turkey. We find that early marriage was more prominent among refugees who were unmarried at the time of migration than among those married before migration; the mean marriage age dropped from 19.6 in prewar Syria to 19.1 in postwar Syria and 18.1 in Turkey. Using the TDHS-S and prewar Syrian surveys, we show that this finding aligns with the observed declines in household income and young women's opportunity cost of marriage. Our duration analysis also reveals a notable shift from traditional arranged marriages to more modern marriage forms among refugees in Turkey. An intergenerational power shift might drive the shift toward nonarranged marriages. After arrival in Turkey, wealth and employment of parents decline among refugees. In contrast, Syrian youth in Turkey have higher age-adjusted employment rates than in prewar Syria. Moreover, nonarranged marriages increase more among demographic groups with stronger intergenerational power shifts than among groups with weaker shifts.

Molecular Mobility and Gas Transport Properties of Mixed Matrix Membranes Based on PIM-1 and a Phosphinine Containing Covalent Organic Framework.

Polymers with intrinsic microporosity (PIMs) are gaining attention as gas separation membranes. Nevertheless, they face limitations due to their pronounced physical aging. In this study, a covalent organic framework containing λ5-phosphinine moieties, CPSF-EtO, was incorporated as a nanofiller (concentration range 0-10 wt %) into a PIM-1 matrix forming dense films with a thickness of ca. 100 µm. The aim of the investigation was to investigate possible enhancements of gas transport properties and mitigating effects on physical aging. The incorporation of the nanofiller occurred on an nanoaggregate level with domains up to 100 nm, as observed by T-SEM and confirmed by X-ray scattering. Moreover, the X-ray data show that the structure of the microporous network of the PIM-1 matrix is changed by the nanofiller. As molecular mobility is fundamental for gas transport as well as for physical aging, the study includes dielectric investigations of pure PIM-1 and PIM-1/CPSF-EtO mixed matrix membranes to establish a correlation between the molecular mobility and the gas transport properties. Using the time-lag method, the gas permeability and the permselectivity were determined for N2, O2, CH4, and CO2 for samples with variation in filler content. A significant increase in the permeability of CH4 and CO2 (50% increase compared to pure PIM-1) was observed for a concentration of 5 wt % of the nanofiller. Furthermore, the most pronounced change in the permselectivity was found for the gas pair CO2/N2 at a filler concentration of 7 wt %.

Purcell-enhanced single photons at telecom wavelengths from a quantum dot in a photonic crystal cavity.

Quantum dots are promising candidates for telecom single photon sources due to their tunable emission across the different low-loss telecommunications bands, making them compatible with existing fiber networks. Their suitability for integration into photonic structures allows for enhanced brightness through the Purcell effect, supporting efficient quantum communication technologies. Our work focuses on InAs/InP QDs created via droplet epitaxy MOVPE to operate within the telecoms C-band. We observe a short radiative lifetime of 340 ps, arising from a Purcell factor of 5, owing to integration of the QD within a low-mode-volume photonic crystal cavity. Through in-situ control of the sample temperature, we show both temperature tuning of the QD's emission wavelength and a preserved single photon emission purity at temperatures up to 25K. These findings suggest the viability of QD-based, cryogen-free C-band single photon sources, supporting applicability in quantum communication technologies.

High Gas Permeability in Aged Superglassy Membranes with Nanosized UiO-66-NH2 /cPIM-1 Network Fillers.

Superglassy membranes synthesised by polymers of intrinsic microporosity (PIMs) suffer from physical aging and show poor gas permeance over time, especially thin membranes, due to the fast rearrangement of nonequilibrium polymer chains. Herein, we constructed a novel PIM-1 thin film nanocomposite membrane (TFN) using nanosized UiO-66-NH2 (≈10 nm)/carboxylated PIM-1 (cPIM-1) as the composite filler. Unlike conventional fillers, which interact with the polymer only via the surface, the UiO-66-NH2 /cPIM-1 forms a stable three-dimensional (3D) network intertwining with the polymer chains, being very effective to impede chain relaxation, and thus physical aging. Nanosizing of UiO-66-NH2 was achieved by regulating the nucleation kinetics using carbon quantum dots (CQD) during the synthesis. This led to increased surface area, and hence more functional groups to bond with cPIM-1 (via hydrogen bonding between -NH2 and -COOH groups), which also improved interfacial compatibility between the 3D network and polymer chains avoiding defect formation. As a result, the novel TFN showed significantly improved performance in gas separation along with reduced aging (i.e. ≈6 % loss in CO2 permeability over 63 days); the aged membranes had a CO2 permeance of 2504 GPU and ideal selectivity values of 37.2 and 23.8 for CO2 /N2 and CO2 /CH4 , respectively.

A population-based epidemiological and health economic analysis of fracture-related infection.

Aims: The aim of this study was to perform the first population-based description of the epidemiological and health economic burden of fracture-related infection (FRI). Methods: This is a retrospective cohort study of operatively managed orthopaedic trauma patients from 1 January 2007 to 31 December 2016, performed in Queensland, Australia. Record linkage was used to develop a person-centric, population-based dataset incorporating routinely collected administrative, clinical, and health economic information. The FRI group consisted of patients with International Classification of Disease 10th Revision diagnosis codes for deep infection associated with an implanted device within two years following surgery, while all others were deemed not infected. Demographic and clinical variables, as well as healthcare utilization costs, were compared. Results: There were 111,402 patients operatively managed for orthopaedic trauma, with 2,775 of these (2.5%) complicated by FRI. The development of FRI had a statistically significant association with older age, male sex, residing in rural/remote areas, Aboriginal or Torres Strait Islander background, lower socioeconomic status, road traffic accident, work-related injuries, open fractures, anatomical region (lower limb, spine, pelvis), high injury severity, requiring soft-tissue coverage, and medical comorbidities (univariate analysis). Patients with FRI had an eight-times longer median inpatient length of stay (24 days vs 3 days), and a 2.8-times higher mean estimated inpatient hospitalization cost (AU$56,565 vs AU$19,773) compared with uninfected patients. The total estimated inpatient cost of the FRI cohort to the healthcare system was AU$156.9 million over the ten-year period. Conclusion: The results of this study advocate for improvements in trauma care and infection management, address social determinants of health, and highlight the upside potential to improve prevention and treatment strategies.

Two Distinct Thermodynamic Gradients for Cellular Metalation of Vitamin B12.

The acquisition of CoII by the corrin component of vitamin B12 follows one of two distinct pathways, referred to as early or late CoII insertion. The late insertion pathway exploits a CoII metallochaperone (CobW) from the COG0523 family of G3E GTPases, while the early insertion pathway does not. This provides an opportunity to contrast the thermodynamics of metalation in a metallochaperone-requiring and a metallochaperone-independent pathway. In the metallochaperone-independent route, sirohydrochlorin (SHC) associates with the CbiK chelatase to form CoII-SHC. CoII-buffered enzymatic assays indicate that SHC binding enhances the thermodynamic gradient for CoII transfer from the cytosol to CbiK. In the metallochaperone-dependent pathway, hydrogenobyrinic acid a,c-diamide (HBAD) associates with the CobNST chelatase to form CoII-HBAD. Here, CoII-buffered enzymatic assays indicate that CoII transfer from the cytosol to HBAD-CobNST must somehow traverse a highly unfavorable thermodynamic gradient for CoII binding. Notably, there is a favorable gradient for CoII transfer from the cytosol to the MgIIGTP-CobW metallochaperone, but further transfer of CoII from the GTP-bound metallochaperone to the HBAD-CobNST chelatase complex is thermodynamically unfavorable. However, after nucleotide hydrolysis, CoII transfer from the chaperone to the chelatase complex is calculated to become favorable. These data reveal that the CobW metallochaperone can overcome an unfavorable thermodynamic gradient for CoII transfer from the cytosol to the chelatase by coupling this process to GTP hydrolysis.

Genomic surveillance uncovers a pandemic clonal lineage of the wheat blast fungus.

Wheat, one of the most important food crops, is threatened by a blast disease pandemic. Here, we show that a clonal lineage of the wheat blast fungus recently spread to Asia and Africa following two independent introductions from South America. Through a combination of genome analyses and laboratory experiments, we show that the decade-old blast pandemic lineage can be controlled by the Rmg8 disease resistance gene and is sensitive to strobilurin fungicides. However, we also highlight the potential of the pandemic clone to evolve fungicide-insensitive variants and sexually recombine with African lineages. This underscores the urgent need for genomic surveillance to track and mitigate the spread of wheat blast outside of South America and to guide preemptive wheat breeding for blast resistance.

Rgs1 is a regulator of effector gene expression during plant infection by the rice blast fungus Magnaporthe oryzae.

To cause rice blast disease, the filamentous fungus Magnaporthe oryzae secretes a battery of effector proteins into host plant tissue to facilitate infection. Effector-encoding genes are expressed only during plant infection and show very low expression during other developmental stages. How effector gene expression is regulated in such a precise manner during invasive growth by M. oryzae is not known. Here, we report a forward-genetic screen to identify regulators of effector gene expression, based on the selection of mutants that show constitutive effector gene expression. Using this simple screen, we identify Rgs1, a regulator of G-protein signaling (RGS) protein that is necessary for appressorium development, as a novel transcriptional regulator of effector gene expression, which acts prior to plant infection. We show that an N-terminal domain of Rgs1, possessing transactivation activity, is required for effector gene regulation and acts in an RGS-independent manner. Rgs1 controls the expression of at least 60 temporally coregulated effector genes, preventing their transcription during the prepenetration stage of development prior to plant infection. A regulator of appressorium morphogenesis is therefore also required for the orchestration of pathogen gene expression required for invasive growth by M. oryzae during plant infection.

Crosslinking of Branched PIM-1 and PIM-Py Membranes for Recovery of Toluene from Dimethyl Sulfoxide by Pervaporation.

Branched forms of the archetypal polymer of intrinsic microporosity PIM-1 and the pyridinecarbonitrile-containing PIM-Py may be crosslinked under ambient conditions by palladium(II) acetate. Branched PIM-1 can arise in polymerizations of 5,5',6,6'-tetrahydroxy-3,3,3',3'-tetramethyl-1,1'-spirobisindane with tetrafluoroterephthalonitrile conducted at a high set temperature (160 °C) under conditions, such as high dilution, that lead to a lower-temperature profile over the course of the reaction. Membranes of PIM-1 and PIM-Py crosslinked with palladium acetate are sufficiently stable in organic solvents for use in the recovery of toluene from its mixture with dimethyl sulfoxide (DMSO) by pervaporation at 65 °C. With both PIM-1 and PIM-Py membranes, pervaporation gives high toluene/DMSO separation factors (around 10 with a 77 vol % toluene feed). Detailed analysis shows that the membranes themselves are slightly selective for DMSO and it is the high driving force for toluene evaporation that drives the separation.

The transcriptional landscape of plant infection by the rice blast fungus Magnaporthe oryzae reveals distinct families of temporally co-regulated and structurally conserved effectors.

The rice blast fungus Magnaporthe oryzae causes a devastating disease that threatens global rice (Oryza sativa) production. Despite intense study, the biology of plant tissue invasion during blast disease remains poorly understood. Here we report a high-resolution transcriptional profiling study of the entire plant-associated development of the blast fungus. Our analysis revealed major temporal changes in fungal gene expression during plant infection. Pathogen gene expression could be classified into 10 modules of temporally co-expressed genes, providing evidence for the induction of pronounced shifts in primary and secondary metabolism, cell signaling, and transcriptional regulation. A set of 863 genes encoding secreted proteins are differentially expressed at specific stages of infection, and 546 genes named MEP (Magnaportheeffector protein) genes were predicted to encode effectors. Computational prediction of structurally related MEPs, including the MAX effector family, revealed their temporal co-regulation in the same co-expression modules. We characterized 32 MEP genes and demonstrate that Mep effectors are predominantly targeted to the cytoplasm of rice cells via the biotrophic interfacial complex and use a common unconventional secretory pathway. Taken together, our study reveals major changes in gene expression associated with blast disease and identifies a diverse repertoire of effectors critical for successful infection.

Methanol Vapor Retards Aging of PIM-1 Thin Film Composite Membranes in Storage.

Physical aging of glassy polymers leads to a decrease in permeability over time when they are used in membranes. This hinders the industrial application of high free volume polymers, such as the archetypal polymer of intrinsic microporosity PIM-1, for membrane gas separation. In thin film composite (TFC) membranes, aging is much more rapid than in thicker self-standing membranes, as rearrangement within the thin active layer is relatively fast. Liquid alcohol treatment, which swells the membrane, is often used in the laboratory to rejuvenate aged self-standing membranes, but this is not easily applied on an industrial scale and is not suitable to refresh TFC membranes because of the risk of membrane delamination. In this work, it is demonstrated that a simple method of storage in an atmosphere of methanol vapor effectively retards physical aging of PIM-1 TFC membranes. The same method can also be utilized to refresh aged PIM-1 TFC membranes, and one-week methanol vapor storage is sufficient to recover most of the original CO2 permeance.

Establishing Recovery Colleges in mental healthcare services: A Royal Commission recommendation.

Recovery Colleges are an innovative education-based approach to support mental health recovery that, following the recent Royal Commission, will have to be established in every area mental health service within the state of Victoria. This paper describes the rationale, benefits and some of the key considerations to successfully establish Recovery Colleges. The establishment of Recovery Colleges has the potential to drive culture change within mental health services and embed recovery orientation within service provision as well as engaging service users in their own recovery journey. There are significant challenges, however, in implementing the collaborative, co-produced model within the constraints of a publicly funded mental health clinical service. This paper considers some of the practice implications for public mental health services in developing and integrating Recovery Colleges. The paper, like everything we do at the Recovery College, is co-produced and co-authored - in this case, by a lived experience expert, a medically trained expert and a research/writing expert.

Metalation calculators for E. coli strain JM109 (DE3): aerobic, anaerobic, and hydrogen peroxide exposed cells cultured in LB media.

Three Web-based calculators, and three analogous spreadsheets, have been generated that predict in vivo metal occupancies of proteins based on known metal affinities. The calculations exploit estimates of the availabilities of the labile buffered pools of different metals inside a cell. Here, metal availabilities have been estimated for a strain of Escherichia coli that is commonly used in molecular biology and biochemistry research, e.g. in the production of recombinant proteins. Metal availabilities have been examined for cells grown in Luria-Bertani (LB) medium aerobically, anaerobically, and in response to H2O2 by monitoring the abundance of a selected set of metal-responsive transcripts by quantitative polymerase chain reaction (qPCR). The selected genes are regulated by DNA-binding metal sensors that have been thermodynamically characterized in related bacterial cells enabling gene expression to be read out as a function of intracellular metal availabilities expressed as free energies for forming metal complexes. The calculators compare these values with the free energies for forming complexes with the protein of interest, derived from metal affinities, to estimate how effectively the protein can compete with exchangeable binding sites in the intracellular milieu. The calculators then inter-compete the different metals, limiting total occupancy of the site to a maximum stoichiometry of 1, to output percentage occupancies with each metal. In addition to making these new and conditional calculators available, an original purpose of this article was to provide a tutorial that discusses constraints of this approach and presents ways in which such calculators might be exploited in basic and applied research, and in next-generation manufacturing.

An Antibiotic-Loaded Hydrogel Demonstrates Efficacy as Prophylaxis and Treatment in a Large Animal Model of Orthopaedic Device-Related Infection.

Local antibiotic therapy is increasingly being recognised for its role in preventing and treating orthopaedic device-related infection (ODRI). A bioresorbable, injectable gentamicin-loaded hydrogel has been developed to deliver local antibiotics at the time of surgery with potential for both prevention and treatment of ODRI. In a prophylaxis model, the antibiotic hydrogel was compared with systemic perioperative antibiotic prophylaxis alone in twelve sheep (six per group) at the time of intramedullary (IM) nail insertion to the tibia, which was inoculated with methicillin-sensitive Staphylococcus aureus (MSSA). In a treatment model of single-stage revision surgery, adjunctive antibiotic-loaded hydrogel was compared with systemic antibiotics alone in a single stage revision of MSSA infection associated with a tibia intramedullary nail in eleven sheep (five/six per group). The primary endpoint was quantitative microbiological results of soft tissue, bone and sonicate fluid from explanted hardware at the time of euthanasia. At euthanasia, the control sheep that received no local antibiotics in the prophylaxis model were all culture-positive (median 1x108, range 7x106-3x108 colony forming units, CFU) while only two of six sheep receiving local gentamicin had any culture positive biopsies (median 1x101, range 0 - 1x105 CFU). For the treatment model, sheep receiving only systemic antibiotics were all culture-positive (median 8x105, range 2x103- 9x106 CFU) while only two of six sheep treated with gentamicin-loaded hydrogel had any culture positive biopsies (median 3x102, range 0 - 7x104 CFU). Local gentamicin concentrations measured in extracellular fluid in the tibial canal show a burst release of gentamicin from the hydrogel. Serum gentamicin concentrations peaked in both models at one day post application and were below detection limit thereafter. This study has demonstrated the effective use of a locally delivered antibiotic hydrogel for both the prevention and treatment of ODRI that is superior to that of systemic antibiotics alone. Future studies will endeavour to translate from preclinical to clinical research trials.

A bibliometric analysis of clinical research on fracture-related infection.

Background: Infection following orthopaedic trauma surgery is increasingly recognized as one of the major research priorities with as primary goal, improving patient care. This increased interest has been anecdotally recognized through published research, research grants, and, finally, with the development of the fracture-related infection (FRI) consensus group. In 2017, the accepted consensus definition of FRI was published, which has been followed by consensus recommendations from both a surgical and medical perspective. A bibliometric analysis was performed to objectively describe the trends in published clinical research related to FRI. Methods: The terms related to FRI were searched in the Web of Science database between 2000 and 2020. The characteristics of clinical research on FRI regarding the author, country, journal, institution, scientific output, top 100 most cited articles, and trend topics were analyzed using Bibliometrix and WPS Office. Results: A total of 2597 records were eligible for inclusion in this bibliometric approach, with studies originating from 89 countries, including eight languages. The United States of America (USA) published the highest number of articles and citations. International collaborations were present between 72 countries, with the most active country being the USA. The most contributive institution was the University of California. The highest number of papers and citations were from the Injury-International Journal of the Care of the Injured and the Journal of Orthopaedic Trauma. The top 100 most cited articles were published in 27 different journals, with the number of citations ranging between 97 and 1004. The latest trend topics were related to the diagnosis of FRI. Conclusion: The present bibliometric analysis shows the research characteristics and trends of FRI from multiple perspectives. The fact that there is an increasing number of studies being published on FRI shows the agreement among scientists and clinicians that standardization with respect to this topic is very important.

Engineering Chiral Light-Matter Interactions in a Waveguide-Coupled Nanocavity.

Spin-dependent, directional light-matter interactions form the basis of chiral quantum networks. In the solid state, quantum emitters commonly possess circularly polarized optical transitions with spin-dependent handedness. We demonstrate numerically that spin-dependent chiral coupling can be realized by embedding such an emitter in a waveguide-coupled nanocavity, which supports two near-degenerate, orthogonally polarized cavity modes. The chiral behavior arises due to direction-dependent interference between the cavity modes upon coupling to two single-mode output waveguides. Notably, an experimentally realistic cavity design simultaneously supports near-unity chiral contrast, efficient (>95%) cavity-waveguide coupling and enhanced light-matter interaction strength (Purcell factor F P > 70). In combination, these parameters enable the development of highly coherent spin-photon interfaces ready for integration into nanophotonic circuits.