Dissecting the stability of Atezolizumab with renewable amino acid-based ionic liquids: Colloidal stability and anticancer activity under thermal stress.

Monoclonal antibodies (mAbs) have revolutionised the biopharmaceutical market. Being proteinaceous, mAbs are prone to chemical and physical instabilities. Various approaches were attempted to stabilise proteins against degradation factors. Ionic liquids (ILs) and deep eutectic solvents (DESs) have been established as green solvents for ever-increasing pharmaceutical and biopharmaceutical applications. Hence, amino acid (AA)-based ILs, were used for the first time, for mAb stabilisation. Choline (Ch)-based DESs were also utilised for comparison purposes. The prepared ILs and DESs were utilised to stabilise Atezolizumab (Amab, anti-PDL-1 mAb). The formulations of Amab in ILs and DESs were incubated at room temperature, 45 or 55 °C. Following this, the structural stability of Amab was appraised. Interestingly, Ch-Valine retained favourable structural stability of Amab with minimal detected aggregation or degradation as confirmed by UV-visible spectroscopy and protein Mass Spectroscopy. The measured hydrodynamic diameter of Amab in Ch-Valine ranged from 10.40 to 11.65 nm. More interestingly, the anticancer activity of Amab was evaluated, and Ch-Valine was found to be optimum in retaining the activity of Amab when compared to other formulations, including the control Amab sample. Collectively, this study has spotlighted the advantages of adopting the Ch-AA ILs for the structural and functional stabilising of mAbs.

VerSoX B07-B: a high-throughput XPS and ambient pressure NEXAFS beamline at Diamond Light Source.

The beamline optics and endstations at branch B of the Versatile Soft X-ray (VerSoX) beamline B07 at Diamond Light Source are described. B07-B provides medium-flux X-rays in the range 45-2200 eV from a bending magnet source, giving access to local electronic structure for atoms of all elements from Li to Y. It has an endstation for high-throughput X-ray photoelectron spectroscopy (XPS) and near-edge X-ray absorption fine-structure (NEXAFS) measurements under ultrahigh-vacuum (UHV) conditions. B07-B has a second endstation dedicated to NEXAFS at pressures from UHV to ambient pressure (1 atm). The combination of these endstations permits studies of a wide range of interfaces and materials. The beamline and endstation designs are discussed in detail, as well as their performance and the commissioning process.

Non-attendance at diabetic retinal screening in Te Tai Tokerau, Northland, Aotearoa New Zealand.

AIMS: To explore socio-demographic characteristics of non-attenders at diabetic retinal screening. METHODS: A retrospective, register-based cross-sectional analysis of 10,275 participants invited to diabetic retinal screening in Te Tai Tokerau (Northland) between May 2011 and June 2020 was performed. Multivariable logistic regression analysis was used to assess the association of age, sex, type of diabetes, ethnicity and socio-economic deprivation with non-attendance at diabetic retinal screening. RESULTS: Median age was 66 years and 54.3% of participants were male. The non-attendance rate was 26.4%, with 46.6% of individuals having at least one non-attendance. Younger age was associated with higher odds of non-attendance (OR 1.84 95% CI 1.41-2.40, <0.001 for odds of non-attendance in those aged under 35 years compared with age over 75 years). Maori (OR 2.69, 95% CI 2.44-2.96, p<0.001) and Pacific peoples (OR 1.71, 95% CI 1.25-2.36, p=0.001) had higher odds of non-attendance compared with NZ Europeans. People living in areas of high socio-economic deprivation had higher odds of non-attendance (OR 1.56, 95% CI 1.33-1.82, p<0.001), as did type 1 diabetics (OR 1.31, p5% CI 1.08-1.59, p=0.006). CONCLUSION: Younger age, socio-economic deprivation, type 1 diabetes and Maori and Pacific ethnicity are risk factors for nonattendance at diabetic retinal screening.

Enhanced detection of severe aortic stenosis via artificial intelligence: a clinical cohort study.

OBJECTIVE: We developed an artificial intelligence decision support algorithm (AI-DSA) that uses routine echocardiographic measurements to identify severe aortic stenosis (AS) phenotypes associated with high mortality. METHODS: 631 824 individuals with 1.08 million echocardiograms were randomly spilt into two groups. Data from 442 276 individuals (70%) entered a Mixture Density Network (MDN) model to train an AI-DSA to predict an aortic valve area <1 cm2, excluding all left ventricular outflow tract velocity or dimension measurements and then using the remainder of echocardiographic measurement data. The optimal probability threshold for severe AS detection was identified at the f1 score probability of 0.235. An automated feature also ensured detection of guideline-defined severe AS. The AI-DSA's performance was independently evaluated in 184 301 (30%) individuals. RESULTS: The area under receiver operating characteristic curve for the AI-DSA to detect severe AS was 0.986 (95% CI 0.985 to 0.987) with 4622/88 199 (5.2%) individuals (79.0±11.9 years, 52.4% women) categorised as 'high-probability' severe AS. Of these, 3566 (77.2%) met guideline-defined severe AS. Compared with the AI-derived low-probability AS group (19.2% mortality), the age-adjusted and sex-adjusted OR for actual 5-year mortality was 2.41 (95% CI 2.13 to 2.73) in the high probability AS group (67.9% mortality)-5-year mortality being slightly higher in those with guideline-defined severe AS (69.1% vs 64.4%; age-adjusted and sex-adjusted OR 1.26 (95% CI 1.04 to 1.53), p=0.021). CONCLUSIONS: An AI-DSA can identify the echocardiographic measurement characteristics of AS associated with poor survival (with not all cases guideline defined). Deployment of this tool in routine clinical practice could improve expedited identification of severe AS cases and more timely referral for therapy.

Staphylococcal Complement Evasion Protein Sbi Stabilises C3d Dimers by Inducing an N-Terminal Helix Swap.

Staphylococcus aureus is an opportunistic pathogen that is able to thwart an effective host immune response by producing a range of immune evasion molecules, including S. aureus binder of IgG (Sbi) which interacts directly with the central complement component C3, its fragments and associated regulators. Recently we reported the first structure of a disulfide-linked human C3d17C dimer and highlighted its potential role in modulating B-cell activation. Here we present an X-ray crystal structure of a disulfide-linked human C3d17C dimer, which undergoes a structurally stabilising N-terminal 3D domain swap when in complex with Sbi. These structural studies, in combination with circular dichroism and fluorescence spectroscopic analyses, reveal the mechanism underpinning this unique helix swap event and could explain the origins of a previously discovered N-terminally truncated C3dg dimer isolated from rat serum. Overall, our study unveils a novel staphylococcal complement evasion mechanism which enables the pathogen to harness the ability of dimeric C3d to modulate B-cell activation.

Discovery of Autoantibodies Targeting Nephrin in Minimal Change Disease Supports a Novel Autoimmune Etiology.

BACKGROUND: Failure of the glomerular filtration barrier, primarily by loss of slit diaphragm architecture, underlies nephrotic syndrome in minimal change disease. The etiology remains unknown. The efficacy of B cell-targeted therapies in some patients, together with the known proteinuric effect of anti-nephrin antibodies in rodent models, prompted us to hypothesize that nephrin autoantibodies may be present in patients with minimal change disease. METHODS: We evaluated sera from patients with minimal change disease, enrolled in the Nephrotic Syndrome Study Network (NEPTUNE) cohort and from our own institutions, for circulating nephrin autoantibodies by indirect ELISA and by immunoprecipitation of full-length nephrin from human glomerular extract or a recombinant purified extracellular domain of human nephrin. We also evaluated renal biopsies from our institutions for podocyte-associated punctate IgG colocalizing with nephrin by immunofluorescence. RESULTS: In two independent patient cohorts, we identified circulating nephrin autoantibodies during active disease that were significantly reduced or absent during treatment response in a subset of patients with minimal change disease. We correlated the presence of these autoantibodies with podocyte-associated punctate IgG in renal biopsies from our institutions. We also identified a patient with steroid-dependent childhood minimal change disease that progressed to end stage kidney disease; she developed a massive post-transplant recurrence of proteinuria that was associated with high pretransplant circulating nephrin autoantibodies. CONCLUSIONS: Our discovery of nephrin autoantibodies in a subset of adults and children with minimal change disease aligns with published animal studies and provides further support for an autoimmune etiology. We propose a new molecular classification of nephrin autoantibody minimal change disease to serve as a framework for instigation of precision therapeutics for these patients.

Insights Into the Structure-Function Relationships of Dimeric C3d Fragments.

Cleavage of C3 to C3a and C3b plays a central role in the generation of complement-mediated defences. Although the thioester-mediated surface deposition of C3b has been well-studied, fluid phase dimers of C3 fragments remain largely unexplored. Here we show C3 cleavage results in the spontaneous formation of C3b dimers and present the first X-ray crystal structure of a disulphide-linked human C3d dimer. Binding studies reveal these dimers are capable of crosslinking complement receptor 2 and preliminary cell-based analyses suggest they could modulate B cell activation to influence tolerogenic pathways. Altogether, insights into the physiologically-relevant functions of C3d(g) dimers gained from our findings will pave the way to enhancing our understanding surrounding the importance of complement in the fluid phase and could inform the design of novel therapies for immune system disorders in the future.

Targeting a Braf/Mapk pathway rescues podocyte lipid peroxidation in CoQ-deficiency kidney disease.

Mutations affecting mitochondrial coenzyme Q (CoQ) biosynthesis lead to kidney failure due to selective loss of podocytes, essential cells of the kidney filter. Curiously, neighboring tubular epithelial cells are spared early in disease despite higher mitochondrial content. We sought to illuminate noncanonical, cell-specific roles for CoQ, independently of the electron transport chain (ETC). Here, we demonstrate that CoQ depletion caused by Pdss2 enzyme deficiency in podocytes results in perturbations in polyunsaturated fatty acid (PUFA) metabolism and the Braf/Mapk pathway rather than ETC dysfunction. Single-nucleus RNA-Seq from kidneys of Pdss2kd/kd mice with nephrotic syndrome and global CoQ deficiency identified a podocyte-specific perturbation of the Braf/Mapk pathway. Treatment with GDC-0879, a Braf/Mapk-targeting compound, ameliorated kidney disease in Pdss2kd/kd mice. Mechanistic studies in Pdss2-depleted podocytes revealed a previously unknown perturbation in PUFA metabolism that was confirmed in vivo. Gpx4, an enzyme that protects against PUFA-mediated lipid peroxidation, was elevated in disease and restored after GDC-0879 treatment. We demonstrate broader human disease relevance by uncovering patterns of GPX4 and Braf/Mapk pathway gene expression in tissue from patients with kidney diseases. Our studies reveal ETC-independent roles for CoQ in podocytes and point to Braf/Mapk as a candidate pathway for the treatment of kidney diseases.

Can preoperative imaging characteristics predict pT3 bladder cancer following cystectomy?

PURPOSE: Imaging characteristics in bladder cancer (BC), such as hydronephrosis, are predictive of ≥ pT3 disease at time of radical cystectomy (RC). The predictive capacity of other findings, such as perivesical stranding (PS), remains unclear. We investigated whether PS was associated with ≥ pT3 BC in patients who did not receive neoadjuvant chemotherapy (NAC). METHODS: We identified 433 patients with BC who underwent RC from 2003 to 2018 of which 128 did not receive NAC. Evidence of PS on pre-TURBT imaging was determined by radiologist review and a stranding grading system was created. Factors associated with PS and hydronephrosis were identified. Multivariable logistic regressions evaluated PS and hydronephrosis as predictors for ≥ pT3 BC. RESULTS: Of the 128 patients who did not receive NAC, 48 (38%) had pT3 and 12 (9%) had pT4 BC. 125 (98%) patients had CT and three (2%) had MRI. PS and hydronephrosis on imaging were identified in 19 (15%) and 45 (35%) patients. PS was not associated with imaging type (p = 0.38), BMI (p = 0.18), or pathologic T stage (p = 0.24). Hydronephrosis was more frequently associated with higher pathologic T stage (p = 0.034). Multivariable analysis demonstrated that PS was not predictive of ≥ pT3 BC (p = 0.457), while hydronephrosis was positively associated (p = 0.003). Stratification by grade of stranding did not improve the predictive capacity of PS (p = 0.667). CONCLUSION: While hydronephrosis is an indicator of higher stage BC, PS failed to be a reliable predictor of ≥ pT3 stage. These observations should give pause in using PS on imaging to guide decisions until further investigations can be explored.

A High-Content Screen for Mucin-1-Reducing Compounds Identifies Fostamatinib as a Candidate for Rapid Repurposing for Acute Lung Injury.

Drug repurposing has the advantage of identifying potential treatments on a shortened timescale. In response to the pandemic spread of SARS-CoV-2, we took advantage of a high-content screen of 3,713 compounds at different stages of clinical development to identify FDA-approved compounds that reduce mucin-1 (MUC1) protein abundance. Elevated MUC1 levels predict the development of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) and correlate with poor clinical outcomes. Our screen identifies fostamatinib (R788), an inhibitor of spleen tyrosine kinase (SYK) approved for the treatment of chronic immune thrombocytopenia, as a repurposing candidate for the treatment of ALI. In vivo, fostamatinib reduces MUC1 abundance in lung epithelial cells in a mouse model of ALI. In vitro, SYK inhibition by the active metabolite R406 promotes MUC1 removal from the cell surface. Our work suggests fostamatinib as a repurposing drug candidate for ALI.

Endoscopic Closure of a Large Rectovesical Fistula Following Robotic Prostatectomy.

Background: Rectovesical fistulae (RVF) are uncommon complications of pelvic surgeries and are a potential cause of significant morbidity. RVF are not typically closed endoscopically but rather require reoperative surgery of the lower pelvis with closure of tract, interposition of fat or omentum, and possible permanent bowel diversion. We present a unique case of a rectovesical fistula developing after robotic prostatectomy that was managed by multimodal multistage endoscopic therapy as an alternative to conventional operative repair. Case Presentation: A healthy 78-year-old Caucasian man underwent a robot-assisted laparoscopic radical prostatectomy with bilateral pelvic lymph node dissection for high-risk adenocarcinoma of the prostate. The patient's postoperative course was complicated by an unrecognized rectal injury culminating in emergent exploration, abdominal washout, creation of a diverting loop transverse colostomy, and resultant development of a large rectovesical fistula. Given the patient's hostile abdomen and desire for conservative management the fistula was managed through a combined cystoscopic and endoscopic procedure that utilized suturing and clipping to close the fistula. This novel technique was followed by a series of three subsequent endoscopic procedures that enabled us to gradually downsize the fistula over time and ultimately achieve complete closure. The patient's colostomy was eventually reversed with return of bowel continuity. Conclusion: Although uncommon, RVF are significant complications of pelvic surgery. The presence of abdominal/pelvic adhesions from previous surgeries or patient comorbidities can make open surgical repair extremely challenging or impracticable. Therefore, it is important to recognize and consider the use of endoscopic techniques as potential options for closure of rectovesical fistula in certain situations.

Monoclonal antibody stability can be usefully monitored using the excitation-energy-dependent fluorescence edge-shift.

Among the major challenges in the development of biopharmaceuticals are structural heterogeneity and aggregation. The development of a successful therapeutic monoclonal antibody (mAb) requires both a highly active and also stable molecule. Whilst a range of experimental (biophysical) approaches exist to track changes in stability of proteins, routine prediction of stability remains challenging. The fluorescence red edge excitation shift (REES) phenomenon is sensitive to a range of changes in protein structure. Based on recent work, we have found that quantifying the REES effect is extremely sensitive to changes in protein conformational state and dynamics. Given the extreme sensitivity, potentially this tool could provide a 'fingerprint' of the structure and stability of a protein. Such a tool would be useful in the discovery and development of biopharamceuticals and so we have explored our hypothesis with a panel of therapeutic mAbs. We demonstrate that the quantified REES data show remarkable sensitivity, being able to discern between structurally identical antibodies and showing sensitivity to unfolding and aggregation. The approach works across a broad concentration range (µg-mg/ml) and is highly consistent. We show that the approach can be applied alongside traditional characterisation testing within the context of a forced degradation study (FDS). Most importantly, we demonstrate the approach is able to predict the stability of mAbs both in the short (hours), medium (days) and long-term (months). The quantified REES data will find immediate use in the biopharmaceutical industry in quality assurance, formulation and development. The approach benefits from low technical complexity, is rapid and uses instrumentation which exists in most biochemistry laboratories without modification.

Ambient-pressure endstation of the Versatile Soft X-ray (VerSoX) beamline at Diamond Light Source.

The ambient-pressure endstation and branchline of the Versatile Soft X-ray (VerSoX) beamline B07 at Diamond Light Source serves a very diverse user community studying heterogeneous catalysts, pharmaceuticals and biomaterials under realistic conditions, liquids and ices, and novel electronic, photonic and battery materials. The instrument facilitates studies of the near-surface chemical composition, electronic and geometric structure of a variety of samples using X-ray photoelectron spectroscopy (XPS) and near-edge X-ray absorption fine-structure (NEXAFS) spectroscopy in the photon energy range from 170 eV to 2800 eV. The beamline provides a resolving power hν/Δ(hν) > 5000 at a photon flux > 1010 photons s-1 over most of its energy range. By operating the optical elements in a low-pressure oxygen atmosphere, carbon contamination can be almost completely eliminated, which makes the beamline particularly suitable for carbon K-edge NEXAFS. The endstation can be operated at pressures up to 100 mbar, whereby XPS can be routinely performed up to 30 mbar. A selection of typical data demonstrates the capability of the instrument to analyse details of the surface composition of solid samples under ambient-pressure conditions using XPS and NEXAFS. In addition, it offers a convenient way of analysing the gas phase through X-ray absorption spectroscopy. Short XPS spectra can be measured at a time scale of tens of seconds. The shortest data acquisition times for NEXAFS are around 0.5 s per data point.

Are we underestimating microplastic abundance in the marine environment? A comparison of microplastic capture with nets of different mesh-size.

Microplastic debris is ubiquitous and yet sampling, classifying and enumerating this prolific pollutant in marine waters has proven challenging. Typically, waterborne microplastic sampling is undertaken using nets with a 333 µm mesh, which cannot account for smaller debris. In this study, we provide an estimate of the extent to which microplastic concentrations are underestimated with traditional sampling. Our efforts focus on coastal waters, where microplastics are predicted to have the greatest influence on marine life, on both sides of the North Atlantic Ocean. Microplastic debris was collected via surface trawls using 100, 333 and 500 µm nets. Our findings show that sampling using nets with a 100 µm mesh resulted in the collection of 2.5-fold and 10-fold greater microplastic concentrations compared with using 333 and 500 µm meshes respectively (P < 0.01). Based on the relationship between microplastic concentrations identified and extrapolation of our data using a power law, we estimate that microplastic concentrations could exceed 3700 microplastics m-3 if a net with a 1 µm mesh size is used. We further identified that use of finer nets resulted in the collection of significantly thinner and shorter microplastic fibres (P < 0.05). These results elucidate that estimates of marine microplastic concentrations could currently be underestimated.

High PD-1/PD-L1 Checkpoint Interaction Infers Tumor Selection and Therapeutic Sensitivity to Anti-PD-1/PD-L1 Treatment.

Many cancers are termed immunoevasive due to expression of immunomodulatory ligands. Programmed death ligand-1 (PD-L1) and cluster of differentiation 80/86 (CD80/86) interact with their receptors, programmed death receptor-1 (PD-1) and cytotoxic T-lymphocyte antigen-4 (CTLA-4), respectively, on tumor-infiltrating leukocytes eliciting immunosuppression. Immunotherapies aimed at blocking these interactions are revolutionizing cancer treatments, albeit in an inadequately described patient subset. To address the issue of patient stratification for immune checkpoint intervention, we quantitatively imaged PD-1/PD-L1 interactions in tumor samples from patients, employing an assay that readily detects these intercellular protein-protein interactions in the less than or equal to 10 nm range. These analyses across multiple patient cohorts demonstrated the intercancer, interpatient, and intratumoral heterogeneity of interacting immune checkpoints. The PD-1/PD-L1 interaction was not correlated with clinical PD-L1 expression scores in malignant melanoma. Crucially, among anti-PD-1-treated patients with metastatic non-small cell lung cancer, those with lower PD-1/PD-L1 interaction had significantly worsened survival. It is surmised that within tumors selecting for an elevated level of PD-1/PD-L1 interaction, there is a greater dependence on this pathway for immune evasion and hence, they exhibit more impressive patient response to intervention. SIGNIFICANCE: Quantitation of immune checkpoint interaction by direct imaging demonstrates that immunotherapy-treated patients with metastatic NSCLC with a low extent of PD-1/PD-L1 interaction show significantly worse outcome.

A High Content Screen for Mucin-1-Reducing Compounds Identifies Fostamatinib as a Candidate for Rapid Repurposing for Acute Lung Injury during the COVID-19 pandemic.

Drug repurposing is the only method capable of delivering treatments on the shortened time-scale required for patients afflicted with lung disease arising from SARS-CoV-2 infection. Mucin-1 (MUC1), a membrane-bound molecule expressed on the apical surfaces of most mucosal epithelial cells, is a biochemical marker whose elevated levels predict the development of acute lung injury (ALI) and respiratory distress syndrome (ARDS), and correlate with poor clinical outcomes. In response to the pandemic spread of SARS-CoV-2, we took advantage of a high content screen of 3,713 compounds at different stages of clinical development to identify FDA-approved compounds that reduce MUC1 protein abundance. Our screen identified Fostamatinib (R788), an inhibitor of spleen tyrosine kinase (SYK) approved for the treatment of chronic immune thrombocytopenia, as a repurposing candidate for the treatment of ALI. In vivo , Fostamatinib reduced MUC1 abundance in lung epithelial cells in a mouse model of ALI. In vitro , SYK inhibition by Fostamatinib promoted MUC1 removal from the cell surface. Our work reveals Fostamatinib as a repurposing drug candidate for ALI and provides the rationale for rapidly standing up clinical trials to test Fostamatinib efficacy in patients with COVID-19 lung injury.