Facile Rebridging Conjugation Approach to Attain Monoclonal Antibody-Targeted Nanoparticles with Enhanced Antigen Binding and Payload Delivery.

Adopting conventional conjugation approaches to construct antibody-targeted nanoparticles (NPs) has demonstrated suboptimal control over the binding orientation and the structural stability of monoclonal antibodies (mAbs). Hitherto, the developed antibody-targeted NPs have shown proof of concept but lack product homogeneity, batch-to-batch reproducibility, and stability, precluding their advancement toward the clinic. To circumvent these limitations and advance toward clinical application, herein, a refined approach based on site-specific construction of mAb-immobilized NPs will be appraised. Initially, the conjugation of atezolizumab (anti-PDL1 antibody, Amab) with polymeric NPs was developed using bis-haloacetamide (BisHalide) rebridging chemistry, followed by click chemistry (NP-Fab BisHalide Ab and NP-Fc BisHalide Ab). For comparison purposes, mAb-immobilized NPs developed utilizing conventional conjugation methods, namely, N-hydroxysuccinimide (NHS) coupling and maleimide chemistry (NP-NHS Ab and NP-Mal Ab), were included. Next, flow cytometry and confocal microscopy experiments evaluated the actively targeted NPs (loaded with fluorescent dye) for cellular binding and uptake. Our results demonstrated the superior and selective binding and uptake of NP-Fab BisHalide Ab and NP-Fc BisHalide Ab into EMT6 cells by 19-fold and 13-fold, respectively. To evaluate the PDL1-dependent cell uptake and the selectivity of the treatments, a blocking step of the PDL1 receptor with Amab was performed prior to incubation with NP-Fab BisHalide Ab and NP-Fc BisHalide Ab. To our delight, the binding and uptake of fluorescent NPs were reduced significantly by 3-fold for NP-Fab BisHalide Ab, demonstrating the PDL1-mediated uptake. Moreover, NP-Fab BisHalide Ab and NP-Fc BisHalide Ab were entrapped with the paclitaxel payload, and their cytotoxicity was evaluated. They showed significant enhancements compared to free paclitaxel and NP-NHS Ab. Overall, this work will provide a facile conjugation method that could be implemented to actively target NPs with a plethora of therapeutic mAbs approved for various malignancies.

Glomus tumor with malignant features: A case report and review of the literature.

Glomus tumors are rare mesenchymal neoplasms of the subcutaneous tissue, most frequently found in the distal extremities. They are typically benign, but malignant glomus tumors have been described in the literature. Here we present a patient found to have a unilateral renal mass with pathology displaying a primary renal glomus tumor with malignant features. Review of the literature reveals only three cases of malignant glomus tumors and five glomus tumors with malignant potential. As such, previous initial presentations, current criteria for glomus tumor malignancy, and previous treatment outcomes of these cases were reviewed.

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.