Digital image correlation and infrared thermography data for seven unique geometries of 304L stainless steel.

Material Testing 2.0 (MT2.0) is a paradigm that advocates for the use of rich, full-field data, such as from digital image correlation and infrared thermography, for material identification. By employing heterogeneous, multi-axial data in conjunction with sophisticated inverse calibration techniques such as finite element model updating and the virtual fields method, MT2.0 aims to reduce the number of specimens needed for material identification and to increase confidence in the calibration results. To support continued development, improvement, and validation of such inverse methods-specifically for rate-dependent, temperature-dependent, and anisotropic metal plasticity models-we provide here a thorough experimental data set for 304L stainless steel sheet metal. The data set includes full-field displacement, strain, and temperature data for seven unique specimen geometries tested at different strain rates and in different material orientations. Commensurate extensometer strain data from tensile dog bones is provided as well for comparison. We believe this complete data set will be a valuable contribution to the experimental and computational mechanics communities, supporting continued advances in material identification methods.

Association Between Self-Reported Infections and Seropositivity Among Pregnant Women With Gastroschisis: A Case Control Study, With Emphasis on Chlamydia trachomatis.

BACKGROUND: Gastroschisis is a birth defect with the greatest risk among women <20 years of age. METHODS: Pregnant women attending the University of Utah's Maternal-Fetal Medicine Diagnostic Center between 2011 and 2017 for either their routine diagnostic ultrasound or referral were recruited (cases: pregnant women with fetal gastroschisis, n = 53 participated/57, 93%; controls: pregnant women without fetal abnormalities, n = 102 participated/120, 85%). A clinic coordinator consented and interviewed women and obtained a blood sample and prenatal medical records. We evaluated self-reported maternal characteristics, risk factors, and infections. To assess pathogen seropositivity we used Serimmune's Serum Epitope Repertoire Analysis validated 35 pathogen panels and Chlamydia trachomatis and compared seropositivity to self-report and prenatal medical record screening to assess sensitivity. RESULTS: Cases were more likely to report a younger age at sexual debut (p = <0.01), more sexual partners (p = 0.02), being unmarried (p < 0.01), changing partners between pregnancies (p = <0.01), smoking cigarettes (<0.01), and a recent sexually transmitted infection (STI) (p = 0.02). No differences were observed for self-report of illicit drug use or periconceptional urinary tract infections. Cases had a higher seropositivity for cytomegalovirus (p = 0.01). No differences were observed for herpes simplex I, II, or Epstein-Barr. Though based on small numbers, C. trachomatis seropositivity was highest in cases (17%) compared to controls (8.8%) with the highest proportion observed in case women <20 years of age (cases 33%; controls 0%). Any STI (self-report or seropositivity) was also highest among cases <20 years of age (cases 47%; controls 0%). Among C. trachomatis seropositive women, self-report and prenatal medical record sensitivity was 27.8% and 3%, respectively. CONCLUSIONS: Cases were more likely to engage in behaviors that can increase their risk of exposure to sexually transmitted pathogens. Case women <20 years of age had the highest proportion of C. trachomatis seropositivity and any STI. Prenatal medical records and self-report were inadequate to identify a recent chlamydial infection whereas, the SERA assay is a novel approach for evaluating subclinical infections that may impact the developing embryo.

Structural insights into Frizzled3 through nanobody modulators.

The Wnt receptor Frizzled3 (FZD3) is important for brain axonal development and cancer progression. We report structures of FZD3 in complex with extracellular and intracellular binding nanobodies (Nb). The crystal structure of Nb8 in complex with the FZD3 cysteine-rich domain (CRD) reveals that the nanobody binds at the base of the lipid-binding groove and can compete with Wnt5a. Nb8 fused with the Dickkopf-1 C-terminal domain behaves as a FZD3-specific Wnt surrogate, activating ß-catenin signalling. The cryo-EM structure of FZD3 in complex with Nb9 reveals partially resolved density for the CRD, which exhibits positional flexibility, and a transmembrane conformation that resembles active GPCRs. Nb9 binds to the cytoplasmic region of FZD3 at the putative Dishevelled (DVL) or G protein-binding site, competes with DVL binding, and inhibits GαS coupling. In combination, our FZD3 structures with nanobody modulators map extracellular and intracellular interaction surfaces of functional, and potentially therapeutic, relevance.

Semmaphorin 3 A causes immune suppression by inducing cytoskeletal paralysis in tumour-specific CD8+ T cells.

Semaphorin-3A (SEMA3A) functions as a chemorepulsive signal during development and can affect T cells by altering their filamentous actin (F-actin) cytoskeleton. The exact extent of these effects on tumour-specific T cells are not completely understood. Here we demonstrate that Neuropilin-1 (NRP1) and Plexin-A1 and Plexin-A4 are upregulated on stimulated CD8+ T cells, allowing tumour-derived SEMA3A to inhibit T cell migration and assembly of the immunological synapse. Deletion of NRP1 in both CD4+ and CD8+ T cells enhance CD8+ T-cell infiltration into tumours and restricted tumour growth in animal models. Conversely, over-expression of SEMA3A inhibit CD8+ T-cell infiltration. We further show that SEMA3A affects CD8+ T cell F-actin, leading to inhibition of immune synapse formation and motility. Examining a clear cell renal cell carcinoma patient cohort, we find that SEMA3A expression is associated with reduced survival, and that T-cells appear trapped in SEMA3A rich regions. Our study establishes SEMA3A as an inhibitor of effector CD8+ T cell tumour infiltration, suggesting that blocking NRP1 could improve T cell function in tumours.

Longitudinal profiling identifies co-occurring BRCA1/2 reversions, TP53BP1, RIF1 and PAXIP1 mutations in PARP inhibitor-resistant advanced breast cancer.

BACKGROUND: Resistance to therapies that target homologous recombination deficiency (HRD) in breast cancer limits their overall effectiveness. Multiple, preclinically validated, mechanisms of resistance have been proposed, but their existence and relative frequency in clinical disease are unclear, as is how to target resistance. PATIENTS AND METHODS: Longitudinal mutation and methylation profiling of circulating tumour (ct)DNA was carried out in 47 patients with metastatic BRCA1-, BRCA2- or PALB2-mutant breast cancer treated with HRD-targeted therapy who developed progressive disease-18 patients had primary resistance and 29 exhibited response followed by resistance. ctDNA isolated at multiple time points in the patient treatment course (before, on-treatment and at progression) was sequenced using a novel >750-gene intron/exon targeted sequencing panel. Where available, matched tumour biopsies were whole exome and RNA sequenced and also used to assess nuclear RAD51. RESULTS: BRCA1/2 reversion mutations were present in 60% of patients and were the most prevalent form of resistance. In 10 cases, reversions were detected in ctDNA before clinical progression. Two new reversion-based mechanisms were identified: (i) intragenic BRCA1/2 deletions with intronic breakpoints; and (ii) intragenic BRCA1/2 secondary mutations that formed novel splice acceptor sites, the latter being confirmed by in vitro minigene reporter assays. When seen before commencing subsequent treatment, reversions were associated with significantly shorter time to progression. Tumours with reversions retained HRD mutational signatures but had functional homologous recombination based on RAD51 status. Although less frequent than reversions, nonreversion mechanisms [loss-of-function (LoF) mutations in TP53BP1, RIF1 or PAXIP1] were evident in patients with acquired resistance and occasionally coexisted with reversions, challenging the notion that singular resistance mechanisms emerge in each patient. CONCLUSIONS: These observations map the prevalence of candidate drivers of resistance across time in a clinical setting, information with implications for clinical management and trial design in HRD breast cancers.

Inflammatory biomarker signatures in post-surgical drain fluid may detect anastomotic leaks within 48 hours of colorectal resection.

BACKGROUND: The optimal treatment of colorectal cancer is surgical resection and primary anastomosis. Anastomotic leak can affect up to 20% of patients and creates significant morbidity and mortality. Current diagnosis of a leak is based on clinical suspicion and subsequent radiology. Peritoneal biomarkers have shown diagnostic utility in other conditions and could be useful in providing earlier diagnosis. This pilot study was designed to assess the practical utility of peritoneal biomarkers after abdominal surgery utilising an automated immunoassay system in routine use for quantifying cytokines. METHODS: Patients undergoing an anterior resection for a rectal cancer diagnosis were recruited at University Hospital of Wales, Cardiff between June 2019 and June 2021. A peritoneal drain was placed in the proximity of the anastomosis during surgery, and peritoneal fluid was collected at days 1 to 3 post-operatively, and analysed using the Siemens IMMULITE platform for interleukin (IL)-1ß, IL-6, IL-10, CXCL8, tumour necrosis factor alpha (TNFα) and C-reactive protein (CRP). RESULTS: A total of 42 patients were recruited (22M:20F, median age 65). Anastomotic leak was detected in four patients and a further five patients had other intra-abdominal complications. The IMMULITE platform was able to provide robust and reliable results from the analysis of the peritoneal fluid. A metric based on the combination of peritoneal IL-6 and CRP levels was able to accurately diagnose three anastomotic leaks, whilst correctly classifying all negative control patients including those with other complications. CONCLUSIONS: This pilot study demonstrates that a simple immune signature in surgical drain fluid could accurately diagnose an anastomotic leak at 48 h postoperatively using instrumentation that is already widely available in hospital clinical laboratories.

Family and occupational functioning following military trauma exposure and moral injury.

INTRODUCTION: Evidence is growing regarding the impact of potentially morally injurious events (PMIEs) on mental health; yet how moral injury may affect an individual's occupational and familial functioning remains poorly understood. METHOD: Thirty male veterans who reported exposure to either traumatic or morally injurious events and 15 clinicians were recruited for semi-structured qualitative interviews. RESULTS: While many veterans experienced psychological distress postevent, those who experienced PMIEs especially reported social withdrawal and engagement in aggressive, risk-taking behaviours. This was highly distressing for family members and created a tense, volatile home and workplace environment that was difficult for others to navigate. Following PMIEs, employment could be used as a cognitive avoidance strategy or as a means to atone for transgressive acts. In cases of moral injury, clinicians considered that targeted support for spouses and accessible guidance to help children to better understand how their military parent may be feeling would be beneficial. CONCLUSIONS: This study provides some of the first evidence of the pervasive negative impact of PMIEs on veterans' familial and occupational functioning. These findings highlight the need to comprehensively screen for the impact of moral injury on daily functioning in future studies that goes beyond just an assessment of psychological symptoms.

Primary and secondary functions of HLA-E are determined by stability and conformation of the peptide-bound complexes.

MHC-E regulates NK cells by displaying MHC class Ia signal peptides (VL9) to NKG2A:CD94 receptors. MHC-E can also present sequence-diverse, lower-affinity, pathogen-derived peptides to T cell receptors (TCRs) on CD8+ T cells. To understand these affinity differences, human MHC-E (HLA-E)-VL9 versus pathogen-derived peptide structures are compared. Small-angle X-ray scatter (SAXS) measures biophysical parameters in solution, allowing comparison with crystal structures. For HLA-E-VL9, there is concordance between SAXS and crystal parameters. In contrast, HLA-E-bound pathogen-derived peptides produce larger SAXS dimensions that reduce to their crystallographic dimensions only when excess peptide is supplied. Further crystallographic analysis demonstrates three amino acids, exclusive to MHC-E, that not only position VL9 close to the α2 helix, but also allow non-VL9 peptide binding with re-configuration of a key TCR-interacting α2 region. Thus, non-VL9-bound peptides introduce an alternative peptide-binding motif and surface recognition landscape, providing a likely basis for VL9- and non-VL9-HLA-E immune discrimination.

Structural Analysis and Development of Notum Fragment Screening Hits.

The Wnt signaling suppressor Notum is a promising target for osteoporosis, Alzheimer's disease, and colorectal cancers. To develop novel Notum inhibitors, we used an X-ray crystallographic fragment screen with the Diamond-SGC Poised Library (DSPL) and identified 59 fragment hits from the analysis of 768 data sets. Fifty-eight of the hits were found bound at the enzyme catalytic pocket with potencies ranging from 0.5 to >1000 µM. Analysis of the fragments' diverse binding modes, enzymatic inhibitory activities, and chemical properties led to the selection of six hits for optimization, and five of these resulted in improved Notum inhibitory potencies. One hit, 1-phenyl-1,2,3-triazole 7, and its related cluster members, have shown promising lead-like properties. These became the focus of our fragment development activities, resulting in compound 7d with IC50 0.0067 µM. The large number of Notum fragment structures and their initial optimization provided an important basis for further Notum inhibitor development.

A smoking cessation induction intervention via virtual reality headset during a dental cleaning: protocol for a randomized controlled trial.

BACKGROUND: Effective smoking cessation programs exist but are underutilized by smokers, especially by disadvantaged smokers. Cessation interventions in dental settings have been shown to be effective, but are not consistently delivered due to provider burden and lack of training, especially on how to counsel smokers who are not motivated to quit. METHODS: This study is a 2-arm, phase III longitudinal randomized controlled efficacy trial to motivate utilization of evidenced based treatments (EBTs) for smoking cessation (e.g., state quitline, clinic-based counseling, the National Cancer Institute's text message program, and pharmacotherapy). Patients attending an urban dental clinic (n = 376) will be randomized to an intervention group (INT; smoking cessation induction video delivered via VR headset during their teeth cleaning, brochure about EBTs, and a 4-week text message program) or control group (CTRL; relaxation video delivered via VR headset during teeth cleaning, the same brochure as INT, and assessment-only text messages). Assessments will occur at baseline, immediately after the clinic appointment, one-month post-appointment and 3-and 6 months later. We hypothesize INT will be more likely to contact EBTs vs CTRL and have greater utilization rates of EBTs. Secondary objectives are to test the efficacy of INT on point-prevalence smoking abstinence, quit smoking attempts, and motivation to quit vs. CTRL. DISCUSSION: Incorporating smoking cessation into a dental clinic visit and targeting all smokers, regardless of motivation to quit, provides proactive reach to cigarette smokers who otherwise may not seek treatment for smoking. TRIAL REGISTRATION: NCT04524533 Registered August 24, 2020.

Design of a Potent, Selective, and Brain-Penetrant Inhibitor of Wnt-Deactivating Enzyme Notum by Optimization of a Crystallographic Fragment Hit.

Notum is a carboxylesterase that suppresses Wnt signaling through deacylation of an essential palmitoleate group on Wnt proteins. There is a growing understanding of the role Notum plays in human diseases such as colorectal cancer and Alzheimer's disease, supporting the need to discover improved inhibitors, especially for use in models of neurodegeneration. Here, we have described the discovery and profile of 8l (ARUK3001185) as a potent, selective, and brain-penetrant inhibitor of Notum activity suitable for oral dosing in rodent models of disease. Crystallographic fragment screening of the Diamond-SGC Poised Library for binding to Notum, supported by a biochemical enzyme assay to rank inhibition activity, identified 6a and 6b as a pair of outstanding hits. Fragment development of 6 delivered 8l that restored Wnt signaling in the presence of Notum in a cell-based reporter assay. Assessment in pharmacology screens showed 8l to be selective against serine hydrolases, kinases, and drug targets.

Mouse and human antibodies bind HLA-E-leader peptide complexes and enhance NK cell cytotoxicity.

The non-classical class Ib molecule human leukocyte antigen E (HLA-E) has limited polymorphism and can bind HLA class Ia leader peptides (VL9). HLA-E-VL9 complexes interact with the natural killer (NK) cell receptors NKG2A-C/CD94 and regulate NK cell-mediated cytotoxicity. Here we report the isolation of 3H4, a murine HLA-E-VL9-specific IgM antibody that enhances killing of HLA-E-VL9-expressing cells by an NKG2A+ NK cell line. Structural analysis reveal that 3H4 acts by preventing CD94/NKG2A docking on HLA-E-VL9. Upon in vitro maturation, an affinity-optimized IgG form of 3H4 showes enhanced NK killing of HLA-E-VL9-expressing cells. HLA-E-VL9-specific IgM antibodies similar in function to 3H4 are also isolated from naïve B cells of cytomegalovirus (CMV)-negative, healthy humans. Thus, HLA-E-VL9-targeting mouse and human antibodies isolated from the naïve B cell antibody pool have the capacity to enhance NK cell cytotoxicity.

Truncated-semaphorin3A is a potential regulatory molecule to restore immune homeostasis in immune-mediated diseases.

Regulatory molecules have recently been recognized for their beneficial effects in the treatment of immune-mediated diseases, rather than using cytotoxic immune-suppressing drugs, which are associated with many unwanted side effects. Semaphorin3A (sema3A), a unique regulatory master of the immune system, was shown to be decreased in the serum of systemic lupus erythematosus (SLE) patients, in association with disease severity. Later, we were able to show its extremely beneficial effect in treating lupus nephritis in the NZB/W mice model. The mechanisms by which sema3A maintains its regulatory effect is by binding the regulatory receptor CD72 on B cells, thereby reducing the threshold of BCR signaling on B cells and reducing the production of pro-inflammatory cytokines. The aim of this study was to generate a stable sema3A molecule, easy to produce with a higher binding capacity to CD72 receptor rather than to Neuropilin-1 (NRP-1) receptor, which is expressed in many cell types. Using the crystallographic structure of parental sema3A, we synthesized a new secreted (shorter) sema3A derivative, which we called truncated sema3A (T-sema3A). The new molecule lacked the NRP-1 binding domain (the C-terminal site) and has an artificial dimerization site at position 257 (serine residue was exchanged with a cysteine residue). To facilitate the purification of this molecule we added Histidine epitope tag in frame upstream to a stop codon. This construct was transfected using a viral vector to 293HEK cells to generate cells stably expressing T-sema3A. T-sema3A is shown to be with a higher binding ability to CD72 than to NRP-1 as demonstrated by a homemade ELISA. In addition, T-sema3A was shown to be a regulatory agent which can induce the expression of IL-10 and TGF-ß and reduce the secretion of pro-inflammatory cytokines such as IL-6, IFN-γ, and IL-17A from human T and B-lymphocytes. Keeping this in mind, T-sema3A is highly effective in maintaining immune homeostasis, therefore, becoming a potential agent in restoring the regulatory status of the immune system in immune-mediated diseases.

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.

NOTUM from Apc-mutant cells biases clonal competition to initiate cancer.

The tumour suppressor APC is the most commonly mutated gene in colorectal cancer. Loss of Apc in intestinal stem cells drives the formation of adenomas in mice via increased WNT signalling1, but reduced secretion of WNT ligands increases the ability of Apc-mutant intestinal stem cells to colonize a crypt (known as fixation)2. Here we investigated how Apc-mutant cells gain a clonal advantage over wild-type counterparts to achieve fixation. We found that Apc-mutant cells are enriched for transcripts that encode several secreted WNT antagonists, with Notum being the most highly expressed. Conditioned medium from Apc-mutant cells suppressed the growth of wild-type organoids in a NOTUM-dependent manner. Furthermore, NOTUM-secreting Apc-mutant clones actively inhibited the proliferation of surrounding wild-type crypt cells and drove their differentiation, thereby outcompeting crypt cells from the niche. Genetic or pharmacological inhibition of NOTUM abrogated the ability of Apc-mutant cells to expand and form intestinal adenomas. We identify NOTUM as a key mediator during the early stages of mutation fixation that can be targeted to restore wild-type cell competitiveness and provide preventative strategies for people at a high risk of developing colorectal cancer.

Small-molecule inhibitors of carboxylesterase Notum.

Notum has recently been identified as a negative regulator of Wnt signaling through the removal of an essential palmitoleate group from Wnt proteins. There are emerging reports that Notum plays a role in human disease, with published data suggesting that targeting Notum could represent a new therapeutic approach for treating cancer, osteoporosis and neurodegenerative disorders. Complementary hit-finding strategies have been applied with successful approaches that include high-throughput screening, activity-based protein profiling, screening of fragment libraries and virtual screening campaigns. Structural studies are accelerating the discovery of new inhibitors of Notum. Three fit-for-purpose examples are LP-922056, ABC99 and ARUK3001185. The application of these small-molecule inhibitors is helping to further advance an understanding of the role Notum plays in human disease.

The in vitro effects of platelet products on the biophysiological functions of human bone marrow mesenchymal stromal cells: a systematic review.

Platelet products (PP) and bone-marrow aspirate are popular sources of osteoinductive signalling molecules and osteogenic bone marrow mesenchymal stromal cells (BM-MSCs) used in the treatment of impaired bone healing. However, the combined use of PP and BM-MSCs in clinical studies has reported mixed results. Understanding the cellular and molecular interactions between PP and BM-MSCs plays the important role of guiding future research and clinical application. This systematic review investigates the effects of PP on the biophysiological functions of BM-MSCs in in vitro human studies, including (i) proliferation, (ii) migration, (iii) differentiation, (iv) growth factor/cytokine/protein expression, (v) immunomodulation, (vi) chemotactic effect on haematopoietic stem cells, (vii) response to apoptotic stress, and (viii) gene expression. In vitro studies in human have demonstrated the multi-faceted 'priming effect' of PP on the biophysiological functions of BM-MSCs. PP has been shown to improve proliferation, migration, osteogenic differentiation, reaction to apoptotic stress as well as immunomodulatory, pro-angiogenic and pro-inflammatory capacities of BM-MSCs. Several factors are highlighted that restrict the transferability of these findings into clinical practice. Therefore, more collaborative in vitro research in humans modelled to reflect clinical practice is required to better understand the effects of PP exposure on the biophysiological function(s) of BM-MSCs in human.

Revisiting Gillies' "Cocked Hat" Reconstruction for Thumb Reconstruction.

A technique for reconstructing a thumb following amputation through the metacarpal phalangeal joint is described. This technique was first described by Gillies and involves bony augmentation with iliac crest bone graft to lengthen the first ray and a random pattern flap to cover and provide durable sensate soft tissue coverage. As opposed to more commonly performed procedures, this technique does not involve microsurgery or any specialized equipment. There are several limitations to this technique, but it remains a good option where resources do not allow more modern reconstructive procedures. We present our use of the technique, which was performed at a forward operating base for a coalition service member.