RASA2 ablation in T cells boosts antigen sensitivity and long-term function.

The efficacy of adoptive T cell therapies for cancer treatment can be limited by suppressive signals from both extrinsic factors and intrinsic inhibitory checkpoints1,2. Targeted gene editing has the potential to overcome these limitations and enhance T cell therapeutic function3-10. Here we performed multiple genome-wide CRISPR knock-out screens under different immunosuppressive conditions to identify genes that can be targeted to prevent T cell dysfunction. These screens converged on RASA2, a RAS GTPase-activating protein (RasGAP) that we identify as a signalling checkpoint in human T cells, which is downregulated upon acute T cell receptor stimulation and can increase gradually with chronic antigen exposure. RASA2 ablation enhanced MAPK signalling and chimeric antigen receptor (CAR) T cell cytolytic activity in response to target antigen. Repeated tumour antigen stimulations in vitro revealed that RASA2-deficient T cells show increased activation, cytokine production and metabolic activity compared with control cells, and show a marked advantage in persistent cancer cell killing. RASA2-knockout CAR T cells had a competitive fitness advantage over control cells in the bone marrow in a mouse model of leukaemia. Ablation of RASA2 in multiple preclinical models of T cell receptor and CAR T cell therapies prolonged survival in mice xenografted with either liquid or solid tumours. Together, our findings highlight RASA2 as a promising target to enhance both persistence and effector function in T cell therapies for cancer treatment.

A single inactivating amino acid change in the SARS-CoV-2 NSP3 Mac1 domain attenuates viral replication in vivo.

Despite unprecedented efforts, our therapeutic arsenal against SARS-CoV-2 remains limited. The conserved macrodomain 1 (Mac1) in NSP3 is an enzyme exhibiting ADP-ribosylhydrolase activity and a possible drug target. To determine the role of Mac1 catalytic activity in viral replication, we generated recombinant viruses and replicons encoding a catalytically inactive NSP3 Mac1 domain by mutating a critical asparagine in the active site. While substitution to alanine (N40A) reduced catalytic activity by ~10-fold, mutations to aspartic acid (N40D) reduced activity by ~100-fold relative to wild-type. Importantly, the N40A mutation rendered Mac1 unstable in vitro and lowered expression levels in bacterial and mammalian cells. When incorporated into SARS-CoV-2 molecular clones, the N40D mutant only modestly affected viral fitness in immortalized cell lines, but reduced viral replication in human airway organoids by 10-fold. In mice, the N40D mutant replicated at >1000-fold lower levels compared to the wild-type virus while inducing a robust interferon response; all animals infected with the mutant virus survived infection. Our data validate the critical role of SARS-CoV-2 NSP3 Mac1 catalytic activity in viral replication and as a promising therapeutic target to develop antivirals.

Ventriculotomy Decreases Agreement Between Assessment of Right Ventricular Function by Echocardiography and Cardiac Magnetic Resonance Imaging in Patients with Hypoplastic Left Heart Syndrome.

Accurate assessment of the right ventricular (RV) volume and function is important in patients with hypoplastic left heart syndrome (HLHS). We sought to investigate the effect of ventriculotomy on the correlation of RV functional assessments by two-dimensional echocardiography (2DE) to cardiac magnetic resonance (CMR)-derived RV ejection fraction (EF) in patients with HLHS. A retrospective re-analysis of CMR imaging with matched 2DE was performed from the institutional HLHS registry. Echocardiographic RV functional parameters were analyzed and correlated with CMR-derived EF. Intraclass correlation coefficient was used to determine interobserver reliability. A total of 58 matched echocardiograms and CMR imaging studies from 46 patients was evaluated. Median duration between CMR imaging and echocardiogram was 1 day (range 0-6 days). No significant difference was seen in CMR RV EF between patients with and without a ventriculotomy (EF - 43.6% vs 44.7%, p = 0.85). The presence of a ventriculotomy significantly decreased the correlation of biplane FAC (r = 0.86 vs 0.52; p = 0.02), triplane FAC (r = 0.84 vs 0.49; p = 0.03), and 2DE visually estimated EF (r = 0.83 vs 0.49; p = 0.02). The correlation of circumferential and longitudinal strains to CMR-derived EF was not significantly affected by the presence of a ventriculotomy. A prior ventriculotomy significantly affected correlation between 2DE FAC and visually estimated EF with CMR-derived EF. The dyskinetic myocardial segment due to ventriculotomy, which is often not visualized by 2DE, may be the reason for this discrepancy.

Inter-observer and Inter-vendor Variability in Strain Measurements in Patients with Single Right Ventricular Anatomy.

Myocardial strain offers new insights into ventricular performance, There are software packages from several different companies used to ascertain this, and little data is available in patients with single right ventricle (sRV) physiology. We aimed to compare the analysis of two strain software applications using a cohort of patients with sRV for both inter-vendor and inter-observer variability. Echocardiograms from 85 patients with sRV (122 separate studies) were prospectively evaluated. All had Glenn and/or Fontan palliation. Longitudinal 4-chamber (4LS), inflow/outflow (IO), circumferential, and radial strain were assessed using Velocity Vector Imaging (VVI, Seimens, Munich) and Automated Functional Imaging (AFI, General Electric, Boston) software. In a subset of 45 patients (61 separate studies), strain measurements were obtained by two sonographers so a paired "inter-observer" analysis could be performed. A moderate correlation between measurements made by the two systems was observed. Circumferential strain assessment had the highest R value (0.77) with all others having R values < 0.6. Both software packages showed modest inter-observer reproducibility for longitudinal and circumferential strain. VVI intraclass correlation coefficients (ICC) for 4LS and average circumferential strain (ACS) were 0.6 and 0.58, compared to 0.68 and 0.59 for AFI. Other than radial strain and VVI IO inferior strain, mean strain differences between AFI and VVI were ≤ 1%. Inter-observer variability is modest, however, mean differences are minimal suggesting reasonable clinical reliability. Inter-vendor variability is greater and not as clinically reliable. In patients with sRV, serial assessments with strain should be performed using the same software.

Negative Impact of the Left Ventricular Remnant Morphology on Systemic Right Ventricular Myocardial Deformation in Hypoplastic Left Heart Syndrome.

Left ventricular (LV) morphology may affect right ventricular (RV) function before and after Fontan palliation in patients with hypoplastic left heart syndrome (HLHS). We sought to assess the potential impact of LV morphology on RV function in patients with HLHS using cardiac magnetic resonance (CMR) imaging. A retrospective analysis of available CMR scans from all patients with HLHS was performed. LV morphology was categorized as absent/slit-like or globular/miniaturized. Volumetric analysis was performed using manual disc-summation method on steady-state free precession (SSFP) stack obtained in short-axis orientation of the ventricles. 4-chamber and short-axis SSFP images were used to measure strain on a semi-automated feature-tracking (FT) module. Two sample t-test was used to compare the groups. A total of 48 CMR scans were analyzed. Of those, 12 patients had absent/slit-like and 36 had globular/miniaturized LV morphology. Averaged 4-chamber longitudinal RV strain was significantly higher for absent/slit-like (- 17.6 ± 4.7%) than globular/miniaturized (- 13.4 ± 3.5; P = 0.002). Averaged 4-chamber radial RV strain was also significantly higher for absent/slit-like (33.1 ± 14.9%) than globular/miniaturized (21.6 ± 7.1; P = 0.001). For globular/miniaturized LV morphology, the decreases of 4-chamber longitudinal and radial strains were mainly attributable to the septal basilar and septal mid-ventricular segments. No differences were found in short-axis RV global circumferential strain between the morphologic subtypes (absent/slit-like - 15.0 ± 6.5, globular/miniaturized - 15.7 ± 4.7; P = 0.68). Larger LV remnants, with globular/miniaturized LV morphology, demonstrated diminished strain in the septal base and mid-ventricle segments. Patients with globular/miniaturized LV morphology may benefit with closer monitoring and lower threshold to start heart failure medications. These results exemplify the utility of including both septal and regional deformation in systemic RV strain analysis.

Placement of Reveal LINQ Device in the Left Anterior Axillary Position.

Implantable loop recorders (ILR) are utilized for long-term rhythm monitoring. Typical placement of the Medtronic Reveal LINQ along the left parasternal border may compromise the quality and/or feasibility of future imaging studies. We sought to evaluate the utility of placing an ILR in the left anterior axillary position and the impact on the quality of cardiac imaging. We reviewed patients from May 2017 to June 2018 who had placement of a Reveal LINQ device in the left anterior axillary position. Demographic, procedural, and clinical data were collected via retrospective review. Cardiac magnetic resonance imaging (MRI) studies were reviewed for image quality after ILR placement. Eight patients met inclusion criteria for this study (median age 6 years, 50% female). Six patients (75%) had an ILR placed in the operating room, while all others were placed in the electrophysiology lab. All patients demonstrated acceptable R waves for diagnostic evaluation (median = 0.85 mV, range 0.24-1.7 mV). Cardiac MRI was obtained in 7 patients following ILR placement with diagnostic image quality and no adverse events. One device was explanted 28 days after placement due to concern for possible infection. No other devices required removal or revision (median follow up duration 11 months, IQR 8-13.5). ILR placement in the left anterior axillary position can record adequate signals in pediatric patients. In addition, axillary ILR device position may allow for completion of cardiac imaging, particularly cardiac MRI, without significant artifacts which is critical for patients with congenital heart disease.

Hypoplastic Left Heart Syndrome: An Overview for Primary Care Providers.

Hypoplastic left heart syndrome is one of the most complex congenital heart diseases and requires several cardiac surgeries for survival. The diagnosis is usually established prenatally or shortly after birth. Each stage of surgery poses a unique hemodynamic situation that requires deeper understanding to manage common pediatric problems such as dehydration and respiratory infections. Careful multidisciplinary involvement in the care of these complex patients is improving their outcome; however, morbidity and mortality are still substantial. In this review, we focus on the hemodynamic aspects of various surgical stages that a primary care provider should know to manage these challenging patients.

Targeting peroxiredoxin 1 impairs growth of breast cancer cells and potently sensitises these cells to prooxidant agents.

BACKGROUND: Our previous work has shown peroxiredoxin-1 (PRDX1), one of major antioxidant enzymes, to be a biomarker in human breast cancer. Hereby, we further investigate the role of PRDX1, compared to its close homolog PRDX2, in mammary malignant cells. METHODS: CRISPR/Cas9- or RNAi-based methods were used for genetic targeting PRDX1/2. Cell growth was assessed by crystal violet, EdU incorporation or colony formation assays. In vivo growth was assessed by a xenotransplantation model. Adenanthin was used to inhibit the thioredoxin-dependent antioxidant defense system. The prooxidant agents used were hydrogen peroxide, glucose oxidase and sodium L-ascorbate. A PY1 probe or HyPer-3 biosensor were used to detect hydrogen peroxide content in samples. RESULTS: PRDX1 downregulation significantly impaired the growth rate of MCF-7 and ZR-75-1 breast cancer cells. Likewise, xenotransplanted PRDX1-deficient MCF-7 cells presented a retarded tumour growth. Furthermore, genetic targeting of PRDX1 or adenanthin, but not PRDX2, potently sensitised all six cancer cell lines studied, but not the non-cancerous cells, to glucose oxidase and ascorbate. CONCLUSIONS: Our study pinpoints the dominant role for PRDX1 in management of exogeneous oxidative stress by breast cancer cells and substantiates further exploration of PRDX1 as a target in this disease, especially when combined with prooxidant agents.

Development of acquired resistance to lapatinib may sensitise HER2-positive breast cancer cells to apoptosis induction by obatoclax and TRAIL.

BACKGROUND: Lapatinib has clinical efficacy in the treatment of trastuzumab-refractory HER2-positive breast cancer. However, a significant proportion of patients develop progressive disease due to acquired resistance to the drug. Induction of apoptotic cell death is a key mechanism of action of lapatinib in HER2-positive breast cancer cells. METHODS: We examined alterations in regulation of the intrinsic and extrinsic apoptosis pathways in cell line models of acquired lapatinib resistance both in vitro and in patient samples from the NCT01485926 clinical trial, and investigated potential strategies to exploit alterations in apoptosis signalling to overcome lapatinib resistance in HER2-positive breast cancer. RESULTS: In this study, we examined two cell lines models of acquired lapatinib resistance (SKBR3-L and HCC1954-L) and showed that lapatinib does not induce apoptosis in these cells. We identified alterations in members of the BCL-2 family of proteins, in particular MCL-1 and BAX, which may play a role in resistance to lapatinib. We tested the therapeutic inhibitor obatoclax, which targets MCL-1. Both SKBR3-L and HCC1954-L cells showed greater sensitivity to obatoclax-induced apoptosis than parental cells. Interestingly, we also found that the development of acquired resistance to lapatinib resulted in acquired sensitivity to TRAIL in SKBR3-L cells. Sensitivity to TRAIL in the SKBR3-L cells was associated with reduced phosphorylation of AKT, increased expression of FOXO3a and decreased expression of c-FLIP. In SKBR3-L cells, TRAIL treatment caused activation of caspase 8, caspase 9 and caspase 3/7. In a second resistant model, HCC1954-L cells, p-AKT levels were not decreased and these cells did not show enhanced sensitivity to TRAIL. Furthermore, combining obatoclax with TRAIL improved response in SKBR3-L cells but not in HCC1954-L cells. CONCLUSIONS: Our findings highlight the possibility of targeting altered apoptotic signalling to overcome acquired lapatinib resistance, and identify potential novel treatment strategies, with potential biomarkers, for HER2-positive breast cancer that is resistant to HER2 targeted therapies.

Heart transplantation after Fontan: Results from a surgical Fontan cohort.

We performed a retrospective review of outcomes after heart transplantation during long-term follow-up of a surgical cohort of 1138 Fontan patients who were followed at the Mayo Clinic. Follow-up information was obtained from medical records and a clinical questionnaire that was mailed to patients not known to be deceased at the initiation of the study. Forty-four of 1138 Fontan patients with initial or subsequent evaluation at Mayo had cardiac transplantation between 1988 and 2014 (mean age at transplantation was 23.2 ± 12 yr, median was 19.8 yr; mean interval between Fontan and transplantation was 13.0 ± 7.7 yr, median was 13.1 yr). Two patients had combined organ transplantation (one heart-lung, one heart-liver). Twelve of the 44 (27%) patients had PLE prior to transplantation. There was no difference in post-bypass Fontan pressures or incidence of late reoperations for AVV repair/replacement between transplanted and non-transplanted patients. There were 16 (36%) deaths in the transplantation cohort; seven occurred within 30 days of transplantation. Overall one, five, 10, and 15 yr post-transplantation survival was 80%, 72%, 69%, and 55%, respectively. Although this is a challenging group of patients, intermediate-term results suggest that cardiac transplantation remains a reasonable option for patients with a failed Fontan circulation.

Electrocardiographic abnormalities in elite high school athletes: comparison to adolescent hypertrophic cardiomyopathy.

BACKGROUND: In athletes, ECG changes from physiological cardiac remodelling are common but can overlap with findings from a pathological disorder. We compared ECG findings in a group of elite high school athletes to a cohort of adolescents with hypertrophic cardiomyopathy (HCM). METHODS/RESULTS: We prospectively performed 15-lead ECGs and echocardiograms in 147 elite high school athletes. Student-athlete ECGs were compared in blinded fashion to ECGs of 148 adolescents with HCM of similar age and ethnicity. Standard ECG hypertrophy criteria and established expert opinion guidelines (European Society of Cardiology, ESC and Seattle criteria) were analysed. All student-athletes had normal echocardiograms. Overall, 77/147 (52%) of student-athletes met standard ECG criteria for ventricular hypertrophy compared to 126/148 (85%) adolescents with HCM (p<0.0001). There were 112/148 (76%) adolescents with HCM who had pathological Q-waves, T-wave inversion and/or ST-segment depression compared to 1/147 (1%) athletes (p<0.0001). Most patients with HCM (84%, 124/148) had ≥1 abnormal ECG finding(s) according to Seattle criteria, compared to 1% of student-athletes (2/147). Similarly, 130/148 (88%) patients with HCM met group-2 ESC criteria (abnormal), compared to 36/147 (24%) student-athletes (p<0.0001). CONCLUSIONS: Over 50% of elite high school athletes with echocardiographically confirmed normal hearts satisfied standard voltage criteria for ventricular hypertrophy. Pathological Q-waves, T-wave inversion or ST-segment depression were most helpful in distinguishing adolescents with HCM from normals. Both ESC and Seattle criteria successfully stratified the student-athlete and HCM cohorts, however each had a false-negative rate >10% for the HCM cohort. The Seattle criteria demonstrated a significantly lower false-positive rate (1%) than the ESC criteria (24%).

Compound heterozygous NOTCH1 mutations underlie impaired cardiogenesis in a patient with hypoplastic left heart syndrome.

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart defect (CHD) that necessitates staged, single ventricle surgical palliation. An increased frequency of bicuspid aortic valve (BAV) has been observed among relatives. We postulated number of mutant alleles as a molecular basis for variable CHD expression in an extended family comprised of an HLHS proband and four family members who underwent echocardiography and whole-genome sequencing (WGS). Dermal fibroblast-derived induced pluripotent stem cells (iPSC) were procured from the proband-parent trio and bioengineered into cardiomyocytes. Cardiac phenotyping revealed aortic valve atresia and a slit-like left ventricular cavity in the HLHS proband, isolated bicuspid pulmonary valve in his mother, BAV in a maternal 4° relative, and no CHD in his father or sister. Filtering of WGS for rare, functional variants that segregated with CHD and were compound heterozygous in the HLHS proband identified NOTCH1 as the sole candidate gene. An unreported missense mutation (P1964L) in the cytoplasmic domain, segregating with semilunar valve malformation, was maternally inherited and a rare missense mutation (P1256L) in the extracellular domain, clinically silent in the heterozygous state, was paternally inherited. Patient-specific iPSCs exhibited diminished transcript levels of NOTCH1 signaling pathway components, impaired myocardiogenesis, and a higher prevalence of heterogeneous myofilament organization. Extended, phenotypically characterized families enable WGS-derived variant filtering for plausible Mendelian modes of inheritance, a powerful strategy to discover molecular underpinnings of CHD. Identification of compound heterozygous NOTCH1 mutations and iPSC-based functional modeling implicate mutant allele burden and impaired myogenic potential as mechanisms for HLHS.

A study of the norcaradiene-cycloheptatriene equilibrium in a series of azulenones by NMR spectroscopy; the impact of substitution on the position of equilibrium.

A systematic investigation of the influence of substitution at positions C-2 and C-3 on the azulenone skeleton, based on NMR characterisation, is discussed with particular focus on the impact of the steric and electronic characteristics of substituents on the position of the norcaradiene-cycloheptatriene (NCD-CHT) equilibrium. Variable temperature (VT) NMR studies, undertaken to enable the resolution of signals for the equilibrating valence tautomers revealed, in addition, interesting shifts in the equilibrium.

Two-dimensional measurement of tricuspid annular plane systolic excursion in children: can it substitute for an m-mode assessment?

BACKGROUND: Tricuspid annular plane systolic excursion measured by M-mode (MM-TAPSE) has been validated as a marker of right ventricular systolic performance. A similar measurement by 2D imaging (2D-TAPSE) can be obtained. We sought to determine the correlation and strength of agreement between MM-TAPSE and 2D-TAPSE in children. METHODS: Echocardiographic studies performed for clinical indications were reviewed retrospectively. All consecutive subjects ≤18 years of age were included. The cohort was divided into those with normal echocardiographic findings and those with disorders affecting the right ventricle. Digitally recorded images were analyzed for both MM-TAPSE and 2D-TAPSE. Measurements of 2D-TAPSE were made in an apical four-chamber view, from the tricuspid valve annulus to a consistent point at the apex of the imaging sector at end-diastole and end-systole, with the difference representing the 2D-TAPSE value. RESULTS: A total of 329 subjects (mean age 9.0 ± 6.1 years) met entry criteria. Correlation coefficient between MM-TAPSE and 2D-TAPSE was 0.90. Bland-Altman analysis showed agreement between the two methods to be within 1.2 ± 2.6 mm (mean percentage difference of 6.5%). About 1 mm difference between MM-TAPSE and 2D-TAPSE was consistently observed in all diagnostic subgroups, and across all age categories. CONCLUSION: MM-TAPSE and 2D-TAPSE correlate strongly, with 2D-TAPSE being consistently about 1 mm less than values obtained by the M-mode technique. We conclude that 2D-TAPSE can provide a reliable alternative to MM-TAPSE to quantitatively measure right ventricular systolic function and may be especially useful in situations where retrospective comparisons are sought.

Peroxiredoxin-1 protects estrogen receptor α from oxidative stress-induced suppression and is a protein biomarker of favorable prognosis in breast cancer.

INTRODUCTION: Peroxiredoxin-1 (PRDX1) is a multifunctional protein, acting as a hydrogen peroxide (H2O2) scavenger, molecular chaperone and immune modulator. Although differential PRDX1 expression has been described in many tumors, the potential role of PRDX1 in breast cancer remains highly ambiguous. Using a comprehensive antibody-based proteomics approach, we interrogated PRDX1 protein as a putative biomarker in estrogen receptor (ER)-positive breast cancer. METHODS: An anti-PRDX1 antibody was validated in breast cancer cell lines using immunoblotting, immunohistochemistry and reverse phase protein array (RPPA) technology. PRDX1 protein expression was evaluated in two independent breast cancer cohorts, represented on a screening RPPA (n = 712) and a validation tissue microarray (n = 498). In vitro assays were performed exploring the functional contribution of PRDX1, with oxidative stress conditions mimicked via treatment with H2O2, peroxynitrite, or adenanthin, a PRDX1/2 inhibitor. RESULTS: In ER-positive cases, high PRDX1 protein expression is a biomarker of improved prognosis across both cohorts. In the validation cohort, high PRDX1 expression was an independent predictor of improved relapse-free survival (hazard ratio (HR) = 0.62, 95% confidence interval (CI) = 0.40 to 0.96, P = 0.032), breast cancer-specific survival (HR = 0.44, 95% CI = 0.24 to 0.79, P = 0.006) and overall survival (HR = 0.61, 95% CI = 0.44 to 0.85, P = 0.004). RPPA screening of cancer signaling proteins showed that ERα protein was upregulated in PRDX1 high tumors. Exogenous H2O2 treatment decreased ERα protein levels in ER-positive cells. PRDX1 knockdown further sensitized cells to H2O2- and peroxynitrite-mediated effects, whilst PRDX1 overexpression protected against this response. Inhibition of PRDX1/2 antioxidant activity with adenanthin dramatically reduced ERα levels in breast cancer cells. CONCLUSIONS: PRDX1 is shown to be an independent predictor of improved outcomes in ER-positive breast cancer. Through its antioxidant function, PRDX1 may prevent oxidative stress-mediated ERα loss, thereby potentially contributing to maintenance of an ER-positive phenotype in mammary tumors. These results for the first time imply a close connection between biological activity of PRDX1 and regulation of estrogen-mediated signaling in breast cancer.

Intersections of Intimate Partner Violence and Natural Disasters: A Systematic Review of the Quantitative Evidence.

Natural disasters and extreme weather events are increasing in both intensity and frequency. Emerging evidence suggests that there is a relationship between intimate partner violence (IPV) and natural disasters. However, there is a scarcity of methodologically sound research in this area with no systematic review to date. To address the gap, this paper systematically assesses the quantitative evidence on the association between IPV with natural disasters between 1990 and March 2023. There were 27 articles that meet the inclusion criteria for the data extraction process. A quantitative critical appraisal tool was used to assess the quality of each study and a narrative synthesis approach to explore the findings. The review found an association between IPV and disasters, across disaster types and countries. However, more research is needed to explore the nuances and gaps within the existing knowledge base. It was unclear whether this relationship was causal or if natural disasters heightened existing risk factors. Further, it is inconclusive as to whether disasters create new cases of IPV or exacerbate existing violence. The majority of studies focused on hurricanes and earthquakes with a dearth of research on "slow onset disasters." These gaps represent the need for further research. Further research can provide a more thorough understanding of IPV and natural disasters, increasing stakeholders' ability to strengthen community capacity and reduce IPV when natural disasters occur.

Biomechanical Motion Changes in Adjacent and Noncontiguous Segments Following Single-Level Anterior Cervical Discectomy and Fusion: A Computed Tomography-Based 3D Motion Capture Study.

BACKGROUND: Anterior cervical discectomy and fusion (ACDF) is known to elicit adverse biomechanical effects on immediately adjacent segments; however, its impact on the kinematics of the remaining nonadjacent cervical levels has not been understood. This study aimed to explore the biomechanical impact of ACDF on kinematics beyond the immediate fusion site. We hypothesized that compensatory motion following single-level ACDF is not predictably distributed to adjacent segments due to compensation from noncontiguous levels. METHODS: Six fresh-frozen cervical spines (C2-T1) underwent fluoroscopic screening and sagittal and coronal reformats from computed tomography scans and were utilized to grade segmental degeneration. Each specimen was tested to 30° of flexion and extension intact and following single-level ACDF at the C5-C6 level. The motions of each vertebral body were tracked using 3-dimensional (3D) motion capture into an inverse kinematics model, facilitating correlations between the 3D reconstruction from computed tomography images and the 3D motion capture data. This model was used to calculate each level's flexion/extension range of motion (ROM). RESULTS: Single-level fusion at the C5-C6 level across all specimens resulted in a significant motion reduction of -6.8° (P = 0.002). No significant change in ROM occurred in the immediate adjacent segments C4-C5 (P = 0.07) or C6-C7 (P = 0.15). Hypermobility was observed in 2 specimens (33%) exclusively in adjacent segments. In contrast, the other 4 spines (66%) displayed hypermobility at noncontiguous segments. Hypermobility occurred in 42% (5/12) of the adjacent segments, 28% (5/18) of the noncontiguous segments, and 50% (3/6) of the cervicothoracic segments. CONCLUSION: Single-level ACDF impacts ROM beyond adjacent segments, extending to noncontiguous levels. Compensatory motion, not limited to adjacent levels, may be influenced by degenerative changes in noncontiguous segments. Surprisingly, hypermobility may not occur in adjacent segments after ACDF. CLINICAL RELEVANCE: Overall, the multifaceted biomechanical effects of ACDF underscore the need for a comprehensive understanding of cervical spine dynamics beyond immediate adjacency, and it needs to be taken into consideration when planning single-level ACDF.

Large tandem duplications in cancer result from transcription and DNA replication collisions.

Despite the abundance of somatic structural variations (SVs) in cancer, the underlying molecular mechanisms of their formation remain unclear. Here, we use 6,193 whole-genome sequenced tumors to study the contributions of transcription and DNA replication collisions to genome instability. After deconvoluting robust SV signatures in three independent pan-cancer cohorts, we detect transcription-dependent replicated-strand bias, the expected footprint of transcription-replication collision (TRC), in large tandem duplications (TDs). Large TDs are abundant in female-enriched, upper gastrointestinal tract and prostate cancers. They are associated with poor patient survival and mutations in TP53, CDK12, and SPOP. Upon inactivating CDK12, cells display significantly more TRCs, R-loops, and large TDs. Inhibition of G2/M checkpoint proteins, such as WEE1, CHK1, and ATR, selectively inhibits the growth of cells deficient in CDK12. Our data suggest that large TDs in cancer form due to TRCs, and their presence can be used as a biomarker for prognosis and treatment.

Systematic antibody generation and validation via tissue microarray technology leading to identification of a novel protein prognostic panel in breast cancer.

BACKGROUND: Although omic-based discovery approaches can provide powerful tools for biomarker identification, several reservations have been raised regarding the clinical applicability of gene expression studies, such as their prohibitive cost. However, the limited availability of antibodies is a key barrier to the development of a lower cost alternative, namely a discrete collection of immunohistochemistry (IHC)-based biomarkers. The aim of this study was to use a systematic approach to generate and screen affinity-purified, mono-specific antibodies targeting progression-related biomarkers, with a view towards developing a clinically applicable IHC-based prognostic biomarker panel for breast cancer. METHODS: We examined both in-house and publicly available breast cancer DNA microarray datasets relating to invasion and metastasis, thus identifying a cohort of candidate progression-associated biomarkers. Of these, 18 antibodies were released for extended analysis. Validated antibodies were screened against a tissue microarray (TMA) constructed from a cohort of consecutive breast cancer cases (n = 512) to test the immunohistochemical surrogate signature. RESULTS: Antibody screening revealed 3 candidate prognostic markers: the cell cycle regulator, Anillin (ANLN); the mitogen-activated protein kinase, PDZ-Binding Kinase (PBK); and the estrogen response gene, PDZ-Domain Containing 1 (PDZK1). Increased expression of ANLN and PBK was associated with poor prognosis, whilst increased expression of PDZK1 was associated with good prognosis. A 3-marker signature comprised of high PBK, high ANLN and low PDZK1 expression was associated with decreased recurrence-free survival (p < 0.001) and breast cancer-specific survival (BCSS) (p < 0.001). This novel signature was associated with high tumour grade (p < 0.001), positive nodal status (p = 0.029), ER-negativity (p = 0.006), Her2-positivity (p = 0.036) and high Ki67 status (p < 0.001). However, multivariate Cox regression demonstrated that the signature was not a significant predictor of BCSS (HR = 6.38; 95% CI = 0.79-51.26, p = 0.082). CONCLUSIONS: We have developed a comprehensive biomarker pathway that extends from discovery through to validation on a TMA platform. This proof-of-concept study has resulted in the identification of a novel 3-protein prognostic panel. Additional biochemical markers, interrogated using this high-throughput platform, may further augment the prognostic accuracy of this panel to a point that may allow implementation into routine clinical practice.