Strand-resolved mutagenicity of DNA damage and repair.

DNA base damage is a major source of oncogenic mutations1. Such damage can produce strand-phased mutation patterns and multiallelic variation through the process of lesion segregation2. Here we exploited these properties to reveal how strand-asymmetric processes, such as replication and transcription, shape DNA damage and repair. Despite distinct mechanisms of leading and lagging strand replication3,4, we observe identical fidelity and damage tolerance for both strands. For small alkylation adducts of DNA, our results support a model in which the same translesion polymerase is recruited on-the-fly to both replication strands, starkly contrasting the strand asymmetric tolerance of bulky UV-induced adducts5. The accumulation of multiple distinct mutations at the site of persistent lesions provides the means to quantify the relative efficiency of repair processes genome wide and at single-base resolution. At multiple scales, we show DNA damage-induced mutations are largely shaped by the influence of DNA accessibility on repair efficiency, rather than gradients of DNA damage. Finally, we reveal specific genomic conditions that can actively drive oncogenic mutagenesis by corrupting the fidelity of nucleotide excision repair. These results provide insight into how strand-asymmetric mechanisms underlie the formation, tolerance and repair of DNA damage, thereby shaping cancer genome evolution.

Mediator kinase inhibition impedes transcriptional plasticity and prevents resistance to ERK/MAPK-targeted therapy in KRAS-mutant cancers.

Acquired resistance remains a major challenge for therapies targeting oncogene activated pathways. KRAS is the most frequently mutated oncogene in human cancers, yet strategies targeting its downstream signaling kinases have failed to produce durable treatment responses. Here, we developed multiple models of acquired resistance to dual-mechanism ERK/MAPK inhibitors across KRAS-mutant pancreatic, colorectal, and lung cancers, and then probed the long-term events enabling survival against this class of drugs. These studies revealed that resistance emerges secondary to large-scale transcriptional adaptations that are diverse and cell line-specific. Transcriptional reprogramming extends beyond the well-established early response, and instead represents a dynamic, evolved process that is refined to attain a stably resistant phenotype. Mechanistic and translational studies reveal that resistance to dual-mechanism ERK/MAPK inhibition is broadly susceptible to manipulation of the epigenetic machinery, and that Mediator kinase, in particular, can be co-targeted at a bottleneck point to prevent diverse, cell line-specific resistance programs.

Endogenous CD28 drives CAR T cell responses in multiple myeloma.

Recent FDA approvals of chimeric antigen receptor (CAR) T cell therapy for multiple myeloma (MM) have reshaped the therapeutic landscape for this incurable cancer. In pivotal clinical trials B cell maturation antigen (BCMA) targeted, 4-1BB co-stimulated (BBζ) CAR T cells dramatically outperformed standard-of-care chemotherapy, yet most patients experienced MM relapse within two years of therapy, underscoring the need to improve CAR T cell efficacy in MM. We set out to determine if inhibition of MM bone marrow microenvironment (BME) survival signaling could increase sensitivity to CAR T cells. In contrast to expectations, blocking the CD28 MM survival signal with abatacept (CTLA4-Ig) accelerated disease relapse following CAR T therapy in preclinical models, potentially due to blocking CD28 signaling in CAR T cells. Knockout studies confirmed that endogenous CD28 expressed on BBζ CAR T cells drove in vivo anti-MM activity. Mechanistically, CD28 reprogrammed mitochondrial metabolism to maintain redox balance and CAR T cell proliferation in the MM BME. Transient CD28 inhibition with abatacept restrained rapid BBζ CAR T cell expansion and limited inflammatory cytokines in the MM BME without significantly affecting long-term survival of treated mice. Overall, data directly demonstrate a need for CD28 signaling for sustained in vivo function of CAR T cells and indicate that transient CD28 blockade could reduce cytokine release and associated toxicities.

Predictive performance of the common red flags in emergency department headache patients: a HEAD and HEAD-Colombia study.

OBJECTIVES: Only a small proportion of patients presenting to an ED with headache have a serious cause. The SNNOOP10 criteria, which incorporates red and orange flags for serious causes, has been proposed but not well studied. This project aims to compare the proportion of patients with 10 commonly accepted red flag criteria (singly and in combination) between patients with and without a diagnosis of serious secondary headache in a large, multinational cohort of ED patients presenting with headache. METHODS: Secondary analysis of data obtained in the HEAD and HEAD-Colombia studies. The outcome of interest was serious secondary headache. The predictive performance of 10 red flag criteria from the SNNOOP10 criteria list was estimated individually and in combination. RESULTS: 5293 patients were included, of whom 6.1% (95% CI 5.5% to 6.8%) had a defined serious cause identified. New neurological deficit, history of neoplasm, older age (>50 years) and recent head trauma (2-7 days prior) were independent predictors of a serious secondary headache diagnosis. After adjusting for other predictors, sudden onset, onset during exertion, pregnancy and immune suppression were not associated with a serious headache diagnosis. The combined sensitivity of the red flag criteria overall was 96.5% (95% CI 93.2% to 98.3%) but specificity was low, 5.1% (95% CI 4.3% to 6.0%). Positive predictive value was 9.3% (95% CI 8.2% to 10.5%) with negative predictive value of 93.5% (95% CI 87.6% to 96.8%). CONCLUSION: The sensitivity and specificity of the red flag criteria in this study were lower than previously reported. Regarding clinical practice, this suggests that red flag criteria may be useful to identify patients at higher risk of a serious secondary headache cause, but their low specificity could result in increased rates of CT scanning. TRIAL REGISTRATION NUMBER: ANZCTR376695.

Fishing for oil and meat drives irreversible defaunation of deepwater sharks and rays.

The deep ocean is the last natural biodiversity refuge from the reach of human activities. Deepwater sharks and rays are among the most sensitive marine vertebrates to overexploitation. One-third of threatened deepwater sharks are targeted, and half the species targeted for the international liver-oil trade are threatened with extinction. Steep population declines cannot be easily reversed owing to long generation lengths, low recovery potentials, and the near absence of management. Depth and spatial limits to fishing activity could improve conservation when implemented alongside catch regulations, bycatch mitigation, and international trade regulation. Deepwater sharks and rays require immediate trade and fishing regulations to prevent irreversible defaunation and promote recovery of this threatened megafauna group.

A Next-Generation Adenoviral Vaccine Elicits Mucosal and Systemic Immunogenicity and Reduces Viral Shedding after SARS-CoV-2 Challenge in Nonhuman Primates.

As new SARS-CoV-2 variants continue to emerge and impact communities worldwide, next-generation vaccines that enhance protective mucosal immunity may have a significant impact on productive infection and transmission. We have developed recombinant non-replicating adenovirus serotype 5 (rAd5) vaccines delivered by mucosal administration that express both target antigen and a novel molecular adjuvant within the same cell. Here, we describe the immunogenicity of three unique SARS-CoV-2 rAd5 vaccine candidates and their efficacy following viral challenge in non-human primates (NHPs). Intranasal immunization with rAd5 vaccines expressing Wuhan, or Beta variant spike alone, or Wuhan spike and nucleocapsid elicited strong antigen-specific serum IgG and IgA with neutralizing activity against multiple variants of concern (VOC). Robust cross-reactive mucosal IgA was detected after a single administration of rAd5, which showed strong neutralizing activity against multiple VOC. Additionally, mucosal rAd5 vaccination increased spike-specific IFN-γ producing circulating T-cells. Upon Beta variant SARS-CoV-2 challenge, all the vaccinated NHPs exhibited significant reductions in viral load and infectious particle shedding in both the nasal passages and lower airways. These findings demonstrate that mucosal rAd5 immunization is highly immunogenic, confers protective cross-reactive antibody responses in the circulation and mucosa, and reduces viral load and shedding after SARS-CoV-2 challenge.

CD8+ T cell metabolic flexibility elicited by CD28-ARS2 axis-driven alternative splicing of PKM supports antitumor immunity.

Metabolic flexibility has emerged as a critical determinant of CD8+ T-cell antitumor activity, yet the mechanisms driving the metabolic flexibility of T cells have not been determined. In this study, we investigated the influence of the nuclear cap-binding complex (CBC) adaptor protein ARS2 on mature T cells. In doing so, we discovered a novel signaling axis that endows activated CD8+ T cells with flexibility of glucose catabolism. ARS2 upregulation driven by CD28 signaling reinforced splicing factor recruitment to pre-mRNAs and affected approximately one-third of T-cell activation-induced alternative splicing events. Among these effects, the CD28-ARS2 axis suppressed the expression of the M1 isoform of pyruvate kinase in favor of PKM2, a key determinant of CD8+ T-cell glucose utilization, interferon gamma production, and antitumor effector function. Importantly, PKM alternative splicing occurred independently of CD28-driven PI3K pathway activation, revealing a novel means by which costimulation reprograms glucose metabolism in CD8+ T cells.

Narcotic analgesics.

There is documentation of the use of opium derived products in the ancient history of the Assyrians: the Egyptians; in the sixth century AD by the Roman Dioscorides; and by Avicenna (980-1037). Reference to opium like products is made by Paracelsus and by Shakespeare. Charles Louis Derosne and Fredrich Wilhelm Adam Serturner isolated morphine from raw opium in 1802 and 1806 respectively, and it was Sertürner who named the substance morphine, after Morpheus, the Greek God of dreams. By the middle 1800s, Opium and related opioid derived products were the source of a major addiction in USA, and to some extent in the United Kingdom. Opioid products are of major therapeutic value in the treatment of pain from injury, post surgery, intractable pain conditions, and some forms of terminal cancer.

Spondyloarthropathies and arthritis post-infection: a historical perspective.

The spondyloarthropathies are a group of conditions characterised by spinal joint pain and have related clinical, epidemiological and genetic-related features. Ankylosing spondylitis, reactive arthritis, the spinal form of psoriatic arthritis and Crohn's and colitis enteropathic arthritis are the major clinical entities of the spondyloarthropathies, and principally occur in HLA-B27 positive individuals. Ankylosing spondylitis is much more common in males than females. Patients are usually seronegative for rheumatoid factor, and extra-articular features including iridocyclitis, mucous membrane and skin lesions: aortitis, may occur in some patients. The reactive arthritis form classically occurs following an infection of the gastrointestinal or genitourinary tract. The Crohn's and colitis enteropathic arthritis forms often have an associated large joint asymmetrical arthritis. Also discussed are acute rheumatic fever and Lyme disease which are conditions where the individual develops arthritis after an infection.

Rheumatoid arthritis.

It is difficult to determine from ancient writings, old human specimens, and from Art over the centuries, as to when Rheumatoid Arthritis first appeared. It may be a relatively modern condition, as it was reasonably well described in the seventeenth century. Augustin Jacob Landre-Beauvais (1772-1840), University of Paris is credited, with the first clear description of the disease in his thesis. In 1859 Sir Alfred Baring Garrod (1819-1907), the "father of rheumatology", gave the disease its current name which was finally adapted in Britain by the Ministry of Health in 1922. Some forms of Juvenile Arthritis are related to adult Rheumatoid Arthritis (aka Still's disease). If untreated Rheumatoid arthritis can result in severe destructive joint damage and often there are associated severe systemic complications. Disease modifying agents have benefited the disease management, but it was the discovery of the anti TNF-alpha agents in the 1990s, and subsequently many additional Biologic agents, which have greatly changed the clinical outcome in Rheumatoid Arthritis.

Surgery of joints.

Crude forms of musculoskeletal surgery have been performed through history for the treatment of deformity, pain and the horrors of battle. In more modern times Muller is credited with the first synovectomy in rheumatoid arthritis in 1884, and a Synovectomy was first performed by Richard von Volkmann (1830-1889) for joint tuberculosis. Chemical synovectomy consisting of the intra-articular injection of various agents was popular for a while but is now largely discarded. Joint resection for sepsis and tuberculosis has been documented since the early 1800s, and also joint arthrodesis, and osteotomy. Modern arthroscopic techniques have added the utility of faster intra-joint inspection and treatment while reduced surgical time exposure and often applied with the use of limb regional anaesthetic nerve blocks, to avoid general anaesthetic. Joint arthroplasty has been developed since1800s, with the use of many artificial joint components. There have been many notable pioneers of this work who are documented in this text, among them Austin T. Moore (1899-1963), George McKee (1906-1991) and Sir John Charnley (1911-1982). The success of joint arthroplasty to the hip, knee, shoulder and other joints has resulted in life-changing benefit for hundreds of arthritis and injury sufferers.

Sjögren's syndrome.

Sjögren's syndrome (SS) is characterised as keratoconjunctivitis sicca (dry eyes), xerostomia (dry mouth) commonly associated with salivary gland enlargement, and is referred to as Primary Sjögren's syndrome. It is known as Secondary Sjögren's syndrome when it occurs in patients, with connective tissue disease, such as rheumatoid arthritis, systemic lupus erythematosus, polyarthritis nodosa, polymyositis, and systemic sclerosis. SS has also been associated with chronic graft-versus-host disease after allogeneic bone marrow transplantation, human immunodeficiency syndrome (AIDS), hepatitis C infection (HCV), chronic biliary cirrhosis, neoplastic and myeloplastic syndromes, fibromyalgia, and chronic fatigue syndrome.

Osteoarthritis.

The clinical appearance and radiological pattern of osteoarthritis have been identified in the skeletons of dinosaurs some 50-70 million years old, and in Egyptian mummies, and in ancient skeletons in England. Osteoarthritis patterns of joint involvement, often referred to as primary osteoarthritis, can be seen in the hands, spinal facet joints, hips, knees and feet, but can also be termed secondary osteoarthritis when seen in any joint that has had trauma, sepsis, surgery or metabolic insult. The prevalence of osteoarthritis increases with age. The histology and pathophysiology both demonstrate an inflammatory process. While there have been studies of genetic predisposition, the basic cause of primary osteoarthritis has not been determined.

Assessing cerebrovascular reactivity (CVR) in rhesus macaques (Macaca mulatta) using a hypercapnic challenge and pseudo-continuous arterial spin labeling (pCASL).

Cerebrovascular reactivity (CVR) is a measure of cerebral small vessels' ability to respond to changes in metabolic demand and can be quantified using magnetic resonance imaging (MRI) coupled with a vasoactive stimulus. Reduced CVR occurs with neurodegeneration and is associated with cognitive decline. While commonly measured in humans, few studies have evaluated CVR in animal models. Herein, we describe methods to induce hypercapnia in rhesus macaques (Macaca mulatta) under gas anesthesia to measure cerebral blood flow (CBF) and CVR using pseudo-continuous arterial spin labeling (pCASL). Fifteen (13 M, 2 F) adult rhesus macaques underwent pCASL imaging that included a baseline segment (100% O2) followed by a hypercapnic challenge (isoflurane anesthesia with 5% CO2, 95% O2 mixed gas). Relative hypercapnia was defined as an end-tidal CO2 (ETCO2) ≥5 mmHg above baseline ETCO2. The mean ETCO2 during the baseline segment of the pCASL sequence was 34 mmHg (range: 23-48 mmHg). During this segment, mean whole-brain CBF was 51.48 ml/100g/min (range: 21.47-77.23 ml/100g/min). Significant increases (p<0.0001) in ETCO2 were seen upon inspiration of the mixed gas (5% CO2, 95% O2). The mean increase in ETCO2 was 8.5 mmHg and corresponded with a mean increase in CBF of 37.1% (p<0.0001). The mean CVR measured was 4.3%/mmHg. No anesthetic complications occurred as a result of the CO2 challenge. Our methods were effective at inducing a state of relative hypercapnia that corresponds with a detectable increase in whole brain CBF using pCASL MRI. Using these methods, a CO2 challenge can be performed in conjunction with pCASL imaging to evaluate CBF and CVR in rhesus macaques. The measured CVR in rhesus macaques is comparable to human CVR highlighting the translational utility of rhesus macaques in neuroscience research. These methods present a feasible means to measure CVR in comparative models of neurodegeneration and cerebrovascular dysfunction.

MET Inhibitor Capmatinib Radiosensitizes MET Exon 14-Mutated and MET-Amplified Non-Small Cell Lung Cancer.

PURPOSE: The objective of this study was to investigate the effects of inhibiting the MET receptor with capmatinib, a potent and clinically relevant ATP-competitive tyrosine kinase inhibitor, in combination with radiation in MET exon 14-mutated and MET-amplified non-small cell lung (NSCLC) cancer models. METHODS AND MATERIALS: In vitro effects of capmatinib and radiation on cell proliferation, colony formation, MET signaling, apoptosis, and DNA damage repair were evaluated. In vivo tumor responses were assessed in cell line xenograft and patient-derived xenograft models. Immunohistochemistry was used to confirm the in vitro results. RESULTS: In vitro clonogenic survival assays demonstrated radiosensitization with capmatinib in both MET exon 14-mutated and MET-amplified NSCLC cell lines. No radiation-enhancing effect was observed in MET wild-type NSCLC and a human bronchial epithelial cell line. Minimal apoptosis was detected with the combination of capmatinib and radiation. Capmatinib plus radiation compared with radiation alone resulted in inhibition of DNA double-strand break repair, as measured by prolonged expression of γH2AX. In vivo, the combination of capmatinib and radiation significantly delayed tumor growth compared with vehicle control, capmatinib alone, or radiation alone. Immunohistochemistry indicated inhibition of phospho-MET and phospho-S6 and a decrease in Ki67 with inhibition of MET. CONCLUSIONS: Inhibition of MET with capmatinib enhances the effect of radiation in both MET exon 14-mutated and MET-amplified NSCLC models.