Structures of the entire human opioid receptor family.

Opioids are effective analgesics, but their use is beset by serious side effects, including addiction and respiratory depression, which contribute to the ongoing opioid crisis. The human opioid system contains four opioid receptors (µOR, δOR, κOR, and NOPR) and a set of related endogenous opioid peptides (EOPs), which show distinct selectivity toward their respective opioid receptors (ORs). Despite being key to the development of safer analgesics, the mechanisms of molecular recognition and selectivity of EOPs to ORs remain unclear. Here, we systematically characterize the binding of EOPs to ORs and present five structures of EOP-OR-Gi complexes, including ß-endorphin- and endomorphin-bound µOR, deltorphin-bound δOR, dynorphin-bound κOR, and nociceptin-bound NOPR. These structures, supported by biochemical results, uncover the specific recognition and selectivity of opioid peptides and the conserved mechanism of opioid receptor activation. These results provide a structural framework to facilitate rational design of safer opioid drugs for pain relief.

Regulation of the RNAPII Pool Is Integral to the DNA Damage Response.

In response to transcription-blocking DNA damage, cells orchestrate a multi-pronged reaction, involving transcription-coupled DNA repair, degradation of RNA polymerase II (RNAPII), and genome-wide transcription shutdown. Here, we provide insight into how these responses are connected by the finding that ubiquitylation of RNAPII itself, at a single lysine (RPB1 K1268), is the focal point for DNA-damage-response coordination. K1268 ubiquitylation affects DNA repair and signals RNAPII degradation, essential for surviving genotoxic insult. RNAPII degradation results in a shutdown of transcriptional initiation, in the absence of which cells display dramatic transcriptome alterations. Additionally, regulation of RNAPII stability is central to transcription recovery-persistent RNAPII depletion underlies the failure of this process in Cockayne syndrome B cells. These data expose regulation of global RNAPII levels as integral to the cellular DNA-damage response and open the intriguing possibility that RNAPII pool size generally affects cell-specific transcription programs in genome instability disorders and even normal cells.

UV Irradiation Induces a Non-coding RNA that Functionally Opposes the Protein Encoded by the Same Gene.

The transcription-related DNA damage response was analyzed on a genome-wide scale with great spatial and temporal resolution. Upon UV irradiation, a slowdown of transcript elongation and restriction of gene activity to the promoter-proximal ∼25 kb is observed. This is associated with a shift from expression of long mRNAs to shorter isoforms, incorporating alternative last exons (ALEs) that are more proximal to the transcription start site. Notably, this includes a shift from a protein-coding ASCC3 mRNA to a shorter ALE isoform of which the RNA, rather than an encoded protein, is critical for the eventual recovery of transcription. The non-coding ASCC3 isoform counteracts the function of the protein-coding isoform, indicating crosstalk between them. Thus, the ASCC3 gene expresses both coding and non-coding transcript isoforms with opposite effects on transcription recovery after UV-induced DNA damage.

Comprehensive Analysis of Hypermutation in Human Cancer.

We present an extensive assessment of mutation burden through sequencing analysis of >81,000 tumors from pediatric and adult patients, including tumors with hypermutation caused by chemotherapy, carcinogens, or germline alterations. Hypermutation was detected in tumor types not previously associated with high mutation burden. Replication repair deficiency was a major contributing factor. We uncovered new driver mutations in the replication-repair-associated DNA polymerases and a distinct impact of microsatellite instability and replication repair deficiency on the scale of mutation load. Unbiased clustering, based on mutational context, revealed clinically relevant subgroups regardless of the tumors' tissue of origin, highlighting similarities in evolutionary dynamics leading to hypermutation. Mutagens, such as UV light, were implicated in unexpected cancers, including sarcomas and lung tumors. The order of mutational signatures identified previous treatment and germline replication repair deficiency, which improved management of patients and families. These data will inform tumor classification, genetic testing, and clinical trial design.

Wireless Optofluidic Systems for Programmable In Vivo Pharmacology and Optogenetics.

In vivo pharmacology and optogenetics hold tremendous promise for dissection of neural circuits, cellular signaling, and manipulating neurophysiological systems in awake, behaving animals. Existing neural interface technologies, such as metal cannulas connected to external drug supplies for pharmacological infusions and tethered fiber optics for optogenetics, are not ideal for minimally invasive, untethered studies on freely behaving animals. Here, we introduce wireless optofluidic neural probes that combine ultrathin, soft microfluidic drug delivery with cellular-scale inorganic light-emitting diode (µ-ILED) arrays. These probes are orders of magnitude smaller than cannulas and allow wireless, programmed spatiotemporal control of fluid delivery and photostimulation. We demonstrate these devices in freely moving animals to modify gene expression, deliver peptide ligands, and provide concurrent photostimulation with antagonist drug delivery to manipulate mesoaccumbens reward-related behavior. The minimally invasive operation of these probes forecasts utility in other organ systems and species, with potential for broad application in biomedical science, engineering, and medicine.

Heavy-element production in a compact object merger observed by JWST.

The mergers of binary compact objects such as neutron stars and black holes are of central interest to several areas of astrophysics, including as the progenitors of gamma-ray bursts (GRBs)1, sources of high-frequency gravitational waves (GWs)2 and likely production sites for heavy-element nucleosynthesis by means of rapid neutron capture (the r-process)3. Here we present observations of the exceptionally bright GRB 230307A. We show that GRB 230307A belongs to the class of long-duration GRBs associated with compact object mergers4-6 and contains a kilonova similar to AT2017gfo, associated with the GW merger GW170817 (refs. 7-12). We obtained James Webb Space Telescope (JWST) mid-infrared imaging and spectroscopy 29 and 61 days after the burst. The spectroscopy shows an emission line at 2.15 microns, which we interpret as tellurium (atomic mass A = 130) and a very red source, emitting most of its light in the mid-infrared owing to the production of lanthanides. These observations demonstrate that nucleosynthesis in GRBs can create r-process elements across a broad atomic mass range and play a central role in heavy-element nucleosynthesis across the Universe.

Heat shock induces premature transcript termination and reconfigures the human transcriptome.

The heat shock (HS) response involves rapid induction of HS genes, whereas transcriptional repression is established more slowly at most other genes. Previous data suggested that such repression results from inhibition of RNA polymerase II (RNAPII) pause release, but here, we show that HS strongly affects other phases of the transcription cycle. Intriguingly, while elongation rates increase upon HS, processivity markedly decreases, so that RNAPII frequently fails to reach the end of genes. Indeed, HS results in widespread premature transcript termination at cryptic, intronic polyadenylation (IPA) sites near gene 5'-ends, likely via inhibition of U1 telescripting. This results in dramatic reconfiguration of the human transcriptome with production of new, previously unannotated, short mRNAs that accumulate in the nucleus. Together, these results shed new light on the basic transcription mechanisms induced by growth at elevated temperature and show that a genome-wide shift toward usage of IPA sites can occur under physiological conditions.

RECQL5 controls transcript elongation and suppresses genome instability associated with transcription stress.

RECQL5 is the sole member of the RECQ family of helicases associated with RNA polymerase II (RNAPII). We now show that RECQL5 is a general elongation factor that is important for preserving genome stability during transcription. Depletion or overexpression of RECQL5 results in corresponding shifts in the genome-wide RNAPII density profile. Elongation is particularly affected, with RECQL5 depletion causing a striking increase in the average rate, concurrent with increased stalling, pausing, arrest, and/or backtracking (transcription stress). RECQL5 therefore controls the movement of RNAPII across genes. Loss of RECQL5 also results in the loss or gain of genomic regions, with the breakpoints of lost regions located in genes and common fragile sites. The chromosomal breakpoints overlap with areas of elevated transcription stress, suggesting that RECQL5 suppresses such stress and its detrimental effects, and thereby prevents genome instability in the transcribed region of genes.

The Higgs boson turns ten.

The discovery of the Higgs boson, ten years ago, was a milestone that opened the door to the study of a new sector of fundamental physical interactions. We review the role of the Higgs field in the Standard Model of particle physics and explain its impact on the world around us. We summarize the insights into Higgs physics revealed so far by ten years of work, discuss what remains to be determined and outline potential connections of the Higgs sector with unsolved mysteries of particle physics.

A kilonova following a long-duration gamma-ray burst at 350 Mpc.

Gamma-ray bursts (GRBs) are divided into two populations1,2; long GRBs that derive from the core collapse of massive stars (for example, ref. 3) and short GRBs that form in the merger of two compact objects4,5. Although it is common to divide the two populations at a gamma-ray duration of 2 s, classification based on duration does not always map to the progenitor. Notably, GRBs with short (≲2 s) spikes of prompt gamma-ray emission followed by prolonged, spectrally softer extended emission (EE-SGRBs) have been suggested to arise from compact object mergers6-8. Compact object mergers are of great astrophysical importance as the only confirmed site of rapid neutron capture (r-process) nucleosynthesis, observed in the form of so-called kilonovae9-14. Here we report the discovery of a possible kilonova associated with the nearby (350 Mpc), minute-duration GRB 211211A. The kilonova implies that the progenitor is a compact object merger, suggesting that GRBs with long, complex light curves can be spawned from merger events. The kilonova of GRB 211211A has a similar luminosity, duration and colour to that which accompanied the gravitational wave (GW)-detected binary neutron star (BNS) merger GW170817 (ref. 4). Further searches for GW signals coincident with long GRBs are a promising route for future multi-messenger astronomy.

Sphingolipid metabolism cooperates with BAK and BAX to promote the mitochondrial pathway of apoptosis.

Mitochondria are functionally and physically associated with heterotypic membranes, yet little is known about how these interactions impact mitochondrial outer-membrane permeabilization (MOMP) and apoptosis. We observed that dissociation of heterotypic membranes from mitochondria inhibited BAK/BAX-dependent cytochrome c (cyto c) release. Biochemical purification of neutral sphingomyelinases that correlated with MOMP sensitization suggested that sphingolipid metabolism coordinates BAK/BAX activation. Using purified lipids and enzymes, sensitivity to MOMP was achieved by in vitro reconstitution of the sphingolipid metabolic pathway. Sphingolipid metabolism inhibitors blocked MOMP from heavy membrane preparations but failed to influence MOMP in the presence of sphingolipid-reconstituted, purified mitochondria. Furthermore, the sphingolipid products, sphingosine-1-PO(4) and hexadecenal, cooperated specifically with BAK and BAX, respectively. Sphingolipid metabolism was also required for cellular responses to apoptosis. Our studies suggest that BAK/BAX activation and apoptosis are coordinated through BH3-only proteins and a specific lipid milieu that is maintained by heterotypic membrane-mitochondrial interactions.

MDM2 Integrates Cellular Respiration and Apoptotic Signaling through NDUFS1 and the Mitochondrial Network.

Signaling diversity and subsequent complexity in higher eukaryotes is partially explained by one gene encoding a polypeptide with multiple biochemical functions in different cellular contexts. For example, mouse double minute 2 (MDM2) is functionally characterized as both an oncogene and a tumor suppressor, yet this dual classification confounds the cell biology and clinical literatures. Identified via complementary biochemical, organellar, and cellular approaches, we report that MDM2 negatively regulates NADH:ubiquinone oxidoreductase 75 kDa Fe-S protein 1 (NDUFS1), leading to decreased mitochondrial respiration, marked oxidative stress, and commitment to the mitochondrial pathway of apoptosis. MDM2 directly binds and sequesters NDUFS1, preventing its mitochondrial localization and ultimately causing complex I and supercomplex destabilization and inefficiency of oxidative phosphorylation. The MDM2 amino-terminal region is sufficient to bind NDUFS1, alter supercomplex assembly, and induce apoptosis. Finally, this pathway is independent of p53, and several mitochondrial phenotypes are observed in Drosophila and murine models expressing transgenic Mdm2.

SARM1 regulates NAD+-linked metabolism and select immune genes in macrophages.

Sterile alpha and HEAT/armadillo motif-containing protein (SARM1) was recently described as a NAD+-consuming enzyme and has previously been shown to regulate immune responses in macrophages. Neuronal SARM1 is known to contribute to axon degeneration due to its NADase activity. However, how SARM1 affects macrophage metabolism has not been explored. Here, we show that macrophages from Sarm1-/- mice display elevated NAD+ concentrations and lower cyclic ADP-ribose, a known product of SARM1-dependent NAD+ catabolism. Further, SARM1-deficient macrophages showed an increase in the reserve capacity of oxidative phosphorylation and glycolysis compared to WT cells. Stimulation of macrophages to a proinflammatory state by lipopolysaccharide (LPS) revealed that SARM1 restricts the ability of macrophages to upregulate glycolysis and limits the expression of the proinflammatory gene interleukin (Il) 1b, but boosts expression of anti-inflammatory Il10. In contrast, we show macrophages lacking SARM1 induced to an anti-inflammatory state by IL-4 stimulation display increased oxidative phosphorylation and glycolysis, and reduced expression of the anti-inflammatory gene, Fizz1. Overall, these data show that SARM1 fine-tunes immune gene transcription in macrophages via consumption of NAD+ and altered macrophage metabolism.

Telomere dysfunction in Tert knockout mice delays BrafV600E -induced melanoma development.

Telomerase activation is a crucial step in melanomagenesis, often occurring because of ultraviolet radiation (UVR)-induced mutations at the telomerase gene (TERT) promoter and rendering TERT transcription in response to the activated Raf-MAP kinase pathway by BRAFV600E mutation. Due to the excessively long telomeres in mice, this process does not occur during melanomagenesis in mouse models. To investigate the impact of telomere dysfunction on melanomagenesis, BrafV600E was induced in generations 1 and 4 (G1 and G4) of Tert-/- mice. Our findings revealed that, regardless of UVR exposure, melanoma development was delayed in G4 mice, which had shorter telomeres compared to G1 and wild-type C57BL/6J (G0) mice. Moreover, many G4 tumors displayed an accumulation of excessive DNA damage, as evidenced by increased γH2A.X staining. Tumors from UVR-exposed mice exhibited elevated p53 protein expression. Cultured tumor cells isolated from G4 mice displayed abundant chromosomal fusions and rearrangements, indicative of telomere dysfunction in these cells. Additionally, tumor cells derived from UVB-exposed mice exhibited constitutively elevated expression of mutant p53 proteins, suggesting that p53 was a target of UVB-induced mutagenesis. Taken together, our findings suggest that telomere dysfunction hampers melanomagenesis, and targeting telomere crisis-mediated genomic instability may hold promise for the prevention and treatment of melanoma.

Receptor Discordance in Metastatic Breast Cancer; a review of clinical and genetic subtype alterations from primary to metastatic disease.

PURPOSE: Receptor and subtype discordance between primary breast tumours and metastases is a frequently reported phenomenon. The aim of this article is to review the current evidence on receptor discordance in metastatic breast cancer and to explore the benefit of performing a repeat biopsy in this context. METHODS: Searches were undertaken on PubMed and Clinicaltrials.gov for relevant publications and trials. CONCLUSION: The current guidelines recommend offering to perform a biopsy of a metastatic lesion to evaluate receptor status. The choice of systemic therapy in metastatic disease is often based on the receptor status of the primary lesion. As therapeutic decision making is guided by subtype, biopsy of the metastatic lesion to determine receptor status may alter treatment. This article discusses discordance rates, the mechanisms of receptor discordance, the effect of discordance on treatment and survival outcomes, as well as highlighting some ongoing clinical trials in patients with metastatic breast cancer.

Digital DNA lifecycle security and privacy: an overview.

DNA sequencing technologies have advanced significantly in the last few years leading to advancements in biomedical research which has improved personalised medicine and the discovery of new treatments for diseases. Sequencing technology advancement has also reduced the cost of DNA sequencing, which has led to the rise of direct-to-consumer (DTC) sequencing, e.g. 23andme.com, ancestry.co.uk, etc. In the meantime, concerns have emerged over privacy and security in collecting, handling, analysing and sharing DNA and genomic data. DNA data are unique and can be used to identify individuals. Moreover, those data provide information on people's current disease status and disposition, e.g. mental health or susceptibility for developing cancer. DNA privacy violation does not only affect the owner but also affects their close consanguinity due to its hereditary nature. This article introduces and defines the term 'digital DNA life cycle' and presents an overview of privacy and security threats and their mitigation techniques for predigital DNA and throughout the digital DNA life cycle. It covers DNA sequencing hardware, software and DNA sequence pipeline in addition to common privacy attacks and their countermeasures when DNA digital data are stored, queried or shared. Likewise, the article examines DTC genomic sequencing privacy and security.

Predictive value of pretreatment circulating inflammatory response markers in the neoadjuvant treatment of breast cancer: meta-analysis.

BACKGROUND: Systemic inflammatory response markers have been found to have a prognostic role in several cancers, but their value in predicting the response to neoadjuvant chemotherapy in breast cancer is uncertain. A systematic review and meta-analysis of the literature was carried out to investigate this. METHODS: A systematic search of electronic databases was conducted to identify studies that explored the predictive value of circulating systemic inflammatory response markers in patients with breast cancer before commencing neoadjuvant therapy. A meta-analysis was undertaken for each inflammatory marker where three or more studies reported pCR rates in relation to the inflammatory marker. Outcome data are reported as ORs and 95% confidence intervals. RESULTS: A total of 49 studies were included, of which 42 were suitable for meta-analysis. A lower pretreatment neutrophil-to-lymphocyte ratio was associated with an increased pCR rate (pooled OR 1.66 (95% c.i. 1.32 to 2.09); P < 0.001). A lower white cell count (OR 1.96 (95% c.i. 1.29 to 2.97); P = 0.002) and a lower monocyte count (OR 3.20 (95% c.i. 1.71 to 5.97); P < 0.001) were also associated with a pCR. A higher lymphocyte count was associated with an increased pCR rate (OR 0.44 (95% c.i. 0.30 to 0.64); P < 0.001). CONCLUSION: The present study found the pretreatment neutrophil-to-lymphocyte ratio, white cell count, lymphocyte count, and monocyte count of value in the prediction of a pCR in the neoadjuvant treatment of breast cancer. Further research is required to determine their value in specific breast cancer subtypes and to establish optimal cut-off values, before their adoption in clinical practice.

Monopolar diathermy versus a vessel-sealing device for reducing postoperative drain output after simple mastectomy: randomized clinical trial.

BACKGROUND: Electrosurgical devices are commonly used during mastectomy for simultaneous dissection and haemostasis, and can provide potential benefits regarding vessel and lymphatic ligation. The aim of this prospective RCT was to assess whether using a vessel-sealing device (LigaSure™) improves perioperative outcomes compared with monopolar diathermy when performing simple mastectomy. METHODS: Patients were recruited prospectively and randomized in a 1 : 1 manner to undergo simple mastectomy using either LigaSure™ or conventional monopolar diathermy at a single centre. The primary outcome was the number of days the drain remained in situ after surgery. Secondary outcomes of interest included operating time and complications. RESULTS: A total of 86 patients were recruited (42 were randomized to the monopolar diathermy group and 44 were randomized to the LigaSure™ group). There was no significant difference in the mean number of days the drain remained in situ between the monopolar diathermy group and the LigaSure™ group (7.75 days versus 8.23 days; P = 0.613) and there was no significant difference in the mean total drain output between the monopolar diathermy group and the LigaSure™ group (523.50 ml versus 572.80 ml; P = 0.694). In addition, there was no significant difference in the mean operating time between the groups, for simple mastectomy alone (88.25 min for the monopolar diathermy group versus 107.20 min for the LigaSure™ group; P = 0.078) and simple mastectomy with sentinel lymph node biopsy (107.20 min for the monopolar diathermy group versus 114.40 min for the LigaSure™ group; P = 0.440). CONCLUSION: In this double-blinded single-centre RCT, there was no difference in the total drain output or the number of days the drain remained in situ between the monopolar diathermy group and the LigaSure™ group. REGISTRATION NUMBER: EudraCT 2018-003191-13 BEAUMONT HOSPITAL REC 18/66.

Exploring High-Purity Multiparton Scattering at Hadron Colliders.

Multiparton interactions are a fascinating phenomenon that occur in almost every high-energy hadron-hadron collision yet are remarkably difficult to study quantitatively. In this Letter, we present a strategy to optimally disentangle multiparton interactions from the primary scattering in a collision. That strategy enables probes of multiparton interactions that are significantly beyond the state of the art, including their characteristic momentum scale, the interconnection between primary and secondary scatters, and the pattern of three and potentially even more simultaneous hard scatterings. This opens a path to powerful new constraints on multiparton interactions for LHC phenomenology and to the investigation of their rich field-theoretical structure.

Neoadjuvant trastuzumab deruxtecan (T-DXd) with response-directed definitive therapy in early stage HER2-positive breast cancer: a phase II study protocol (SHAMROCK study).

BACKGROUND: The current standard of care in the neoadjuvant setting for high-risk HER2-positive (HER2 +) breast cancer is to combine systemic chemotherapy with dual HER2 blockade, trastuzumab and pertuzumab. Targeted therapies have significantly improved outcomes for patients with HER2-positive breast cancer. To improve treatment-associated toxicity, chemotherapy-sparing approaches are currently being investigated. Trastuzumab deruxtecan (T-DXd) is an HER2-directed antibody-drug-conjugate (ADC) with promising results in the metastatic setting for HER2-positive breast cancer. The SHAMROCK study investigates neoadjuvant T-DXd in early stage HER2-positive breast cancer, using pathological complete response (pCR) rate as the primary endpoint. METHODS: This is a phase II open-label, single arm, adaptive multi-centre trial of T-DXd in the neoadjuvant setting in stage 2-3 HER2-positive breast cancer. Eligible patients will receive 5.4 mg/kg of T-DXd intravenously every 3 weeks for up to 6 cycles. A repeat biopsy will performed after 2 cycles for the RNA disruption index (RDI) score assessment. According to their likelihood of pCR, as determined by the RDI score, patients will either undergo 4 or 6 cycles of T-DXd prior to imaging. Patients with imaging complete response (iCR) after either 4 or 6 cycles will proceed to surgery. Patients who do not achieve iCR will either undergo further systemic therapy or proceed to surgery. DISCUSSION: The SHAMROCK study is a chemotherapy-sparing approach to curative intent treatment, investigating neoadjuvant T-DXd. We hypothesise that neoadjuvant T-DXd will have a high pCR rate and be associated low toxicity in early stage HER2-positive breast cancer. TRIAL REGISTRATION: EudraCT Number: 2022-002485-32; ClinicalTrials.gov identifier: NCT05710666; Cancer Trials Ireland study number: CTRIAL-IE 22-01.