Influenza Infection in Humans Induces Broadly Cross-Reactive and Protective Neuraminidase-Reactive Antibodies.

Antibodies to the hemagglutinin (HA) and neuraminidase (NA) glycoproteins are the major mediators of protection against influenza virus infection. Here, we report that current influenza vaccines poorly display key NA epitopes and rarely induce NA-reactive B cells. Conversely, influenza virus infection induces NA-reactive B cells at a frequency that approaches (H1N1) or exceeds (H3N2) that of HA-reactive B cells. NA-reactive antibodies display broad binding activity spanning the entire history of influenza A virus circulation in humans, including the original pandemic strains of both H1N1 and H3N2 subtypes. The antibodies robustly inhibit the enzymatic activity of NA, including oseltamivir-resistant variants, and provide robust prophylactic protection, including against avian H5N1 viruses, in vivo. When used therapeutically, NA-reactive antibodies protected mice from lethal influenza virus challenge even 48 hr post infection. These findings strongly suggest that influenza vaccines should be optimized to improve targeting of NA for durable and broad protection against divergent influenza strains.

The lung fibroblast as "soil fertilizer" in breast cancer metastasis.

Fibroblasts strongly impact tumor progression, but whether they prime the pre-metastatic niche is poorly understood. In this issue of Immunity, Gong and Li et al. identify lung-specific immunosuppressive fibroblasts, which are hijacked by breast cancer cells to facilitate metastasis.

Polyreactive Broadly Neutralizing B cells Are Selected to Provide Defense against Pandemic Threat Influenza Viruses.

Polyreactivity is the ability of a single antibody to bind to multiple molecularly distinct antigens and is a common feature of antibodies induced upon pathogen exposure. However, little is known about the role of polyreactivity during anti-influenza virus antibody responses. By analyzing more than 500 monoclonal antibodies (mAbs) derived from B cells induced by numerous influenza virus vaccines and infections, we found mAbs targeting conserved neutralizing influenza virus hemagglutinin epitopes were polyreactive. Polyreactive mAbs were preferentially induced by novel viral exposures due to their broad viral binding breadth. Polyreactivity augmented mAb viral binding strength by increasing antibody flexibility, allowing for adaption to imperfectly conserved epitopes. Lastly, we found affinity-matured polyreactive B cells were typically derived from germline polyreactive B cells that were preferentially selected to participate in B cell responses over time. Together, our data reveal that polyreactivity is a beneficial feature of antibodies targeting conserved epitopes.

SARS-CoV-2 infection and persistence in the human body and brain at autopsy.

Coronavirus disease 2019 (COVID-19) is known to cause multi-organ dysfunction1-3 during acute infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with some patients experiencing prolonged symptoms, termed post-acute sequelae of SARS-CoV-2 (refs. 4,5). However, the burden of infection outside the respiratory tract and time to viral clearance are not well characterized, particularly in the brain3,6-14. Here we carried out complete autopsies on 44 patients who died with COVID-19, with extensive sampling of the central nervous system in 11 of these patients, to map and quantify the distribution, replication and cell-type specificity of SARS-CoV-2 across the human body, including the brain, from acute infection to more than seven months following symptom onset. We show that SARS-CoV-2 is widely distributed, predominantly among patients who died with severe COVID-19, and that virus replication is present in multiple respiratory and non-respiratory tissues, including the brain, early in infection. Further, we detected persistent SARS-CoV-2 RNA in multiple anatomic sites, including throughout the brain, as late as 230 days following symptom onset in one case. Despite extensive distribution of SARS-CoV-2 RNA throughout the body, we observed little evidence of inflammation or direct viral cytopathology outside the respiratory tract. Our data indicate that in some patients SARS-CoV-2 can cause systemic infection and persist in the body for months.

Cancer-Cell-Intrinsic Mechanisms Shaping the Tumor Immune Landscape.

Owing to their tremendous diversity and plasticity, immune cells exert multifaceted functions in tumor-bearing hosts, ranging from anti-tumor to pro-tumor activities. Tumor immune landscapes differ greatly between and within cancer types. Emerging evidence suggests that genetic aberrations in cancer cells dictate the immune contexture of tumors. Here, we review the current understanding of the mechanisms whereby common drivers of tumorigenesis modulate the tumor immune milieu. We discuss these findings in the context of clinical observations and examine how cancer-cell-intrinsic properties can be exploited to maximize the benefit of immunomodulatory therapies. Understanding the relationship between cancer cell-intrinsic genetic events and the immune response may enable personalized immune intervention strategies for cancer patients.

Somatostatin: Linking Cognition and Alzheimer's Disease to Therapeutic Targeting.

Over four decades of research support the link between Alzheimer's disease (AD) and somatostatin (somatotropin-releasing inhibitory factor, SRIF). SRIF and SRIF-expressing neurons play an essential role in brain function, modulating hippocampal activity and memory formation. Loss of SRIF and SRIF-expressing neurons in the brain rests at the center of a series of interdependent pathological events driven by amyloid-beta peptide (Aß), culminating in cognitive decline and dementia. The connection between the SRIF and AD further extends to the neuropsychiatric symptoms, seizure activity, and inflammation. Whereas, preclinical AD investigations show SRIF or SRIF-receptor agonist administration capable of enhancing cognition. SRIF receptor subtype-4 activation in particular presents unique attributes, with the potential to mitigate learning and memory decline, reduce comorbid symptoms, and enhance enzymatic degradation of Aß in the brain. Here we review the links between SRIF and AD, along with the therapeutic implications. Significance Statement Somatostatin and somatostatin-expressing neurons in the brain are extensively involved in cognition. Loss of somatostatin and somatostatin-expressing neurons in Alzheimer's disease rests at the center of a series of interdependent pathological events contributing to cognitive decline and dementia. Targeting somatostatin mediated processes has significant therapeutic potential for the treatment of Alzheimer's disease.

N4-Hydroxycytidine/molnupiravir inhibits RNA virus-induced encephalitis by producing less fit mutated viruses.

A diverse group of RNA viruses have the ability to gain access to the central nervous system (CNS) and cause severe neurological disease. Current treatment for people with this type of infection is generally limited to supportive care. To address the need for reliable antivirals, we utilized a strategy of lethal mutagenesis to limit virus replication. We evaluated ribavirin (RBV), favipiravir (FAV) and N4-hydroxycytidine (NHC) against La Crosse virus (LACV), which is one of the most common causes of pediatric arboviral encephalitis cases in North America and serves as a model for viral CNS invasion during acute infection. NHC was approximately 3 to 170 times more potent than RBV or FAV in neuronal cells. Oral administration of molnupiravir (MOV), the prodrug of NHC, decreased neurological disease development (assessed as limb paralysis, ataxia and weakness, repeated seizures, or death) by 31% (4 mice survived out of 13) when treatment was started on the day of infection. MOV also reduced disease by 23% when virus was administered intranasally (IN). NHC and MOV produced less fit viruses by incorporating predominantly G to A or C to U mutations. Furthermore, NHC also inhibited virus production of two other orthobunyaviruses, Jamestown Canyon virus and Cache Valley virus. Collectively, these studies indicate that NHC/MOV has therapeutic potential to inhibit viral replication and subsequent neurological disease caused by orthobunyaviruses and potentially as a generalizable strategy for treating acute viral encephalitis.

Outcomes of younger patients with mantle cell lymphoma experiencing late relapse (>24 months): the LATE-POD study.

ABSTRACT: Patients with mantle cell lymphoma (MCL) who experience first relapse/refractoriness can be categorized into early or late progression-of-disease (POD) groups, with a threshold of 24 months from MCL diagnosis. Bruton tyrosine kinase inhibitors (BTKi) are the established standard treatment at first relapse, but their effectiveness compared with chemoimmunotherapy (CIT) in late-POD patients remains unknown. In this international, observational cohort study, we evaluated outcomes among patients at first, late POD beyond 24 months. The primary objective was progression-free survival from the time of second-line therapy (PFS-2) of BTKi vs CIT. Overall, 385 late-POD patients were included from 10 countries. Their median age was 59 years (range, 19-70), and 77% were male. Median follow-up from the time of second-line therapy was 53 months (range, 12-144). Overall, 114 patients had second-line BTKi, whereas 271 had CIT, consisting of rituximab-bendamustine (R-B; n = 101), R-B and cytarabine (R-BAC; n = 70), or other regimens (mostly cyclophosphamide-hydroxydaunorubicin-vincristine-prednisone]- or platinum-based; n = 100). The 2 groups were balanced in clinicopathological features and median time to first relapse. Overall, BTKi was associated with significantly prolonged median PFS-2 than CIT (not reached [NR] vs 26 months, respectively; P = .0003) and overall survival (NR and 56 months, respectively; P = .03). Multivariate analyses showed that BTKi was associated with lower risk of death than R-B and other regimens (hazard ratio, 0.41 for R-B and 0.46 for others), but similar to R-BAC. These results may establish BTKi as the preferable second-line approach in patients with BTKi-naïve MCL.

Imbalance of APOB Lipoproteins and Large HDL in Type 1 Diabetes Drives Atherosclerosis.

BACKGROUND: Individuals with type 1 diabetes (T1D) generally have normal or even higher HDL (high-density lipoprotein)-cholesterol levels than people without diabetes yet are at increased risk for atherosclerotic cardiovascular disease (CVD). Human HDL is a complex mixture of particles that can vary in cholesterol content by >2-fold. To investigate if specific HDL subspecies contribute to the increased atherosclerosis associated with T1D, we created mouse models of T1D that exhibit human-like HDL subspecies. We also measured HDL subspecies and their association with incident CVD in a cohort of people with T1D. METHODS: We generated LDL receptor-deficient (Ldlr-/-) mouse models of T1D expressing human APOA1 (apolipoprotein A1). Ldlr-/-APOA1Tg mice exhibited the main human HDL subspecies. We also generated Ldlr-/-APOA1Tg T1D mice expressing CETP (cholesteryl ester transfer protein), which had lower concentrations of large HDL subspecies versus mice not expressing CETP. HDL particle concentrations and sizes and proteins involved in lipoprotein metabolism were measured by calibrated differential ion mobility analysis and targeted mass spectrometry in the mouse models of T1D and in a cohort of individuals with T1D. Endothelial transcytosis was analyzed by total internal reflection fluorescence microscopy. RESULTS: Diabetic Ldlr-/-APOA1Tg mice were severely hyperglycemic and hyperlipidemic and had markedly elevated plasma APOB levels versus nondiabetic littermates but were protected from the proatherogenic effects of diabetes. Diabetic Ldlr-/-APOA1Tg mice expressing CETP lost the atheroprotective effect and had increased lesion necrotic core areas and APOB accumulation, despite having lower plasma APOB levels. The detrimental effects of low concentrations of larger HDL particles in diabetic mice expressing CETP were not explained by reduced cholesterol efflux. Instead, large HDL was more effective than small HDL in preventing endothelial transcytosis of LDL mediated by scavenger receptor class B type 1. Finally, in humans with T1D, increased concentrations of larger HDL particles relative to APOB100 negatively predicted incident CVD independently of HDL-cholesterol levels. CONCLUSIONS: Our results suggest that the balance between APOB lipoproteins and the larger HDL subspecies contributes to atherosclerosis progression and incident CVD in the setting of T1D and that larger HDLs exert atheroprotective effects on endothelial cells rather than by promoting macrophage cholesterol efflux.

A lower X-gate in TASK channels traps inhibitors within the vestibule.

TWIK-related acid-sensitive potassium (TASK) channels-members of the two pore domain potassium (K2P) channel family-are found in neurons1, cardiomyocytes2-4 and vascular smooth muscle cells5, where they are involved in the regulation of heart rate6, pulmonary artery tone5,7, sleep/wake cycles8 and responses to volatile anaesthetics8-11. K2P channels regulate the resting membrane potential, providing background K+ currents controlled by numerous physiological stimuli12-15. Unlike other K2P channels, TASK channels are able to bind inhibitors with high affinity, exceptional selectivity and very slow compound washout rates. As such, these channels are attractive drug targets, and TASK-1 inhibitors are currently in clinical trials for obstructive sleep apnoea and atrial fibrillation16. In general, potassium channels have an intramembrane vestibule with a selectivity filter situated above and a gate with four parallel helices located below; however, the K2P channels studied so far all lack a lower gate. Here we present the X-ray crystal structure of TASK-1, and show that it contains a lower gate-which we designate as an 'X-gate'-created by interaction of the two crossed C-terminal M4 transmembrane helices at the vestibule entrance. This structure is formed by six residues (243VLRFMT248) that are essential for responses to volatile anaesthetics10, neurotransmitters13 and G-protein-coupled receptors13. Mutations within the X-gate and the surrounding regions markedly affect both the channel-open probability and the activation of the channel by anaesthetics. Structures of TASK-1 bound to two high-affinity inhibitors show that both compounds bind below the selectivity filter and are trapped in the vestibule by the X-gate, which explains their exceptionally low washout rates. The presence of the X-gate in TASK channels explains many aspects of their physiological and pharmacological behaviour, which will be beneficial for the future development and optimization of TASK modulators for the treatment of heart, lung and sleep disorders.

p53 deficient breast cancer cells reprogram preadipocytes toward tumor-protective immunomodulatory cells.

The TP53 gene is mutated in approximately 30% of all breast cancer cases. Adipocytes and preadipocytes, which constitute a substantial fraction of the stroma of normal mammary tissue and breast tumors, undergo transcriptional, metabolic, and phenotypic reprogramming during breast cancer development and play an important role in tumor progression. We report here that p53 loss in breast cancer cells facilitates the reprogramming of preadipocytes, inducing them to acquire a unique transcriptional and metabolic program that combines impaired adipocytic differentiation with augmented cytokine expression. This, in turn, promotes the establishment of an inflammatory tumor microenvironment, including increased abundance of Ly6C+ and Ly6G+ myeloid cells and elevated expression of the immune checkpoint ligand PD-L1. We also describe a potential gain-of-function effect of common p53 missense mutations on the inflammatory reprogramming of preadipocytes. Altogether, our study implicates p53 deregulation in breast cancer cells as a driver of tumor-supportive adipose tissue reprogramming, expanding the network of non-cell autonomous mechanisms whereby p53 dysfunction may promote cancer. Further elucidation of the interplay between p53 and adipocytes within the tumor microenvironment may suggest effective therapeutic targets for the treatment of breast cancer patients.

Flipped C-Terminal Ends of APOA1 Promote ABCA1-Dependent Cholesterol Efflux by Small HDLs.

BACKGROUND: Cholesterol efflux capacity (CEC) predicts cardiovascular disease independently of high-density lipoprotein (HDL) cholesterol levels. Isolated small HDL particles are potent promoters of macrophage CEC by the ABCA1 (ATP-binding cassette transporter A1) pathway, but the underlying mechanisms are unclear. METHODS: We used model system studies of reconstituted HDL and plasma from control and lecithin-cholesterol acyltransferase (LCAT)-deficient subjects to investigate the relationships among the sizes of HDL particles, the structure of APOA1 (apolipoprotein A1) in the different particles, and the CECs of plasma and isolated HDLs. RESULTS: We quantified macrophage and ABCA1 CEC of 4 distinct sizes of reconstituted HDL. CEC increased as particle size decreased. Tandem mass spectrometric analysis of chemically cross-linked peptides and molecular dynamics simulations of APOA1, the major protein of HDL, indicated that the mobility of C-terminus of that protein was markedly higher and flipped off the surface in the smallest particles. To explore the physiological relevance of the model system studies, we isolated HDL from LCAT-deficient subjects, whose small HDLs (like reconstituted HDLs) are discoidal and composed of APOA1, cholesterol, and phospholipid. Despite their very low plasma levels of HDL particles, these subjects had normal CEC. In both the LCAT-deficient subjects and control subjects, the CEC of isolated extra-small HDL (a mixture of extra-small and small HDL by calibrated ion mobility analysis) was 3- to 5-fold greater than that of the larger sizes of isolated HDL. Incubating LCAT-deficient plasma and control plasma with human LCAT converted extra-small and small HDL particles into larger particles, and it markedly inhibited CEC. CONCLUSIONS: We present a mechanism for the enhanced CEC of small HDLs. In smaller particles, the C-termini of the 2 antiparallel molecules of APOA1 are "flipped" off the lipid surface of HDL. This extended conformation allows them to engage with ABCA1. In contrast, the C-termini of larger HDLs are unable to interact productively with ABCA1 because they form a helical bundle that strongly adheres to the lipid on the particle. Enhanced CEC, as seen with the smaller particles, predicts decreased cardiovascular disease risk. Thus, extra-small and small HDLs may be key mediators and indicators of the cardioprotective effects of HDL.

Clinical impact of additional findings detected by genome-wide non-invasive prenatal testing: Follow-up results of the TRIDENT-2 study.

In the TRIDENT-2 study, all pregnant women in the Netherlands are offered genome-wide non-invasive prenatal testing (GW-NIPT) with a choice of receiving either full screening or screening solely for common trisomies. Previous data showed that GW-NIPT can reliably detect common trisomies in the general obstetric population and that this test can also detect other chromosomal abnormalities (additional findings). However, evidence regarding the clinical impact of screening for additional findings is lacking. Therefore, we present follow-up results of the TRIDENT-2 study to determine this clinical impact based on the laboratory and perinatal outcomes of cases with additional findings. Between April 2017 and April 2019, additional findings were detected in 402/110,739 pregnancies (0.36%). For 358 cases, the origin was proven to be either fetal (n = 79; 22.1%), (assumed) confined placental mosaicism (CPM) (n = 189; 52.8%), or maternal (n = 90; 25.1%). For the remaining 44 (10.9%), the origin of the aberration could not be determined. Most fetal chromosomal aberrations were pathogenic and associated with severe clinical phenotypes (61/79; 77.2%). For CPM cases, occurrence of pre-eclampsia (8.5% [16/189] vs 0.5% [754/159,924]; RR 18.5), and birth weight <2.3rd percentile (13.6% [24/177] vs 2.5% [3,892/155,491]; RR 5.5) were significantly increased compared to the general obstetric population. Of the 90 maternal findings, 12 (13.3%) were malignancies and 32 (35.6%) (mosaic) pathogenic copy number variants, mostly associated with mild or no clinical phenotypes. Data from this large cohort study provide crucial information for deciding if and how to implement GW-NIPT in screening programs. Additionally, these data can inform the challenging interpretation, counseling, and follow-up of additional findings.

Germline-encoded specificities and the predictability of the B cell response.

Antibodies result from the competition of B cell lineages evolving under selection for improved antigen recognition, a process known as affinity maturation. High-affinity antibodies to pathogens such as HIV, influenza, and SARS-CoV-2 are frequently reported to arise from B cells whose receptors, the precursors to antibodies, are encoded by particular immunoglobulin alleles. This raises the possibility that the presence of particular germline alleles in the B cell repertoire is a major determinant of the quality of the antibody response. Alternatively, initial differences in germline alleles' propensities to form high-affinity receptors might be overcome by chance events during affinity maturation. We first investigate these scenarios in simulations: when germline-encoded fitness differences are large relative to the rate and effect size variation of somatic mutations, the same germline alleles persistently dominate the response of different individuals. In contrast, if germline-encoded advantages can be easily overcome by subsequent mutations, allele usage becomes increasingly divergent over time, a pattern we then observe in mice experimentally infected with influenza virus. We investigated whether affinity maturation might nonetheless strongly select for particular amino acid motifs across diverse genetic backgrounds, but we found no evidence of convergence to similar CDR3 sequences or amino acid substitutions. These results suggest that although germline-encoded specificities can lead to similar immune responses between individuals, diverse evolutionary routes to high affinity limit the genetic predictability of responses to infection and vaccination.

Revving Up Dendritic Cells while Braking PD-L1 to Jump-Start the Cancer-Immunity Cycle Motor.

Although it is successful for some, most melanoma patients are refractory to T cell checkpoint inhibition. In this issue of Immunity, Merad and colleagues (2016) describe a dendritic-cell-based strategy to heighten the efficacy of therapeutic anti-PD-L1 and BRAF inhibitors in mouse melanoma models.

Inflammasomes and Atherosclerosis: a Mixed Picture.

The CANTOS (Canakinumab Anti-inflammatory Thrombosis Outcome Study) and colchicine trials suggest an important role of inflammasomes and their major product IL-1ß (interleukin 1ß) in human atherosclerotic cardiovascular disease. Moreover, studies in mouse models indicate a causal role of inflammasomes and IL-1ß in atherosclerosis. However, recent studies have led to a more granular view of the role of inflammasomes in atherosclerosis. Studies in hyperlipidemic mouse models suggest that prominent activation of the NLRP3 inflammasome requires a second hit such as defective cholesterol efflux, defective DNA repair, clonal hematopoiesis or diabetes. Similarly in humans some mutations promoting clonal hematopoiesis increase coronary artery disease risk in part by promoting inflammasome activation. Recent studies in mice and humans point to a wider role of the AIM2 (absent in melanoma 2) inflammasome in promoting cardiovascular disease including in some forms of clonal hematopoiesis and diabetes. These developments suggest a precision medicine approach in which treatments targeting inflammasomes or IL-1ß might be best employed in clinical settings involving increased inflammasome activation.

Loss of p53 triggers WNT-dependent systemic inflammation to drive breast cancer metastasis.

Cancer-associated systemic inflammation is strongly linked to poor disease outcome in patients with cancer1,2. For most human epithelial tumour types, high systemic neutrophil-to-lymphocyte ratios are associated with poor overall survival3, and experimental studies have demonstrated a causal relationship between neutrophils and metastasis4,5. However, the cancer-cell-intrinsic mechanisms that dictate the substantial heterogeneity in systemic neutrophilic inflammation between tumour-bearing hosts are largely unresolved. Here, using a panel of 16 distinct genetically engineered mouse models for breast cancer, we uncover a role for cancer-cell-intrinsic p53 as a key regulator of pro-metastatic neutrophils. Mechanistically, loss of p53 in cancer cells induced the secretion of WNT ligands that stimulate tumour-associated macrophages to produce IL-1ß, thus driving systemic inflammation. Pharmacological and genetic blockade of WNT secretion in p53-null cancer cells reverses macrophage production of IL-1ß and subsequent neutrophilic inflammation, resulting in reduced metastasis formation. Collectively, we demonstrate a mechanistic link between the loss of p53 in cancer cells, secretion of WNT ligands and systemic neutrophilia that potentiates metastatic progression. These insights illustrate the importance of the genetic makeup of breast tumours in dictating pro-metastatic systemic inflammation, and set the stage for personalized immune intervention strategies for patients with cancer.

Impact of cancer cell-intrinsic features on neutrophil behavior.

Neutrophils are multifaceted innate immune cells that play a significant role in the progression of cancer by exerting both pro- and anti-tumorigenic functions. The crosstalk between cancer cells and neutrophils is complex and emerging evidence is pointing at cancer cell-intrinsic programs regulating neutrophil abundance, phenotype and function. Cancer cell-derived soluble mediators are key players in modulating the interaction with neutrophils. Here, we review how intrinsic features of cancer cells, including cancer cell genetics, epigenetics, signaling, and metabolism, manipulate neutrophil behavior and how to target these processes to impact cancer progression. A molecular understanding of cancer cell-intrinsic properties that shape the crosstalk with neutrophils will provide novel therapeutic strategies for personalized immunomodulation in cancer patients.

Tick-borne Encephalitis Clinical Characteristics in Adult Patients: A 10-year Retrospective Study in Stockholm, Sweden.

BACKGROUND: The incidence of Tick-borne encephalitis (TBE) has increased during the last decades in Europe. Our aim was to assess the clinical characteristics and outcome of TBE patients in Region Stockholm, as a high-risk area in Sweden. METHODS: The notification database at the regional Department of Communicable Disease Control and Prevention was used to identify TBE cases during 2006-2015. Clinical data was retrieved from the included patients' medical records. The associations of specific variables to predefined outcomes of disease severity were evaluated with multivariate logistic regression models. RESULTS: Of 1004 identified TBE cases, 703 adult patients were included. Sixty-one percent were men, and the median age was 50 years (range 18-94). The majority were non-vaccinated. Comorbidity was present in 34%, and 4% had immunomodulatory therapy. Seventy-five percent were hospitalised, and 11% had severe disease. More than 70% of the 79 patients followed up for more than 6 months had persisting symptoms. The case fatality rate was 1.4%, with 15% in the group with immunomodulatory treatment. In the multivariate analysis, severe disease was associated with underlying comorbidities, age ≥50 years, and previous complete TBE vaccination. CONCLUSION: This is the largest cohort of TBE patients in Scandinavia. Our findings of a more severe course of disease in patients of older age, with immunomodulatory therapy, with comorbidities, and vaccination breakthrough infections must be interpreted in the context of hospitalised patients. Optimised prevention is needed for patients with immunomodulatory therapy, given the considerable case fatality rate. Follow-up visits and rehabilitation should be better standardised.