Familial atrial fibrillation mutation M1875T-SCN5A increases early sodium current and dampens the effect of flecainide.

AIMS: Atrial fibrillation (AF) is the most common cardiac arrhythmia. Pathogenic variants in genes encoding ion channels are associated with familial AF. The point mutation M1875T in the SCN5A gene, which encodes the α-subunit of the cardiac sodium channel Nav1.5, has been associated with increased atrial excitability and familial AF in patients. METHODS AND RESULTS: We designed a new murine model carrying the Scn5a-M1875T mutation enabling us to study the effects of the Nav1.5 mutation in detail in vivo and in vitro using patch clamp and microelectrode recording of atrial cardiomyocytes, optical mapping, electrocardiogram, echocardiography, gravimetry, histology, and biochemistry. Atrial cardiomyocytes from newly generated adult Scn5a-M1875T+/- mice showed a selective increase in the early (peak) cardiac sodium current, larger action potential amplitude, and a faster peak upstroke velocity. Conduction slowing caused by the sodium channel blocker flecainide was less pronounced in Scn5a-M1875T+/- compared to wildtype atria. Overt hypertrophy or heart failure in Scn5a-M1875T+/- mice could be excluded. CONCLUSION: The Scn5a-M1875T point mutation causes gain-of-function of the cardiac sodium channel. Our results suggest increased atrial peak sodium current as a potential trigger for increased atrial excitability.

A systematic scoping review of multidisciplinary cancer team and decision-making in the management of men with advanced prostate cancer.

PURPOSE: The early diagnosis of prostate cancer and subsequent access to the treatment options helps to achieve optimal cancer outcomes. As the treatment options for patients with advanced prostate cancer continues to evolve, patients need to access a multidisciplinary team (MDT) meeting to receive best-practice care. METHODS: In this paper a MEDLINE review was performed to assess clinical decision making in the context of MDT discussions for patients with advanced prostate cancer. RESULTS: From 441 returned articles and abstracts, 50 articles were assessed for eligibility and 16 articles included for analysis. Sixteen articles were identified, 9 of the 16 articles used quantitative methodology including three retrospective analysis of clinical registry data, patient medical records and/or MDT meeting notes and three cross-sectional surveys. Other study designs included one observation study and one study using a combination of qualitative and quantitative methodologies and one mini-review. There were also four editorials included in the review and two consensus statements. CONCLUSION: This paper highlights the important role the inter-disciplinary MDT has on shared decision making for men with advanced prostate cancer. The application of MDT care is a rapidly growing trend in uro-oncology and an efficient MDT service requires further research to assess its efficiency so that it may expand through all aspect of uro-oncology.

Bioinformatic analysis of eosinophil activity and its implications for model and target species.

Eosinophils are important immune cells that have been implicated in resistance to gastrointestinal nematode (GIN) infections in both naturally and experimentally infected sheep. Proteins of particular importance appear to be IgA-Fc alpha receptor (FcαRI), C-C chemokine receptor type 3 (CCR3), proteoglycan 3 (PRG3, major basic protein 2) and EPX (eosinophil peroxidase). We used known human nucleotide sequences to search the ruminant genomes, followed by translation to protein and sequence alignments to visualize differences between sequences and species. Where a sequence was retrieved for cow, but not for sheep and goat, this was used additionally as a reference sequence. In this review, we show that eosinophil function varies among host species. Consequently, investigations into the mechanisms of ruminant immune responses to GIN should be conducted using the natural host. Specifically, we address differences in protein sequence and structure for eosinophil proteins.

The Portsmouth protocol for intra-operative ultrasound of the small bowel in Crohn's disease.

AIM: Bowel preservation is paramount in Crohn's disease surgery as affected patients are typically young adults at risk of having several abdominal surgical procedures during their lifetime. Intra-operative assessment of the extent and location of Crohn's disease is not standardized and is left to a mixture of the surgeon's experience, tactile feedback, macroscopic appearance and preoperative imaging. The aim of this study was to describe the technical steps of a standardized protocol for intra-operative ultrasound assessment of the small bowel in patients undergoing surgery for ileocolic Crohn's disease. METHOD: After laparoscopic mobilization of the bowel, a periumbilical incision is performed for extracorporeal division of the mesentery and the resection and anastomosis. A gastrointestinal consultant radiologist, with expertise in Crohn's disease imaging and abdominal ultrasound, performs full intra-operative assessment of the small bowel by applying a sterile ultrasound probe directly to the bowel, prior to resection being performed by the surgeon. The bowel is assessed through the wound protector with a sterile technique and the length, location and number of segments is documented together with further quantitative assessment using the METRIC (MR enterography or ultrasound in Crohn's disease) scoring guide. RESULTS: A step-by-step protocol for intra-operative ultrasound evaluation of the entire small bowel is described. CONCLUSIONS: A standardized approach to intra-operative evaluation of the extent and location of Crohn's disease is desirable. Intra-operative ultrasound may provide added value for assessment of proximal and multifocal Crohn's disease.

The Differences in Antibiotic Decision-making Between Acute Surgical and Acute Medical Teams: An Ethnographic Study of Culture and Team Dynamics.

BACKGROUND: Cultural and social determinants influence antibiotic decision-making in hospitals. We investigated and compared cultural determinants of antibiotic decision-making in acute medical and surgical specialties. METHODS: An ethnographic observational study of antibiotic decision-making in acute medical and surgical teams at a London teaching hospital was conducted (August 2015-May 2017). Data collection included 500 hours of direct observations, and face-to-face interviews with 23 key informants. A grounded theory approach, aided by Nvivo 11 software, analyzed the emerging themes. An iterative and recursive process of analysis ensured saturation of the themes. The multiple modes of enquiry enabled cross-validation and triangulation of the findings. RESULTS: In medicine, accepted norms of the decision-making process are characterized as collectivist (input from pharmacists, infectious disease, and medical microbiology teams), rationalized, and policy-informed, with emphasis on de-escalation of therapy. The gaps in antibiotic decision-making in acute medicine occur chiefly in the transition between the emergency department and inpatient teams, where ownership of the antibiotic prescription is lost. In surgery, team priorities are split between 3 settings: operating room, outpatient clinic, and ward. Senior surgeons are often absent from the ward, leaving junior staff to make complex medical decisions. This results in defensive antibiotic decision-making, leading to prolonged and inappropriate antibiotic use. CONCLUSIONS: In medicine, the legacy of infection diagnosis made in the emergency department determines antibiotic decision-making. In surgery, antibiotic decision-making is perceived as a nonsurgical intervention that can be delegated to junior staff or other specialties. Different, bespoke approaches to optimize antibiotic prescribing are therefore needed to address these specific challenges.

Supervised machine learning for the prediction of infection on admission to hospital: a prospective observational cohort study.

BACKGROUND: Infection diagnosis can be challenging, relying on clinical judgement and non-specific markers of infection. We evaluated a supervised machine learning (SML) algorithm for diagnosing bacterial infection using routinely available blood parameters on presentation to hospital. METHODS: An SML algorithm was developed to classify cases into infection versus no infection using microbiology records and six available blood parameters (C-reactive protein, white cell count, bilirubin, creatinine, ALT and alkaline phosphatase) from 160203 individuals. A cohort of patients admitted to hospital over a 6 month period had their admission blood parameters prospectively inputted into the SML algorithm. They were prospectively followed up from admission to classify those who fulfilled clinical case criteria for a community-acquired bacterial infection within 72 h of admission using a pre-determined definition. Predictive ability was assessed using receiver operating characteristics (ROC) with cut-off values for optimal sensitivity and specificity explored. RESULTS: One hundred and four individuals were included prospectively. The median (range) cohort age was 65 (21-98) years. The majority were female (56/104; 54%). Thirty-six (35%) were diagnosed with infection in the first 72 h of admission. Overall, 44/104 (42%) individuals had microbiological investigations performed. Treatment was prescribed for 33/36 (92%) of infected individuals and 4/68 (6%) of those with no identifiable bacterial infection. Mean (SD) likelihood estimates for those with and without infection were significantly different. The infection group had a likelihood of 0.80 (0.09) and the non-infection group 0.50 (0.29) (P < 0.01; 95% CI: 0.20-0.40). ROC AUC was 0.84 (95% CI: 0.76-0.91). CONCLUSIONS: An SML algorithm was able to diagnose infection in individuals presenting to hospital using routinely available blood parameters.

Middle Hepatic Vein Roadmap for a Safe Laparoscopic Right Hepatectomy.

BACKGROUND: When performing a right hepatectomy, the middle hepatic vein (MHV) should guide the parenchymal transection. MHV hotspots for bleeding can be anticipated when applying the previously developed MHV Roadmap to a minimally invasive approach.1 This video demonstrates application of the MHV Roadmap to perform a safe laparoscopic right hepatectomy. PATIENT: A 44-year-old woman with a solitary and large breast cancer liver metastasis in the right liver was considered for a laparoscopic right hepatectomy following an excellent response to neoadjuvant chemotherapy. The MHV anatomy was reconstructed using automated vascular reconstruction software (Synapse, Fuji) ahead of surgery. TECHNIQUE: With the patient in the French position, the hilar vessels are exposed and the inflow is controlled. Parenchymal transection begins along the demarcation line.2,3 The constant relationship between the portal bifurcation and the V5 ventral and dorsal allows for easy intraparenchymal identification of the MHV. The parenchymal transection is performed in a convex fashion to optimize exposure of the MHV. Using MHV guidance, the parenchymal transection is continued and V8 is safely identified. The operation is completed with division of the anterior fissure and right hepatic vein. CONCLUSION: Outlining the MHV anatomy according to the MHV Roadmap preoperatively helps to anticipate hotspots of bleeding. Guidance along the MHV through the parenchymal transection allows for early identification of tributaries, thereby preventing injury and remnant liver ischemia.

Methylomic profiling and replication implicates deregulation of PCSK9 in alcohol use disorder.

Alcohol use disorder (AUD) is a common and chronic disorder with substantial effects on personal and public health. The underlying pathophysiology is poorly understood but strong evidence suggests significant roles of both genetic and epigenetic components. Given that alcohol affects many organ systems, we performed a cross-tissue and cross-phenotypic analysis of genome-wide methylomic variation in AUD using samples from 3 discovery, 4 replication, and 2 translational cohorts. We identified a differentially methylated region in the promoter of the proprotein convertase subtilisin/kexin 9 (PCSK9) gene that was associated with disease phenotypes. Biological validation showed that PCSK9 promoter methylation is conserved across tissues and positively correlated with expression. Replication in AUD datasets confirmed PCSK9 hypomethylation and a translational mouse model of AUD showed that alcohol exposure leads to PCSK9 downregulation. PCSK9 is primarily expressed in the liver and regulates low-density lipoprotein cholesterol (LDL-C). Our finding of alcohol-induced epigenetic regulation of PCSK9 represents one of the underlying mechanisms between the well-known effects of alcohol on lipid metabolism and cardiovascular risk, with light alcohol use generally being protective while chronic heavy use has detrimental health outcomes.

Delivering precision antimicrobial therapy through closed-loop control systems.

Sub-optimal exposure to antimicrobial therapy is associated with poor patient outcomes and the development of antimicrobial resistance. Mechanisms for optimizing the concentration of a drug within the individual patient are under development. However, several barriers remain in realizing true individualization of therapy. These include problems with plasma drug sampling, availability of appropriate assays, and current mechanisms for dose adjustment. Biosensor technology offers a means of providing real-time monitoring of antimicrobials in a minimally invasive fashion. We report the potential for using microneedle biosensor technology as part of closed-loop control systems for the optimization of antimicrobial therapy in individual patients.

Partitioning heritability analysis reveals a shared genetic basis of brain anatomy and schizophrenia.

This corrects the article DOI: 10.1038/mp.2017.42.

Effect of adding a mobile health intervention to a multimodal antimicrobial stewardship programme across three teaching hospitals: an interrupted time series study.

Objectives: To evaluate the impact of adding a mobile health (mHealth) decision support system for antibiotic prescribing to an established antimicrobial stewardship programme (ASP). Methods: In August 2011, the antimicrobial prescribing policy was converted into a mobile application (app). A segmented regression analysis of interrupted time series was used to assess the impact of the app on prescribing indicators, using data (2008-14) from a biannual point prevalence survey of medical and surgical wards. There were six data points pre-implementation and six data points post-implementation. Results: There was an increase in compliance with policy (e.g. compliance with empirical therapy or expert advice) in the two specialties of medicine (6.48%, 95% CI = -1.25 to 14.20) and surgery (6.63%, 95% CI = 0.15-13.10) in the implementation period, with a significant sudden change in level in surgery ( P < 0.05). There was an increase, though not significant, in medicine (15.20%, 95% CI = -17.81 to 48.22) and surgery (35.97%, 95% CI = -3.72 to 75.66) in the percentage of prescriptions that had a stop/review date documented. The documentation of indication decreased in both medicine (-16.25%, 95% CI = -42.52 to 10.01) and surgery (-14.62%, 95% CI = -42.88 to 13.63). Conclusions: Introducing the app into an existing ASP had a significant impact on the compliance with policy in surgery, and a positive, but not significant, effect on documentation of stop/review date in both specialties. The negative effect on the third indicator may reflect a high level of compliance pre-intervention, due to existing ASP efforts. The broader value of providing an antimicrobial policy on a digital platform, e.g. the reach and access to the policy, should be measured using indicators more sensitive to mHealth interventions.

Partitioning heritability analysis reveals a shared genetic basis of brain anatomy and schizophrenia.

Schizophrenia is a devastating neurodevelopmental disorder with a complex genetic etiology. Widespread cortical gray matter loss has been observed in patients and prodromal samples. However, it remains unresolved whether schizophrenia-associated cortical structure variations arise due to disease etiology or secondary to the illness. Here we address this question using a partitioning-based heritability analysis of genome-wide single-nucleotide polymorphism (SNP) and neuroimaging data from 1750 healthy individuals. We find that schizophrenia-associated genetic variants explain a significantly enriched proportion of trait heritability in eight brain phenotypes (false discovery rate=10%). In particular, intracranial volume and left superior frontal gyrus thickness exhibit significant and robust associations with schizophrenia genetic risk under varying SNP selection conditions. Cross-disorder comparison suggests that the neurogenetic architecture of schizophrenia-associated brain regions is, at least in part, shared with other psychiatric disorders. Our study highlights key neuroanatomical correlates of schizophrenia genetic risk in the general population. These may provide fundamental insights into the complex pathophysiology of the illness, and a potential link to neurocognitive deficits shaping the disorder.

Durable fear memories require PSD-95.

Traumatic fear memories are highly durable but also dynamic, undergoing repeated reactivation and rehearsal over time. Although overly persistent fear memories underlie anxiety disorders, such as posttraumatic stress disorder, the key neural and molecular mechanisms underlying fear memory durability remain unclear. Postsynaptic density 95 (PSD-95) is a synaptic protein regulating glutamate receptor anchoring, synaptic stability and certain types of memory. Using a loss-of-function mutant mouse lacking the guanylate kinase domain of PSD-95 (PSD-95(GK)), we analyzed the contribution of PSD-95 to fear memory formation and retrieval, and sought to identify the neural basis of PSD-95-mediated memory maintenance using ex vivo immediate-early gene mapping, in vivo neuronal recordings and viral-mediated knockdown (KD) approaches. We show that PSD-95 is dispensable for the formation and expression of recent fear memories, but essential for the formation of precise and flexible fear memories and for the maintenance of memories at remote time points. The failure of PSD-95(GK) mice to retrieve remote cued fear memory was associated with hypoactivation of the infralimbic (IL) cortex (but not the anterior cingulate cortex (ACC) or prelimbic cortex), reduced IL single-unit firing and bursting, and attenuated IL gamma and theta oscillations. Adeno-associated virus-mediated PSD-95 KD in the IL, but not the ACC, was sufficient to impair recent fear extinction and remote fear memory, and remodel IL dendritic spines. Collectively, these data identify PSD-95 in the IL as a critical mechanism supporting the durability of fear memories over time. These preclinical findings have implications for developing novel approaches to treating trauma-based anxiety disorders that target the weakening of overly persistent fear memories.

Secretory component mediates Candida albicans binding to epithelial cells.

OBJECTIVES: Candida albicans attaches to oral surfaces via a number of mechanisms including adherence mediated by salivary components adsorbed to the C. albicans cell surface. Our goal was to identify the salivary molecules involved. MATERIALS AND METHODS: Biotinylated salivary polypeptides that were bound by C. albicans were detected in extracts from washed, saliva-treated yeast cells by polyacrylamide gel electrophoresis and electroblot or immunoblot transfer analysis and purified by electroelution. Purified material was tested for the ability to promote the adherence of radiolabelled C. albicans yeast cells to cultured epithelial monolayers. RESULTS: Three of the polypeptides bound by C. albicans cells were identified as components of secretory IgA, including secretory component. Using non-denaturing polyacrylamide gel electrophoresis, we demonstrated that secretory component could be detected in its free form in saliva, and was bound by yeast cells. Secretory component which was purified by electroelution from non-denaturing PAGE-separated saliva, without detectable complete IgA, promoted adherence of yeast cells to cultured epithelial monolayers in a dose-dependent fashion. CONCLUSION: These results indicate that despite the inhibitory effect on adherence of IgA specific to C. albicans, IgA components, in particular secretory component, also promote binding to cultured epithelial monolayers.

Innovative tools for quality assessment: integrated quality criteria for review of multiple study designs (ICROMS).

OBJECTIVES: With the aim to facilitate a more comprehensive review process in public health including patient safety, we established a tool that we have termed ICROMS (Integrated quality Criteria for the Review Of Multiple Study designs), which unifies, integrates and refines current quality criteria for a large range of study designs including qualitative research. STUDY DESIGN: Review, pilot testing and expert consensus. METHODS: The tool is the result of an iterative four phase process over two years: 1) gathering of established criteria for assessing controlled, non-controlled and qualitative study designs; 2) pilot testing of a first version in two systematic reviews on behavioural change in infection prevention and control and in antibiotic prescribing; 3) further refinement and adding of additional study designs in the context of the European Centre for Disease Prevention and Control funded project 'Systematic review and evidence-based guidance on organisation of hospital infection control programmes' (SIGHT); 4) scrutiny by the pan-European expert panel of the SIGHT project, which had the objective of ensuring robustness of the systematic review. RESULTS: ICROMS includes established quality criteria for randomised studies, controlled before-and-after studies and interrupted time series, and incorporates criteria for non-controlled before-and-after studies, cohort studies and qualitative studies. The tool consists of two parts: 1) a list of quality criteria specific for each study design, as well as criteria applicable across all study designs by using a scoring system; 2) a 'decision matrix', which specifies the robustness of the study by identifying minimum requirements according to the study type and the relevance of the study to the review question. The decision matrix directly determines inclusion or exclusion of a study in the review. ICROMS was applied to a series of systematic reviews to test its feasibility and usefulness in the appraisal of multiple study designs. The tool was applicable across a wide range of study designs and outcome measures. CONCLUSION: ICROMS is a comprehensive yet feasible appraisal of a large range of study designs to be included in systematic reviews addressing behaviour change studies in patient safety and public health. The tool is sufficiently flexible to be applied to a variety of other domains in health-related research. Beyond its application to systematic reviews, we envisage that ICROMS can have a positive effect on researchers to be more rigorous in their study design and more diligent in their reporting.

Exploring the epidemiology of carbapenem-resistant Gram-negative bacteria in west London and the utility of routinely collected hospital microbiology data.

OBJECTIVES: The objective of this study was to identify carbapenem-resistant organisms using routinely collected local microbiology data and describe the epidemiology of carbapenem resistance in two London teaching hospitals. METHODS: Data on inpatients infected or colonized with Gram-negative organisms between March 2009 and February 2012 were extracted. A computer algorithm was developed incorporating internationally recognized criteria to distinguish carbapenem-resistant organisms. Multivariable analysis was conducted to identify factors associated with infection or colonization with carbapenem-resistant organisms. Binomial regression was performed to detect changes in resistance trends over time. RESULTS: Yearly incidence of carbapenem resistance was observed to be increasing, with significant increasing trends in Acinetobacter baumannii (47.1% in 2009-10 to 77.2% in 2011-12; P<0.001) and Enterobacter spp. (2.2% in 2009-10 to 11.5% in 2011-12; P<0.001). Single-variable and multivariable analysis demonstrated differences in the proportion of carbapenem-resistant isolates across all variables investigated, including age, sex and clinical specialty; in the latter organism-specific niches were identified. Patients in the youngest age group (16-24 years old) had the highest odds of being infected or colonized with carbapenem-resistant isolates of Escherichia coli, Klebsiella spp. or Pseudomonas aeruginosa. Furthermore, proportions of carbapenem-resistant organisms differed between the hospitals. CONCLUSIONS: Carbapenem resistance is an emerging problem within the UK inpatient healthcare setting. This is not an issue confined to the Enterobacteriaceae and fine-resolution surveillance is needed to identify at-risk groups. Regular analysis of routinely collected data can provide insight into the evolving carbapenem-resistance threat, with the ability to inform efforts to prevent the spread of resistance.

Dissociable cortico-striatal connectivity abnormalities in major depression in response to monetary gains and penalties.

BACKGROUND: Individuals with major depressive disorder (MDD) are characterized by maladaptive responses to both positive and negative outcomes, which have been linked to localized abnormal activations in cortical and striatal brain regions. However, the exact neural circuitry implicated in such abnormalities remains largely unexplored. METHOD: In this study 26 unmedicated adults with MDD and 29 matched healthy controls (HCs) completed a monetary incentive delay task during functional magnetic resonance imaging (fMRI). Psychophysiological interaction (PPI) analyses probed group differences in connectivity separately in response to positive and negative outcomes (i.e. monetary gains and penalties). RESULTS: Relative to HCs, MDD subjects displayed decreased connectivity between the caudate and dorsal anterior cingulate cortex (dACC) in response to monetary gains, yet increased connectivity between the caudate and a different, more rostral, dACC subregion in response to monetary penalties. Moreover, exploratory analyses of 14 MDD patients who completed a 12-week, double-blind, placebo-controlled clinical trial after the baseline fMRI scans indicated that a more normative pattern of cortico-striatal connectivity pre-treatment was associated with greater improvement in symptoms 12 weeks later. CONCLUSIONS: These results identify the caudate as a region with dissociable incentive-dependent dACC connectivity abnormalities in MDD, and provide initial evidence that cortico-striatal circuitry may play a role in MDD treatment response. Given the role of cortico-striatal circuitry in encoding action-outcome contingencies, such dysregulated connectivity may relate to the prominent disruptions in goal-directed behavior that characterize MDD.

Chronic treatment with a tryptophan-rich protein hydrolysate improves emotional processing, mental energy levels and reaction time in middle-aged women.

Common pharmacological treatments of mood disorders aim to modulate serotonergic neurotransmission and enhance serotonin levels in the brain. Brain serotonin levels are dependent on the availability of its food-derived precursor essential amino acid tryptophan (Trp). We tested the hypothesis that delivery of Trp via food may serve as an alternative treatment, and examined the effects of a Trp-rich, bioavailable dietary supplement from egg protein hydrolysate on cognitive and emotional functions, mood state, and sleep quality. In a randomised, placebo-controlled, parallel trial, fifty-nine mentally and physically healthy women aged 45-65 years received placebo (n 30) or the supplement (n 29) (both as 0.5 g twice per d) for 19 d. Emotional processing was significantly changed by supplementation, exhibiting a shift in bias away from negative stimuli. The results for the Affective Go/No-Go Task exhibited a slowing of responses to negative words, suggesting reduced attention to negative emotional stimuli. The results for the Facial Emotional Expression Rating Task also supported a shift away from attention to negative emotions and a bias towards happiness. An increase in arousal-like symptoms, labelled 'high energy', shorter reaction times and a slight benefit to sustained attention were observed in the treated subjects. Finally, when the supplement was taken 60-90 min before bedtime, a feeling of happiness before going to bed was consistently reported. In summary, daily consumption of a low-dose supplement containing bioavailable Trp may have beneficial effects on emotional and cognitive functions.

Inter-laboratory comparison of multi-locus variable-number tandem repeat analysis (MLVA) for verocytotoxin-producing Escherichia coli O157 to facilitate data sharing.

Multi-locus variable number tandem repeat analysis (MLVA) is used in clinical and reference laboratories for subtyping verocytotoxin-producing Escherichia coli O157 (VTEC O157). However, as yet there is no common allelic or profile nomenclature to enable laboratories to easily compare data. In this study, we carried out an inter-laboratory comparison of an eight-loci MLVA scheme using a set of 67 isolates of VTEC O157. We found all but two isolates were identical in profile in the two laboratories, and repeat units were homogeneous in size but some were incomplete. A subset of the isolates (n = 17) were sequenced to determine the actual copy number of representative alleles, thereby enabling alleles to be named according to international consensus guidelines. This work has enabled us to realize the potential of MLVA as a portable, highly discriminatory and convenient subtyping method.