How Does Escherichia coli Allocate Proteome?

Microorganisms are shown to actively partition their intracellular resources, such as proteins, for growth optimization. Recent experiments have begun to reveal molecular components unpinning the partition; however, quantitatively, it remains unclear how individual parts orchestrate to yield precise resource allocation that is both robust and dynamic. Here, we developed a coarse-grained mathematical framework that centers on guanosine pentaphosphate (ppGpp)-mediated regulation and used it to systematically uncover the design principles of proteome allocation in Escherichia coli. Our results showed that the cellular ability of resource partition lies in an ultrasensitive, negative feedback-controlling topology with the ultrasensitivity arising from zero-order amino acid kinetics and the negative feedback from ppGpp-controlled ribosome synthesis. In addition, together with the time-scale separation between slow ribosome kinetics and fast turnovers of ppGpp and amino acids, the network topology confers the organism an optimization mechanism that mimics sliding mode control, a nonlinear optimization strategy that is widely used in man-made systems. We further showed that such a controlling mechanism is robust against parameter variations and molecular fluctuations and is also efficient for biomass production over time. This work elucidates the fundamental controlling mechanism of E. coli proteome allocation, thereby providing insights into quantitative microbial physiology as well as the design of synthetic gene networks.

Recruitment of CTCF to the SIRT1 promoter after Oxidative Stress mediates Cardioprotective Transcription.

Because most DNA-binding transcription factors (dbTFs), including the architectural regulator CTCF, bind RNA and exhibit di-/multimerization, a central conundrum is whether these distinct properties are regulated post-transcriptionally to modulate transcriptional programs. Here, investigating stress-dependent activation of SIRT1, encoding an evolutionarily-conserved protein deacetylase, we show that induced phosphorylation of CTCF acts as a rheostat to permit CTCF occupancy of low-affinity promoter DNA sites to precisely the levels necessary. This CTCF recruitment to the SIRT1 promoter is eliciting a cardioprotective cardiomyocyte transcriptional activation program and provides resilience against the stress of the beating heart in vivo . Mice harboring a mutation in the conserved low-affinity CTCF promoter binding site exhibit an altered, cardiomyocyte-specific transcriptional program and a systolic heart failure phenotype. This transcriptional role for CTCF reveals that a covalent dbTF modification regulating signal-dependent transcription serves as a previously unsuspected component of the oxidative stress response.

Distribution of Acute and Chronic Kidney Disease Across Clinical Phenotypes for Sepsis.

BACKGROUND: Sepsis is the most common cause of acute kidney injury (AKI) in critically ill patients. Four phenotypes (α, ß, γ, δ) for sepsis, which have different outcomes and responses to treatment, were described using routine clinical data in the electronic health record. RESEARCH QUESTION: Do the frequencies of AKI, acute kidney disease (AKD), chronic kidney disease (CKD), and AKI on CKD differ by sepsis phenotype? STUDY DESIGN AND METHODS: This was a secondary analysis of a randomized clinical trial of early resuscitation, including patients with septic shock at 31 sites. After excluding patients with end-stage kidney disease and missing data, we determined frequencies of the following clinical outcomes: AKI (defined within 24 h as Kidney Disease: Improving Global Outcomes stages 2 or 3 or stage 1 with tissue inhibitor of metalloproteinases-2 × insulin-like growth factor binding protein 7 value of > 2.0), CKD, and AKD (persistence of AKI at any stage on day 7 after enrollment) across four phenotypes. We performed multivariable logistic regression to assess the risk-adjusted association between development of AKI and AKD and phenotype. RESULTS: Among 1,090 eligible patients, 543 patients (50%) had AKI. Across phenotypes, the frequencies of AKI varied, being highest in the δ and ß phenotypes (78% and 71%, respectively) and the lowest in the α phenotype (26%; P < .001). AKD occurred most often in the δ phenotype (41%) and least often in the α phenotype (8%; P < .001). The highest frequencies of CKD and of AKI on CKD were found in the ß phenotype (53% and 38% respectively; P < .001 for both). In the multivariable logistic regression models (α phenotype as reference), δ phenotype showed the strongest association with AKI (OR, 12.33; 95% CI, 7.81-19.47; P < .001) and AKD (OR, 9.18; 95% CI, 5.44-15.51; P < .001). INTERPRETATION: The rates of AKI and AKD differed across clinical sepsis phenotypes and are more common among patients with phenotypes ß and δ. Phenotype ß showed a higher level of underlying CKD that predisposed patients to new AKI. The α and γ phenotypes showed lower frequencies of AKI and less progression to AKD.

Recovery after AKI: Effects on outcomes over 15 years.

PURPOSE: To examine the effect of kidney recovery on mortality, dialysis and kidney transplantation up to 15 years after AKI. MATERIALS AND METHODS: We studied 29,726 survivors of critical illness and compared these outcomes stratified by AKI and recovery status at hospital discharge. Kidney recovery was defined as a return of serum creatinine to ≤150% of baseline without dialysis prior to hospital discharge. RESULTS: Overall AKI occurred in 59.2% in which two thirds developed stage 2-3 AKI. Recovery rate of AKI at hospital discharge was 80.8%. Patients who did not recover experienced the worst 15-year mortality compared to those who recovered and those without AKI (57.8% vs 45.2% vs 30.3%, p < 0.001). This pattern was also found in subgroups of patients with suspected sepsis-associated (57.1% vs 47.9% vs 36.5%, p < 0.001) and cardiac surgery-associated AKI (60.1% vs 41.8% vs 25.9%, p < 0.001). The rates of dialysis and transplantation at 15 years were low and not associated with recovery status. CONCLUSIONS: Recovery of AKI in critically ill patients at hospital discharge had an effect on long-term mortality for up to 15 years. These results have implications for acute care, follow-up and choice of endpoints for clinical trials.

Extraintestinal pathogenic Escherichia coli (ExPEC) reservoirs, and antibiotics resistance trends: a one-health surveillance for risk analysis from "farm-to-fork".

Extraintestinal pathogenic Escherichia coli (ExPEC) associated infections are significant health concerns for both animals and humans. ExPEC strains are associated with various infections in humans, i.e. urinary tract infections, meningitis, septicemia, and other infections. Over the few years, several studies revealed, food animals act as a reservoir for ExPEC pathovars, but there is no information about the agricultural sector. In particular, the extensive use of antibiotics in food animals and agricultural settings could be significantly contributed to the emergence of antibiotic-resistant pathogens. However, global outbreaks of food-borne illnesses from contaminated food have made a significant concern for both public health and food safety. This review focuses on the reservoirs for ExPEC and their potential circulation between animals, humans, and environment. In this, we first report that the agricultural setting could be the reservoir of ExPEC and can play a role in disseminating antimicrobial-resistant ExPEC. A thorough understanding of ExPEC ecology, reservoirs, and transmission dynamics can significantly contribute to reducing the burden of ExPEC-associated infections. Overall, the study provides the important data on the current state of knowledge for different reservoirs with dynamic, dissemination, and transmission of antimicrobial-resistance ExPEC in animals, humans, and environment in the "One-Health" context.

Urinary tract infection and sepsis causing potential of multidrug-resistant Extraintestinal pathogenic E. coli isolated from plant-origin foods.

The dissemination of Extraintestinal pathogenic Escherichia coli (ExPEC) in food is a critical concern for human health and food safety. The present study is the first to systematically examine the diverse plant-origin foods such as cucumber, carrot, tomato, radish, chilli, fenugreek, coriander, peppermint, spring onion, cabbage, and spinach for the presence of ExPEC or specific putative ExPEC pathotypes with an in-depth assessment of their phylogenetics, virulence, and drug resistance. A total of 77 (15.9 %) ExPEC isolates were recovered from 1780 samples of the diverse plant-origin foods of distinct environments. Specific putative ExPEC pathotypes such as Uropathogenic E. coli (UPEC, 23.3 %) and Septicemia-associated E. coli (SEPEC, 24.6 %) were identified among ExPEC isolates. The Clermont revisited new phylotyping method revealed the varied distribution (1-27 %) of specific putative ExPEC pathotypes in the different phylogenetic lineages such as A, D/E, B1, and Clade 1, etc. All putative ExPEC pathotypes possess multiple genes (4.3-92.8 %) or phenotypes (3.3-100 %) associated with their virulence. In-vitro antimicrobial susceptibility testing of all putative ExPEC pathotypes demonstrated the presence of 100 % multidrug resistance with moderate to high (52-100 %) resistance to drugs used as last-resorts (chloramphenicol, colistin) or frontline (nitrofurantoin, sulfamethoxazole, ampicillin, gentamicin) in ExPEC-associated infections in humans. Overall, the present findings significantly contribute to our better understanding of the presence of ExPEC in the non-clinical niche, such as plant-origin foods with a possible consequence on human health and food safety.

Epidemiology, etiology and clinical associations of congenital heart disease identified during congenital rubella syndrome surveillance.

BACKGROUND: Congenital heart disease (CHD) is a common congenital malformation. Antenatal rubella infection in the mother and genetic defects are important causes to which CHD are attributed. Exact contribution of antenatal rubella infection or genetic causes to CHD is still unknown. OBJECTIVE: To study the epidemiology, etiology and clinical associations of echocardiographically confirmed congenital heart disease in infants in Western Rajasthan enrolled in the congenital rubella syndrome (CRS) surveillance project. To study the utility of clinical diagnostic criteria in identifying congenital rubella infection. METHOD: This was a prospective observational study, in which 251 patients with echocardiographically confirmed CHD were enrolled. Detailed clinical evaluation was done in all patients. Rubella serology was done in all patients. Genetic and other testing was done as appropriate. RESULT: The hospital-based prevalence of CHD in infants was 1% at our center. Fifty-seven percent of the babies had acyanotic septal heart defects of which ventricular septal defect (VSD) was the most common (35%). Anti-rubella immunoglobulin M (IgM) antibodies were positive in 8.5% of the CHD patients. A clinically identifiable genetic cause was present in 3.6% of the cases. In patients who tested positive for anti-rubella IgM antibodies also, VSD was the most common (33%) CHD followed by Tetralogy of Fallot (13.2%). CONCLUSION: CRS contributes to 8.5% of CHD. CRS is associated with a wide spectrum of CHD. The etiology of a large number of CHD remains elusive. Detailed studies on the cause and mechanism of development of CHD need to be undertaken.

VASOPRESSOR-RESISTANT HYPOTENSION, COMBINATION VASOPRESSOR THERAPY, AND SHOCK PHENOTYPES IN CRITICALLY ILL ADULTS WITH VASODILATORY SHOCK.

ABSTRACT: Objective: To examine the risk factors, resource utilization, and 1-year mortality associated with vasopressor-resistant hypotension (VRH) compared with vasopressor-sensitive hypotension (VSH) among critically ill adults with vasodilatory shock. We also examined whether combination vasopressor therapy and patient phenotype were associated with mortality. Design: Retrospective cohort study. Setting: Eight medical-surgical intensive care units at the University of Pittsburgh Medical Center, Pittsburgh, PA. Patients : Critically ill patients with vasodilatory shock admitted between July 2000 and October 2008. Interventions : None. Measurements and Main Results: Vasopressor-resistant hypotension was defined as those requiring greater than 0.2 µg/kg per minute of norepinephrine equivalent dose of vasopressor consecutively for more than 6 h, and VSH was defined as patients requiring ≤0.2 µg/kg per minute to maintain MAP between 55 and 70 mm Hg after adequate fluid resuscitation. Of 5,313 patients with vasodilatory shock, 1,291 patients (24.3%) developed VRH. Compared with VSH, VRH was associated with increased risk of acute kidney injury (72.7% vs. 65.0%; P < 0.001), use of kidney replacement therapy (26.0% vs. 11.0%; P < 0.001), longer median (interquartile range [IQR]) intensive care unit length of stay (10 [IQR, 4.0-20.0] vs. 6 [IQR, 3.0-13.0] days; P < 0.001), and increased 1-year mortality (64.7% vs. 34.8%; P < 0.001). Vasopressor-resistant hypotension was associated with increased odds of risk-adjusted mortality (adjusted odds ratio [aOR], 2.93; 95% confidence interval [CI], 2.52-3.40; P < 0.001). When compared with monotherapy, combination vasopressor therapy with two (aOR, 0.91; 95% CI, 0.78-1.06) and three or more vasopressors was not associated with lower mortality (aOR, 0.93; 95% CI, 0.68-1.27). Using a finite mixture model, we identified four unique phenotypes of patient clusters that differed with respect to demographics, severity of illness, processes of care, vasopressor use, and outcomes. Conclusions: Among critically ill patients with vasodilatory shock, VRH compared with VSH is associated with increased resource utilization and long-term risk of death. However, combination vasopressor therapy was not associated with lower risk of death. We identified four unique phenotypes of patient clusters that require further validation.

Listeria monocytogenes is a solvent tolerant organism secreting a solvent stable lipase: potential biotechnological applications.

PURPOSE: The emerging biobased economy will require robust, adaptable, organisms for the production and processing of biomaterials as well as for bioremediation. Recently, the search for solvent tolerant organisms and solvent tolerant enzymes has intensified. Resilient organisms secreting solvent stable lipases are of particular interest for biotechnological applications. METHODS: Screening of soil samples for lipase-producing organisms was carried out on Rhodamine B plates. The most productive lipase-producing organisms were further screened for their resistance to solvents commonly used in biotechnological applications. RESULTS: In the course of screening, one of the isolated organisms that exhibited extracellular lipase activity, was identified as the human pathogen Listeria monocytogenes through 16S rRNA sequencing. Further exploration revealed that this organism was resistant to solvents ranging from log P - 0.81 to 4.0. Moreover, in the presence of these solvents, L. monocytogenes secreted an extracellular, solvent tolerant, lipase activity. This lipase retained approximately 80% activity when incubated in 30% (v/v) methanol for 24 h. CONCLUSION: These findings identify L. monocytogenes as a potentially useful organism for biotechnological applications. However, the fact that Listeria is a pathogen is problematic and it will require the use of non-pathogenic or attenuated Listeria strains for practical applications. Nonetheless, the ability to adapt to rapidly changing environmental conditions, to grow at low temperatures, to resist solvents and to secrete an extracellular solvent tolerant lipase are unique and highly useful characteristics. The potential application of L. monocytogenes in wastewater bioremediation and plastics degradation is discussed.

E2F1-induced lncRNA, EMSLR regulates lncRNA LncPRESS1.

E2F1 induces hundreds of protein-coding genes influencing diverse signaling pathways but much less is known about its non-coding RNA targets. For identifying E2F1-dependent oncogenic long non-coding RNAs (lncRNAs), we carried out genome-wide transcriptome analysis and discovered an lncRNA, EMSLR, which is induced both in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). EMSLR depletion blocks the cells in G1 phase and inhibits the clonogenic ability indicating that it is essential for the tumor-related phenotypes. We discovered that EMSLR represses the promoter activity of another lncRNA, LncPRESS1, which is located 6.9 kb upstream of EMSLR and they display an inverse expression pattern in lung cancer cell lines. Depletion of C-MYC results in downregulation of EMSLR and simultaneous upregulation of EMSLR target LncPRESS1, exemplifying how C-MYC and E2F1 signal transduction pathways control the network of lncRNA genes to modulate cell proliferation and differentiation.

Guardian-Reported Impact of the COVID-19 Pandemic on the Lifestyle of Children with Diabetes Mellitus.

INTRODUCTION: The coronavirus disease-2019 (COVID-19) pandemic has had an unprecedented impact on the lives and lifestyles of people of all ages worldwide. Lifestyle has an essential role in the management of diabetes mellitus in children. METHODS: The study was carried out at a tertiary care centre in India. A telehealth survey was conducted among the parents/guardians of children with diabetes to study the impact of the COVID-19 pandemic. The survey evaluated the effects on lifestyle, diabetes management and challenges in connecting to a new telemedicine programme. RESULTS: The survey was completed by guardians of 91 patients. The mean age of the patients was 13.0 ± 3.8 years in boys and 11.9 ± 4.5 years in girls. Fifty-seven per cent of them were boys, and 63.7% stayed in rural areas. The pandemic has resulted in a significant increase in screen time and sleep duration. The median non-educational screen time has gone up from 1.00 (0.5-2.0) to 2.50 (1.0-4.0) h. The mean sleep duration in children increased from 9.1 ± 1.4 to 9.7 ± 1.4 h. Telemedicine services have been established with minimum resources, but they have limitations, and awareness about them is also limited. CONCLUSION: The COVID-19 pandemic has made the lifestyle of children with diabetes more sedentary. Some of them have also faced challenges with regard to diabetes-related supplies and management. It would be fair to anticipate more complications related to this sedentary lifestyle in the future and work towards identifying and treating them.

Early versus delayed initiation of renal replacement therapy in cardiac-surgery associated acute kidney injury: an economic perspective.

BACKGROUND: Timing for renal replacement therapy (RRT) initiation for cardiac-surgery associated acute kidney surgery (CSA-AKI) is subject to debate. Evidence suggests earlier initiation leads to shorter length of stay (LoS). We investigated differences in healthcare costs associated with timing of RRT initiation in CSA-AKI. METHODS: A cost-consequences model compared costs of Early (<24 h) vs. Delayed (>24 h) RRT initiation. Data were from the ELAIN trial in Germany, and the HiDenIC database, a US multi-hospital database. Resource utilization was determined by RRT duration, ICU, and hospital LoS. All resources were costed from a US healthcare perspective. Extensive sensitivity analyses (SA) were conducted, notably regarding the proportion of patients not initiated on RRT with the Delayed strategy. RESULTS: Early RRT initiation exhibited cost savings compared to Delayed RRT initiation. With ELAIN data, savings reached -$122,188 (ranging from -$157,707 to -$74,763 in the SA). Findings were confirmed with HiDenIC data; Early RRT initiation showed savings of -$77,303 (ranging from -$108,971 to -$47,012 in the SA). CONCLUSIONS: Our costing model indicates that Early RRT initiation for CSA-AKI may result in appreciable cost savings. Delaying RRT, in the setting of CSA-AKI, may lead to longer LoS and increased healthcare costs.

Association of Metformin Use During Hospitalization and Mortality in Critically Ill Adults With Type 2 Diabetes Mellitus and Sepsis.

OBJECTIVES: Whether metformin exposure is associated with improved outcomes in patients with type 2 diabetes mellitus and sepsis. DESIGN: Retrospective cohort study. SETTING: Patients admitted to ICUs in 16 hospitals in Pennsylvania from October 2008 to December 2014. PATIENTS: Adult critical ill patients with type 2 diabetes mellitus and sepsis. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We conducted a retrospective cohort study to compare 90-day mortality in diabetic patients with sepsis with and without exposure to metformin during hospitalization. Data were obtained from the electronic health record of a large healthcare system in Pennsylvania from October 2008 to December 2014, on patients admitted to the ICU at any of the 16 hospitals within the system. The primary outcome was mortality at 90 days. The absolute and adjusted odds ratio (OR) with 95% CI were calculated in a propensity score-matched cohort. Among 14,847 patients with type 2 diabetes mellitus and sepsis, 682 patients (4.6%) were exposed to metformin during hospitalization and 14,165 (95.4%) were not. Within a total of 2,691 patients subjected to propensity score-matching at a 1:4 ratio, exposure to metformin (n = 599) was associated with decreased 90-day mortality (71/599, 11.9% vs 475/2,092, 22.7%; OR, 0.46; 95% CI, 0.35-0.60), reduced severe acute kidney injury (50% vs 57%; OR, 0.75; 95% CI, 0.62-0.90), less Major Adverse Kidney Events at 1 year (OR, 0.27; 95% CI, 0.22-0.68), and increased renal recovery (95% vs 86%; OR, 6.43; 95% CI, 3.42-12.1). CONCLUSIONS: Metformin exposure during hospitalization is associated with a decrease in 90-day mortality in patients with type 2 diabetes mellitus and sepsis.

Kidney and Mortality Outcomes Associated with Ondansetron in Critically Ill Patients.

Background: Ondansetron is a preferred anti-emetic in critical care to treat nausea and vomiting, and has historically been considered a largely safe option. A recent pharmacoepidemiology study reported that ondansetron may be associated with an increased risk for acute kidney injury (AKI). Methods: We interrogated the High-Density Intensive Care (HiDenIC-15) database containing intensive care data for 13 hospitals across Western Pennsylvania between Oct 2008-Dec 2014. AKI was defined using the Kidney Disease, Improving Global Outcomes 2012 guidelines. Ondansetron use was considered as receiving any form of ondansetron within 24 h of admission. The subsequent 48 h (hours 25-72 after admission) were analyzed for outcomes. Primary outcome was development of AKI; secondary outcomes included 90-day mortality and time to AKI. Propensity-matched, multivariate logistic regression was applied for both outcomes. Comparator groups were metoclopramide and prochlorperazine using the same exposure criteria. Results:AKI occurred in 965 (5.6%), 12 (3.0%), and 61 (6.5%) patients receiving ondansetron, prochlorperazine, and metoclopramide, respectively. In the adjusted analysis, no anti-emetic was associated with a significant change in the odds of developing AKI. Ondansetron was associated with a 5.48% decrease (CI -6.17--4.79) in death within 90 days of ICU-admission, which was independent of AKI status; an effect not seen with other anti-emetics. Anti-emetic usage was not associated with a change in the time to first AKI. Conclusion:Anti-emetic usage did not alter AKI risk. Ondansetron was associated with a significant decrease in 90-day mortality that was not seen by other anti-emetics, which requires further exploration.

Cole-Cole modeling of real-time capacitance data for estimation of cell physiological properties in recombinant Escherichia coli cultivation.

Real-time estimation of physiological properties of the cell during recombinant protein production would ensure enhanced process monitoring. In this study, we explored the application of dielectric spectroscopy to track the fed-batch phase of recombinant Escherichia coli cultivation for estimating the physiological properties, namely, cell diameter and viable cell concentration (VCC). The scanning capacitance data from the dielectric spectroscopy were pre-processed using moving average. Later, it was modeled through a nonlinear theoretical Cole-Cole model and further solved using a global evolutionary genetic algorithm (GA). The parameters obtained from the GA were further applied for the estimation of the aforementioned physiological properties. The offline cell diameter and cell viability data were obtained from particle size analyzer and flow cytometry measurements to validate the Cole-Cole model. The offline VCC was calculated from the cell viability % from flow cytometry data and dry cell weight concentration. The Cole-Cole model predicted the cell diameter and VCC with an error of 1.03% and 7.72%, respectively. The proposed approach can enable the operator to take real-time process decisions to achieve desired productivity and product quality.

Acute kidney disease predicts chronic kidney disease in pediatric non-kidney solid organ transplant patients.

BACKGROUND: Acute kidney disease (AKD) is defined as impaired kidney function present for <90 days with or without an acute kidney injury (AKI) event. Adults with AKD have an increased risk for progression to chronic kidney disease (CKD) and mortality. There are no data on the epidemiology of AKD in children after transplant. The aim of this study was to evaluate the incidence and risk factors for AKI, AKD, and CKD in children after transplantation. METHODS: This is a retrospective cohort study of all children undergoing non-kidney solid organ transplant between 2011 and 2019 at UPMC Children's Hospital of Pittsburgh. AKI and AKD were defined using the Kidney Disease Improving Global Outcomes criteria. Patients with a new estimated glomerular filtration rate <60 ml/min/1.73m2 persisting for >3 months met criteria for new CKD. Variables associated with AKI, AKD, and CKD were analyzed. RESULTS: Among 338 patients, 37.9% met criteria for severe AKI, 13% for AKD, and 8% for a new diagnosis of CKD. Stage 3 AKI was independently associated with AKD (OR: 5.35; 95% CI: 2.23-12.86). Severe AKI was not associated with new-onset CKD, whereas AKD was associated with new-onset CKD (OR: 29.74; CI: 11.22-78.82). CONCLUSION: AKD may be superior to AKI in predicting risk of CKD in children after non-kidney solid organ transplantation.

Bioprocess Control: Current Progress and Future Perspectives.

Typical bioprocess comprises of different unit operations wherein a near optimal environment is required for cells to grow, divide, and synthesize the desired product. However, bioprocess control caters to unique challenges that arise due to non-linearity, variability, and complexity of biotech processes. This article presents a review of modern control strategies employed in bioprocessing. Conventional control strategies (open loop, closed loop) along with modern control schemes such as fuzzy logic, model predictive control, adaptive control and neural network-based control are illustrated, and their effectiveness is highlighted. Furthermore, it is elucidated that bioprocess control is more than just automation, and includes aspects such as system architecture, software applications, hardware, and interfaces, all of which are optimized and compiled as per demand. This needs to be accomplished while keeping process requirement, production cost, market value of product, regulatory constraints, and data acquisition requirements in our purview. This article aims to offer an overview of the current best practices in bioprocess control, monitoring, and automation.