Generation of a bloodstream form Trypanosoma brucei double glycosyltransferase null mutant competent in receptor-mediated endocytosis of transferrin.

The bloodstream form of Trypanosoma brucei expresses large poly-N-acetyllactosamine (pNAL) chains on complex N-glycans of a subset of glycoproteins. It has been hypothesised that pNAL may be required for receptor-mediated endocytosis. African trypanosomes contain a unique family of glycosyltransferases, the GT67 family. Two of these, TbGT10 and TbGT8, have been shown to be involved in pNAL biosynthesis in bloodstream form Trypanosoma brucei, raising the possibility that deleting both enzymes simultaneously might abolish pNAL biosynthesis and provide clues to pNAL function and/or essentiality. In this paper, we describe the creation of a TbGT10 null mutant containing a single TbGT8 allele that can be excised upon the addition of rapamycin and, from that, a TbGT10 and TbGT8 double null mutant. These mutants were analysed by lectin blotting, glycopeptide methylation linkage analysis and flow cytometry. The data show that the mutants are defective, but not abrogated, in pNAL synthesis, suggesting that other GT67 family members can compensate to some degree for loss of TbGT10 and TbGT8. Despite there being residual pNAL synthesis in these mutants, certain glycoproteins appear to be particularly affected. These include the lysosomal CBP1B serine carboxypeptidase, cell surface ESAG2 and the ESAG6 subunit of the essential parasite transferrin receptor (TfR). The pNAL deficient TfR in the mutants continued to function normally with respect to protein stability, transferrin binding, receptor mediated endocytosis of transferrin and subcellular localisation. Further the pNAL deficient mutants were as viable as wild type parasites in vitro and in in vivo mouse infection experiments. Although we were able to reproduce the inhibition of transferrin uptake with high concentrations of pNAL structural analogues (N-acetylchito-oligosaccharides), this effect disappeared at lower concentrations that still inhibited tomato lectin uptake, i.e., at concentrations able to outcompete lectin-pNAL binding. Based on these findings, we recommend revision of the pNAL-dependent receptor mediated endocytosis hypothesis.

Cell proliferation and carcinogenesis: an approach to screening for potential human carcinogens.

Cancer arises from multiple genetic errors occurring in a single stem cell (clonality). Every time DNA replicates, mistakes occur. Thus, agents can increase the risk of cancer either by directly damaging DNA (DNA-reactive carcinogens) or increasing the number of DNA replications (increased cell proliferation). Increased cell proliferation can be achieved either by direct mitogenesis or cytotoxicity with regenerative proliferation. Human carcinogens have a mode of action of DNA reactivity, immunomodulation (mostly immunosuppression), increased estrogenic activity (mitogenesis), or cytotoxicity and regeneration. By focusing on screening for these four effects utilizing in silico, in vitro, and short-term in vivo assays, a biologically based screening for human chemical carcinogens can be accomplished with greater predictivity than the traditional 2-year bioassay with considerably less cost, less time, and the use of fewer animals.

Portable Saliva Sensor Based on Dual Recognition Elements for Detection of Caries Pathogenic Bacteria.

Dental caries is one of the most common diseases affecting more than 2 billion people's health worldwide. In a clinical setting, it is challenging to predict and proactively guard against dental cavities prior to receiving a confirmed diagnosis. Streptococcus mutans (S. mutans) in saliva has been recognized as the main causative bacterial agent that causes dental caries. High sensitivity, good selectivity, and a wide detection range are incredibly important factors to affect S. mutans detection in practical applications. In this study, we present a portable saliva biosensor designed for the early detection of S. mutans with the potential to predict the occurrence of dental cavities. The biosensor was fabricated using a S. mutans-specific DNA aptamer and S. mutans-imprinted polymers. Methylene blue was utilized as a redox probe in the sensor to generate current signals for analysis. When S. mutans enters complementarily S. mutans cavities, it blocks electron transfer between methylene blue and the electrode, resulting in decreases in the reduction current signal. The signal variations are associated with S. mutans concentrations that are useful for quantitative analysis. The linear detection range of S. mutans is 102-109 cfu mL-1, which covers the critical concentration of high caries risk. The biosensor exhibited excellent selectivity toward S. mutans in the presence of other common oral bacteria. The biosensor's wide detection range, excellent selectivity, and low limit of detection (2.6 cfu mL-1) are attributed to the synergistic effect of aptamer and S. mutans-imprinted polymers. The sensor demonstrates the potential to prevent dental caries.

Mammalian Meat Allergy and IgE to Alpha-gal in in Central Virginia: findings from a COVID-19 vaccine and patient cohort.

BACKGROUND: IgE to galactose-alpha-1,3-galactose (alpha-gal) is linked with tick bites and an important cause of anaphylaxis and urticarial reactions to mammalian meat. The "alpha-gal syndrome" (AGS) is recognized as being common in the southeastern USA, however prevalence studies are lacking and open questions remain about risk factors and clinical presentation of alpha-gal sensitization. OBJECTIVE: Here we characterized the prevalence, as well as presentation and risk factors, of AGS and alpha-gal IgE sensitization in adults in central Virginia recruited without regards to history of allergic disease. METHODS: Adults in central Virginia, primarily University of Virginia Health employees, were recruited as part of a COVID-19 vaccine study. Subjects provided at least one blood sample and answered questionnaires about medical and dietary history. ImmunoCAP was used for IgE assays and ABO blood group was assessed by reverse typing using stored serum. Biobanked serum from COVID-19 patients was also investigated. RESULTS: Of 267 enrollees, median age was 42, 76% were female and 43 (16%) were sensitized to alpha-gal (cut-off 0.1 IU/mL), of which mammalian meat allergy was reported by 7 (2.6%). Sensitized subjects were i) older, ii) had higher total IgE levels but similar frequency of IgE to common respiratory allergens, and iii) were more likely to report tick bites than non-sensitized subjects. Among those who were sensitized, alpha-gal IgE levels were higher among meat allergic than non-allergic subjects (GM 9.0 vs 0.5 IU/mL, P<0.001). Mammalian meat and dairy consumption was common in individuals with low-level sensitization. CONCLUSION: In central Virginia AGS is a dominant cause of adult food allergy with a prevalence approaching or exceeding 2%.

Extreme Ultraviolet Reflection Spectroscopy of Lanthanides and Actinides Using a High Harmonic Generation Light Source.

Absorption spectroscopy probing transitions from shallow-core d and f orbitals in lanthanides and actinides reveals information about bonding and the electronic structure in compounds containing these elements. However, spectroscopy in this photon energy range is challenging because of the limited availability of light sources and extremely short penetration depths. In this work, we address these challenges using a tabletop extreme ultraviolet (XUV), ultrafast, laser-driven, high harmonic generation light source, which generates femtosecond pulses in the 40-140 eV range. We present reflection spectroscopy measurements at the N4,5 (i.e., predominantly 4d to 5f transitions) and O4,5 (i.e., 5d to 5f transitions) absorption edges on several lanthanide and uranium oxide crystals. We compare these results to density functional theory calculations to assign the electronic transitions and predict the spectra for other lanthanides. This work paves the way for laboratory-scale XUV absorption experiments for studying crystalline and molecular f-electron systems, with applications ranging from surface chemistry, photochemistry, and electronic or chemical structure determination to nuclear forensics.

Identifying trigger cues for hospital blood transfusions based on ensemble of machine learning methods.

BACKGROUND: Traumatic shock is the leading cause of preventable death with most patients dying within the first six hours from arriving to the hospital. This underscores the importance of prehospital interventions, and growing evidence suggests prehospital transfusion improves survival. Optimizing transfusion triggers in the prehospital setting is key to improving outcomes for patients in hemorrhagic shock. Our objective was to identify factors associated with early in-hospital transfusion requirements available to prehospital clinicians in the field to develop a simple algorithm for prehospital transfusion, particularly for patients with occult shock. METHODS: We included trauma patients transported by a single critical care transport service to a level I trauma center between 2012 and 2019. We used logistic regression, Fast and Frugal Trees (FFTs), and Bayesian analysis to identify factors associated with early in-hospital blood transfusion as a potential trigger for prehospital transfusion. RESULTS: We included 2,157 patients transported from the scene or emergency department (ED) of whom 207 (9.60%) required blood transfusion within four hours of admission. The mean age was 47 (IQR = 28 - 62) and 1,480 (68.6%) patients were male. From 13 clinically relevant factors for early hospital transfusions, four were incorporated into the FFT in following order: 1) SBP, 2) prehospital lactate concentration, 3) Shock Index, 4) AIS of chest (sensitivity = 0.81, specificity = 0.71). The chosen thresholds were similar to conventional ones. Using conventional thresholds resulted in lower model sensitivity. Consistently, prehospital lactate was among most decisive factors of hospital transfusions identified by Bayesian analysis (OR = 2.31; 95% CI 1.55 - 3.37). CONCLUSIONS: Using an ensemble of frequentist statistics, Bayesian analysis and machine learning, we developed a simple, clinically relevant prehospital algorithm to help identify patients requiring transfusion within 4 h of hospital arrival.

Design and synthesis of oxytocin glycosides for the treatment of pain and substance use disorder.

Peptide drugs are a promising alternative to classical small molecule therapeutics with diverse applications, ranging from antibiotic resistant infection to prostate cancer. Oxytocin (OT) is a highly evolutionarily conserved peptide neurohormone and has been of interest for pharmaceutical use since 1909. Despite their increased safety profile relative to most small molecule drugs, peptides are poor candidates based on the pharmacokinetic (PK) properties from their peptide nature. Broad application of OT as a drug has been limited by these same PK issues. Several strategies have been proposed to overcome these limitations, among them glycosylation, which was used in combination with other sequence modifications to produce robust antinociception in mouse models, increased selectivity and potency at the OT receptor, and improved stability in rats.

Profiling Bacteria in the Lungs of Patients with Severe Influenza Versus COVID-19 with or without Aspergillosis.

RATIONALE: The influence of the lung bacterial microbiome, including potential pathogens, in patients with influenza- or COVID-19-associated pulmonary aspergillosis (IAPA or CAPA) is yet to be explored. OBJECTIVES: To explore the composition of the lung bacterial microbiome and its association with viral and fungal infection, immunity and outcome in severe influenza versus COVID-19 with or without aspergillosis. METHODS: We performed a retrospective study in mechanically ventilated influenza and COVID-19 patients with or without invasive aspergillosis in whom bronchoalveolar lavage (BAL) for bacterial culture (with or without PCR) was obtained within two weeks after ICU admission. Additionally, 16S rRNA gene sequencing data and viral and bacterial load of BAL samples from a subset of these patients, and of patients requiring non-invasive ventilation, were analyzed. We integrated 16S rRNA gene sequencing data with existing immune parameter datasets. MEASUREMENTS AND MAIN RESULTS: Potential bacterial pathogens were detected in 20% (28/142) of influenza and 37% (104/281) of COVID-19 patients, while aspergillosis was detected in 38% (54/142) of influenza and 31% (86/281) of COVID-19 patients. A significant association between bacterial pathogens in BAL and 90-day mortality was found only in influenza patients, particularly IAPA patients. COVID-19 but not influenza patients showed increased pro-inflammatory pulmonary cytokine responses to bacterial pathogens. CONCLUSIONS: Aspergillosis is more frequently detected in lungs of severe influenza patients than bacterial pathogens. Detection of bacterial pathogens associates with worse outcome in influenza patients, particularly in those with IAPA, but not in COVID-19 patients. The immunological dynamics of tripartite viral-fungal-bacterial interactions deserve further investigation. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Millimeter-Wave and High-Resolution Infrared Spectroscopy of 3-Furonitrile.

The rotational spectrum of 3-furonitrile has been collected from 85 to 500 GHz, spanning the most intense rotational transitions observable at room temperature. The large dipole moment imparted by the nitrile substituent confers substantial intensity to the rotational spectrum, enabling the observation of over 5600 new rotational transitions. Combined with previously published transitions, the available data set was least-squares fit to partial-octic, distorted-rotor A- and S-reduced Hamiltonian models with low statistical uncertainty (σfit < 0.031 MHz) for the ground vibrational state. Similar to its isomer 2-furonitrile, the two lowest-energy vibrationally excited states of 3-furonitrile (ν17, ν24), which correspond to the in-plane and out-of-plane nitrile bending vibrations, form an a- and b-axis Coriolis-coupled dyad. Rotationally resolved infrared transitions (30-600 cm-1) and over 4200 pure rotational transitions for both ν17 and ν24 were fit to a partial-octic, Coriolis-coupled, two-state Hamiltonian with low statistical uncertainty (σfit rot < 0.045 MHz, σfit IR < 6.1 MHz). The least-squares fitting of these vibrationally excited states provides their accurate and precise vibrational frequencies (ν17 = 168.193 164 8 (67) cm-1 and ν24 = 169.635 831 5 (77) cm-1) and seven Coriolis-coupling terms (Ga, GaJ, GaK, Fbc, FbcK, Gb, and Fac). The two fundamental states exhibit a notably small energy gap (1.442 667 (10) cm-1) and an inversion of the relative energies of ν17 and ν24 compared to those of the isomer 2-furonitrile. The rotational frequencies and spectroscopic constants of 3-furonitrile that we present herein provide a sufficient basis for conducting radioastronomical searches for this molecule across the majority of the frequency range available to current radiotelescopes.

An autoinhibitory switch of the LSD1 disordered region controls enhancer silencing.

Transcriptional coregulators and transcription factors (TFs) contain intrinsically disordered regions (IDRs) that are critical for their association and function in gene regulation. More recently, IDRs have been shown to promote multivalent protein-protein interactions between coregulators and TFs to drive their association into condensates. By contrast, here we demonstrate how the IDR of the corepressor LSD1 excludes TF association, acting as a dynamic conformational switch that tunes repression of active cis-regulatory elements. Hydrogen-deuterium exchange shows that the LSD1 IDR interconverts between transient open and closed conformational states, the latter of which inhibits partitioning of the protein's structured domains with TF condensates. This autoinhibitory switch controls leukemic differentiation by modulating repression of active cis-regulatory elements bound by LSD1 and master hematopoietic TFs. Together, these studies unveil alternative mechanisms by which disordered regions and their dynamic crosstalk with structured regions can shape coregulator-TF interactions to control cis-regulatory landscapes and cell fate.

Phase 1 randomized trials to assess safety, pharmacokinetics, and vaginal bleeding associated with use of extended duration dapivirine and levonorgestrel vaginal rings.

BACKGROUND: Vaginal rings formulated to deliver two drugs simultaneously have potential as user-controlled, long-acting methods for dual prevention of HIV and pregnancy. METHODS: Two phase 1 randomized trials (MTN-030/IPM 041 and MTN-044/IPM 053/CCN019) respectively enrolled 24 and 25 healthy, HIV-negative participants to evaluate safety, pharmacokinetics, and vaginal bleeding associated with use of a vaginal ring containing 200mg dapivirine (DPV) and 320mg levonorgestrel (LNG) designed for 90-day use. MTN-030/IPM 041 compared the DPV/LNG ring to a DPV-only ring (200mg) over 14 days of use. MTN-044/IPM 053/CCN019 compared continuous or cyclic use of the DPV/LNG ring over 90 days of use. Safety was assessed by recording adverse events (AEs). DPV and LNG concentrations were quantified in plasma, cervicovaginal fluid, and cervical tissue. Vaginal bleeding was self-reported. RESULTS: There were no differences in the proportion of participants with grade ≥2 genitourinary AEs or grade ≥3 AEs with DPV/LNG ring vs. DPV ring use (p = .22), or with DPV/LNG ring continuous vs. cyclic use (p = .67). Higher plasma DPV concentrations were observed in users of DPV/LNG compared to DPV-only rings (Cmax p = 0.049; AUC p = 0.091). Plasma DPV and LNG concentrations were comparable with continuous and cyclic use (Cmax p = 0.74; AUC p = 0.25). With cyclic use, median nadir plasma DPV concentration was approximately 300 pg/mL two days after removal and median t1/2 for cervicovaginal fluid DPV concentration was 5.76 hours (n = 3). Overall bleeding experiences did not differ between continuous and cyclic users (p = 0.12). CONCLUSIONS: The extended duration DPV/ LNG rings were well tolerated and the observed DPV concentrations in plasma and cervicovaginal fluid when used continuously exceeded concentrations observed in previous DPV ring efficacy studies. LNG concentrations in plasma were comparable with other efficacious LNG-based contraceptives. Genital DPV concentrations had a short half-life and were thus not well sustained following ring removal.

Evaluating a Novel AI Tool for Automated Measurement of the Aortic Root and Valve in Cardiac Magnetic Resonance Imaging.

Objective Evaluating an artificial intelligence (AI) tool (AIATELLA, version 1.0; AIATELLA Oy, Helsinki, Finland) in interpreting cardiac magnetic resonance (CMR) imaging to produce measurements of the aortic root and valve by comparison of accuracy and efficiency with that of three National Health Service (NHS) cardiologists. Methods AI-derived aortic root and valve measurements were recorded alongside manual measurements from three experienced NHS consultant cardiologists (CCs) over three separate sites in the northeast part of the United Kingdom. The study utilised a comprehensive dataset of CMR images, with the intraclass correlation coefficient (ICC) being the primary measure of concordance between the AI and the cardiologist assessments. Patient imaging was anonymised and blinded at the point of transfer to a secure data server.  Results The study demonstrates a high level of concordance between AI assessment of the aortic root and valve with NHS cardiologists (ICC of 0.98). Notably, the AI delivered results in 2.6 seconds (+/- 0.532) compared to a mean of 334.5 seconds (+/- 61.9) by the cardiologists, a statistically significant improvement in efficiency without compromising accuracy. Conclusion AI's accuracy and speed of analysis suggest that it could be a valuable tool in cardiac diagnostics, addressing the challenges of time-consuming and variable clinician-based assessments. This research reinforces AI's role in optimising the patient journey and improving the efficiency of the diagnostic pathway.

TRPV4 affects visual signals in photoreceptors and rod bipolar cells.

Introduction: Mechanical sensitive channels expressed in mammalian retinas are effectors of elevated pressure stresses, but it is unclear how their activation affects visual function in pressure-related retinal disorders. Methods: This study investigated the role of the transient potential channel vanilloid TRPV4 in photoreceptors and rod bipolar cells (RBCs) with immunohistochemistry, confocal microscopy, electroretinography (ERG), and patch-clamp techniques. Results: TRPV4 immunoreactivity (IR) was found in the outer segments of photoreceptors, dendrites and somas of PKCα-positive RBCs and other BCs, plexiform layers, and retinal ganglion cells (RGCs) in wild-type mice. TRPV4-IR was largely diminished in the retinas of homozygous TRPV4 transgenic mice. Genetically suppressing TRPV4 expression moderately but significantly enhanced the amplitude of ERG a- and b-waves evoked by scotopic and mesopic lights (0.55 to 200 Rh*rod-1 s-1) and photopic lights (105-106 Rh*rod-1 s-1) compared to wild-type mice in fully dark-adapted conditions. The implicit time evoked by dim lights (0.55 to 200 Rh*rod-1 s-1) was significantly decreased for b-waves and elongated for a-waves in the transgenic mice. ERG b-wave evoked by dim lights is primarily mediated by RBCs, and under voltage-clamp conditions, the latency of the light-evoked cation current in RBCs of the transgenic mice was significantly shorter compared to wild-type mice. About 10% of the transgenic mice had one eye undeveloped, and the percentage was significantly higher than in wild-type mice. Conclusions: The data indicates that TRPV4 involves ocular development and is expressed and active in outer retinal neurons, and interventions of TRPV4 can variably affect visual signals in rods, cones, RBCs, and cone ON BCs.

Single-Use, Metabolite Absorbing, Resonant Transducer (SMART) Culture Vessels for Label-Free, Continuous Cell Culture Progression Monitoring.

Secreted metabolites are an important class of bio-process analytical technology (PAT) targets that can correlate to cell conditions. However, current strategies for measuring metabolites are limited to discrete measurements, resulting in limited understanding and ability for feedback control strategies. Herein, a continuous metabolite monitoring strategy is demonstrated using a single-use metabolite absorbing resonant transducer (SMART) to correlate with cell growth. Polyacrylate is shown to absorb secreted metabolites from living cells containing hydroxyl and alkenyl groups such as terpenoids, that act as a plasticizer. Upon softening, the polyacrylate irreversibly conformed into engineered voids above a resonant sensor, changing the local permittivity which is interrogated, contact-free, with a vector network analyzer. Compared to sensing using the intrinsic permittivity of cells, the SMART approach yields a 20-fold improvement in sensitivity. Tracking growth of many cell types such as Chinese hamster ovary, HEK293, K562, HeLa, and E. coli cells as well as perturbations in cell proliferation during drug screening assays are demonstrated. The sensor is benchmarked to show continuous measurement over six days, ability to track different growth conditions, selectivity to transducing active cell growth metabolites against other components found in the media, and feasibility to scale out for high throughput campaigns.

Effectiveness of Updated 2023-2024 (Monovalent XBB.1.5) COVID-19 Vaccination Against SARS-CoV-2 Omicron XBB and BA.2.86/JN.1 Lineage Hospitalization and a Comparison of Clinical Severity - IVY Network, 26 Hospitals, October 18, 2023-March 9, 2024.

Background: Assessing COVID-19 vaccine effectiveness (VE) and severity of SARS-CoV-2 variants can inform public health risk assessments and decisions about vaccine composition. BA.2.86 and its descendants, including JN.1 (referred to collectively as "JN lineages"), emerged in late 2023 and exhibited substantial genomic divergence from co-circulating XBB lineages. Methods: We analyzed patients hospitalized with COVID-19-like illness at 26 hospitals in 20 U.S. states admitted October 18, 2023-March 9, 2024. Using a test-negative, case-control design, we estimated the effectiveness of an updated 2023-2024 (Monovalent XBB.1.5) COVID-19 vaccine dose against sequence-confirmed XBB and JN lineage hospitalization using logistic regression. Odds of severe outcomes, including intensive care unit (ICU) admission and invasive mechanical ventilation (IMV) or death, were compared for JN versus XBB lineage hospitalizations using logistic regression. Results: 585 case-patients with XBB lineages, 397 case-patients with JN lineages, and 4,580 control-patients were included. VE in the first 7-89 days after receipt of an updated dose was 54.2% (95% CI = 36.1%-67.1%) against XBB lineage hospitalization and 32.7% (95% CI = 1.9%-53.8%) against JN lineage hospitalization. Odds of ICU admission (adjusted odds ratio [aOR] 0.80; 95% CI = 0.46-1.38) and IMV or death (aOR 0.69; 95% CI = 0.34-1.40) were not significantly different among JN compared to XBB lineage hospitalizations. Conclusions: Updated 2023-2024 COVID-19 vaccination provided protection against both XBB and JN lineage hospitalization, but protection against the latter may be attenuated by immune escape. Clinical severity of JN lineage hospitalizations was not higher relative to XBB lineage hospitalizations.

Scanning the aged to minimize missed injury: An EAST multicenter study.

BACKGROUND: Despite the high incidence of blunt trauma in older adults, there is a lack of evidence-based guidance for computed tomography (CT) imaging in this population. We aimed to identify an algorithm to guide use of a Pan-Scan (Head/C-spine/Torso) or a Selective Scan (Head/C-spine ± Torso). We hypothesized that a patient's initial history and exam could be used to guide imaging. METHODS: We prospectively studied blunt trauma patients aged 65+ at 18 Level I/II trauma centers. Patients presenting >24 h after injury or who died upon arrival were excluded. We collected history and physical elements and final injury diagnoses. Injury diagnoses were categorized into CT body regions of Head/C-spine or Torso (chest, abdomen/pelvis, and T/L spine). Using machine learning and regression modeling as well as a priori clinical algorithms based, we tested various decision rules against our dataset. Our priority was to identify a simple rule which could be applied at the bedside, maximizing sensitivity (Sens) and negative predictive value (NPV) to minimize missed injuries. RESULTS: We enrolled 5,498 patients with 3,082 injuries. Nearly half (47.1%, n = 2,587) had an injury within the defined CT body regions. No rule to guide a Pan-Scan could be identified with suitable Sens/NPV for clinical use. A clinical algorithm to identify patients for Pan-Scan, using a combination of physical exam findings and specific high-risk criteria, was identified and had a Sens of 0.94 and NPV of 0.86 This rule would have identified injuries in all but 90 patients (1.6%) and would theoretically spare 11.9% (655) of blunt trauma patients a torso CT. CONCLUSIONS: Our findings advocate for Head/Cspine CT in all geriatric patients with the addition of torso CT in the setting of positive clinical findings and high-risk criteria. Prospective validation of this rule could lead to streamlined diagnostic care of this growing trauma population. LEVEL OF EVIDENCE: Level 2, Diagnostic Tests or Criteria.