A diazeniumdiolate signal in Pseudomonas syringae upregulates virulence factors and promotes survival in plants.

Pseudomonas syringae infects a wide variety of crops. The mangotoxin-generating operon (mgo) is conserved across many P. syringae strains and is responsible for producing an extracellular chemical signal, leudiazen. Disruption of the mgoA gene in P. syringae pv. syringae (Pss) UMAF0158 alleviated tomato chlorosis caused by this bacterium. We showed that deletion of entire mgo reduced Pss UMAF0158 population in tomato leaflets. Leudiazen restored the signaling activity of the deletion mutant at a concentration as low as 10 nM. Both the diazeniumdiolate and isobutyl groups of leudiazen are critical for this potent signaling activity. Transcriptional analysis showed that mgo and leudiazen induce the expression of mangotoxin biosynthetic operon as well as an uncharacterized gene cluster, RS17235-RS17245. We found that this cluster enhances survival of Pss UMAF0158 in planta and is widely distributed in P. syringae strains. Our results demonstrate that mgo plays prominent roles in the virulence and growth of P. syringae. The mgo and mgo-like signaling systems in different bacteria likely regulate diverse microbe-host interactions.

Ultrafiltration: Impact of process temperature (7 and 50°C) on process performance and protein beverage physical, chemical, and sensory properties.

Our objectives were to determine the impact of ultrafiltration (UF) of skim milk at 7 and 50°C on UF processing, lactose removal, mineral partitioning, and skim milk retentate physical, chemical, and sensory properties at 3 (3.4 7.5, and 10.5%) protein concentration with 2 different heat processing treatments high temperature short time (HTST) pasteurization and autoclave). Pasteurized skim milk was split into 2 portions and the 7°C UF processing run was done on one day and the 50°C UF processing run was done on the next day. Skim milk was ultrafiltered and diafiltered at 7 and 50°C and as permeate was removed, deionized water at 7 or 50°C was added in an equal amount by weight as permeate removed to maintain constant protein concentration in the retentate during UF until 98% or more of lactose and low molecular weight soluble milk components were removed. All skim milk-based beverage bases from the 7 and 50°C UF of skim milk were HTST (78°C for 15 s) processed or autoclaved (116°C for 6 min). The physical, chemical, and sensory properties of all treatments were measured. This process was replicated twice with a new batch of pasteurized skim milk in a different week with the 7 and 50°C UF processing runs ran in reverse order. Overall, lactose-free skim milk at 3.4, 7.5, and 10.5% protein produced by UF with DF, was more bland, more white and less heat stable (i.e., stable to retorting but not direct steam injection at 142°C for 2 to 3 s) than skim milk based on both sensory scores and instrumental measures. A 98 to 99% removal of lactose from skim milk was achieved (final lactose concentration <0.06 g/100g) with a diafiltration ratio of water to milk of about 4 to 1 was used at both 7°C and 50°C. The processing time to achieve that lactose removal from the same starting weight of milk was about twice as long when filtering at 7°C than 50°C because of the lower flux (23 versus 48 kg/m2/h). The continuous DF at constant protein concentration maintained constant flux for a processing time of 4 and 8 h at 50 and 7°C, respectively. The final freezing point of the lactose and soluble mineral reduced milk was close to that of water (-0.015°C versus -0.525°C for skim milk) and the pH of the lactose-free milk at 20°C increased from about 6.5 for skim to about 7.33 and 7.46 for UF/DF skim milk at 7 and 50°C, respectively. Removal of compounds that absorb light (in the range of 360 to 500 nm) from milk in the permeate, increased light reflectance and whiteness and decreased yellowness relative to the starting skim milk.

Consumer perception of Cheddar cheese color.

The color of Cheddar cheese in the United States is influenced by many factors, primarily the amount of annatto added as a colorant. The US Food and Drug Administration is currently reviewing its definition of the term "natural" on food labels, which may result in the use of colorants being restricted in natural cheeses. The objective of this study was to evaluate how consumers perceive Cheddar cheese color to better understand how changes to legislation surrounding colorants in natural Cheddar cheese may affect consumption. We were also interested in determining if a relationship exists between color and other perceived characteristics of Cheddar cheese. Two online surveys on Cheddar cheese color and flavor attributes (n = 1,226 and n = 1,183, respectively) were conducted, followed by a consumer acceptance test on 6 commercially available Cheddar cheeses (n = 196). Overall, consumers preferred light orange color in Cheddar cheese over dark orange or white Cheddar cheese, but segmentation was observed for Cheddar color preference. Light orange Cheddar and white Cheddar were perceived as approximately equal in terms of "naturalness." White and light orange Cheddars were perceived as more natural than dark orange Cheddars conceptually and in consumer acceptance testing. White Cheddar was considered most natural by 50.3% of n = 1,283 survey participants and 43.4% of n = 196 consumer acceptance test participants, whereas light orange Cheddar was perceived as most natural by 40.6% and 45.9% of these groups, respectively. A bimodal distribution was observed in both the online survey and in consumer acceptance testing for the naturalness of Cheddar cheese color, with a subset of consumers (31.4% of n = 1,183 survey participants and 30.6% of n = 196 consumer testing participants) indicating that white Cheddar was the least natural option. Consumers associated orange color in Cheddar cheese with a sharper flavor both in an online survey format and consumer acceptance testing.

Association of prior treatment with nitrogen-containing bisphosphonates on outcomes of COVID-19 positive patients.

COVID-19 infection has resulted in significant morbidity and mortality globally, especially among older adults. Repurposed drugs have demonstrated activity in respiratory illnesses, including nitrogen-containing bisphosphonates. In this retrospective longitudinal study at 4 academic medical centers, we show no benefit of nitrogen-containing bisphosphonates regarding ICU admission, ventilator use, and mortality among older adults with COVID-19 infection. We specifically evaluated the intravenous bisphosphonate zoledronic acid and found no difference compared to oral bisphosphonates. BACKGROUND: Widely used in osteoporosis treatment, nitrogen-containing bisphosphonates (N-BP) have been associated with reduced mortality and morbidity among older adults. Based on prior studies, we hypothesized that prior treatment with N-BP might reduce intensive care unit (ICU) admission, ventilator use, and death among older adults diagnosed with COVID-19. METHODS: This retrospective analysis of the PCORnet Common Data Model across 4 academic medical centers through 1 September 2021 identified individuals age >50 years with a diagnosis of COVID-19. The composite outcome included ICU admission, ventilator use, or death within 15, 30, and 180 days of COVID-19 diagnosis. Use of N-BP was defined as a prescription within 3 years prior. ICU admission and ventilator use were determined using administrative codes. Death included both in-hospital and out-of-hospital events. Patients treated with N-BP were matched 1:1 by propensity score to patients without prior N-BP use. Secondary analysis compared outcomes among those prescribed zoledronic acid (ZOL) to those prescribed oral N-BPs. RESULTS: Of 76,223 COVID-19 patients identified, 1,853 were previously prescribed N-BP, among whom 559 were prescribed ZOL. After propensity score matching, there were no significant differences in the composite outcome at 15 days (HR 1.22, 95% CI: 0.89-1.67), 30 days (HR 1.24, 95% CI: 0.93-1.66), or 180 days (HR 1.17, 95% CI: 0.93-1.48), comparing those prescribed and not prescribed N-BP. Compared to those prescribed oral N-BP, there were no significant differences in outcomes among those prescribed ZOL. CONCLUSION: Among older COVID-19 patients, prior exposure to N-BP including ZOL was not associated with a reduction in ICU admission, ventilator use, or death.

Milk beverage base with lactose removed with ultrafiltration: Effect of fat and protein concentration on sensory and physical properties.

Our objectives were to determine the effect of fat (skim to whole milk) and protein (3.4%-10.5%) concentration on the sensory and physical properties of milk beverage base that had lactose and other low molecular components removed by ultrafiltration (UF). In experiment 1, a matrix of 16 treatments was produced to achieve 4 levels of lactose removal (0%, 30%, 70%, and 97%) at each of 4 fat levels (skim, 1%, 2%, and whole milk). In experiment 2, a matrix of 12 treatments was produced to achieve 4 levels of lactose removal (0%, 30%, 70%, and 97%) at each of 3 protein concentrations (3.4%, 6.5%, and 10.5% protein). Physical and sensory properties of these products were determined. Removal of >95% of milk lactose by UF required a diafiltration volume of approximately 3 times the milk volume. Lactose and low molecular weight solute removal increased whiteness across the range from skim to whole milk while decreasing viscosity and making milk flavor blander. In addition, lactose (and other low molecular weight solute) removal by UF decreased titratable acidity by more than 50% and increased milk pH at 20°C to >7.0. Future work on milk and milk-based beverages with lactose removed by UF needs to focus on interaction of the remaining milk solids with added flavorings, changing casein to whey protein ratio before removal of lactose by UF, and the effect of lactose and low molecular weight solute removal on heat stability, particularly for neutral-pH, shelf-stable milk-based beverages.

Parental perception of children's school lunch milk.

School lunch programs are mandated by the US Code of Federal Regulations to serve pasteurized milk that is skim or 1% fat and fortified with vitamins A and D. In recent years, proposals have been made to alter nutritional requirements for school lunches and school lunch milk, including changes to the milk fat and flavor options available. The objective of this study was to evaluate parental understanding and perception of school lunch milk to better understand how changes to school lunch milk are perceived by parents. Four focus groups (n = 34) were conducted with parents of school-aged children (5-13 yr) who purchased milk as part of a lunch at school. Participants were asked about school lunch milk, including nutritional content, packaging, and flavoring. Focus groups included a build-your-own milk activity and discussion of children's milk products currently available on the market. Two subsequent online surveys were conducted with parents of school-aged children (survey 1, n = 216; survey 2, n = 133). Maximum difference scaling was used to evaluate what beverages parents would prefer their child to drink at school (survey 1) and which attributes of chocolate milk for children were most important to parents (survey 2). An adaptive choice-based conjoint activity (survey 1) included flavor, milk fat, heat treatment, label claims, and packaging type. Both surveys included questions to evaluate knowledge of milk nutrition and attitudes regarding milk and flavored milk. Agree/disagree questions were used in both surveys to assess parental opinions of school lunch milk. Survey 2 also included semantic differential (sliding scale) questions to assess parental opinions of chocolate milk and their acceptance of sugar alternatives in chocolate milk served in schools. Parents were familiar with the flavor options and packaging of school lunch milk, but expressed limited familiarity with school lunch milk fat content. Parents perceived milk to be healthy and a good source of vitamin D and calcium. From survey results, parents placed the highest importance on school lunch milk packaging, followed by milk fat percentage and flavoring over label claims and heat treatment. The ideal school lunch milk for parents was unflavored (white milk) or chocolate, 2% fat, and packaged in a cardboard gabletop carton. For school lunch chocolate milk, 3 distinct clusters of parents with differing opinions for children's chocolate milk were identified. Parents are largely unfamiliar with the specific attributes and nutritional profile of milk served in schools but believe that schools should offer milk to their children as part of breakfast and lunch. Parents in both surveys also displayed a preference for 2% fat milk over low-fat options, which provides actionable insight for both governmental bodies determining educational and nutritional policies for school meals and fluid milk producers seeking to optimize their products intended for schools.

Sensory Analysis and Consumer Preference: Best Practices.

Sensory science is a multidisciplinary field that encompasses a wide variety of established and newly developed tests to document human responses to stimuli. Sensory tests are not limited to the area of food science but they find wide application within the diverse areas of the food science arena. Sensory tests can be divided into two basic groups: analytical tests and affective tests. Analytical tests are generally product-focused, and affective tests are generally consumer-focused. Selection of the appropriate test is critical for actionable results. This review addresses an overview of sensory tests and best practices.

Predicting and influencing PrEP use: the role of motivational and attitudinal factors.

This study examined PrEP attitude and HIV risk factors associated with PrEP motivation and evaluated the efficacy of a mock public health video that addresses both motivational and stigma issues for improving PrEP uptake over a standard educational video. Gay, bisexual, and other men who have sex with men (GBMSM; N = 604) were enrolled. One-way between subject analysis of variance (ANOVA) and post-hoc comparisons revealed that GBMSM in later stages of change had significantly higher endorsement of positive PrEP attitudes, PrEP stigma, objective and perceived HIV risk, HIV worry, and valued health benefits of PrEP more. Stepwise multiple regression revealed five significant predictors of PrEP motivation: HIV worry, objective HIV risk, anticipated PrEP stigma, positive PrEP attitudes, and perceived social consequences of PrEP use. Video conditions did not differ in their impact on PrEP attitudes or motivation; however, regardless of video condition, participants experienced a pre- to post-video increase in positive PrEP attitudes and motivation. Findings suggest HIV worry, objective HIV risk, positive PrEP attitudes, and perceived PrEP health benefits are important factors to optimize PrEP motivation, which can inform secondary prevention efforts. Further study is needed on promotional PrEP campaigns that disconfirm stigmatizing PrEP misconceptions.

The role of packaging on the flavor of fluid milk.

Few studies have addressed the effects of package material in the absence of light on contributions to fluid milk flavor. The objective of this study was to compare the sensory and chemical properties of fluid milk packaged in paperboard cartons, low-density polyethylene, high-density polyethylene (HDPE), polyethylene terephthalate (PET), linear low-density polyethylene (LLDPE), and glass. Pasteurized (high temperature short time, 77°C for 25 s) skim and whole milk were filled (280 mL ± 10 mL) into paperboard cartons, low-density polyethylene, HDPE, PET, LLDPE, and glass (control). Milks were stored at 4°C in the dark and sampled at d 0, 5, 10, and 15. Descriptive analysis was applied to document sensory profiles at each time point, and volatile compounds were extracted and identified by solid-phase microextraction with gas chromatography mass spectrometry and gas chromatography-olfactometry. Tetrad tests with consumers were conducted at d 10. Both skim and whole milks packaged in cartons had noticeable paperboard flavor by d 5 and higher levels of hexanal than skim and whole milks in other package types at d 5. Skim milks packaged in paperboard cartons and LLDPE had distinct refrigerator/stale flavor compared with milks in the other package types, concurrent with increased levels of refrigerator/package-related compounds including styrene, acetophenone and 2-ethyl-1-hexanol. Milks packaged in glass, PET and HDPE were not distinguished by consumers at d 10 post-processing. Package type influences fluid milk flavor, and these effects are greater in skim milk compared with whole milk. Paperboard cartons do not preserve milk freshness, as well as PET, HDPE, or glass, due to flavor migration and scalping. Glass remains an ideal barrier to preserve fluid milk flavor, but in the absence of light, HDPE and PET provide additional benefits while also maintaining fluid milk flavor.

Proper understanding of recurrent stress urinary incontinence treatment in women (PURSUIT): a randomised controlled trial of endoscopic and surgical treatment.

BACKGROUND: Women with stress urinary incontinence (SUI) experience urine leakage with physical activity. Currently, the interventional treatments for SUI are surgical, or endoscopic bulking injection(s). However, these procedures are not always successful, and symptoms can persist or come back after treatment, categorised as recurrent SUI. There are longstanding symptoms and distress associated with a failed primary treatment, and currently, there is no consensus on how best to treat women with recurrent, or persistent, SUI. METHODS: A two-arm trial, set in at least 20 National Health Service (NHS) urology and urogynaecology referral units in the UK, randomising 250 adult women with recurrent or persistent SUI 1:1 to receive either an endoscopic intervention (endoscopic bulking injections) or a standard NHS surgical intervention, currently colposuspension, autologous fascial sling or artificial urinary sphincter. The aim of the trial is to determine whether surgical treatment is superior to endoscopic bulking injections in terms of symptom severity at 1 year after randomisation. This primary outcome will be measured using the patient-reported International Consultation on Incontinence Questionnaire - Urinary Incontinence - Short Form (ICIQ-UI-SF). Secondary outcomes include assessment of longer-term clinical impact, improvement of symptoms, safety, operative assessments, sexual function, cost-effectiveness and an evaluation of patients' and clinicians' views and experiences of the interventions. DISCUSSION: There is a lack of high-quality, randomised, scientific evidence for which treatment is best for women presenting with recurrent SUI. The PURSUIT study will benefit healthcare professionals and patients and provide robust evidence to guide further treatment and improve symptoms and quality of life for women with this condition. TRIAL REGISTRATION: International Standard Randomised Controlled Trials Number (ISRCTN) registry ISRCTN12201059. Registered on 09 January 2020.

Self-Masked Aldehyde Inhibitors of Human Cathepsin L Are Potent Anti-CoV-2 Agents.

Cysteine proteases comprise an important class of drug targets, especially for infectious diseases such as Chagas disease (cruzain) and COVID-19 (3CL protease, cathepsin L). Peptide aldehydes have proven to be potent inhibitors for all of these proteases. However, the intrinsic, high electrophilicity of the aldehyde group is associated with safety concerns and metabolic instability, limiting the use of aldehyde inhibitors as drugs. We have developed a novel class of compounds, self-masked aldehyde inhibitors (SMAIs) which are based on the dipeptide aldehyde inhibitor (Cbz-Phe-Phe-CHO, 1), for which the P1 Phe group contains a 1'-hydroxy group, effectively, an o-tyrosinyl aldehyde (Cbz-Phe-o-Tyr-CHO, 2; (Li et al. (2021) J. Med. Chem. 64, 11,267-11,287)). Compound 2 and other SMAIs exist in aqueous mixtures as stable δ-lactols, and apparent catalysis by the cysteine protease cruzain, the major cysteine protease of Trypanosoma cruzi, results in the opening of the lactol ring to afford the aldehydes which then form reversible thiohemiacetals with the enzyme. These SMAIs are also potent, time-dependent inhibitors of human cathepsin L (K i = 11-60 nM), an enzyme which shares 36% amino acid identity with cruzain. As inactivators of cathepsin L have recently been shown to be potent anti-SARS-CoV-2 agents in infected mammalian cells (Mellott et al. (2021) ACS Chem. Biol. 16, 642-650), we evaluated SMAIs in VeroE6 and A549/ACE2 cells infected with SARS-CoV-2. These SMAIs demonstrated potent anti-SARS-CoV-2 activity with values of EC50 = 2-8 µM. We also synthesized pro-drug forms of the SMAIs in which the hydroxyl groups of the lactols were O-acylated. Such pro-drug SMAIs resulted in significantly enhanced anti-SARS-CoV-2 activity (EC50 = 0.3-0.6 µM), demonstrating that the O-acylated-SMAIs afforded a level of stability within infected cells, and are likely converted to SMAIs by the action of cellular esterases. Lastly, we prepared and characterized an SMAI in which the sidechain adjacent to the terminal aldehyde is a 2-pyridonyl-alanine group, a mimic of both phenylalanine and glutamine. This compound (9) inhibited both cathepsin L and 3CL protease at low nanomolar concentrations, and also exerted anti-CoV-2 activity in an infected human cell line.

Effects of micellar casein concentrate purity and milk fat on sulfur/eggy flavor in ultrapasteurized milk-based beverages.

Our objectives were to determine the level of milk-derived whey protein (MDWP) removal necessary to achieve no detectable sulfur/eggy flavor in ultrapasteurized fat-free micellar casein concentrate (MCC) beverages (6.5% protein) and in the same beverages containing 1 and 2% milk fat. Micellar casein concentrate with 95% MDWP removal was produced from skim milk (50°C) with a 3×, 3-stage ceramic microfiltration (MF) process using 0.1-µm pore size graded permeability membranes (n = 3). In experiment 1, MCC-based beverages at about 6.5% (wt/wt) true protein were formulated at a fat content of 0.15% fat (wt/wt) at 4 different levels of MDWP removal percentages (95.2%, 91.0%, 83.2%, and 69.3%). In experiment 2, a similar series of beverages at 3 MDWP removal percentages (95.2%, 83.2%, and 69.3%) with 0.1, 1, and 2% fat content were produced. The purity (or completeness of removal of whey protein by MF) of MCC was determined by the Kjeldahl method and sodium dodecyl sulfate (SDS)-PAGE. Sensory properties of beverages were documented by descriptive sensory analysis, and volatile sulfur compounds were evaluated using solid-phase microextraction followed by gas chromatography-triple quadrupole mass spectrometry. The purity of MCC measured by the Kjeldahl method (casein as a percentage of true protein) was higher after thermal treatment than before, whereas MCC purity evaluated by SDS-PAGE was unchanged by heat treatment. The purity of MCC had an effect on the flavor profile of thermally processed beverages at 6.5% protein made with fresh liquid MCC. No sulfur/eggy flavor was detected in MCC beverages when 95% of the MDWP was removed (MCC purity about 93 to 94%) from skim milk by microfiltration at 0.1, 1, and 2% fat. As the fat content of 6.5% protein beverages produced with MCC increased, sulfur/eggy flavor intensity and hydrogen sulfide concentration decreased. However, the effect of increasing milk fat on reducing sulfur/eggy flavor in MCC-based beverages at 6.5% protein was less than that of increasing MDWP removal from MCC. Sulfur off-flavors in neutral-pH dairy protein beverages can be mitigated by use of high-purity MCC or by incorporation of fat in the beverage, or both.

Identification of aroma-active compounds in Cheddar cheese imparted by wood smoke.

Cheddar cheese is the most popular cheese in the United States, and the demand for specialty categories of cheese, such as smoked cheese, are rising. The objective of this study was to characterize the flavor differences among Cheddar cheeses smoked with hickory, cherry, or apple woods, and to identify important aroma-active compounds contributing to these differences. First, the aroma-active compound profiles of hickory, cherry, and apple wood smokes were analyzed by solid-phase microextraction (SPME) gas chromatography-olfactometry (GCO) and gas chromatography-mass spectrometry (GC-MS). Subsequently, commercial Cheddar cheeses smoked with hickory, cherry, or apple woods, as well as an unsmoked control, were evaluated by a trained sensory panel and by SPME GCO and GC-MS to identify aroma-active compounds. Selected compounds were quantified with external standard curves. Seventy-eight aroma-active compounds were identified in wood smokes. Compounds included phenolics, carbonyls, and furans. The trained panel identified distinct sensory attributes and intensities among the 3 cheeses exposed to different wood smokes (P < 0.05). Hickory smoked cheeses had the highest intensities of flavors associated with characteristic "smokiness" including smoke aroma, overall smoke flavor intensity, and meaty, smoky flavor. Cherry wood smoked cheeses were distinguished by the presence of a fruity flavor. Apple wood smoked cheeses were characterized by the presence of a waxy, green flavor. Ninety-nine aroma-active compounds were identified in smoked cheeses. Phenol, guaiacol, 4-methylguaiacol, and syringol were identified as the most important compounds contributing to characteristic "smokiness." Benzyl alcohol contributed to the fruity flavor in cherry wood smoked cheeses, and 2-methyl-2-butenal and 2-ethylfuran were responsible for the waxy, green flavor identified in apple wood smoked cheeses. These smoke flavor compounds, in addition to diacetyl and acetoin, were deemed important to the flavor of cheeses in this study. Results from this study identified volatile aroma-active compounds contributing to differences in sensory perception among Cheddar cheeses smoked with different wood sources.

Migraine-Associated Common Genetic Variants Confer Greater Risk of Posterior vs. Anterior Circulation Ischemic Stroke☆.

OBJECTIVE: To examine potential genetic relationships between migraine and the two distinct phenotypes posterior circulation ischemic stroke (PCiS) and anterior circulation ischemic stroke (ACiS), we generated migraine polygenic risk scores (PRSs) and compared these between PCiS and ACiS, and separately vs. non-stroke control subjects. METHODS: Acute ischemic stroke cases were classified as PCiS or ACiS based on lesion location on diffusion-weighted MRI. Exclusion criteria were lesions in both vascular territories or uncertain territory; supratentorial PCiS with ipsilateral fetal posterior cerebral artery; and cases with atrial fibrillation. We generated migraine PRS for three migraine phenotypes (any migraine; migraine without aura; migraine with aura) using publicly available GWAS data and compared mean PRSs separately for PCiS and ACiS vs. non-stroke control subjects, and between each stroke phenotype. RESULTS: Our primary analyses included 464 PCiS and 1079 ACiS patients with genetic European ancestry. Compared to non-stroke control subjects (n=15396), PRSs of any migraine were associated with increased risk of PCiS (p=0.01-0.03) and decreased risk of ACiS (p=0.010-0.039). Migraine without aura PRSs were significantly associated with PCiS (p=0.008-0.028), but not with ACiS. When comparing PCiS vs. ACiS directly, migraine PRSs were higher in PCiS vs. ACiS for any migraine (p=0.001-0.010) and migraine without aura (p=0.032-0.048). Migraine with aura PRS did not show a differential association in our analyses. CONCLUSIONS: Our results suggest a stronger genetic overlap between unspecified migraine and migraine without aura with PCiS compared to ACiS. Possible shared mechanisms include dysregulation of cerebral vessel endothelial function.

Parents' implicit perceptions of dairy milk and plant-based milk alternatives.

Dairy product consumption is motivated by both familiarity and habit. Milk consumption decreases with age, but milk consumption during childhood and adolescence increases the chances of lifetime milk consumption. Understanding how parents perceive dairy milk and other dairy foods further enables development of dairy-positive messaging that aligns with their perceptions. The objective of this research was to understand parent belief systems around fluid dairy milk and plant-based alternatives (PBA). This goal was accomplished by assessing parents' implicit attitudes toward dairy milk and PBA with an implicit bias exercise (n = 331), followed by qualitative interviews to understand explicitly stated purchase motivations and guided recall of information heard about dairy milk and PBA to better understand external influences on milk perception (n = 88). The majority of parents (73.4%) implicitly associated dairy milk with positive attributes compared with those with a positive association with PBA (13.8%) or with a neutral bias (12.7%). The stronger a parent's implicit bias toward PBA, the more likely they were to purchase these products either alongside or as a replacement for dairy milk. Eighty-five percent of parents in our study could recall drinking milk at home as a child, and 58% remembered encouragement from their parents to drink milk. However, only 38% encouraged their own children to drink milk (the majority, 55%, were neutral toward their children's milk consumption). Generally negative media messaging toward dairy milk and positive messaging toward PBA may contribute to this trend, even if consumers are not explicitly aware of their perception changes. Seventy-seven percent of parents felt generally confident in choosing dairy milk or PBA for their children. However, only 26% of parents felt that nothing about dairy milk or PBA information or messaging was confusing. Sources of uncertainty about dairy milk included hormones and antibiotics, animal welfare, ecological sustainability, potential contamination, and intolerances or allergies. By addressing the most commonly encountered and recalled concerns about milk from parents, dairy producers may be able to increase trust and implicit bias toward dairy milk compared with PBA.

Children's perceptions of fluid milk with varying levels of milkfat.

Schools participating in federal meal programs are limited to serving skim or low-fat (≤1%) flavored and unflavored milk. Few studies have directly addressed child perceptions and preferences for milk containing different amounts of milkfat. The objective of this study was to determine whether children can differentiate between flavored and unflavored fluid milk containing varying levels of milkfat and whether preferences for certain levels of milkfat exist. Flavored and unflavored milks containing 4 different percentages of milkfat (≤0.5, 1, 2, and 3.25%) were high-temperature, short-time processed, filled into half-gallon light-shielded milk jugs, and stored at 4°C in the dark. Milks were evaluated by children (ages 8-13 yr) following 7 d at 4°C. Acceptance testing and tetrad difference testing were conducted on flavored and unflavored milks with and without visual cues to determine if differences were driven by visual or flavor or mouthfeel cues. Child acceptance testing (n = 138 unflavored; n = 123 flavored) was conducted to evaluate liking and perception of selected attributes. Tetrad testing (n = 127 unflavored; n = 129 flavored) was conducted to determine if children could differentiate between different fat levels even in the absence of a difference in acceptance. The experiment was replicated twice. When visual cues were present, children had higher overall liking for 1% and 2% milks than skim for unflavored milk and higher liking for chocolate milks containing at least 1% milk fat than for skim. Differences in liking were driven by appearance, viscosity, and flavor. In the absence of visual cues, no differences were observed in liking or flavor or mouthfeel attributes for unflavored milk but higher liking for at least 1% milk fat in chocolate milk compared with skim was consistent with the presence of visual cues. From tetrad testing, children could visually tell a difference between all unflavored pairs except 2% versus whole milk and could not detect consistent differences between milkfat pairs in the absence of visual cues. For chocolate milk, children could tell a difference between all milk fat pairs with visual cues and could tell a difference between skim versus 2% and skim versus whole milk without visual cues. These results demonstrate that in the absence of package-related flavors, school-age children like unflavored skim milk as well as milk with higher fat content in the absence of visual cues. In contrast, appearance as well as flavor and mouthfeel attributes play a role in children's liking as well as their ability to discriminate between chocolate milks containing different amounts of fat, with chocolate milk containing at least 1% fat preferred. The sensory quality of school lunch milk is vital to child preference, and processing efforts are needed to maximize school milk sensory quality.

Consumer perception of natural hot-pepper cheeses.

Hot-pepper cheese (HPC) is a growing category of flavored natural cheese. The objective of this study was to evaluate consumer perception of HPC using a combination of quantitative survey methods and consumer evaluation of HPC. An online survey (n = 510) was conducted to understand drivers of purchase for the HPC category. Consumers of HPC answered maximum difference exercises and an adaptive choice-based conjoint activity focused on HPC attributes. Subsequently, natural HPC were manufactured in duplicate with 5 different hot-pepper blends with a range of heat intensities and distinct color differences. Trained panel profiling and consumer-acceptance testing (n = 194 consumers) were conducted on the cheeses. Three clusters of consumers were identified from the online survey. Cluster 1 (n = 175) were traditional HPC consumers, and cluster 2 (n = 152) preferred milder HPC. Cluster 3 (n = 183) showed preference for spicier HPC as well as novel HPC, such as those made with habanero peppers or white Cheddar cheese. Conceptually, the overall ideal HPC was a Monterey Jack with medium-sized, multicolored pieces of jalapeno peppers and a medium heat and spiciness. Heat and spiciness intensity and type of cheese were the most important attributes. The 5 HPC used in consumer testing had a distinct range (low to high) of hot-pepper burn and heat intensity by trained panel profiling. Consumer overall liking increased as hot-pepper burn and heat intensity increased to a certain point, indicating HPC consumers may have an optimal point for heat and spiciness in HPC. Consumers also preferred HPC with multicolored pepper pieces over those with a single pepper color, consistent with survey results. Consumers who self-reported that they prefer mild- or medium-spicy foods (mild consumers) preferred HPC that were less intense in hot-pepper burn than consumers who self-reported preference for hot or spicy foods (hot consumers). Most HPC consumers preferred HPC with higher heat intensity and were also motivated by visual characteristics of HPC.

Consumer perception of ice cream and frozen desserts in the "better-for-you" category.

The consumption of ice cream and frozen desserts in the "better-for-you" (BFY) category has grown rapidly over the past few years, even as traditional ice cream sales remain stagnant. To better understand consumer preferences within the BFY category, an online survey (n = 1,051) was conducted with ice cream and frozen dessert consumers, followed by consumer acceptance testing of commercial BFY frozen dairy desserts. Consumers of BFY frozen desserts (n = 578) completed an adaptive choice-based conjoint survey and MaxDiff exercise to identify the attributes that drive purchase of BFY frozen desserts. MaxDiff exercises were also used to determine which attributes all frozen dessert consumers (n = 1,051) perceived to make a frozen dessert BFY and which stabilizers or emulsifiers were most attractive on an ice cream or frozen dessert label. Subsequently, a consumer acceptance test (n = 186) was conducted using 4 commercial vanilla-flavored frozen dairy desserts made with different sweetening systems (sugar, sucralose + acesulfame K, monk fruit + allulose, and stevia + erythritol). Half of consumers were primed or informed with the sweeteners and basic nutritional information for the frozen desserts before tasting, and the other half of consumers evaluated samples blinded, where they were only informed that they were tasting a vanilla-flavored frozen dessert. Sweetener type and base (dairy vs. plant) were the most important attributes to BFY consumers when selecting a BFY frozen dessert (n = 578). For all ice cream and BFY dessert consumers (n = 1,051), sweetener-related claims (naturally sweetened, reduced sugar, no added sugar), along with "all natural" and a short ingredient list, were the top attributes that contributed to perception of a "healthier" frozen dessert. When BFY frozen desserts were tasted by consumers, purchase intent decreased after tasting, suggesting that frozen desserts made with natural non-nutritive sweeteners did not meet consumer expectations. Flavor of BFY frozen desserts remains more important than perceived healthiness. Consumers perceive frozen desserts, even those in the BFY category, as an indulgence. Frozen dessert manufacturers should focus on naturally sweetened, dairy-based desserts with minimal sweetener-related flavor defects when designing products for the BFY category.

Enzymatic Synthesis of Diverse Heterocycles by a Noncanonical Nonribosomal Peptide Synthetase.

Nonribosomal peptide synthetases (NRPSs) are typically multimodular enzymes that assemble amino acids or carboxylic acids into complex natural products. Here, we characterize a monomodular NRPS, PvfC, encoded by the Pseudomonas virulence factor (pvf) gene cluster that is essential for virulence and signaling in different bacterial species. PvfC exhibits a unique adenylation-thiolation-reductase (ATR) domain architecture that is understudied in bacteria. We show that the activity of PvfC is essential in the production of seven leucine-derived heterocyclic natural products, including two pyrazines, a pyrazinone, and a rare disubstituted imidazole, as well as three pyrazine N-oxides that require an additional N-oxygenation step. Mechanistic studies reveal that PvfC, without a canonical peptide-forming domain, makes a dipeptide aldehyde intermediate en route to both the pyrazinone and imidazole. Our work identifies a novel biosynthetic route for the production of pyrazinones, an emerging class of signaling molecules and virulence factors. Our discovery also showcases the ability of monomodular NRPSs to generate amino acid- and dipeptide-aldehydes that lead to diverse natural products. The diversity-prone biosynthesis by the pvf-encoded enzymes sets the stage for further understanding the functions of pvf in bacterial cell-to-cell signaling.