Sulfoquinovosyl diacylglycerol is required for dimerisation of the Rhodobacter sphaeroides reaction centre-light harvesting 1 core complex.

The reaction centre-light harvesting 1 (RC-LH1) core complex is indispensable for anoxygenic photosynthesis. In the purple bacterium Rhodobacter (Rba.) sphaeroides RC-LH1 is produced both as a monomer, in which 14 LH1 subunits form a C-shaped antenna around 1 RC, and as a dimer, where 28 LH1 subunits form an S-shaped antenna surrounding 2 RCs. Alongside the five RC and LH1 subunits, an additional polypeptide known as PufX provides an interface for dimerisation and also prevents LH1 ring closure, introducing a channel for quinone exchange that is essential for photoheterotrophic growth. Structures of Rba. sphaeroides RC-LH1 complexes revealed several new components; protein-Y, which helps to form the quinone channel; protein-Z, of unknown function and seemingly unique to dimers; and a tightly bound sulfoquinovosyl diacylglycerol (SQDG) lipid that interacts with two PufX arginine residues. This lipid lies at the dimer interface alongside weak density for a second molecule, previously proposed to be an ornithine lipid. In this work we have generated strains of Rba. sphaeroides lacking protein-Y, protein-Z, SQDG or ornithine lipids to assess the roles of these previously unknown components in the assembly and activity of RC-LH1. We show that whilst the removal of either protein-Y, protein-Z or ornithine lipids has only subtle effects, SQDG is essential for the formation of RC-LH1 dimers but its absence has no functional effect on the monomeric complex.

Attitudes and beliefs about how chefs can promote nutrition and sustainable food systems among students at a US culinary school.

OBJECTIVE: Chefs have the potential to influence diet quality and food systems sustainability through their work. We aimed to assess the attitudes and perceptions of culinary students about nutrition and sustainability as part of their roles, responsibilities and future work as chefs. DESIGN: We surveyed students attending the Culinary Institute of America (CIA) in the fall of 2019 (n 546). Descriptive statistics compared food priority rankings and Likert-scale distributions of nutrition and sustainability attitudes and beliefs. Adjusted generalised linear models were used to evaluate whether there were differences in attitudes and beliefs across demographic groups. SETTING: The CIA, a private, not-for-profit college and culinary school with US campuses in New York, California and Texas. PARTICIPANTS: Students >18 years old currently enrolled in any of the school's associate's or bachelor's degree programs. RESULTS: Students agreed that chefs should be knowledgeable about nutrition (96·0 %) and the environmental impact of their ingredients (90·8 %) but fewer considered healthfulness (57·8 %) and environmental impact (60·2 %) of their food to be primary considerations in their career as a chef. Taste was the primary factor influencing culinary students' food choices but food priorities differed by race/ethnicity. CONCLUSIONS: Culinary students believe nutrition and sustainability are important. Opportunities exist to empower them with knowledge and skills for promoting public health and sustainable food systems in their future work as chefs.

Reproductive coercion, intimate partner violence, and pregnancy risk among adolescent women with a history of foster care involvement.

BACKGROUND: The current study is the first to explore the prevalence of reproductive coercion among adolescent women currently or previously involved in the U.S. foster care system. Reproductive coercion (RC), a form of intimate partner violence (IPV) involving exertion of power over a partner by controlling their reproductive health and decision making, is a significant public health concern. Existing research on RC has primarily been conducted in either healthcare settings or on college campuses. Foster youth are disproportionately impacted by both adolescent pregnancy and interpersonal violence. RC may contribute to this elevated risk. METHODS: We conducted a cross-sectional survey in 2015 and 2016 of adolescent women (n=136), ages 16-24 years old, seeking services from youth-serving agencies affiliated with a child welfare system in Pennsylvania, United States. Participants completed measures assessing RC, experiences of physical and sexual violence, sexual behaviors, and pregnancy. We used multivariable logistic regression to assess associations between RC and study outcomes. RESULTS: The sample was predominantly African American (67.4%) and largely identified as something other than heterosexual (46.6%). Nearly one-third of the sample (30.1%) reported a history of RC, with the most common being male partners telling them not to use birth control. High rates of IPV (62.1%), lifetime pregnancy (43.4%), and unwanted pregnancy (30.9%) were also reported. RC was associated with significantly higher odds of IPV (Adjusted Odds Ratio (AOR) = 4.22, 95% Confidence Interval (CI): 1.60, 11.13), multi-perpetrator rape (AOR 3.56, 95% CI: 1.04, 12.24), pregnancy (AOR = 5.39, 95% CI: 2.14, 13.60), and unintended pregnancy (AOR 5.39, 95% CI: 2.04, 14.25). Young women reporting RC also had elevated odds for using alcohol or drugs before sex (AOR = 4.34, 95% CI: 1.72, 10.97) and having sex with a male partner 5 years or more older (AOR = 7.32, 95% CI: 2.84, 18.87). No significant differences emerged between RC and sociodemographic characteristics. IMPLICATIONS: These data suggest women involved in the U.S. foster care system, particularly women of color and/or LGBTQ+ identified who comprised the majority of participants in the current study, may be at an increased risk for experiencing RC and other forms of IPV associated with adolescent pregnancy. In addition to efforts to prevent IPV and sexual violence, assessment for RC, healthy relationships education, and access to sexual and reproductive health care may mitigate these risks and improve outcomes for these young women.

Sexual Assault, Alcohol Use, and Gender of Sexual Partners Among Cisgender Women Seeking Care at US College Health Centers, 2015-2018.

Objectives. To assess differences by gender of sexual partner in the association between sexual assault and alcohol use among women seeking care in college health centers.Methods. This longitudinal study comprised 1578 women aged 18 to 24 years visiting 28 college health centers in Pennsylvania and West Virginia from 2015 to 2018. We used multilevel logistic regression and negative binomial regression, testing for interactions of gender of sexual partners, sexual assault, and prevalence and frequency of alcohol use and binge drinking.Results. Sexual assault was reported by 87.3% of women who had sex with women or with women and men (WSWM), 68.2% of women who had sex with men only (WSM), and 47.5% of women with no penetrative sexual partners. The relative associations between sexual assault and alcohol outcomes were smaller for WSWM (prevalence: odds ratios from 0.04 to 0.06; frequency: incidence rate ratios [IRRs] from 0.24 to 0.43) and larger for women who had no penetrative sexual partners (IRRs from 1.55 to 2.63), compared with WSM.Conclusions. Alcohol use patterns among women who have experienced sexual assault differ by gender of sexual partners.

Mechanism of microtubule lumen entry for the α-tubulin acetyltransferase enzyme αTAT1.

Microtubules are structural polymers inside of cells that are subject to posttranslational modifications. These posttranslational modifications create functionally distinct subsets of microtubule networks in the cell, and acetylation is the only modification that takes place in the hollow lumen of the microtubule. Although it is known that the α-tubulin acetyltransferase (αTAT1) is the primary enzyme responsible for microtubule acetylation, the mechanism for how αTAT1 enters the microtubule lumen to access its acetylation sites is not well understood. By performing biochemical assays, fluorescence and electron microscopy experiments, and computational simulations, we found that αTAT1 enters the microtubule lumen through the microtubule ends, and through bends or breaks in the lattice. Thus, microtubule structure is an important determinant in the acetylation process. In addition, once αTAT1 enters the microtubule lumen, the mobility of αTAT1 within the lumen is controlled by the affinity of αTAT1 for its acetylation sites, due to the rapid rebinding of αTAT1 onto highly concentrated α-tubulin acetylation sites. These results have important implications for how acetylation could gradually accumulate on stable subsets of microtubules inside of the cell.

Suppression of microtubule assembly kinetics by the mitotic protein TPX2.

TPX2 is a widely conserved microtubule-associated protein that is required for mitotic spindle formation and function. Previous studies have demonstrated that TPX2 is required for the nucleation of microtubules around chromosomes; however, the molecular mechanism by which TPX2 promotes microtubule nucleation remains a mystery. In this study, we found that TPX2 acts to suppress tubulin subunit off-rates during microtubule assembly and disassembly, thus allowing for the support of unprecedentedly slow rates of plus-end microtubule growth, and also leading to a dramatically reduced microtubule shortening rate. These changes in microtubule dynamics can be explained in computational simulations by a moderate increase in tubulin-tubulin bond strength upon TPX2 association with the microtubule lattice, which in turn acts to reduce the departure rate of tubulin subunits from the microtubule ends. Thus, the direct suppression of tubulin subunit off-rates by TPX2 during microtubule growth and shortening could provide a molecular mechanism to explain the nucleation of new microtubules in the presence of TPX2.

Rapid binding to protofilament edge sites facilitates tip tracking of EB1 at growing microtubule plus-ends.

EB1 is a key cellular protein that delivers regulatory molecules throughout the cell via the tip-tracking of growing microtubule plus-ends. Thus, it is important to understand the mechanism for how EB1 efficiently tracks growing microtubule plus-ends. It is widely accepted that EB1 binds with higher affinity to GTP-tubulin subunits at the growing microtubule tip, relative to GDP-tubulin along the microtubule length. However, it is unclear whether this difference in affinity alone is sufficient to explain the tip-tracking of EB1 at growing microtubule tips. Previously, we found that EB1 binds to exposed microtubule protofilament-edge sites at a ~70 fold faster rate than to closed-lattice sites, due to diffusional steric hindrance to binding. Thus, we asked whether rapid protofilament-edge binding could contribute to efficient EB1 tip tracking. A computational simulation with differential EB1 on-rates based on closed-lattice or protofilament-edge binding, and with EB1 off-rates that were dependent on the tubulin hydrolysis state, robustly recapitulated experimental EB1 tip tracking. To test this model, we used cell-free biophysical assays, as well as live-cell imaging, in combination with a Designed Ankyrin Repeat Protein (DARPin) that binds exclusively to protofilament-edge sites, and whose binding site partially overlaps with the EB1 binding site. We found that DARPin blocked EB1 protofilament-edge binding, which led to a decrease in EB1 tip tracking on dynamic microtubules. We conclude that rapid EB1 binding to microtubule protofilament-edge sites contributes to robust EB1 tip tracking at the growing microtubule plus-end.

Testing the feasibility of the QuitAid smoking cessation intervention in a randomized factorial design in an independent, rural community pharmacy.

BACKGROUND: Adult smoking rates in the USA are highest in economically depressed rural Appalachia. Pharmacist-delivered tobacco cessation support that incorporates medication therapy management (such as the QuitAid intervention) is a promising approach to address this need. METHODS: Twenty-four adult smokers recruited between September and November 2021 through an independent pharmacy in rural Appalachia were randomized in a non-blinded 2 × 2 × 2 factorial design to (1) pharmacist delivered QuitAid intervention (yes vs. no); (2) combination nicotine replacement therapy (NRT) gum + NRT patch (vs. NRT patch); and/or (3) 8 weeks of NRT (vs. standard 4 weeks). Participants received 4 weeks of NRT patch in addition to the components to which they were assigned. Participants completed baseline and 3-month follow-up assessments. Primary outcomes were feasibility of recruitment and randomization, retention, treatment adherence, and fidelity. RESULTS: Participants were recruited in 7 weeks primarily through a referral process, commonly referred to as ask-advise-connect (61%). Participants were on average 52.4 years old, 29.2% were male and the majority were white (91.6%) and Non-Hispanic (91.7%). There was a high level of adherence to the interventions, with 85% of QuitAid sessions completed, 83.3% of the patch used, and 54.5% of gum used. Participants reported a high level of satisfaction with the program, and there was a high level of retention (92%). CONCLUSIONS: This demonstration pilot randomized controlled study indicates that an ask-advise-connect model for connecting rural smokers to smoking cessation support and providing QuitAid for smoking cessation is feasible and acceptable among rural Appalachian smokers and independent pharmacists. Further investigation into the efficacy of a pharmacist-delivered approach for smoking cessation is needed. TRIAL REGISTRATION: The trial was retrospectively registered at ClinicalTrials.gov. Trial #: NCT05649241.

Contextualizing Cisgender Women's Histories of Intimate Partner Violence Victimization With Men and Women.

Sexual minoritized women (SMW) are more likely than exclusively heterosexual women to experience intimate partner violence (IPV). We conducted in-depth interviews with a clinic-based sample of plurisexual SMW (n = 25) ages 18 to 34 about the gender of their perpetrators. Participants primarily experienced physical and sexual IPV in relationships with men and emotional abuse in relationships with women. IPV perpetrated by men often included weapons with women fearing for their lives. Offering patients information about IPV resources and supports that do not make assumptions about women's sexualities may create more opportunity for empathic and effective communication with SMW experiencing IPV.

Rationale, design, and methods for the development of a youth adapted Brief Tobacco Intervention plus automated text messaging for high school students.

BACKGROUND: Tobacco use is increasing among youth in the U.S. The current study seeks to develop and pilot test a universal group-based Youth Brief Tobacco Intervention (Y-BTI) plus mobile phone automated text messaging (ATM) for 9th grade students to both prevent initiation among non-users and promote cessation among current users. METHODS: This study will use a sequential, multi-method research design beginning with mixed methods formative work with 9th grade students to adapt the existing young adult Brief Tobacco Intervention (BTI) for youth and develop automated text messages. The formative work with students will identify salient themes and strategies for the interventions and optimal delivery schedules for the ATM intervention. The second phase of the study evaluates the Y-BTI and ATM through a pilot cluster randomized controlled trial that compares four treatment combinations: (1) Y-BTI + ATM, (2) Y-BTI alone, (3) ATM alone, or (4) standard of care. The Y-BTI is a single session, group-based intervention delivered in schools. The ATM intervention will provide comparable content to the Y-BTI but be delivered via text messaging 3-5 times per week for four weeks. CONCLUSION: In order to curb the rise of tobacco use among youth, interventions that are easily implemented and disseminated need to be developed. We aim to build upon previous research by showing that a universal group-based Y-BTI and ATM are effective in reducing tobacco use among a 9th graders by preventing initiation among tobacco naïve youth and promoting cessation among current users.

"They are Assuming That We are Going to Accuse Them of Rape, and We are Assuming That They are Going to Rape us": A Developmental Perspective on Emerging Adults' Consent Conversations Post #MeToo.

Prevalence of sexual assault remains high on American college campuses, and sexual consent education is lacking within school-based sexual health education programming. Much empirical research has aimed to reduce sexual violence through a deeper understanding of college students' perceptions of sexual consent. However, researchers have not yet examined the impact of broader social discourse, such as that initiated by the #MeToo movement, on emerging adults' conceptualizations of sexual consent. Gendered focus groups were conducted with 34 college students at a large midwestern university in spring of 2019. Qualitative analyses using a phenomenological framework revealed a developmental process of consent education shaped by socialized sexual scripts and public discourse of the #MeToo movement. Four distinct themes emerged: (1) Introductions to Consent in Childhood, (2) Lack of Sexual Consent Education in Adolescence, (3) The Nuanced College Context, and (4) Consent in the Era of #MeToo. Findings reveal that consent is introduced in childhood, outside the context of sexuality, but is generally not revisited within the context of sexual consent by parents or educators during adolescence, leaving media messaging and socialized sexual scripts to serve as guides for sexual consent. This lack of sexual consent education in adolescence then leaves emerging adults unprepared for nuanced sexual experiences in the college context and unable to critically engage with public discourse surrounding consent such as the #MeToo movement, which has caused both fearful and positive outcomes. Findings support the need for earlier and more comprehensive education about sexual consent in childhood and adolescence and the need for college sexual assault prevention programs to include further instruction on navigating ambiguous sexual consent experiences.

Non-enzymatic Activity of the α-Tubulin Acetyltransferase αTAT Limits Synaptic Bouton Growth in Neurons.

Neuronal axons terminate as synaptic boutons that form stable yet plastic connections with their targets. Synaptic bouton development relies on an underlying network of both long-lived and dynamic microtubules that provide structural stability for the boutons while also allowing for their growth and remodeling. However, a molecular-scale mechanism that explains how neurons appropriately balance these two microtubule populations remains a mystery. We hypothesized that α-tubulin acetyltransferase (αTAT), which both stabilizes long-lived microtubules against mechanical stress via acetylation and has been implicated in promoting microtubule dynamics, could play a role in this process. Using the Drosophila neuromuscular junction as a model, we found that non-enzymatic dαTAT activity limits the growth of synaptic boutons by affecting dynamic, but not stable, microtubules. Loss of dαTAT results in the formation of ectopic boutons. These ectopic boutons can be similarly suppressed by resupplying enzyme-inactive dαTAT or by treatment with a low concentration of the microtubule-targeting agent vinblastine, which acts to suppress microtubule dynamics. Biophysical reconstitution experiments revealed that non-enzymatic αTAT1 activity destabilizes dynamic microtubules but does not substantially impact the stability of long-lived microtubules. Further, during microtubule growth, non-enzymatic αTAT1 activity results in increasingly extended tip structures, consistent with an increased rate of acceleration of catastrophe frequency with microtubule age, perhaps via tip structure remodeling. Through these mechanisms, αTAT enriches for stable microtubules at the expense of dynamic ones. We propose that the specific suppression of dynamic microtubules by non-enzymatic αTAT activity regulates the remodeling of microtubule networks during synaptic bouton development.

Structural state recognition facilitates tip tracking of EB1 at growing microtubule ends.

The microtubule binding protein EB1 specifically targets the growing ends of microtubules in cells, where EB1 facilitates the interactions of cellular proteins with microtubule plus-ends. Microtubule end targeting of EB1 has been attributed to high-affinity binding of EB1 to GTP-tubulin that is present at growing microtubule ends. However, our 3D single-molecule diffusion simulations predicted a ~ 6000% increase in EB1 arrivals to open, tapered microtubule tip structures relative to closed lattice conformations. Using quantitative fluorescence, single-molecule, and electron microscopy experiments, we found that the binding of EB1 onto opened, structurally disrupted microtubules was dramatically increased relative to closed, intact microtubules, regardless of hydrolysis state. Correspondingly, in cells, the blunting of growing microtubule plus-ends by Vinblastine was correlated with reduced EB1 targeting. Together, our results suggest that microtubule structural recognition, based on a fundamental diffusion-limited binding model, facilitates the tip tracking of EB1 at growing microtubule ends.

Manipulation and quantification of microtubule lattice integrity.

Microtubules are structural polymers that participate in a wide range of cellular functions. The addition and loss of tubulin subunits allows the microtubule to grow and shorten, as well as to develop and repair defects and gaps in its cylindrical lattice. These lattice defects act to modulate the interactions of microtubules with molecular motors and other microtubule-associated proteins. Therefore, tools to control and measure microtubule lattice structure will be invaluable for developing a quantitative understanding of how the structural state of the microtubule lattice may regulate its interactions with other proteins. In this work, we manipulated the lattice integrity of in vitro microtubules to create pools of microtubules with common nucleotide states, but with variations in structural states. We then developed a series of novel semi-automated analysis tools for both fluorescence and electron microscopy experiments to quantify the type and severity of alterations in microtubule lattice integrity. These techniques will enable new investigations that explore the role of microtubule lattice structure in interactions with microtubule-associated proteins.

A novel role for SALL4 during scar-free wound healing in axolotl.

The human response to serious cutaneous damage is limited to relatively primitive wound healing, whereby collagenous scar tissue fills the wound bed. Scars assure structural integrity at the expense of functional regeneration. In contrast, axolotls have the remarkable capacity to functionally regenerate full thickness wounds. Here, we identified a novel role for SALL4 in regulating collagen transcription after injury that is essential for perfect skin regeneration in axolotl. Furthermore, we identify miR-219 as a molecular regulator of Sall4 during wound healing. Taken together, our work highlights one molecular mechanism that allows for efficient cutaneous wound healing in the axolotl.

Reconstitution of the glycosylphosphatidylinositol-anchored protein Thy-1: interaction with membrane phospholipids and galactosylceramide.

Glycosylphosphatidylinositol (GPI)-anchored membrane proteins are proposed to interact preferentially with glycosphingolipids and cholesterol to form microdomains, which may play an important role in apical targeting and signal transduction. The objective of the present study was to investigate the interaction of the GPI-anchored protein Thy-1 with phospholipids and a glycosphingolipid. Purified Thy-1 was reconstituted into lipid bilayer vesicles of dimyristoyl-phosphatidylcholine (DMPC) alone or in combination with galactosylceramide (GC). The ability of Thy-1 to perturb the gel to a liquid-crystalline phase transition of DMPC was examined by differential scanning calorimetry. As the mole fraction of Thy-1 increased, the phase transition enthalpy, deltaH, declined. Analysis indicated that each molecule of Thy-1 perturbed over 50 phospholipids, suggesting that, in addition to the anchor insertion into the bilayer, the protein itself may interact with the membrane surface. Inclusion of 5% w/w GC in the bilayer resulted in a striking change in the interaction of Thy-1 with phospholipids. At low Thy-1 content, there was a reduction in the phase transition temperature and an increase in phospholipid cooperativity, suggesting the formation of Thy-1/GC-enriched domains. DeltaH initially decreased with increasing Thy-1 content of the bilayer; however, at higher Thy-1 mole ratios, deltaH rose again. These results are interpreted in terms of a model whereby, at low protein:lipid mole ratios, Thy-1 preferentially sequesters GC to form enriched microdomains. At high protein:lipid mole ratios, Thy-1 may alter its conformation in response to steric crowding within these domains such that its interaction with the bilayer surface is reduced.