Rp3: Ribosome profiling-assisted proteogenomics improves coverage and confidence during microprotein discovery.

There has been a dramatic increase in the identification of non-canonical translation and a significant expansion of the protein-coding genome. Among the strategies used to identify unannotated small Open Reading Frames (smORFs) that encode microproteins, Ribosome profiling (Ribo-Seq) is the gold standard for the annotation of novel coding sequences by reporting on smORF translation. In Ribo-Seq, ribosome-protected footprints (RPFs) that map to multiple genomic sites are removed since they cannot be unambiguously assigned to a specific genomic location. Furthermore, RPFs necessarily result in short (25-34 nucleotides) reads, increasing the chance of multi-mapping alignments, such that smORFs residing in these regions cannot be identified by Ribo-Seq. Moreover, it has been challenging to identify protein evidence for Ribo-Seq. To solve this, we developed Rp3, a pipeline that integrates proteogenomics and Ribosome profiling to provide unambiguous evidence for a subset of microproteins missed by current Ribo-Seq pipelines. Here, we show that Rp3 maximizes proteomics detection and confidence of microprotein-encoding smORFs.

The ancestral environment of teosinte populations shapes their root microbiome.

BACKGROUND: The composition of the root microbiome affects the host's growth, with variation in the host genome associated with microbiome variation. However, it is not known whether this intra-specific variation of root microbiomes is a consequence of plants performing targeted manipulations of them to adapt to their local environment or varying passively with other traits. To explore the relationship between the genome, environment and microbiome, we sampled seeds from teosinte populations across its native range in Mexico. We then grew teosinte accessions alongside two modern maize lines in a common garden experiment. Metabarcoding was performed using universal bacterial and fungal primers to profile their root microbiomes. RESULTS: The root microbiome varied between the two modern maize lines and the teosinte accessions. We further found that variation of the teosinte genome, the ancestral environment (temperature/elevation) and root microbiome were all correlated. Multiple microbial groups significantly varied in relative abundance with temperature/elevation, with an increased abundance of bacteria associated with cold tolerance found in teosinte accessions taken from high elevations. CONCLUSIONS: Our results suggest that variation in the root microbiome is pre-conditioned by the genome for the local environment (i.e. non-random). Ultimately, these claims would be strengthened by confirming that these differences in the root microbiome impact host phenotype, for example, by confirming that the root microbiomes of high-elevation teosinte populations enhance cold tolerance.

Structural basis of transcription: RNA polymerase II substrate binding and metal coordination using a free-electron laser.

Catalysis and translocation of multisubunit DNA-directed RNA polymerases underlie all cellular mRNA synthesis. RNA polymerase II (Pol II) synthesizes eukaryotic pre-mRNAs from a DNA template strand buried in its active site. Structural details of catalysis at near-atomic resolution and precise arrangement of key active site components have been elusive. Here, we present the free-electron laser (FEL) structures of a matched ATP-bound Pol II and the hyperactive Rpb1 T834P bridge helix (BH) mutant at the highest resolution to date. The radiation-damage-free FEL structures reveal the full active site interaction network, including the trigger loop (TL) in the closed conformation, bonafide occupancy of both site A and B Mg2+, and, more importantly, a putative third (site C) Mg2+ analogous to that described for some DNA polymerases but not observed previously for cellular RNA polymerases. Molecular dynamics (MD) simulations of the structures indicate that the third Mg2+ is coordinated and stabilized at its observed position. TL residues provide half of the substrate binding pocket while multiple TL/BH interactions induce conformational changes that could allow translocation upon substrate hydrolysis. Consistent with TL/BH communication, a FEL structure and MD simulations of the T834P mutant reveal rearrangement of some active site interactions supporting potential plasticity in active site function and long-distance effects on both the width of the central channel and TL conformation, likely underlying its increased elongation rate at the expense of fidelity.

The gendered effect of status-striving in sleep deprivation disclosure.

OBJECTIVES: In this research, we conceptualize status-striving sleep deprivation disclosure as talking about one's lack of sleep with the intention of enhancing one's image. We propose that workers may disclose discretionary information about their sleep deprivation to highlight their potential contributions to the group because of the personal costs of sleep deprivation (e.g., physiological and psychological impediments), which in turn, predicts higher status conferral. We further propose that perceptions of status-striving sleep deprivation disclosure are influenced by gender stereotypes. METHODS: To test our theory, we designed three studies: an experimental study (study 1), a two-wave panel multisource field study (study 2), and another experimental study (study 3). RESULTS: The results showed that status-striving sleep deprivation disclosure predicted status conferral, and this indirect effect was explained by perceptions of strength for men. We also found support that when team performance ambiguity is lower, which meant that members' contributions are more well-known within the group, the indirect effect was explained by perceptions of self-sacrifice for women. CONCLUSION: People talk about sleep deprivation in their workplaces and can gain status for having done so. For men, status-striving sleep deprivation disclosure enhances their status by enhancing others' perception of their strength. For women, status-striving sleep deprivation disclosure enhances their status by enhancing others' perception of their self-sacrifice, but only under conditions of low team performance ambiguity.

Psychometric testing of the Chinese version of the Perceived Maternal Parenting Self-Efficacy Scale among postpartum women.

BACKGROUND: Maternal parenting self-efficacy plays a critical role in facilitating positive parenting practices and successful adaption to motherhood. The Perceived Maternal Parenting Self-Efficacy Scale (PMPS-E), as a task-specific measure, confirms its psychometric properties in cultural contexts. Compared with other tools, the advantages of the PMPS-E are as follows: (i) specific context or time period during the lifespan of a child, (ii) explicitly assess parenting self-efficacy across a diverse enough range of parenting tasks or activities during the perinatal/postnatal period and (iii) having robust psychometric properties. The aim of this study was to translate and determine the psychometric properties of the PMPS-E among Chinese postpartum women (C-PMPS-E). METHOD: The cross-cultural adaptation process followed Beaton et al.'s intercultural debugging guidelines. A total of 471 women were included to establish the psychometric properties of the C-PMPS-E. Mothers were asked to complete the C-PMPS-E, Edinburgh Postnatal Depression Scale (EPDS), the Generalized Anxiety Disorder-7 (GAD-7) and several demographic questions. The psychometric testing of the C-PMPS-E was established through item analysis, construct validity and internal consistency reliability. RESULTS: Item analysis showed that the critical ratios of all items were greater than 3 between the low-score group and high-score group, and all item-total correlation coefficients were greater than 0.4. The fit indices showed that the original correlated four-factor model of C-PMPS-E was observed to be an excellent fit to the data. The PMPS-E was negatively correlated with the EPDS and GAD-7 demonstrating its discriminant validity. As expected, no significant correlation was found between PMPS-E total or subscale scores and mothers' age. In addition, statistically significant differences for parity were detected for C-PMPS-E total and subscale scores with multipara having higher scores. This was taken as further evidence of the scale known-groups discriminant validity. In terms of internal consistency, the Cronbach's alpha of the C-PMPS-E total scale was 0.950, and subscales ranged from 0.76 to 0.89. Furthermore, a ROC curve analysis was conducted to establish the ability of the C-PMPS-E to distinguish between symptoms of depression and symptoms of anxiety. A cut-off value of 55 was identified that resulted in good specificity and fair sensitivity. CONCLUSION: The C-PMPS-E is a reliable and valid tool to assess maternal parenting self-efficacy in a Chinese context.

Bispecific antibodies with broad neutralization potency against SARS-CoV-2 variants of concern.

The ongoing emergence of SARS-CoV-2 variants of concern (VOCs) that reduce the effectiveness of antibody therapeutics necessitates development of next-generation antibody modalities that are resilient to viral evolution. Here, we characterized N-terminal domain (NTD) and receptor binding domain (RBD)-specific monoclonal antibodies previously isolated from COVID-19 convalescent donors for their activity against emergent SARS-CoV-2 VOCs. Among these, the NTD-specific antibody C1596 displayed the greatest breadth of binding to VOCs, with cryo-EM structural analysis revealing recognition of a distinct NTD epitope outside of the site i antigenic supersite. Given C1596's favorable binding profile, we designed a series of bispecific antibodies (bsAbs) termed CoV2-biRNs, that featured both NTD and RBD specificities. Notably, two of the C1596-inclusive bsAbs, CoV2-biRN5 and CoV2-biRN7, retained potent in vitro neutralization activity against all Omicron variants tested, including XBB.1.5, EG.5.1, and BA.2.86, contrasting the diminished potency of parental antibodies delivered as monotherapies or as a cocktail. Furthermore, prophylactic delivery of CoV2-biRN5 significantly reduced the viral load within the lungs of K18-hACE2 mice following challenge with SARS-CoV-2 XBB.1.5. In conclusion, our NTD-RBD bsAbs offer promising potential for the design of resilient, next-generation antibody therapeutics against SARS-CoV-2 VOCs.

Selection, engineering, and in vivo testing of a human leukocyte antigen-independent T-cell receptor recognizing human mesothelin.

BACKGROUND: Canonical α/ß T-cell receptors (TCRs) bind to human leukocyte antigen (HLA) displaying antigenic peptides to elicit T cell-mediated cytotoxicity. TCR-engineered T-cell immunotherapies targeting cancer-specific peptide-HLA complexes (pHLA) are generating exciting clinical responses, but owing to HLA restriction they are only able to target a subset of antigen-positive patients. More recently, evidence has been published indicating that naturally occurring α/ß TCRs can target cell surface proteins other than pHLA, which would address the challenges of HLA restriction. In this proof-of-concept study, we sought to identify and engineer so-called HLA-independent TCRs (HiTs) against the tumor-associated antigen mesothelin. METHODS: Using phage display, we identified a HiT that bound well to mesothelin, which when expressed in primary T cells, caused activation and cytotoxicity. We subsequently engineered this HiT to modulate the T-cell response to varying levels of mesothelin on the cell surface. RESULTS: The isolated HiT shows cytotoxic activity and demonstrates killing of both mesothelin-expressing cell lines and patient-derived xenograft models. Additionally, we demonstrated that HiT-transduced T cells do not require CD4 or CD8 co-receptors and, unlike a TCR fusion construct, are not inhibited by soluble mesothelin. Finally, we showed that HiT-transduced T cells are highly efficacious in vivo, completely eradicating xenografted human solid tumors. CONCLUSION: HiTs can be isolated from fully human TCR-displaying phage libraries against cell surface-expressed antigens. HiTs are able to fully activate primary T cells both in vivo and in vitro. HiTs may enable the efficacy seen with pHLA-targeting TCRs in solid tumors to be translated to cell surface antigens.

Harnessing root-soil-microbiota interactions for drought-resilient cereals.

Cereal plants form complex networks with their associated microbiome in the soil environment. A complex system including variations of numerous parameters of soil properties and host traits shapes the dynamics of cereal microbiota under drought. These multifaceted interactions can greatly affect carbon and nutrient cycling in soil and offer the potential to increase plant growth and fitness under drought conditions. Despite growing recognition of the importance of plant microbiota to agroecosystem functioning, harnessing the cereal root microbiota remains a significant challenge due to interacting and synergistic effects between root traits, soil properties, agricultural practices, and drought-related features. A better mechanistic understanding of root-soil-microbiota associations could lead to the development of novel strategies to improve cereal production under drought. In this review, we discuss the root-soil-microbiota interactions for improving the soil environment and host fitness under drought and suggest a roadmap for harnessing the benefits of these interactions for drought-resilient cereals. These methods include conservative trait-based approaches for the selection and breeding of plant genetic resources and manipulation of the soil environments.

Risk factors and health behaviors associated with loneliness among cancer survivors during the COVID-19 pandemic.

Loneliness may exacerbate poor health outcomes particularly among cancer survivors during the COVID-19 pandemic. Little is known about the risk factors of loneliness among cancer survivors. We evaluated the risk factors of loneliness in the context of COVID-19 pandemic-related prevention behaviors and lifestyle/psychosocial factors among cancer survivors. Cancer survivors (n = 1471) seen at Huntsman Cancer Institute completed a survey between August-September 2020 evaluating health behaviors, medical care, and psychosocial factors including loneliness during COVID-19 pandemic. Participants were classified into two groups: 'lonely' (sometimes, usually, or always felt lonely in past month) and 'non-lonely' (never or rarely felt lonely in past month). 33% of cancer survivors reported feeling lonely in the past month. Multivariable logistic regression showed female sex, not living with a spouse/partner, poor health status, COVID-19 pandemic-associated lifestyle factors including increased alcohol consumption and marijuana/CBD oil use, and psychosocial stressors such as disruptions in daily life, less social interaction, and higher perceived stress and financial stress were associated with feeling lonely as compared to being non-lonely (all p < 0.05). A significant proportion of participants reported loneliness, which is a serious health risk among vulnerable populations, particularly cancer survivors. Modifiable risk factors such as unhealthy lifestyle behaviors and psychosocial stress were associated with loneliness. These results highlight the need to screen for unhealthy lifestyle factors and psychosocial stressors to identify cancer survivors at increased risk of loneliness and to develop effective management strategies.

Examining the relationship between anxiety and regional brain volumes in the National Alzheimer's Coordinating Center uniform, imaging, and biomarker datasets.

Anxiety has been associated with a greater risk of Alzheimer's disease (AD). Existing research has identified structural differences in regional brain tissue in participants with anxiety, but results have been inconsistent. We sought to determine the association between anxiety and regional brain volumes, and the moderation effect of APOE ε4. Using data from participants in the National Alzheimer's Coordinating Center (NACC) Uniform Data Set, with complete imaging (MRI) and biomarker data (n = 1533), multiple linear regression estimated the adjusted effect of anxiety on 30 structural MRI regions. The moderation effect of APOE ε4 on the relation between structural MRI regions and anxiety was assessed as was the moderation effect of cognitive status. False discovery rate was used to adjust for multiple comparisons. After controlling for intracranial volume, age, sex, years of education, race, Hispanic ethnicity, and cognitive status, seven MRI regions demonstrated lower volumes among participants with anxiety: total cerebrum gray matter volume, right hippocampus volume, hippocampal volume (total), right and left frontal lobe cortical gray matter volume, and right and total temporal lobe cortical gray matter volume. Findings suggest that anxiety is associated with significant atrophy in multiple brain regions, with corresponding ventricular enlargement. Future research should investigate if anxiety-related changes to brain morphology contribute to greater AD risk.

Mapping an undergraduate medical education curriculum against national and international palliative care reference learning objectives.

BACKGROUND: The teaching of palliative care competencies is an essential component of undergraduate medical education. There is significant variance in the palliative care content delivered in undergraduate medical curricula, revealing the utility of reference standards to guide curricular development and assessment. To evaluate our university's undergraduate palliative care teaching, we undertook a curriculum mapping exercise, comparing official learning objectives to the national Educating Future Physicians in Palliative and End-of-Life Care (EFPPEC) and the international Palliative Education Assessment Tool (PEAT) reference objectives. METHODS: Multiple assessors independently compared our university's UGME learning objectives with EFPPEC and PEAT reference objectives to determine the degree-of-coverage. Visual curriculum maps were created to depict in which part of the curriculum each objective is delivered and by which medical specialty. RESULTS: Of 122 EFPPEC objectives, 55 (45.1%) were covered fully, 42 (34.4%) were covered partially, and 25 (20.5%) were not covered by university objectives. Of 89 PEAT objectives, 40 (44.9%) were covered fully, 35 (39.3%) were covered partially, and 14 (15.7%) were not covered by university objectives. CONCLUSIONS: The majority of EFPPEC and PEAT reference objectives are fully or partially covered in our university's undergraduate medical curriculum. Our approach could serve as a guide for others who endeavour to review their universities' specialty-specific medical education against reference objectives. Future curriculum development should target the elimination of identified gaps and evaluate the attainment of palliative care competencies by medical learners.

Discovery and engineering of the antibody response against a prominent skin commensal.

The ubiquitous skin colonist Staphylococcus epidermidis elicits a CD8 + T cell response pre-emptively, in the absence of an infection 1 . However, the scope and purpose of this anti-commensal immune program are not well defined, limiting our ability to harness it therapeutically. Here, we show that this colonist also induces a potent, durable, and specific antibody response that is conserved in humans and non-human primates. A series of S. epidermidis cell-wall mutants revealed that the cell surface protein Aap is a predominant target. By colonizing mice with a strain of S. epidermidis in which the parallel ß-helix domain of Aap is replaced by tetanus toxin fragment C, we elicit a potent neutralizing antibody response that protects mice against a lethal challenge. A similar strain of S. epidermidis expressing an Aap-SpyCatcher chimera can be conjugated with recombinant immunogens; the resulting labeled commensal elicits high titers of antibody under conditions of physiologic colonization, including a robust IgA response in the nasal mucosa. Thus, immunity to a common skin colonist involves a coordinated T and B cell response, the latter of which can be redirected against pathogens as a novel form of topical vaccination.

Vaccine design via antigen reorientation.

A major challenge in creating universal influenza vaccines is to focus immune responses away from the immunodominant, variable head region of hemagglutinin (HA-head) and toward the evolutionarily conserved stem region (HA-stem). Here we introduce an approach to control antigen orientation via site-specific insertion of aspartate residues that facilitates antigen binding to alum. We demonstrate the generalizability of this approach with antigens from Ebola, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses and observe enhanced neutralizing antibody responses in all cases. We then reorient an H2 HA in an 'upside-down' configuration to increase the exposure and immunogenicity of HA-stem. The reoriented H2 HA (reoH2HA) on alum induced stem-directed antibodies that cross-react with both group 1 and group 2 influenza A subtypes. Electron microscopy polyclonal epitope mapping (EMPEM) revealed that reoH2HA (group 1) elicits cross-reactive antibodies targeting group 2 HA-stems. Our results highlight antigen reorientation as a generalizable approach for designing epitope-focused vaccines.

A breath of toxic air: The relationship between appraised air pollution, abusive supervision, and laissez-faire leadership through the dual-mediating pathways of negative affect and somatic complaints.

Air pollution has become a global public health hazard leading to debilitating effects on physical, mental, and emotional health. Management research has just begun to explore the effects of air pollution on employees' work life. Drawing from the transactional theory of stress (Lazarus & Folkman, 1984) and crossover theory (Westman, 2001), we argue that appraisal of air pollution is an important factor that influences leaders and their behavior with subordinates. Specifically, we propose that when leaders appraise severe air pollution, they are more likely to behave abusively toward their subordinates and engage in laissez-faire leadership. We also propose that this relationship is mediated by leaders' experience of somatic complaints and negative affect. We test our model using an experience sampling study in India of leaders and followers who were located in different cities from each other. Overall, our results highlight how air pollution appraisals can harm not only the leader experiencing the pollution but also subordinates of those leaders. In other words, our counterintuitive finding is that subordinates may be harmed by air pollution to which they are not even directly exposed. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Amphetamine Positivity Prior to Burn Surgery Does not Adversely Affect Intraoperative Outcomes.

The treatment of burn patients using amphetamines is challenging due hemodynamic liabilty and altered physiology. Wide variation exists in the operative timing for this patient population. We hypothesize that burn excision in patients admitted with amphetamine positivity is safe regardless of timing. Data from two verified burn centers between 2017 and 2022 with differing practice patterns in operative timing for amphetamine-positive patients. Center A obtains toxicology only on admission and proceeds with surgery based on hemodynamic status and operative urgency, whereas Center B sends daily toxicology until a negative test results. The primary outcome was the use of vasoactive agents during the index operation, modeled using logistic regression adjusting for burn severity and hospital days to index operation. Secondary outcomes included death and inpatient complications. A total of 270 patients were included, and there were no significant differences in demographics or burn characteristics between centers. Center A screened once and Center B obtained a median of four screens prior to the surgery. The adjusted OR of requiring vasoactive support intraoperatively was not associated with negative toxicology result (P = .821). Having a body surface area burned >20% conferred a significantly higher risk of vasoactive support (adj. OR 13.42 [3.90-46.23], P < .001). Mortality, number of operations, stroke, and hospital length of stay were similar between cohorts. Comparison between two verified burn centers indicates that waiting until a negative amphetamine toxicology result does not impact intraoperative management or subsequent burn outcomes. Serial toxicology tests are unnecessary to guide operative timing of burn patients with amphetamine use.

Co-designing a nature-based intervention to promote postnatal mental health for mothers and their infants: a complex intervention development study in England.

OBJECTIVES: There is burgeoning evidence for the potential of nature-based interventions to improve wellbeing. However, the role of nature in enhancing maternal mental health, child development and early relationships remains relatively unexplored. This study aimed to develop a co-designed nature-based intervention to improve postnatal mothers' and infants' wellbeing. DESIGN, SETTING AND PARTICIPANTS: Person-based and co-design approaches informed the planning and design of the postnatal nature-based intervention. In stage 1, a multidisciplinary team was formed to agree research questions and appropriate methodology, and a scoping review was conducted. Six qualitative focus groups were then held with 30 mothers and 15 professional stakeholders. In stage 2, intervention guiding principles and a logic model were developed, and a stakeholder consensus meeting was convened to finalise the prototype intervention. The research was conducted in Bristol, UK, across voluntary, educational and community-based healthcare settings. RESULTS: Stakeholder consultation indicated significant enthusiasm for a postnatal nature-based intervention. A scoping review identified little existing research in this area. Focus group data are reported according to four broad themes: (1) perceived benefits of a group postnatal nature-based intervention, (2) potential drawbacks and barriers to access, (3) supporting attendance and implementation, and (4) ideas for intervention content. The developed intervention was tailored for mothers experiencing, or at risk of, postnatal mental health difficulties. It was identified that the intervention should facilitate engagement with the natural world through the senses, while taking into account a broad range of postnatal-specific practical and psychological needs. CONCLUSIONS: To our knowledge, this is the first reported use of person-based and co-design approaches to develop a postnatal nature-based intervention. The resulting intervention was perceived by target users to address their needs and preferences. Further research is needed to determine the feasibility, clinical and cost-effectiveness of this approach.

Structural basis of transcription: RNA Polymerase II substrate binding and metal coordination at 3.0 Å using a free-electron laser.

Catalysis and translocation of multi-subunit DNA-directed RNA polymerases underlie all cellular mRNA synthesis. RNA polymerase II (Pol II) synthesizes eukaryotic pre-mRNAs from a DNA template strand buried in its active site. Structural details of catalysis at near atomic resolution and precise arrangement of key active site components have been elusive. Here we present the free electron laser (FEL) structure of a matched ATP-bound Pol II, revealing the full active site interaction network at the highest resolution to date, including the trigger loop (TL) in the closed conformation, bonafide occupancy of both site A and B Mg2+, and a putative third (site C) Mg2+ analogous to that described for some DNA polymerases but not observed previously for cellular RNA polymerases. Molecular dynamics (MD) simulations of the structure indicate that the third Mg2+ is coordinated and stabilized at its observed position. TL residues provide half of the substrate binding pocket while multiple TL/bridge helix (BH) interactions induce conformational changes that could propel translocation upon substrate hydrolysis. Consistent with TL/BH communication, a FEL structure and MD simulations of the hyperactive Rpb1 T834P bridge helix mutant reveals rearrangement of some active site interactions supporting potential plasticity in active site function and long-distance effects on both the width of the central channel and TL conformation, likely underlying its increased elongation rate at the expense of fidelity.

The Integration of Proteogenomics and Ribosome Profiling Circumvents Key Limitations to Increase the Coverage and Confidence of Novel Microproteins.

There has been a dramatic increase in the identification of non-conical translation and a significant expansion of the protein-coding genome and proteome. Among the strategies used to identify novel small ORFs (smORFs), Ribosome profiling (Ribo-Seq) is the gold standard for the annotation of novel coding sequences by reporting on smORF translation. In Ribo-Seq, ribosome-protected footprints (RPFs) that map to multiple sites in the genome are computationally removed since they cannot unambiguously be assigned to a specific genomic location, or to a specific transcript in the case of multiple isoforms. Furthermore, RPFs necessarily result in short (25-34 nucleotides) reads, increasing the chance of ambiguous and multi-mapping alignments, such that smORFs that reside in these regions cannot be identified by Ribo-Seq. Here, we show that the inclusion of proteogenomics to create a Ribosome Profiling and Proteogenomics Pipeline (RP3) bypasses this limitation to identify a group of microprotein-encoding smORFs that are missed by current Ribo-Seq pipelines. Moreover, we show that the microproteins identified by RP3 have different sequence compositions from the ones identified by Ribo-Seq-only pipelines, which can affect proteomics identification. In aggregate, the development of RP3 maximizes the detection and confidence of protein-encoding smORFs and microproteins.