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Summary: Misfolded endoplasmic reticulum (ER) proteins are degraded through a process called ER-associated degradation (ERAD). Soluble, lumenal ERAD targets are recognized, retrotranslocated across the ER membrane, ubiquitinated, extracted from the membrane, and degraded by the proteasome using an ERAD pathway containing a ubiquitin ligase called Hrd1. To determine how Hrd1 mediates these processes, we developed a deep mutational scanning approach to identify residues involved in Hrd1 function, including those exclusively required for lumenal degradation. We identify several regions required for different Hrd1 functions. Most surprisingly, we find two cytosolic regions of Hrd1 required for lumenal ERAD substrate degradation. Using in vivo and in vitro approaches, we define roles for disordered regions between structural elements that are required for Hrd1 autoubiquitination and substrate interaction. Our results demonstrate that disordered cytosolic regions promote substrate retrotranslocation by controlling Hrd1 activation and establishing directionality of retrotranslocation for lumenal substrate across the ER membrane.

Summary: The miR-15/16 family targets a large network of genes in T cells to restrict their cell cycle, memory formation, and survival. Upon T cell activation, miR-15/16 are downregulated, allowing rapid expansion of differentiated effector T cells to mediate a sustained response. Here, we used conditional deletion of miR-15/16 in regulatory T cells (Tregs) to identify immune functions of the miR-15/16 family in T cells. miR-15/16 are indispensable to maintain peripheral tolerance by securing efficient suppression by a limited number of Tregs. miR-15/16 deficiency alters expression of critical Treg proteins and results in accumulation of functionally impaired FOXP3loCD25loCD127hi Tregs. Excessive proliferation in the absence of miR-15/16 shifts Treg fate and produces an effector Treg phenotype. These Tregs fail to control immune activation, leading to spontaneous multi-organ inflammation and increased allergic inflammation in a mouse model of asthma. Together, our results demonstrate that miR-15/16 expression in Tregs is essential to maintain immune tolerance.

Objective Older adults may be particularly interested in health-related content on Facebook, especially those who have chronic health conditions. The purpose of this study was to compare older adult Facebook users with and without a chronic health condition on their frequency of posting and exposure to health-related content. Methods Participants, recruited via Qualtrics, were regular Facebook users aged 50 + years. Participants were asked separately if they had seen, posted, and shared: Health-related information; about others’/their own health behaviors (e.g., exercise); and about others’/their own medical condition. Six logistic regression models, controlling for demographics and Facebook login frequency, were run to assess whether viewing and/or posting health-related messages differed by chronic health condition status. Results Respondents ( N = 697; 77.9% female) were on average 61.2 (SD = 7.9) years old and ( n = 625; 89.7%) were White. One-half reported a chronic health condition ( n = 351; 50.4%). In adjusted models, those with a chronic health condition had a higher likelihood of seeing posts containing health information (OR = 1.41; 95% CI: 1.04, 1.93) and about others’ medical conditions (OR = 1.67; 95% CI: 1.22, 2.27) at least once a month compared to those with no chronic health conditions. People with and without chronic health conditions did not differ in terms of how often they see others’ post about health behaviors. Those with a chronic health condition had a higher likelihood of posting or sharing health information (OR = 1.67; 95% CI: 1.22, 2.27), posting about their own health behaviors (OR = 1.55; 95% CI: 1.00, 2.44; p = 0.048), and about their health condition (OR = 1.96; 95% CI: 1.17, 3.27) at least once a month. Conclusion Most older adults on Facebook are exposed to and post multiple forms of health-related content. Therefore, Facebook may be an appropriate channel for conducting health-related communication targeting older adults.

Measurement(s) Species biomass densities • Species Interactions Technology Type(s) Field sampling • Direct observation and rule based filters Factor Type(s) Forest fire burn severity Sample Characteristic - Organism Consumers and producers Sample Characteristic - Environment Mixed-conifer montane forest Sample Characteristic - Location Eldorado National Forest

Abstract We hypothesized that the scarcity of N-nucleotides might contribute to the inability of the neonate to mount a robust allergic immune response. To test this, we used terminal deoxyribunucleotidyl Transferase deficient (TdT−/−) mice, which express “fetal-like” T cell receptor and immunoglobulin repertoires with largely germline-encoded CDR3 regions. Intraperitoneal sensitization was followed by aerosol provocation with either PBS or the allergen OVA in both TdT−/− mice and wild-type mice to develop allergic respiratory inflammation. The effects of this procedure were investigated by lung function test, immunological analysis of serum and brochoalveolar lavage. The local TH2 cytokine milieu was significantly attenuated in TdT−/− mice. Within this group, the induction of total IgE levels was also significantly reduced after sensitization. TdT−/− mice showed a tendency toward reduced eosinophilic inflow into the bronchial tubes, which was associated with the elimination of respiratory hyperreactivity. In conclusion, in a murine model of allergic airway inflammation, the expression of fetal-like antigen receptors was associated with potent indications of a reduced ability to mount an asthma phenotype. This underlines the importance of somatically-generated antigen-receptor repertoire diversity in type one allergic immune responses and suggests that the fetus may be protected from allergic responses, at least in part, by controlling N addition.

The early B cell protein λ5 is an essential component of the surrogate light chain and the preB cell receptor (preBCR), which is critical for optimal B cell development. To investigate the effect of λ5 and/or B cells on bone acquisition over time, we developed a panel of JH-/-, λ5-/-, JH-/- λ5-/-, and wild-type (WT) BALB/c mice and then studied postnatal bone development and aging in these mice at one, six, twelve, and twenty-two months of age. The trabecular bone volume over total volume (BV/TV) in JH-/- mice was similar to WT mice at all ages. In contrast, at six months of age and thereafter, λ5-/- and JH-/-λ5-/- mice demonstrated a severe decrease in trabecular bone mass. Surprisingly, bone mass in six-month-old λ5-/- and JH-/-λ5-/- mice was similar to or even lower than in aged (twenty-two-months) WT mice, suggesting accelerated skeletal aging. The postnatal development and the acquisition of cortical bone mass in JH-/-λ5-/- mice were generally comparable to WT. However, JH-/-λ5-/- mice showed a significant decrease in cortical BV/TV at six- and twelve months of age. To examine the contribution of λ5 and B cells to postnatal bone synthesis, we separately transplanted whole bone marrow cells from JH-/-λ5-/- and WT mice into irradiated JH-/-λ5-/- and WT recipients. WT recipients of JH-/-λ5-/- marrow cells failed to show acquisition of trabecular bone mass, whereas transplanting WT marrow cells into JH-/-λ5-/- recipients led to the recovery of trabecular bone mass. Transfer of WT marrow cells into JH-/-λ5-/- mice promoted synthesis of new cortical and trabecular bone. Our findings indicate that λ5 plays a major role in preserving bone mass during postnatal development and skeletal aging which is distinct from its role in B cell development. The absence of both λ5 and B cells in JH-/-λ5-/- mice leads to delayed acquisition of cortical bone during postnatal development. Dissecting the mechanism(s) by which λ5 regulates bone homeostasis may provide new avenues for the treatment of age-related loss of bone mass and osteoporosis.

BackgroundBehavioral weight loss programs typically enroll 12-40 people into groups that then suffer from declining engagement over time. Web-based patient communities, on the other hand, typically offer no limits on capacity and membership is fluid. This model may be useful for boosting engagement in behavioral weight loss interventions, which could lead to better outcomes. ObjectiveIn this study, we aimed to examine the feasibility and acceptability of continuously enrolling participants into a Facebook-delivered weight loss intervention for the first 8 of 16 weeks relative to the same intervention where no new participants were enrolled after randomization. MethodsWe conducted a randomized pilot trial to compare a Facebook weight loss group that used open enrollment with a group that used closed enrollment on feasibility and acceptability in adults with BMI 27-45 kg/m2. The feasibility outcomes included retention, engagement, and diet tracking adherence. We described the percentage loss of ≥5% weight in both groups as an exploratory outcome. We also explored the relationship between total volume of activity in the group and weight loss. The participants provided feedback via web-based surveys and focus groups. ResultsRandomized participants (68/80, 85% women) were on average, aged 40.2 (SD 11.2) years with a mean BMI of 34.4 (SD 4.98) kg/m2. We enrolled an additional 54 participants (50/54, 93% female) in the open enrollment condition between weeks 1 and 8, resulting in a total group size of 94. Retention was 88% and 98% under the open and closed conditions, respectively. Randomized participants across conditions did not differ in engagement (P=.72), or diet tracking adherence (P=.42). Participant feedback in both conditions revealed that sense of community was what they liked most about the program and not enough individualized feedback was what they liked the least. Weight loss of ≥5% was achieved by 30% (12/40) of the participants randomized to the open enrollment condition and 18% (7/40) of the participants in the closed enrollment condition. Exploratory analyses revealed that the open condition (median 385, IQR 228-536.5) had a greater volume of engagement than the closed condition (median 215, IQR 145.5-292; P=.007). Furthermore, an increase of 100 in the total volume of engagement in the Facebook group each week was associated with an additional 0.1% weekly weight loss among the randomized participants (P=.02), which was independent of time, individual participant engagement, and sociodemographic characteristics. ConclusionsOpen enrollment was as feasible and acceptable as closed enrollment. A greater volume of engagement in the Facebook group was associated with weight loss, suggesting that larger groups that produce more engagement overall may be beneficial. Future research should examine the efficacy of the open enrollment approach for weight loss in a fully powered randomized trial. Trial RegistrationClinicalTrials.gov NCT02656680; https://clinicaltrials.gov/ct2/show/NCT02656680

Microbial plant-soil feedbacks (PSF) are fundamentally important for plant diversity. The authors present a spatially explicit dynamic model that separates the effects of microbial mutualists and pathogens, thereby presenting a testable mechanistic framework to reconcile previously puzzling observations of the strength and direction of PSF with diversity maintenance.

The COVID-19 pandemic introduced mandatory stay-at-home orders and concerns about contracting a virus that impacted the physical and mental health of much of the world’s population. This study compared the rates of depression and anxiety in a sample of older primary care patients (aged ≥65 years old) and their family members recruited for a clinical trial before and during the COVID-19 pandemic. Participants were dyads enrolled in the Caregiver Outcomes of Alzheimer’s Disease Screening (COADS) trial, which included 1,809 dyads of older primary care patients and one of their family members. Mean scores on the Patient Health Questionnaire-9 (PHQ-9) and the Generalized Anxiety Disorder Scale-7 (GAD-7) were measured and compared before and during the pandemic. We found no difference in depression and anxiety among dyads of older primary care patients and their family members recruited before and during COVID-19. Additionally, we found that older primary care patients and family members who reported their income as comfortable had significantly lower depression and anxiety compared to those who reported having not enough to make ends meet. Along with this, older primary care patients with a high school education or less were more likely to have anxiety compared to those with a postgraduate degree. Moreover, our findings support the notion that certain demographics of older primary care patients and family members are at a higher risk for depression and anxiety, indicating who should be targeted for psychological health interventions that can be adapted during COVID-19. Future research should continue monitoring older primary care patients and their family members through the remainder of the COVID-19 pandemic.

Lowland tropical rainforests harbor the most diverse plant communities in the world, but the herbaceous plants of the understory are often overlooked. To address this knowledge gap, we asked to what extent the understory herbaceous community contributes to the species richness and phylogenetic diversity of plant communities by surveying a neotropical rainforest at Los Tuxtlas, Mexico. We used Gentry transects to characterize the woody overstory community, and line-intercepts within the same transects to survey understory herbs and subshrubs. We also used published phylogenies to calculate community phylogenetic diversity with and without the understory stratum. We found that the understory contained a diverse (23 species, or 22.1% of all species surveyed) and phylogenetically distinct plant community dominated by aroids (13 species) and ferns (4 species). Inclusion of the understory stratum increased total species richness by 28.4% but increased phylogenetic diversity by 41.4%. Additionally, in contrast to temperate forests, the understory plant community was much less diverse than the overstory, which contained 81 species > 1 cm dbh (77.9% of all species surveyed). This survey adds to the hitherto small body of literature comparing understory and overstory strata in tropical rainforests and reveals previously overlooked patterns of floristic diversity.

Enrichment for tyrosine in immunoglobulin CDR-H3 is due in large part to natural selection of germline immunoglobulin DH sequence. We have previously shown that when DH sequence is modified to reduce the contribution of tyrosine codons, epitope recognition is altered and B cell development, antibody production, autoantibody production, and morbidity and mortality following pathogen challenge are adversely affected. TCRβ diversity (Dβ) gene segment sequences are even more highly conserved than DH, with trout Dβ1 identical to human and mouse Dβ1. We hypothesized that natural selection of Dβ sequence also shapes CDR-B3 diversity and influences T cell development and T cell function. To test this, we used a mouse strain that lacked Dβ2 and contained a novel Dβ1 allele (DβYTL) that replaces Dβ1 with an immunoglobulin DH, DSP2.3. Unlike Dβ1, wherein glycine predominates in all three reading frames (RFs), in DSP2.3 there is enrichment for tyrosine in RF1, threonine in RF2, and leucine in RF3. Mature T cells using DβYTL expressed TCRs enriched at particular CDR-B3 positions for tyrosine but depleted of leucine. Changing Dβ sequence altered thymocyte and peripheral T cell numbers and the T cell response to an ovalbumin immunodominant epitope. The differences in tyrosine content might explain, at least in part, why TCRs are more polyspecific and of lower affinity for their cognate antigens than their immunoglobulin counterparts.

We have previously shown that the sequence of the immunoglobulin diversity gene segment (DH) helps dictate the structure and composition of complementarity determining region 3 of the immunoglobulin heavy chain (CDR-H3). In order to test the role of germline D sequence on the diversity of the preimmune TCRβ repertoire of T cells, we generated a mouse with a mutant TCRβ DJC locus wherein the Dβ2-Jβ2 gene segment cluster was deleted and the remaining diversity gene segment, Dβ1 (IMGT:TRDB1), was replaced with DSP2.3 (IMGT:IGHD2-02), a commonly used B cell immunoglobulin DH gene segment. Crystallographic studies have shown that the length and thus structure of TCR CDR-B3 places amino acids at the tip of CDR-B3 in a position to directly interact with peptide bound to an MHC molecule. The length distribution of complementarity determining region 3 of the T cell receptor beta chain (CDR-B3) has been proposed to be restricted largely by MHC-specific selection, disfavoring CDR-B3 that are too long or too short. Here we show that the mechanism of control of CDR-B3 length depends on the Dβ sequence, which in turn dictates exonucleolytic nibbling. By contrast, the extent of N addition and the variance of created CDR3 lengths are regulated by the cell of origin, the thymocyte. We found that the sequence of the D and control of N addition collaborate to bias the distribution of CDR-B3 lengths in the pre-immune TCR repertoire and to focus the diversity provided by N addition and the sequence of the D on that portion of CDR-B3 that is most likely to interact with the peptide that is bound to the presenting MHC.

Replication-transcription conflicts promote mutagenesis and give rise to evolutionary signatures, with fundamental importance to genome stability ranging from bacteria to metastatic cancer cells. This review focuses on the interplay between replication-transcription conflicts and the evolution of gene directionality. In most bacteria, the majority of genes are encoded on the leading strand of replication such that their transcription is co-directional with the direction of DNA replication fork movement. This gene strand bias arises primarily due to negative selection against deleterious consequences of head-on replication-transcription conflict. However, many genes remain head-on. Can head-on orientation provide some benefit? We combine insights from both mechanistic and evolutionary studies, review published work, and analyze gene expression data to evaluate an emerging model that head-on genes are temporal targets for adaptive mutagenesis during stress. We highlight the alternative explanation that genes in the head-on orientation may simply be the result of genomic inversions and relaxed selection acting on nonessential genes. We seek to clarify how the mechanisms of replication-transcription conflict, in concert with other mutagenic mechanisms, balanced by natural selection, have shaped bacterial genome evolution.

Indonesia and Malaysia have emerged as leading producers of palm oil in the past several decades, expanding production through the conversion of tropical forests to industrial plantations. Efforts to produce sustainable palm oil, including certification by the Roundtable on Sustainable Palm Oil (RSPO), include guidelines designed to reduce the environmental impact of palm oil production. Fire-driven deforestation is prohibited by law in both countries and a stipulation of RSPO certification, yet the degree of environmental compliance is unclear, especially during El Niño events when drought conditions increase fire risk. Here, we used time series of satellite data to estimate the spatial and temporal patterns of fire-driven deforestation on and around oil palm plantations. In Indonesia, fire-driven deforestation accounted for one-quarter of total forest losses on both certified and noncertified plantations. After the first plantations in Indonesia received RSPO certification in 2009, forest loss and fire-driven deforestation declined on certified plantations but did not stop altogether. Oil palm expansion in Malaysia rarely involved fire; only 5 % of forest loss on certified plantations had coincident active fire detections. Interannual variability in fire detections was strongly influenced by El Niño and the timing of certification. Fire activity during the 2002, 2004, and 2006 El Niño events was similar among oil palm plantations in Indonesia that would later become certified, noncertified plantations, and surrounding areas. However, total fire activity was 75 % and 66 % lower on certified plantations than noncertified plantations during the 2009 and 2015 El Niño events, respectively. The decline in fire activity on certified plantations, including during drought periods, highlights the potential for RSPO certification to safeguard carbon stocks in peatlands and remaining forests in accordance with legislation banning fires. However, aligning certification standards with satellite monitoring capabilities will be critical to realize sustainable palm oil production and meet industry commitments to zero deforestation.

Systemic lupus erythematosus (SLE) is an autoimmune disease that reflects a failure to block the production of self-reactive antibodies, especially those that bind double-stranded DNA (dsDNA). Backcrossing the lupus-prone NZM2410 genome onto C57BL/6 led to the identification of three genomic intervals, termed sle1, sle2 and sle3, which are associated with lupus susceptibility. We previously generated a C57BL/6 strain congenic for an immunoglobulin DH locus (ΔD–iD) that enriches for arginine at dsDNA-binding positions. We individually introduced the ΔD–iD allele into the three sle strains to test whether one or more of these susceptibility loci could affect the developmental fate of B cells bearing arginine-enriched CDR-H3s, the CDR-H3 repertoire created by the DH and the prevalence of dsDNA-binding antibodies. We found that the combination of the ΔD–iD allele and the sle1 locus led to a decrease in mature, recirculating B cell numbers and an increase in marginal zone cell numbers while maintaining a highly charged CDR-H3 repertoire. ΔD–iD and sle2 had no effect on peripheral B cell numbers, but the CDR-H3 repertoire was partially normalized. ΔD–iD and sle3 led to an increase in marginal zone B cell numbers, with some normalization of hydrophobicity. Mice with ΔD–iD combined with either sle1 or sle3 had increased production of dsDNA-binding IgM and IgG by 12 months of age. These findings indicate that the peripheral CDR-H3 repertoire can be categorically manipulated by the effects of nonimmunoglobulin genes.

In the bone marrow, preB cells are found adjacent to the bone endosteum where bone synthesizing osteoblast and bone resorbing osteoclasts reside. Although there is evidence of interactions between preB and bone cells, the factors that contribute to such interactions are poorly understood. A critical checkpoint for preB cell development assesses the integrity of the nascent immunoglobulin μ heavy chain (HC) by testing whether it can participate in the formation of a preB cell receptor (preBCR), composed of the μ HC and surrogate light chain (LC). In this work, we tested whether loss of preBCR components can affect bone synthesis. A panel of gene targeted mice with sequential blocks in preBCR formation or function [surrogate light chain component lambda 5 deleted (λ5−/−), transmembrane domain of μHC deleted (IgM-mem−/−), and CD19 preBCR co-receptor deleted (CD19−/−)] were evaluated for effects on postnatal bone synthesis. Postnatal bone mass was analyzed in 6 month old mice using μ-CT, histomorphometry and double calcein labeling. Both cortical and trabecular bone mass were significantly decreased in the femurs of the λ5 and IgM-mem deficient mice. Histomorphometric analysis showed a decrease in the numbers of osteoblasts and osteoclasts in all three mutant strains. Double calcein labeling revealed a significant decrease in dynamic synthesis and mineralization of bone in λ5−/− mice. Our data strongly suggest that interference with preBCR formation or function affects bone homeostasis independent of the presence or absence of mature B cells, and that components of the preBCR play important, and potentially distinct, roles in regulating adult bone mass.

ABSTRACT Biofilms are structured communities of tightly associated cells that constitute the predominant state of bacterial growth in natural and human-made environments. Although the core genetic circuitry that controls biofilm formation in model bacteria such as Bacillus subtilis has been well characterized, little is known about the role that metabolism plays in this complex developmental process. Here, we performed a time-resolved analysis of the metabolic changes associated with pellicle biofilm formation and development in B. subtilis by combining metabolomic, transcriptomic, and proteomic analyses. We report surprisingly widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. Most of these metabolic alterations were hitherto unrecognized as biofilm associated. For example, we observed increased activity of the tricarboxylic acid (TCA) cycle during early biofilm growth, a shift from fatty acid biosynthesis to fatty acid degradation, reorganization of iron metabolism and transport, and a switch from acetate to acetoin fermentation. Close agreement between metabolomic, transcriptomic, and proteomic measurements indicated that remodeling of metabolism during biofilm development was largely controlled at the transcriptional level. Our results also provide insights into the transcription factors and regulatory networks involved in this complex metabolic remodeling. Following upon these results, we demonstrated that acetoin production via acetolactate synthase is essential for robust biofilm growth and has the dual role of conserving redox balance and maintaining extracellular pH. This report represents a comprehensive systems-level investigation of the metabolic remodeling occurring during B. subtilis biofilm development that will serve as a useful road map for future studies on biofilm physiology. IMPORTANCE Bacterial biofilms are ubiquitous in natural environments and play an important role in many clinical, industrial, and ecological settings. Although much is known about the transcriptional regulatory networks that control biofilm formation in model bacteria such as Bacillus subtilis, very little is known about the role of metabolism in this complex developmental process. To address this important knowledge gap, we performed a time-resolved analysis of the metabolic changes associated with bacterial biofilm development in B. subtilis by combining metabolomic, transcriptomic, and proteomic analyses. Here, we report a widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. This report serves as a unique hypothesis-generating resource for future studies on bacterial biofilm physiology. Outside the biofilm research area, this work should also prove relevant to any investigators interested in microbial physiology and metabolism.