N-terminal (Nt) acetylation (NTA) is an ample and irreversible cotranslational protein modification catalyzed by ribosome-associated Nt-acetyltransferases. NTA on specific proteins can act as a degradation signal (called an Ac/N-degron) for proteolysis in yeast and mammals. However, in plants, the biological relevance of NTA remains largely unexplored. In this study, we reveal that Arabidopsis (Arabidopsis thaliana) SIGMA FACTOR-BINDING PROTEIN1 (SIB1), a transcription coregulator and a positive regulator of salicylic acid-primed cell death, undergoes an absolute NTA on the initiator Met; Nt-acetyltransferase B (NatB) partly contributes to this modification. While NTA results in destabilization of certain target proteins, our genetic and biochemical analyses revealed that plant NatB-involved NTA instead renders SIB1 more stable. Given that the ubiquitin/proteasome system stimulates SIB1 degradation, it seems that the NTA-conferred stability ensures the timely expression of SIB1-dependent genes, mostly related to immune responses. Taking our findings together, here we report a noncanonical NTA-driven protein stabilization in land plants.

Hydrogen sulfide (H₂S), a plant gasotransmitter, functions in the plant response to cadmium (Cd) stress, implying a role for cysteine desulfhydrase in producing H₂S in this process. Whether d-CYSTEINE DESULFHYDRASE (DCD) acts in the plant Cd response remains to be identified, and if it does, how DCD is regulated in this process is also unknown. Here, we report that DCD-mediated H₂S production enhances plant Cd tolerance in Arabidopsis (Arabidopsis thaliana). When subjected to Cd stress, a dcd mutant accumulated more Cd and reactive oxygen species and showed increased Cd sensitivity, whereas transgenic lines overexpressing DCD had decreased Cd and reactive oxygen species levels and were more tolerant to Cd stress compared with wild-type plants. Furthermore, the expression of DCD was stimulated by Cd stress, and this up-regulation was mediated by a Cd-induced transcription factor, WRKY13, which bound to the DCD promoter. Consistently, the higher Cd sensitivity of the wrky13-3 mutant was rescued by the overexpression of DCD. Together, our results demonstrate that Cd-induced WRKY13 activates DCD expression to increase the production of H₂S, leading to higher Cd tolerance in plants.

Phosphoinositides function as lipid signals in plant development and stress tolerance by binding with partner proteins. We previously reported that Arabidopsis (Arabidopsis thaliana) phosphoinositide-specific phospholipase C2 functions in the endoplasmic reticulum (ER) stress response. However, the underlying molecular mechanisms of how phosphoinositides act in the ER stress response remain elusive. Here, we report that a phosphoinositide-binding protein, SMALLER TRICHOMES WITH VARIABLE BRANCHES (SVB), is involved in the ER stress tolerance. SVB contains a DUF538 domain with unknown function; orthologs are exclusively found in Viridiplantae. We established that SVB is ubiquitously expressed in plant tissues and is localized to the ER, Golgi apparatus, prevacuolar compartment, and plasma membrane. The knockout mutants of svb showed enhanced tolerance to ER stress, which was genetically complemented by transducing genomic SVB. SVB showed time-dependent induction after tunicamycin-induced ER stress, which depended on IRE1 and bZIP60 but not bZIP17 and bZIP28 in the unfolded protein response (UPR). A protein–lipid overlay assay showed specific binding of SVB to phosphatidylinositol 3,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate. SVB is therefore suggested to be the plant-specific phosphoinositide-binding protein whose expression is controlled by the UPR through the IRE1-bZIP60 pathway in Arabidopsis.

Members of the light-harvesting complex protein family participate in multiple processes connected with light sensing, light absorption, and pigment binding within the thylakoid membrane. Amino acid residues of the light-harvesting chlorophyll a/b-binding proteins involved in pigment binding have been precisely identified through x-ray crystallography experiments. In vitro pigment-binding studies have been performed with LIGHT-HARVESTING-LIKE3 proteins, and the pigment-binding ability of cyanobacterial high-light-inducible proteins has been studied in detail. However, analysis of pigment binding by plant high-light-inducible protein homologs, called ONE-HELIX PROTEINS (OHPs), is lacking. Here, we report on successful in vitro reconstitution of Arabidopsis (Arabidopsis thaliana) OHPs with chlorophylls and carotenoids and show that pigment binding depends on the formation of OHP1/OHP2 heterodimers. Pigment-binding capacity was completely lost in each of the OHPs when residues of the light-harvesting complex chlorophyll-binding motif required for chlorophyll binding were mutated. Moreover, the mutated OHP variants failed to rescue the respective knockout (T-DNA insertion) mutants, indicating that pigment-binding ability is essential for OHP function in vivo. The scaffold protein HIGH CHLOROPHYLL FLUORESCENCE244 (HCF244) is tethered to the thylakoid membrane by the OHP heterodimer. We show that HCF244 stability depends on OHP heterodimer formation and introduce the concept of a functional unit consisting of OHP1, OHP2, and HCF244, in which each protein requires the others. Because of their pigment-binding capacity, we suggest that OHPs function in the delivery of pigments to the D1 subunit of PSII.

Despite the ecological relevance of diatoms, many aspects of their photosynthetic machinery remain poorly understood. Diatoms differ from the green lineage of oxygenic organisms by their photosynthetic pigments and light-harvesting complex (Lhc) proteins, the latter of which are also called fucoxanthin-chlorophyll proteins (FCP). These are composed of three groups of proteins: Lhcf as the main group, Lhcr that are PSI associated, and Lhcx that are involved in photoprotection. The FCP complexes are assembled in trimers and higher oligomers. Several studies have investigated the biochemical properties of purified FCP complexes, but limited knowledge is available about their interaction with the photosystem cores. In this study, isolation of stable supercomplexes from the centric diatom Thalassiosira pseudonana was achieved. To preserve in vivo structure, the separation of thylakoid complexes was performed by native PAGE and sucrose density centrifugation. Different subpopulations of PSI and PSII supercomplexes were isolated and their subunits identified. Analysis of Lhc antenna composition identified Lhc(s) specific for either PSI (Lhcr 1, 3, 4, 7, 10–14, and Lhcf10) or PSII (Lhcf 1–7, 11, and Lhcr2). Lhcx6_1 was reproducibly found in PSII supercomplexes, whereas its association with PSI was unclear. No evidence was found for the interaction between photosystems and higher oligomeric FCPs, comprising Lhcf8 as the main component. Although the subunit composition of the PSII supercomplexes in comparison with that of the trimeric FCP complexes indicated a close mutual association, the higher oligomeric pool is only weakly associated with the photosystems, albeit its abundance in the thylakoid membrane.

Terpene volatiles are found in many important fruit crops, but their relationship to flavor is poorly understood. Here, we demonstrate using sensory descriptive and discriminant analysis that 1,8-cineole contributes a key floral/eucalyptus note to the aroma of ripe 'Hort16A’ kiwifruit (Actinidia chinensis). Two quantitative trait loci (QTLs) for 1,8-cineole production were identified on linkage groups 27 and 29a in a segregating A. chinensis population, with the QTL on LG29a colocating with a complex cluster of putative terpene synthase (TPS)-encoding genes. Transient expression in Nicotiana benthamiana and analysis of recombinant proteins expressed in Escherichia coli showed four genes in the cluster (AcTPS1a–AcTPS1d) encoded functional TPS enzymes, which produced predominantly sabinene, 1,8-cineole, geraniol, and springene, respectively. The terpene profile produced by AcTPS1b closely resembled the terpenes detected in red-fleshed A. chinensis. AcTPS1b expression correlated with 1,8-cineole content in developing/ripening fruit and also showed a positive correlation with 1,8-cineole content in the mapping population, indicating the basis for segregation is an expression QTL. Transient overexpression of AcTPS1b in Actinidia eriantha fruit confirmed this gene produced 1,8-cineole in Actinidia. Structure-function analysis showed AcTPS1a and AcTPS1b are natural variants at key TPS catalytic site residues previously shown to change enzyme specificity in vitro. Together, our results indicate that AcTPS1b is a key gene for production of the signature flavor terpene 1,8-cineole in ripe kiwifruit. Using a sensory-directed strategy for compound identification provides a rational approach for applying marker-aided selection to improving flavor in kiwifruit as well as other fruits.

Heat stress (HS) has serious effects on plant development, resulting in heavy agricultural losses. A critical transcription factor network is involved in plant adaptation to high temperature. DEHYDRATION RESPONSIVE ELEMENT-BINDING PROTEIN2A (DREB2A) is a key transcription factor that functions in plant thermotolerance. The DREB2A protein is unstable under normal temperature and is degraded by the 26S proteasome; however, the mechanism by which DREB2A protein stability dramatically increases in response to HS remains poorly understood. In this study, we found that the DREB2A protein of Arabidopsis (Arabidopsis thaliana) is stabilized under high temperature by the posttranslational modification SUMOylation. Biochemical data indicated that DREB2A is SUMOylated at K163, a conserved residue adjacent to the negative regulatory domain during HS. SUMOylation of DREB2A suppresses its interaction with BPM2, a ubiquitin ligase component, consequently increasing DREB2A protein stability under high temperature. In addition, analysis of plant heat tolerance and marker gene expression indicated that DREB2A SUMOylation is essential for its function in the HS response. Collectively, our data reveal a role for SUMOylation in the maintenance of DREB2A stability under high temperature, thus improving our understanding of the regulatory mechanisms underlying HS response in plant cells.

The aim of this study was to identify factors associated with the death of patients with COVID-19 pneumonia caused by the novel coronavirus SARS-CoV-2.

All clinical and laboratory parameters were collected prospectively from a cohort of patients with COVID-19 pneumonia who were hospitalised to Wuhan Pulmonary Hospital (Wuhan City, Hubei Province, China) between 25 December 2019 and 7 February 2020. Univariate and multivariate logistic regression was performed to investigate the relationship between each variable and the risk of death of COVID-19 pneumonia patients.

In total, 179 patients with COVID-19 pneumonia (97 male and 82 female) were included in the present prospective study, of whom 21 died. Univariate and multivariate logistic regression analysis revealed that age ≥65 years (OR 3.765, 95% CI 1.146-17.394; p=0.023), pre-existing concurrent cardiovascular or cerebrovascular diseases (OR 2.464, 95% CI 0.755-8.044; p=0.007), CD3⁺CD8⁺ T-cells ≤75 cells·μL^–1 (OR 3.982, 95% CI 1.132-14.006; p<0.001) and cardiac troponin I ≥0.05 ng·mL^–1 (OR 4.077, 95% CI 1.166-14.253; p<0.001) were associated with an increase in risk of mortality from COVID-19 pneumonia. In a sex-, age- and comorbid illness-matched case–control study, CD3⁺CD8⁺ T-cells ≤75 cells·μL^–1 and cardiac troponin I ≥0.05 ng·mL^–1 remained as predictors for high mortality from COVID-19 pneumonia.

We identified four risk factors: age ≥65 years, pre-existing concurrent cardiovascular or cerebrovascular diseases, CD3⁺CD8⁺ T-cells ≤75 cells·μL^–1 and cardiac troponin I ≥0.05 ng·mL^–1. The latter two factors, especially, were predictors for mortality of COVID-19 pneumonia patients.

The call for the European Respiratory Society (ERS) to change their e-cigarette and vaping policy, from honourable people with decades of experience fighting the evils of tobacco, is unfortunately misconceived. The three issues of greatest concern are acute toxicity, chronic toxicity and, most importantly, the effects on children and young people. The efficacy of e-cigarettes as an adjunct to smoking cessation are outwith the expertise of paediatric specialists, but we would ask for assurances that any benefits really do outweigh the risks to children and young people (below). Our comments on these key issues are as follows:

We wish to thank J. Britton and co-workers for responding to our editorial and giving us an opportunity to clarify our position as well as correct a few misunderstandings. We definitely share the same goal, which is to relieve Europe and the rest of the world from the terrible results of the tobacco epidemic. We also do not "blankly oppose e-cigarettes"; however, we strongly advocate against a harm reduction strategy including e-cigarettes as well as heated tobacco products [1]. As clinicians we all see reluctant smokers where e-cigarettes can be tried as a last resort for getting off cigarette smoking, but that is of little relevance for a general harm reduction strategy. We also agree that the UK has achieved a lot in the area of smoking cessation but would argue that this has been achieved by impressive tobacco control, not by the use of e-cigarettes, and that a country such as Australia, which has banned nicotine-containing e-cigarettes, has achieved similar results.

The respiratory community is united in its desire to reduce and eliminate the harm caused by tobacco smoking, which is at present on course to kill one billion people in the 21st century. The stated policy of the European Respiratory Society is to strive "constantly to promote strong and evidence-based policies to reduce the burden of tobacco related diseases". In our view, the recent ERS Tobacco Control Committee statement on tobacco harm reduction [1], though well-intentioned, appears to be based on a number of false premises and draws its conclusions from a partial account of available data. It also presents a false dichotomy between the provision of "conventional" tobacco control and harm reduction approaches. We therefore respond, in turn, to the seven arguments presented against the adoption of harm reduction in the Committee's statement.

Purpose of review

Diverse adverse events have been associated with administration of glatiramer acetate (GA), mainly local reactions at the injection site. Other, less frequent generalized reactions include isolated postinjection reactions and anaphylaxis, which may lead to discontinuation of GA.

Objective

To explore practice differences in the diagnosis and management of autoimmune encephalitis (AE), which is complicated by issues with sensitivity/specificity of antibody testing, nonspecific MRI/EEG/CSF findings, and competing differential diagnoses.

Consider consumption of annonacin-containing plant products, including pawpaw, as a possible environmental risk factor for atypical parkinsonism.

Surgical procedures are performed in the United States in a wide variety of clinical settings and with variation in clinical outcomes. In May 2012, the Task Force for Children’s Surgical Care, an ad hoc multidisciplinary group comprising physicians representing specialties relevant to pediatric perioperative care, was convened to generate recommendations to optimize the delivery of children’s surgical care. This group generated a white paper detailing the consensus opinions of the involved experts. Following these initial recommendations, the American College of Surgeons (ACS), Children’s Hospital Association, and Task Force for Children’s Surgical Care, with input from all related perioperative specialties, developed and published specific and detailed resource and quality standards designed to improve children’s surgical care (https://www.facs.org/quality-programs/childrens-surgery/childrens-surgery-verification). In 2015, with the endorsement of the American Academy of Pediatrics (https://pediatrics.aappublications.org/content/135/6/e1538), the ACS established a pilot verification program. In January 2017, after completion of the pilot program, the ACS Children’s Surgery Verification Quality Improvement Program was officially launched. Verified sites are listed on the program Web site at https://www.facs.org/quality-programs/childrens-surgery/childrens-surgery-verification/centers, and more than 150 are interested in verification. This report provides an update on the ACS Children’s Surgery Verification Quality Improvement Program as it continues to evolve.

Pediatricians are encouraged to address male adolescent sexual and reproductive health on a regular basis, including taking a sexual history, discussing healthy sexuality, performing an appropriate physical examination, providing patient-centered and age-appropriate anticipatory guidance, and administering appropriate vaccinations. These services can be provided to male adolescent patients in a confidential and culturally appropriate manner, can promote healthy sexual relationships and responsibility, can and involve parents in age-appropriate discussions about sexual health.

The American Academy of Pediatrics (AAP) believes that current data show that hospitals and accredited birth centers are the safest settings for birth in the United States. The AAP does not recommend planned home birth, which has been reported to be associated with a twofold to threefold increase in infant mortality in the United States. The AAP recognizes that women may choose to plan a home birth. This statement is intended to help pediatricians provide constructive, informed counsel to women considering home birth while retaining their role as child advocates and to summarize appropriate care for newborn infants born at home that is consistent with care provided for infants born in a medical care facility. Regardless of the circumstances of his or her birth, including location, every newborn infant deserves health care consistent with that highlighted in this statement, which is more completely described in other publications from the AAP, including Guidelines for Perinatal Care and the Textbook of Neonatal Resuscitation. All health care clinicians and institutions should promote communications and understanding on the basis of professional interaction and mutual respect.

BACKGROUND:

Pediatric subspecialists routinely provide procedural sedation outside the operating room. No large study has reported trends in outpatient pediatric procedural sedation. Our purpose in this study was to identify significant trends in outpatient procedural sedation using the Pediatric Sedation Research Consortium.

Electronic cigarette or vaping product use–associated lung injury (EVALI) is a newly emerging diagnosis in the United States, yet the incidence has surged greatly in the past year. With the trend of using electronic cigarettes (e-cigarettes) and vaping rising at an alarming rate among teenagers, many are resorting to friends, illicit drug dealers, and other informal sources to obtain their e-cigarettes, which is greatly contributing to the national outbreak of EVALI. The incidence of adolescents presenting with the constellation of respiratory, gastrointestinal, and constitutional symptoms characteristic of EVALI has been widely reported within the nation. We present one such case of an adolescent boy with a 2-year history of daily vaping who presented with nausea, vomiting, weight loss, and fever but lacked the respiratory symptoms that have been reported in the majority of EVALI cases reported thus far. Computed tomography scan of the abdomen and pelvis revealed an incidental finding of lung pathology characteristic of EVALI, prompting further workup and diagnosis of EVALI. In this case, it is demonstrated that the presentation of EVALI can be variable and is still poorly defined. The rising morbidity and mortality from EVALI reveal the importance of considering EVALI in all patients with a history of vaping or e-cigarette use, regardless of the presence or absence of respiratory symptoms.

Cystinuria is an autosomal recessive disorder characterized by excessive urinary excretion of cystine, resulting in recurrent cystine kidney stones, often presenting in childhood. Current treatment options for cystinuria include dietary and/or fluid measures and potassium citrate to reduce cystine excretion and/or increase solubility. Tiopronin and D-penicillamine are used in refractory cases to bind cystine in urine, albeit with serious side effects. A recent study revealed efficacy of nutritional supplement α-lipoic acid (ALA) treatment in preventing kidney stones in a mouse model of cystinuria. Here, we report 2 pediatric patients (6 and 15 years old) with cystinuria who received regular doses of ALA in addition to conventional therapy with potassium citrate. Both patients tolerated ALA without any adverse effects and had reduced frequency of symptomatic and asymptomatic kidney stones with disappearance of existing kidney stones in 1 patient after 2 months of ALA therapy. ALA treatment markedly improved laboratory markers of cystine solubility in urine with increased cystine capacity (–223 to –1 mg/L in patient 1 and +140 to +272 mg/L in patient 2) and decreased cystine supersaturation (1.7 to 0.88 in patient 1 and 0.64 to 0.48 in patient 2) without any changes in cystine excretion or urine pH. Our findings suggest that ALA improves solubility of cystine in urine and prevents stone formation in patients with cystinuria who do not respond to diet and citrate therapy.

Multiple births are increasing in frequency related to advanced maternal age and fertility treatments, and they have an increased risk for congenital anomalies compared to singleton births. However, twins have the same congenital anomalies <15% of the time. Thus, having multiple births with discordant anomalies is a growing challenge for neonatologists. Although external anomalies can often be spotted quickly at delivery or sex differences between multiples can rapidly identify those with internal anomalies described on prenatal ultrasound, we present a case of male multiples, who would optimally receive different initial resuscitation strategies on the basis of the presence or absence of an internal anomaly. The similar size of 4 extremely preterm quadruplets raises concern for whether accurate, immediate identification of 1 neonate with a congenital diaphragmatic hernia will be reliable in the delivery room. Clinicians discuss the ethical considerations of an "all for one" approach to this resuscitation.

BACKGROUND:

There is a dearth of evidence regarding the association of use of electronic cigarettes (e-cigarettes) with certain product characteristics and adolescent and young adult risk of unhealthy tobacco use patterns (eg, frequency of combustible cigarette smoking), which is needed to inform the regulation of e-cigarettes.

Secondary branch number per panicle plays a crucial role in regulating grain number and yield in rice. Here, we report the positional cloning and functional characterization for SECONDARY BRANCH NUMBER7 (qSBN7), a quantitative trait locus affecting secondary branch per panicle and grain number. Our research revealed that the causative variants of qSBN7 are located in the distal promoter region of FRIZZLE PANICLE (FZP), a gene previously associated with the repression of axillary meristem development in rice spikelets. qSBN7 is a novel allele of FZP that causes an ~56% decrease in its transcriptional level, leading to increased secondary branch and grain number, and reduced grain length. Field evaluations showed that qSBN7 increased grain yield by 10.9% in a temperate japonica variety, TN13, likely due to its positive effect on sink capacity. Our findings suggest that incorporation of qSBN7 can increase yield potential and improve the breeding of elite rice varieties.

Single-cross hybrids have been critical to the improvement of maize (Zea mays L.), but the characterization of their genetic architectures remains challenging. Previous studies of hybrid maize have shown the contribution of within-locus complementation effects (dominance) and their differential importance across functional classes of loci. However, they have generally considered panels of limited genetic diversity, and have shown little benefit from genomic prediction based on dominance or functional enrichments. This study investigates the relevance of dominance and functional classes of variants in genomic models for agronomic traits in diverse populations of hybrid maize. We based our analyses on a diverse panel of inbred lines crossed with two testers representative of the major heterotic groups in the U.S. (1106 hybrids), as well as a collection of 24 biparental populations crossed with a single tester (1640 hybrids). We investigated three agronomic traits: days to silking (DTS), plant height (PH), and grain yield (GY). Our results point to the presence of dominance for all traits, but also among-locus complementation (epistasis) for DTS and genotype-by-environment interactions for GY. Consistently, dominance improved genomic prediction for PH only. In addition, we assessed enrichment of genetic effects in classes defined by genic regions (gene annotation), structural features (recombination rate and chromatin openness), and evolutionary features (minor allele frequency and evolutionary constraint). We found support for enrichment in genic regions and subsequent improvement of genomic prediction for all traits. Our results suggest that dominance and gene annotations improve genomic prediction across diverse populations in hybrid maize.

The question of the relative evolutionary roles of adaptive and nonadaptive processes has been a central debate in population genetics for nearly a century. While advances have been made in the theoretical development of the underlying models, and statistical methods for estimating their parameters from large-scale genomic data, a framework for an appropriate null model remains elusive. A model incorporating evolutionary processes known to be in constant operation, genetic drift (as modulated by the demographic history of the population) and purifying selection, is lacking. Without such a null model, the role of adaptive processes in shaping within- and between-population variation may not be accurately assessed. Here, we investigate how population size changes and the strength of purifying selection affect patterns of variation at "neutral" sites near functional genomic components. We propose a novel statistical framework for jointly inferring the contribution of the relevant selective and demographic parameters. By means of extensive performance analyses, we quantify the utility of the approach, identify the most important statistics for parameter estimation, and compare the results with existing methods. Finally, we reanalyze genome-wide population-level data from a Zambian population of Drosophila melanogaster, and find that it has experienced a much slower rate of population growth than was inferred when the effects of purifying selection were neglected. Our approach represents an appropriate null model, against which the effects of positive selection can be assessed.

The vertebrate limb serves as an experimental paradigm to study mechanisms that regulate development of the stereotypical skeletal elements. In this study, we simultaneously inactivated Sall4 using Hoxb6Cre and Plzf in mouse embryos, and found that their combined function regulates development of the proximal-anterior skeletal elements in hindlimbs. The Sall4; Plzf double knockout exhibits severe defects in the femur, tibia, and anterior digits, distinct defects compared to other allelic series of Sall4; Plzf. We found that Sall4 regulates Plzf expression prior to hindlimb outgrowth. Further expression analysis indicated that Hox10 genes and GLI3 are severely downregulated in the Sall4; Plzf double knockout hindlimb bud. In contrast, PLZF expression is reduced but detectable in Sall4; Gli3 double knockout limb buds, and SALL4 is expressed in the Plzf; Gli3 double knockout limb buds. These results indicate that Plzf, Gli3, and Hox10 genes downstream of Sall4, regulate femur and tibia development. In the autopod, we show that Sall4 negatively regulates Hedgehog signaling, which allows for development of the most anterior digit. Collectively, our study illustrates genetic systems that regulate development of the proximal-anterior skeletal elements in hindlimbs.

BEAF (Boundary Element-Associated Factor) was originally identified as a Drosophila melanogaster chromatin domain insulator-binding protein, suggesting a role in gene regulation through chromatin organization and dynamics. Genome-wide mapping found that BEAF usually binds near transcription start sites, often of housekeeping genes, suggesting a role in promoter function. This would be a nontraditional role for an insulator-binding protein. To gain insight into molecular mechanisms of BEAF function, we identified interacting proteins using yeast two-hybrid assays. Here, we focus on the transcription factor Serendipity (Sry-). Interactions were confirmed in pull-down experiments using bacterially expressed proteins, by bimolecular fluorescence complementation, and in a genetic assay in transgenic flies. Sry- interacted with promoter-proximal BEAF both when bound to DNA adjacent to BEAF or > 2-kb upstream to activate a reporter gene in transient transfection experiments. The interaction between BEAF and Sry- was detected using both a minimal developmental promoter (y) and a housekeeping promoter (RpS12), while BEAF alone strongly activated the housekeeping promoter. These two functions for BEAF implicate it in playing a direct role in gene regulation at hundreds of BEAF-associated promoters.

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The spirochete Borrelia burgdorferi sensu lato is the causative agent of Lyme disease (LD). The spirochetes produce the CspZ protein that binds to a complement regulator, factor H (FH). Such binding downregulates activation of host complement to facilitate spirochete evasion of complement killing. However, vaccination with CspZ does not protect against LD infection. In this study, we demonstrated that immunization with CspZ-YA, a CspZ mutant protein with no FH-binding activity, protected mice from infection by several spirochete genotypes introduced via tick feeding. We found that the sera from CspZ-YA-vaccinated mice more efficiently eliminated spirochetes and blocked CspZ FH-binding activity than sera from CspZ-immunized mice. We also found that vaccination with CspZ, but not CspZ-YA, triggered the production of anti-FH antibodies, justifying CspZ-YA as an LD vaccine candidate. The mechanistic and efficacy information derived from this study provides insights into the development of a CspZ-based LD vaccine.

LuxS/AI-2 is an important quorum sensing system which affects the growth, biofilm formation, virulence, and metabolism of bacteria. LuxS is encoded by the luxS gene, but how this gene is associated with a diverse array of physiological activities in Edwardsiella piscicida (E. piscicida) is not known. Here, we constructed an luxS gene mutant strain, the luxS strain, to identify how LuxS/AI-2 affects pathogenicity. The results showed that LuxS was not found in the luxS gene mutant strain, and this gene deletion decreased E. piscicida growth compared to that of the wild-type strain. Meanwhile, the wild-type strain significantly increased penetration and motility in mucin compared to levels with the luxS strain. The 50% lethal dose (LD₅₀) of the E. piscicida luxS strain for zebrafish was significantly higher than that of the wild-type strain, which suggested that the luxS gene deletion could attenuate the strain’s virulence. The AI-2 activities of EIB202 were 56-fold higher than those in the luxS strain, suggesting that the luxS gene promotes AI-2 production. Transcriptome results demonstrated that between cells infected with the luxS strain and those infected with the wild-type strain 46 genes were significantly differentially regulated, which included 34 upregulated genes and 12 downregulated genes. Among these genes, the largest number were closely related to cell immunity and signaling systems. In addition, the biofilm formation ability of EIB202 was significantly higher than that of the luxS strain. The supernatant of EIB202 increased the biofilm formation ability of the luxS strain, which suggested that the luxS gene and its product LuxS enhanced biofilm formation in E. piscicida. All results indicate that the LuxS/AI-2 quorum sensing system in E. piscicida promotes its pathogenicity through increasing a diverse array of physiological activities.

Localized skin lesions are characteristic of cutaneous leishmaniasis (CL); however, Leishmania (Viannia) species, which are responsible for most CL cases in the Americas, can spread systemically, sometimes resulting in mucosal disease. Detection of Leishmania has been documented in healthy mucosal tissues (conjunctiva, tonsils, and nasal mucosa) and healthy skin of CL patients and in individuals with asymptomatic infection in areas of endemicity of L. (V.) panamensis and L. (V.) braziliensis transmission. However, the conditions and mechanisms that favor parasite persistence in healthy mucosal tissues are unknown. In this descriptive study, we compared the cell populations of the nasal mucosa (NM) of healthy donors and patients with active CL and explored the immune gene expression signatures related to molecular detection of Leishmania in this tissue in the absence of clinical signs or symptoms of mucosal disease. The cellular composition and gene expression profiles of NM samples from active CL patients were similar to those of healthy volunteers, with a predominance of epithelial over immune cells, and within the CD45⁺ cell population, a higher frequency of CD66b⁺ followed by CD14⁺ and CD3⁺ cells. In CL patients with molecular evidence of Leishmania persistence in the NM, genes characteristic of an anti-inflammatory and tissue repair responses (IL4R, IL5RA, POSTN, and SATB1) were overexpressed relative to NM samples from CL patients in which Leishmania was not detected. Here, we report the first immunological description of subclinically infected NM tissues of CL patients and provide evidence of a local anti-inflammatory environment favoring parasite persistence in the NM.

Implanted medical device-associated infections pose significant health risks, as they are often the result of bacterial biofilm formation. Staphylococcus aureus is a leading cause of biofilm-associated infections which persist due to mechanisms of device surface adhesion, biofilm accumulation, and reprogramming of host innate immune responses. We found that the S. aureus fibronectin binding protein A (FnBPA) is required for normal biofilm development in mammalian serum and that the SaeRS two-component system is required for functional FnBPA activity in serum. Furthermore, serum-developed biofilms deficient in FnBPA were more susceptible to macrophage invasion, and in a model of biofilm-associated implant infection, we found that FnBPA is crucial for the establishment of infection. Together, these findings show that S. aureus FnBPA plays an important role in physical biofilm development and represents a potential therapeutic target for the prevention and treatment of device-associated infections.

A Yersinia pestis mutant synthesizing an adjuvant form of lipid A (monophosphoryl lipid A, MPLA) displayed increased biogenesis of bacterial outer membrane vesicles (OMVs). To enhance the immunogenicity of the OMVs, we constructed an Asd-based balanced-lethal host-vector system that oversynthesized the LcrV antigen of Y. pestis, raised the amounts of LcrV enclosed in OMVs by the type II secretion system, and eliminated harmful factors like plasminogen activator (Pla) and murine toxin from the OMVs. Vaccination with OMVs containing MPLA and increased amounts of LcrV with diminished toxicity afforded complete protection in mice against subcutaneous challenge with 8 x 10⁵ CFU (80,000 50% lethal dose [LD₅₀]) and intranasal challenge with 5 x 10³ CFU (50 LD₅₀) of virulent Y. pestis. This protection was significantly superior to that resulting from vaccination with LcrV/alhydrogel or rF1-V/alhydrogel. At week 4 postimmunization, the OMV-immunized mice showed more robust titers of antibodies against LcrV, Y. pestis whole-cell lysate (YPL), and F1 antigen and more balanced IgG1:IgG2a/IgG2b-derived Th1 and Th2 responses than LcrV-immunized mice. Moreover, potent adaptive and innate immune responses were stimulated in the OMV-immunized mice. Our findings demonstrate that self-adjuvanting Y. pestis OMVs provide a novel plague vaccine candidate and that the rational design of OMVs could serve as a robust approach for vaccine development.

Mycoplasma gallisepticum is the primary etiological agent of chronic respiratory disease in chickens. Live attenuated vaccines are most commonly used in the field to control the disease, but current vaccines have some limitations. Vaxsafe MG (strain ts-304) is a new vaccine candidate that is efficacious at a lower dose than the current commercial vaccine strain ts-11, from which it is derived. In this study, the transcriptional profiles of the trachea of unvaccinated chickens and chickens vaccinated with strain ts-304 were compared 2 weeks after challenge with M. gallisepticum strain Ap3AS during the chronic stage of infection. After challenge, genes, gene ontologies, pathways, and protein classes involved in inflammation, cytokine production and signaling, and cell proliferation were upregulated, while those involved in formation and motor movement of cilia, formation of intercellular junctional complexes, and formation of the cytoskeleton were downregulated in the unvaccinated birds compared to the vaccinated birds, reflecting immune dysregulation and the pathological changes induced in the trachea by infection with M. gallisepticum. Vaccination appears to protect the structural and functional integrity of the tracheal mucosa 2 weeks after infection with M. gallisepticum.

Programmed death-ligand 1 (PD-L1/B7-H1) serves as a cosignaling molecule in cell-mediated immune responses and contributes to chronicity of inflammation and the escape of tumor cells from immunosurveillance. Here, we investigated the molecular mechanisms leading to PD-L1 upregulation in human oral carcinoma cells and in primary human gingival keratinocytes in response to infection with Porphyromonas gingivalis (P. gingivalis), a keystone pathogen for the development of periodontitis. The bacterial cell wall component peptidoglycan uses bacterial outer membrane vesicles to be taken up by cells. Internalized peptidoglycan triggers cytosolic receptors to induce PD-L1 expression in a myeloid differentiation primary response 88 (Myd88)-independent and receptor-interacting serine/threonine-protein kinase 2 (RIP2)-dependent fashion. Interference with the kinase activity of RIP2 or mitogen-activated protein (MAP) kinases interferes with inducible PD-L1 expression.

Bovine digital dermatitis (BDD), an infectious disease of the bovine foot with a predominant treponemal etiology, is a leading cause of lameness in dairy and beef herds worldwide. BDD is poorly responsive to antimicrobial therapy and exhibits a relapsing clinical course; an effective vaccine is therefore urgently sought. Using a reverse vaccinology approach, the present study surveyed the genomes of the three BDD-associated Treponema phylogroups for putative β-barrel outer membrane proteins and considered their potential as vaccine candidates. Selection criteria included the presence of a signal peptidase I cleavage site, a predicted β-barrel fold, and cross-phylogroup homology. Four candidate genes were overexpressed in Escherichia coli BL21(DE3), refolded, and purified. Consistent with their classification as β-barrel OMPs, circular-dichroism spectroscopy revealed the adoption of a predominantly β-sheet secondary structure. These recombinant proteins, when screened for their ability to adhere to immobilized extracellular matrix (ECM) components, exhibited a diverse range of ligand specificities. All four proteins specifically and dose dependently adhered to bovine fibrinogen. One recombinant protein was identified as a candidate diagnostic antigen (disease specificity, 75%). Finally, when adjuvanted with aluminum hydroxide and administered to BDD-naive calves using a prime-boost vaccination protocol, these proteins were immunogenic, eliciting specific IgG antibodies. In summary, we present the description of four putative treponemal β-barrel OMPs that exhibit the characteristics of multispecific adhesins. The observed interactions with fibrinogen may be critical to host colonization and it is hypothesized that vaccination-induced antibody blockade of these interactions will impede treponemal virulence and thus be of therapeutic value.

The intracellular lifestyle of bacteria is widely acknowledged to be an important mechanism in chronic and recurring infection. Among the Staphylococcus genus, only Staphylococcus aureus and Staphylococcus pseudintermedius have been clearly identified as intracellular in nonprofessional phagocytic cells (NPPCs), for which the mechanism is mainly fibronectin-binding dependent. Here, we used bioinformatics tools to search for possible new fibronectin-binding proteins (FnBP-like) in other Staphylococcus species. We found a protein in Staphylococcus delphini called Staphylococcus delphini surface protein Y (SdsY). This protein shares 68% identity with the Staphylococcus pseudintermedius surface protein D (SpsD), 36% identity with S. aureus FnBPA, and 39% identity with S. aureus FnBPB. The SdsY protein possesses the typical structure of FnBP-like proteins, including an N-terminal signal sequence, an A domain, a characteristic repeated pattern, and an LPXTG cell wall anchor motif. The level of adhesion to immobilized fibronectin was significantly higher in all S. delphini strains tested than in the fibronectin-binding-deficient S. aureus DU5883 strain. By using a model of human osteoblast infection, the level of internalization of all strains tested was significantly higher than with the invasive-incompetent S. aureus DU5883. These findings were confirmed by phenotype restoration after transformation of DU5883 by a plasmid expression vector encoding the SdsY repeats. Additionally, using fibronectin-depleted serum and murine osteoblast cell lines deficient for the β₁ integrin, the involvement of fibronectin and β₁ integrin was demonstrated in S. delphini internalization. The present study demonstrates that additional staphylococcal species are able to invade NPPCs and proposes a method to identify FnBP-like proteins.

African swine fever (ASF) is a highly contagious hemorrhagic viral disease of domestic and wild pigs that is responsible for serious economic and production losses. It is caused by the African swine fever virus (ASFV), a large and complex icosahedral DNA virus of the Asfarviridae family. Currently, there is no effective treatment or approved vaccine against the ASFV. pS273R, a specific SUMO-1 cysteine protease, catalyzes the maturation of the pp220 and pp62 polyprotein precursors into core-shell proteins. Here, we present the crystal structure of the ASFV pS273R protease at a resolution of 2.3 Å. The overall structure of the pS273R protease is represented by two domains named the "core domain" and the N-terminal "arm domain." The "arm domain" contains the residues from M1 to N83, and the "core domain" contains the residues from N84 to A273. A structure analysis reveals that the "core domain" shares a high degree of structural similarity with chlamydial deubiquitinating enzyme, sentrin-specific protease, and adenovirus protease, while the "arm domain" is unique to ASFV. Further, experiments indicated that the "arm domain" plays an important role in maintaining the enzyme activity of ASFV pS273R. Moreover, based on the structural information of pS273R, we designed and synthesized several peptidomimetic aldehyde compounds at a submolar 50% inhibitory concentration, which paves the way for the design of inhibitors to target this severe pathogen.

IMPORTANCE African swine fever virus, a large and complex icosahedral DNA virus, causes a deadly infection in domestic pigs. In addition to Africa and Europe, countries in Asia, including China, Vietnam, and Mongolia, were negatively affected by the hazards posed by ASFV outbreaks in 2018 and 2019, at which time more than 30 million pigs were culled. Until now, there has been no vaccine for protection against ASFV infection or effective treatments to cure ASF. Here, we solved the high-resolution crystal structure of the ASFV pS273R protease. The pS273R protease has a two-domain structure that distinguishes it from other members of the SUMO protease family, while the unique "arm domain" has been proven to be essential for its hydrolytic activity. Moreover, the peptidomimetic aldehyde compounds designed to target the substrate binding pocket exert prominent inhibitory effects and can thus be used in a potential lead for anti-ASFV drug development.