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Knowledge about time trends of cancer incidence and cancer survival in a defined region is an essential prerequisite for the planning of regional healthcare infrastructure. The aim of the study was to provide population-based analyses of all common tumour sites to assess the cancer burden in the Augsburg study region. Total population of the study region of Augsburg (668 522 residents), Southern Germany. The data obtained from the Cancer Registry Augsburg comprised 37 487 incident cases of malignant tumours (19 313 men and 18 174 women) diagnosed between 2005 and 2016 in the Augsburg region's resident population. We calculated sex-specific, age-standardised incidence rates and annual percent change to assess time trends. In men and in women, 3-year and 5-year relative survival was calculated and results were compared with the latest German estimates. Survival trends were presented for the most common cancers only. Decreasing age-standardised incidence rates were observed for prostate cancer and for colorectal cancer in men. For oropharyngeal cancer, rates declined in men, but significantly increased in women. Incidence for female breast cancer remained stable. Five-year relative survival ranged between 6.4% (95% CI: 4.1% to 10.1%) for pancreatic cancer and 97.7% (95% CI: 96.0% to 99.4%) for prostate cancer in men and between 10.2% (95% CI: 7.1% to 14.6%) for pancreatic cancer and 96.6% (95% CI: 93.6% to 99.6%) for malignant melanoma in women. Trends in 3-year survival of the five most common tumour sites in men showed a significant increase for lung and oropharyngeal cancer. In women, continuously rising survival trends were observed for breast cancer. Survival of cancer patients in the Augsburg study region was largely concordant with the situation in Germany as a whole, while incidence showed slight deviations in some cancer sites. Regional evaluations on cancer survival are a valuable instrument for identifying deficits and determining advances in oncological health management.

Trends in cancer incidence and survival in the Augsburg study region-results from the Augsburg cancer registry.

BMJ open

medicine

To examine the prevalence of dehydration without diarrhoea among admitted children aged 1-59 months and to describe fluid management practices in such cases. A multisite observational study that used routine in-patient data collected prospectively between October 2013 and December 2018. Study conducted in 13 county referral hospitals in Kenya. Children aged 1-59 months with admission or discharge diagnosis of dehydration but had no diarrhoea as a symptom or diagnosis. Children aged <28 days and those with severe acute malnutrition were excluded. The prevalence of dehydration in children without diarrhoea was 3.0% (2019/68 204) and comprised 15.9% (2019/12 702) of all dehydration cases. Only 55.8% (1127/2019) of affected children received either oral or intravenous fluid therapy. Where fluid treatment was given, the volumes, type of fluid, duration of fluid therapy and route of administration were similar to those used in the treatment of dehydration secondary to diarrhoea. Pneumonia (1021/2019, 50.6%) and malaria (715/2019, 35.4%) were the two most common comorbid diagnoses. Overall case fatality in the study population was 12.9% (260/2019). Sixteen per cent of children hospitalised with dehydration do not have diarrhoea but other common illnesses. Two-fifths do not receive fluid therapy; a regimen similar to that used in diarrhoeal cases is used in cases where fluid is administered. Efforts to promote compliance with guidance in routine clinical settings should recognise special circumstances where guidelines do not apply, and further studies on appropriate management for dehydration in the absence of diarrhoea are required.

Prevalence and fluid management of dehydration in children without diarrhoea admitted to Kenyan hospitals: a multisite observational study.

BMJ open

medicine

Suicidality among sexual minority adolescents has generated worldwide concern in recent decades, and previous Western studies have demonstrated that sexual minority status is associated with adolescent suicidality. However, whether this association exists in Chinese adolescents remains largely unknown. This study aimed to estimate the associations between sexual minority status and suicidal behaviour among Chinese adolescents. Cross-sectional survey. A total of 506 high schools in 7 provinces of China. A total of 150 822 students in grades 7-12 who completed the questionnaires (response rate of 95.9%) were included. Suicidal ideation and suicide attempts were used to measure suicidal behaviour, and sexual attraction (opposite sex, same sex or both sex) was used as a measure for sexual minority status. Of the 150 822 adolescents analysed, 4.1% self-reported as sexual minorities and 17.3% were unsure. Compared with heterosexual and unsure adolescents, same-sex romantic attraction (SSA) and both-sex romantic attraction (BSA) adolescents reported a higher prevalence of past-year suicidal ideation (SSA: 21.6% for males and 30.4% for females; BSA: 34.7% for males and 42.3% for females) and suicide attempts (SSA: 6.9% for males and 8.9% for females; BSA: 12.2% for males and 10.9% for females). After adjustment for covariates, SSA and BSA adolescents were more likely to have past-year suicidal ideation and suicide attempts than their heterosexual and unsure peers. BSA adolescents reported the highest risk of suicidal ideation (males: adjusted OR (AOR) 2.42, 95% CI 2.03 to 2.88; females: AOR 2.61, 95% CI 2.41 to 2.82) and suicide attempts (males: AOR 3.83, 95% CI 2.85 to 5.14; females: AOR 2.59, 95% CI 2.19 to 3.06). Our study suggested that Chinese sexual minority adolescents were at increased risk of suicidality, and those with BSA had an especially high risk in this population. These findings emphasised the urgent need to develop targeted interventions to effectively address suicide-related problems among Chinese sexual minority adolescents.

Sexual minority status and suicidal behaviour among Chinese adolescents: a nationally representative cross-sectional study.

BMJ open

medicine

To determine whether there is a relation between statin utilisation and coronary heart disease (CHD) mortality in populations with different levels of coronary risk, and whether the relation changes over time. Ecological study using national databases of dispensed medicines and mortality rates. Western European countries with similar public health systems. Population CHD mortality rates (rate/100,000) as a proxy for population coronary risk level, and statin utilisation expressed as Defined Daily Dose per one Thousand Inhabitants per Day (DDD/TID), in each country, for each year between 2000 and 2012. Spearman's correlation coefficients between CHD mortality and statin utilisation were calculated. Linear regression analysis was used to assess the relation between changes in CHD mortality and statin utilisation over the years. 12 countries were included in the study. There was a wide range of CHD mortality reduction between the years 2000 and 2012 (from 25.9% in Italy to 57.9% in Denmark) and statin utilisation increase (from 121% in Belgium to 1263% in Denmark). No statistically significant relations were found between CHD mortality rates and statin utilisation, nor between changes in CHD and changes in statin utilisation in the countries over the years 2000 and 2012. Among the Western European countries studied, the large increase in statin utilisation between 2000 and 2012 was not associated with CHD mortality, nor with its rate of change over the years. Factors different from the individual coronary risk, such as population ageing, health authority programmes, guidelines, media attention and pharmaceutical industry marketing, may have influenced the large increase in statin utilisation. These need to be re-examined with a greater emphasis on prevention strategies.

Time trends in statin utilisation and coronary mortality in Western European countries.

BMJ open

medicine

The quality of ambulatory care in Switzerland is widely unknown. Therefore, this study aimed to evaluate the recently proposed quality indicators (QIs) based on a nationwide healthcare claims database and determine their association with the risk of subsequent hospitalisation at patient-level. Retrospective cohort study. Inpatient and outpatient claims data of a large health insurance in Switzerland covering all regions and population strata. 520 693 patients continuously insured during 2015 and 2016. A total of 24 QIs were obtained by adapting the existing instruments to the Swiss national context and measuring at patient-level. The association between each QI and hospitalisation in the subsequent year was assessed using multiple logistic regression models. The proportion of patients with good adherence to QIs was high for the secondary prevention of diabetes and myocardial infarction (glycated haemoglobin (HbA1c) control, 89%; aspirin use, 94%) but relatively low for polypharmacy (53%) or using potentially inappropriate medications (PIMs) in the elderly (PIM, 33%). Diabetes-related indicators such as the HbA1c control were significantly associated with a lower risk of hospitalisation (OR, 0.87; 95% CI, 0.80 to 0.95), whereas the occurrence of polypharmacy and PIM increased the risk of hospitalisation in the following year (OR, 1.57/1.08; 95% CI, 1.51 to 1.64/1.05 to 1.12). This is the first study to evaluate the recently presented QIs in Switzerland using nationwide real-life data. Our study suggests that the quality of healthcare, as measured by these QIs, varied. The majority of QIs, in particular QIs reflecting chronic care and medication use, are considered beneficial markers of healthcare quality as they were associated with reduced risk of hospitalisation in the subsequent year. Results from this large practical test on real-life data show the feasibility of these QIs and are beneficial in selecting the appropriate QIs for healthcare implementation in general practice.

Evidence-based quality indicators for primary healthcare in association with the risk of hospitalisation: a population-based cohort study in Switzerland.

BMJ open

medicine

(1) Assess the population-level probability of prenatal diagnosis and termination of pregnancy for fetal anomaly for four major congenital heart defects; (2) Examine, using population-based data, the relation between timing of (prenatal vs postnatal) diagnosis and risk of infant (ie, < 1 year) mortality for four major congenital heart defects (CHDs). Population-based cohort (the EPIdémiologie des CARDiopathies congénitales) study. Greater Paris area (Paris and its surrounding suburbs). Three hundred and fifty-four cases of four major CHDs, including functionally univentricular heart (FUH, N=132), d-transposition of great arteries (d-TGA, N=85), tetralogy of Fallot (TOF, N=60) and coarctation of aorta (CoA, N=77). Statistical analysis included the Mantel-Haenszel method and a test of homogeneity of risk ratios. Approximately 95% of FUH, more than two-thirds of d-TGA and TOF, and 40% of CoA were prenatally diagnosed. Overall, we did not find any statistically significant association between timing of (prenatal vs postnatal) diagnosis of CHD and risk of infant mortality (Mantel-Haenszel risk ratio 1.1, 95% CI 0.5 - 2.7); and the differences between the risk ratios of the association between prenatal diagnosis and infant mortality across the four CHDs was not statistically significant. These results imply that at least in the settings where specialised services are readily available, survival may no longer be the most relevant outcome, or the best criterion, for evaluating the impact of prenatal diagnosis on the outcome of CHD. The beneficial effects of prenatal diagnosis may be better sought by looking at more 'subtle' or long-term neurodevelopmental outcomes.

Impact of prenatal diagnosis on survival of newborns with four congenital heart defects: a prospective, population-based cohort study in France (the EPICARD Study).

BMJ open

medicine

To identify the research priorities for medical nutrition education worldwide. A 5-step stakeholder engagement process based on methodological guidelines for identifying research priorities in health. 277 individuals were identified as representatives for 30 different stakeholder organisations across 86 countries. The stakeholder organisations represented the views of medical educators, medical students, doctors, patients and researchers in medical education. Each stakeholder representative was asked to provide up to three research questions that should be deemed as a priority for medical nutrition education. Research questions were critically appraised for answerability, sustainability, effectiveness, potential for translation and potential to impact on disease burden. A blinded scoring system was used to rank the appraised questions, with higher scores indicating higher priority (range of scores possible 36-108). 37 submissions were received, of which 25 were unique research questions. Submitted questions received a range of scores from 62 to 106 points. The highest scoring questions focused on (1) increasing the confidence of medical students and doctors in providing nutrition care to patients, (2) clarifying the essential nutrition skills doctors should acquire, (3) understanding the effectiveness of doctors at influencing dietary behaviours and (4) improving medical students' attitudes towards the importance of nutrition. These research questions can be used to ensure future projects in medical nutrition education directly align with the needs and preferences of research stakeholders. Funders should consider these priorities in their commissioning of research.

Setting priorities for research in medical nutrition education: an international approach.

BMJ open

medicine

The creatine kinase circuit is central to the regulation of high-energy phosphate metabolism and the maintenance of cellular energy turnover. This circuit is fuelled by creatine, an amino acid derivative that can be obtained from a diet containing animal products, and by synthesis in the body de novo. A recent retrospective study conducted in a cohort of 287 pregnant women determined that maternal excreted levels of creatine may be associated with fetal growth. This prospective study aims to overcome some of the limitations associated with the previous study and thoroughly characterise creatine homeostasis throughout gestation in a low-risk pregnant population. This study is recruiting women with a singleton low-risk pregnancy who are attending Monash Health, in Melbourne, Australia. Maternal blood and urine samples, along with dietary surveys, are collected at five time points during pregnancy and then at delivery. Cord blood and placenta (including membranes and cord) are collected at birth. A biobank of tissue samples for future research is being established. Primary outcome measures will include creatine, creatine kinase and associated metabolites in antenatal bloods and urine, cord bloods and placenta, along with molecular analysis of the creatine transporter (SLC6A8) and synthesising enzymes L - arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT) in placental tissues. Secondary outcome measures include dietary protein intake over pregnancy and any associations with maternal creatine, pregnancy events and birth outcomes. Ethical approval was granted in August 2015 from Monash Health (Ref: 14140B) and Monash University (Ref: 7785). Study outcomes will be disseminated at international conferences and published in peer-reviewed scientific journals. ACTRN12618001558213; Pre-results.

Creatine and pregnancy outcomes, a prospective cohort study in low-risk pregnant women: study protocol.

BMJ open

medicine

Evaluating the efficacy of a laparoscopically guided, surgical transversus abdominis plane (TAP) and rectus sheath (RS) block in reducing analgesic consumption while improving functional outcomes in patients undergoing laparoscopic bariatric surgery. 150 patients Living with obesity undergoing elective laparoscopic Roux-En-Y gastric bypass for obesity will be recruited to this double-blinded, placebo-controlled randomised controlled trial from a Bariatric Centre of Excellence over a period of 6 months. Patients will be electronically randomised on a 1:1 basis to either an intervention or placebo group. Those on the intervention arm will receive a total of 60 mL 0.25% ropivacaine, divided into four injections: two for TAP and two for RS block under laparoscopic visualisation. The placebo arm will receive normal saline in the same manner. A standardised surgical and anaesthetic protocol will be followed, with care in adherence to the Enhanced Recovery after Bariatric Surgery guidelines. Demographic information and relevant medical history will be collected from the 150 patients enrolled in the study. Our primary efficacy endpoint is cumulative postoperative narcotic use. Secondary outcomes are peak expiratory flow, postoperative pain score and the 6 min walk test. Quality of recovery (QoR) will be assessed using a validated questionnaire (QoR-40). Statistical analysis will be conducted to assess differences within and between the two groups. The repeated measures will be analysed by a mixed modelling approach and results reported through publication. Ethics approval was obtained (20170749-01H) through our institutional research ethics board (Ottawa Health Science Network Research Ethics Board) and the study results, regardless of the outcome, will be reported in a manuscript submitted for a medical/surgical journal. Pre-results NCT03367728.

Randomised, double-blinded, placebo-controlled trial to investigate the role of laparoscopic transversus abdominis plane block in gastric bypass surgery: a study protocol.

BMJ open

medicine

CD8+ T cell immune monitoring aims at measuring the size and functions of antigen-specific CD8+ T cell populations, thereby providing insights into cell-mediated immunity operational in a test subject. The selection of peptides for ex vivo CD8+ T cell detection is critical because within a complex antigen exists a multitude of potential epitopes that can be presented by HLA class I molecules. Further complicating this task, there is HLA class I polygenism and polymorphism which predisposes CD8+ T cell responses towards individualized epitope recognition profiles. In this study, we compare the actual CD8+ T cell recognition of a well-characterized model antigen, human cytomegalovirus (HCMV) pp65 protein, with its anticipated epitope coverage. Due to the abundance of experimentally defined HLA-A * 02:01-restricted pp65 epitopes, and because in silico epitope predictions are most advanced for HLA-A * 02:01, we elected to focus on subjects expressing this allele. In each test subject, every possible CD8+ T cell epitope was systematically covered testing 553 individual peptides that walk the sequence of pp65 in steps of single amino acids. Highly individualized CD8+ T cell response profiles with aleatory epitope recognition patterns were observed. No correlation was found between epitopes' ranking on the prediction scale and their actual immune dominance. Collectively, these data suggest that accurate CD8+ T cell immune monitoring may necessitate reliance on agnostic mega peptide pools, or brute force mapping, rather than electing individual peptides as representative epitopes for tetramer and other multimer labeling of surface antigen receptors.

Discordance Between the Predicted Versus the Actually Recognized CD8+ T Cell Epitopes of HCMV pp65 Antigen and Aleatory Epitope Dominance.

Frontiers in immunology

immunology

Natural killer (NK) cells are cytotoxic innate lymphocytes that are well-known for their ability to kill infected or malignant cells. Beyond their roles in tumor surveillance and anti-pathogen defense, more recent studies have highlighted key roles for NK cells in a broad range of biological processes, including metabolic homeostasis, immunomodulation of T cells, contact hypersensitivity, and pregnancy. Consistent with the breadth and diversity of these functions, it is now appreciated that NK cells are a heterogeneous population, comprised of specialized and sometimes tissue-specific subsets with distinct phenotypes and effector functions. Indeed, in addition to the conventional NK cells (cNKs) that are abundant and have been well-studied in the blood and spleen, distinct subsets of tissue-resident NK cells (trNKs) and "helper" Group 1 innate lymphoid cells (ILC1s) have now been described in multiple organs and tissues, including the liver, uterus, thymus, adipose tissue, and skin, among others. The cNK, trNK, and/or helper ILC1 populations that co-exist in these various tissues exhibit both common and distinct developmental requirements, suggesting that a combination of lineage-, subset-, and tissue-specific differentiation processes may contribute to the unique functional properties of these various populations. Here, we provide an overview of the transcriptional regulatory pathways known to instruct the development and differentiation of cNK, trNK, and helper ILC1 populations in specific tissues in mice.

Transcriptional Regulation of Mouse Tissue-Resident Natural Killer Cell Development.

Frontiers in immunology

immunology

Conjunctival epithelium forms a barrier between the ocular surface microbial flora and the ocular mucosa. In addition to secreting gel-forming mucins, goblet cells, located in the conjunctival epithelium, help maintain local immune homeostasis by secreting active TGFβ2 and promoting tolerogenic phenotype of dendritic cells in the vicinity. Although dendritic cell subsets, characteristic of mucosal tissues, are found in the conjunctiva, previous studies provided limited information about their location within the tissue. In this study, we examine immunostained conjunctiva explants to determine the location of CD11c-positive dendritic cells in the context of MUC5AC-positive goblet cells. Considering that conjunctival goblet cells are responsive to signaling induced by pathogen recognition receptors, we also assess if their responses to microbial product, flagellin, can contribute to the disruption of ocular mucosal homeostasis that promotes activation of dendritic cells and results in chronic ocular surface inflammation. We find that dendritic cells in the conjunctiva with an increased microbial colonization are located adjacent to goblet cells. While their cell bodies in the stromal layer are immediately below the epithelial layer, several extensions of dendritic cells are projected across the epithelium towards the ocular surface. Such trans-epithelial dendrites are not detectable in healthy ocular mucosa. In response to topically applied flagellin, increased proportion of CD11c-positive cells in the conjunctiva strongly express MHC class II relative to the untreated conjunctiva. This change is accompanied by reduced immunoreactivity to TGFβ-activating Thrombospondin-1 in the conjunctival epithelium. These findings are supported by in vitro observations in primary cultures of goblet cells that respond to the TLR5 stimulation with an increased expression of IL-6 and reduced level of active TGFβ. The observed changes in the conjunctiva after flagellin application correspond with the development of clinical signs of chronic ocular mucosal inflammation including corneal epitheliopathy. Collectively, these findings demonstrate the ability of ocular mucosal dendritic cells to extend trans-epithelial dendrites in response to increased microbial colonization at the ocular surface. Moreover, this study provides key insight into how goblet cell responses to microbial stimuli may contribute to the disruption of ocular mucosal homeostasis and chronic ocular mucosal inflammation.

Conjunctival Goblet Cell Responses to TLR5 Engagement Promote Activation of Local Antigen-Presenting Cells.

Frontiers in immunology

immunology

Myeloid cells are a major heterogeneous cell population in the tumor immune microenvironment (TIME). Imbalance of myeloid response remains a major obstacle to a favorable prognosis and successful immune therapy. Therefore, we aimed to construct a risk model to evaluate the myeloid contexture, which may facilitate the prediction of prognosis and immune infiltration in patients with head and neck squamous cell carcinoma (HNSCC). In our study, six myeloid signature genes (including CCL13 , CCR7 , CD276 , IL1B , LYVE1 and VEGFC ) analyzed from 52 differentially expressed myeloid signature genes were finally pooled to establish a prognostic risk model, termed as myeloid gene score (MGS) in a training cohort and validated in a test cohort and an independent external cohort. Furthermore, based on the MGS subgroups, we were able to effectively identify patients with a poor prognosis, aggressive clinical parameters, immune cell infiltration status and immunotherapy response. Thus, MGS may serve as an effective prognostic signature and predictive indicator for immunotherapy response in patients with HNSCC.

Comprehensive Analysis of Myeloid Signature Genes in Head and Neck Squamous Cell Carcinoma to Predict the Prognosis and Immune Infiltration.

Frontiers in immunology

immunology

Dramatic progress in the outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT) from alternative sources in pediatric patients has been registered over the past decade, providing a chance to cure children and adolescents in need of a transplant. Despite these advances, transplant-related mortality due to infectious complications remains a major problem, principally reflecting the inability of the depressed host immune system to limit infection replication and dissemination. In addition, development of multiple infections, a common occurrence after high-risk allo-HSCT, has important implications for overall survival. Prophylactic and preemptive pharmacotherapy is limited by toxicity and, to some extent, by lack of efficacy in breakthrough infections. T-cell reconstitution is a key requirement for effective infection control after HSCT. Consequently, T-cell immunotherapeutic strategies to boost pathogen-specific immunity may complement or represent an alternative to drug treatments. Pioneering proof of principle studies demonstrated that the administration of donor-derived T cells directed to human herpesviruses, on the basis of viral DNA monitoring, could effectively restore specific immunity and confer protection against viral infections. Since then, the field has evolved with implementation of techniques able to hasten production, allow for selection of specific cell subsets, and target multiple pathogens. This review provides a brief overview of current cellular therapeutic strategies to prevent or treat pathogen-related complications after HSCT, research carried out to increase efficacy and safety, including T-cell production for treatment of infections in patients with virus-naïve donors, results from clinical trials, and future developments to widen adoptive T-cell therapy access in the HSCT setting.

Harnessing T Cells to Control Infections After Allogeneic Hematopoietic Stem Cell Transplantation.

Frontiers in immunology

immunology

Emerging evidence indicates that gut dysbiosis may play a regulatory role in the onset and progression of Huntington's disease (HD). However, any alterations in the fecal microbiome of HD patients and its relation to the host cytokine response remain unknown. The present study investigated alterations and host cytokine responses in patients with HD. We enrolled 33 HD patients and 33 sex- and age- matched healthy controls. Fecal microbiota communities were determined through 16S ribosomal DNA gene sequencing, from which we analyzed fecal microbial richness, evenness, structure, and differential abundance of individual taxa between HD patients and healthy controls. HD patients were evaluated for their clinical characteristics, and the relationships of fecal microbiota with these clinical characteristics were analyzed. Plasma concentrations of interferon gamma (IFN-γ), interleukin 1 beta (IL-1β), IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and tumor necrosis factor alpha were measured by Meso Scale Discovery (MSD) assays, and relationships between microbiota and cytokine levels were analyzed in the HD group. HD patients showed increased α-diversity (richness), β-diversity (structure), and altered relative abundances of several taxa compared to those in healthy controls. HD-associated clinical characteristics correlated with the abundances of components of fecal microbiota at the genus level. Genus Intestinimonas was correlated with total functional capacity scores and IL-4 levels. Our present study also revealed that genus Bilophila were negatively correlated with proinflammatory IL-6 levels. Taken together, our present study represents the first to demonstrate alterations in fecal microbiota and inflammatory cytokine responses in HD patients. Further elucidation of interactions between microbial and host immune responses may help to better understand the pathogenesis of HD.

Altered Gut Microbiota Related to Inflammatory Responses in Patients With Huntington's Disease.

Frontiers in immunology

immunology

Accumulation of somatic hypermutation (SHM) is the primary mechanism to enhance the binding affinity of antibodies to antigens in vivo . However, the structural basis of the effects of many SHMs remains elusive. Here, we integrated atomistic molecular dynamics (MD) simulation and data mining to build a high-throughput structural bioinformatics pipeline to study the effects of individual and combination SHMs on antibody conformation, flexibility, stability, and affinity. By applying this pipeline, we characterized a common mechanism of modulation of heavy-light pairing orientation by frequent SHMs at framework positions 39 H , 91 H , 38 L , and 87 L through disruption of a conserved hydrogen-bond network. Q39L H alone and in combination with light chain framework 4 (FWR4 L ) insertions further modulated the elbow angle between variable and constant domains of many antibodies, resulting in improved binding affinity for a subset of anti-HIV-1 antibodies. Q39L H also alleviated aggregation induced by FWR4 L insertion, suggesting remote epistasis between these SHMs. Altogether, this study provides tools and insights for understanding antibody affinity maturation and for engineering functionally improved antibodies.

Structural Basis of Antibody Conformation and Stability Modulation by Framework Somatic Hypermutation.

Frontiers in immunology

immunology

Neutrophils are key pathogen exterminators of the innate immune system endowed with oxidative and non-oxidative defense mechanisms. More recently, a more complex role for neutrophils as decision shaping cells that instruct other leukocytes to fine-tune innate and adaptive immune responses has come into view. Under homeostatic conditions, neutrophils are short-lived cells that are continuously released from the bone marrow. Their development starts with undifferentiated hematopoietic stem cells that pass through different immature subtypes to eventually become fully equipped, mature neutrophils capable of launching fast and robust immune responses. During severe (systemic) inflammation, there is an increased need for neutrophils. The hematopoietic system rapidly adapts to this increased demand by switching from steady-state blood cell production to emergency granulopoiesis. During emergency granulopoiesis, the de novo production of neutrophils by the bone marrow and at extramedullary sites is augmented, while additional mature neutrophils are rapidly released from the marginated pools. Although neutrophils are indispensable for host protection against microorganisms, excessive activation causes tissue damage in neutrophil-rich diseases. Therefore, tight regulation of neutrophil homeostasis is imperative. In this review, we discuss the kinetics of neutrophil ontogenesis in homeostatic conditions and during emergency myelopoiesis and provide an overview of the different molecular players involved in this regulation. We substantiate this review with the example of an autoinflammatory disease, i.e. systemic juvenile idiopathic arthritis.

Neutrophil Homeostasis and Emergency Granulopoiesis: The Example of Systemic Juvenile Idiopathic Arthritis.

Frontiers in immunology

immunology

Despite increasing evidence to indicate that long non-coding RNAs (lncRNAs) are novel regulators of immunity, there has been no systematic attempt to identify and characterize the lncRNAs whose expression is changed following the induction of the innate immune response. To address this issue, we have employed next-generation sequencing data to determine the changes in the lncRNA profile in four human (monocytes, macrophages, epithelium, and chondrocytes) and four mouse cell types (RAW 264.7 macrophages, bone marrow-derived macrophages, peritoneal macrophages, and splenic dendritic cells) following exposure to the pro-inflammatory mediators, lipopolysaccharides (LPS), or interleukin-1β. We show differential expression of 204 human and 210 mouse lncRNAs, with positional analysis demonstrating correlation with immune-related genes. These lncRNAs are predominantly cell-type specific, composed of large regions of repeat sequences, and show poor evolutionary conservation. Comparison within the human and mouse sequences showed less than 1% sequence conservation, although we identified multiple conserved motifs. Of the 204 human lncRNAs, 21 overlapped with syntenic mouse lncRNAs, of which five were differentially expressed in both species. Among these syntenic lncRNA was IL7-AS (antisense), which was induced in multiple cell types and shown to regulate the production of the pro-inflammatory mediator interleukin-6 in both human and mouse cells. In summary, we have identified and characterized those lncRNAs that are differentially expressed following activation of the human and mouse innate immune responses and believe that these catalogs will provide the foundation for the future analysis of the role of lncRNAs in immune and inflammatory responses.

Catalog of Differentially Expressed Long Non-Coding RNA following Activation of Human and Mouse Innate Immune Response.

Frontiers in immunology

immunology

The number of diabetic patients in Europe and world-wide is growing. Diabetes confers a 2-fold higher risk for vascular disease. Lack of insulin production (Type 1 diabetes, T1D) or lack of insulin responsiveness (Type 2 diabetes, T2D) causes systemic metabolic changes such as hyperglycemia (HG) which contribute to the pathology of diabetes. Monocytes and macrophages are key innate immune cells that control inflammatory reactions associated with diabetic vascular complications. Inflammatory programming of macrophages is regulated and maintained by epigenetic mechanisms, in particular histone modifications. The aim of our study was to identify the epigenetic mechanisms involved in the hyperglycemia-mediated macrophage activation. Using Affymetrix microarray profiling and RT-qPCR we identified that hyperglycemia increased the expression of S100A9 and S100A12 in primary human macrophages. Expression of S100A12 was sustained after glucose levels were normalized. Glucose augmented the response of macrophages to Toll-like receptor (TLR)-ligands Palmatic acid (PA) and Lipopolysaccharide (LPS) i.e., pro-inflammatory stimulation. The abundance of activating histone Histone 3 Lysine 4 methylation marks (H3K4me1, H3K4me3) and general acetylation on histone 3 (AceH3) with the promoters of these genes was analyzed by chromatin immunoprecipitation. Hyperglycemia increased acetylation of histones bound to the promoters of S100A9 and S100A12 in M1 macrophages. In contrast, hyperglycemia caused a reduction in total H3 which correlated with the increased expression of both S100 genes. The inhibition of histone methyltransferases SET domain-containing protein (SET)7/9 and SET and MYND domain-containing protein (SMYD)3 showed that these specifically regulated S100A12 expression. We conclude that hyperglycemia upregulates expression of S100A9, S100A12 via epigenetic regulation and induces an activating histone code on the respective gene promoters in M1 macrophages. Mechanistically, this regulation relies on action of histone methyltransferases SMYD3 and SET7/9. The results define an important role for epigenetic regulation in macrophage mediated inflammation in diabetic conditions.

Epigenetic Regulation of S100A9 and S100A12 Expression in Monocyte-Macrophage System in Hyperglycemic Conditions.

Frontiers in immunology

immunology

Human CD52 is a small glycopeptide (12 amino acid residues) with one N- linked glycosylation site at asparagine 3 (Asn3) and several potential O- glycosylation serine/threonine sites. Soluble CD52 is released from the surface of activated T cells and mediates immune suppression via its glycan moiety. In suppressing activated T cells, it first sequesters the pro-inflammatory high mobility group Box 1 (HMGB1) protein, which facilitates its binding to the inhibitory sialic acid-binding immunoglobulin-like lectin-10 (Siglec-10) receptor. We aimed to identify the features of CD52 glycan that underlie its bioactivity. Analysis of native CD52 purified from human spleen revealed extensive heterogeneity in N- glycosylation and multi-antennary sialylated N- glycans with abundant polyLacNAc extensions, together with mainly di-sialylated O- glycosylation type structures. Glycomic (porous graphitized carbon-ESI-MS/MS) and glycopeptide (C8-LC-ESI-MS) analysis of recombinant soluble human CD52-immunoglobulin Fc fusion proteins revealed that CD52 bioactivity was correlated with a high abundance of tetra-antennary α-2,3/6 sialylated N- glycans. Removal of α-2,3 sialylation abolished bioactivity, which was restored by re-sialylation with α-2,3 sialyltransferases. When glycoforms of CD52-Fc were fractionated by anion exchange MonoQ-GL chromatography, bioactive fractions displayed mainly tetra-antennary, α-2,3 sialylated N- glycan structures and a lower relative abundance of bisecting GlcNAc structures compared to non-bioactive fractions. In addition, O- glycan core type-2 di-sialylated structures at Ser12 were more abundant in bioactive CD52 fractions. Understanding the structural features of CD52 glycan required for its bioactivity will aid its development as an immunotherapeutic agent.

Specific Sialoforms Required for the Immune Suppressive Activity of Human Soluble CD52.

Frontiers in immunology

immunology

The peripheral nervous system consists of sensory circuits that respond to external and internal stimuli and effector circuits that adapt physiologic functions to environmental challenges. Identifying neurotransmitters and neuropeptides and the corresponding receptors on immune cells implies an essential role for the nervous system in regulating immune reactions. Vice versa, neurons express functional cytokine receptors to respond to inflammatory signals directly. Recent advances in single-cell and single-nuclei sequencing have provided an unprecedented depth in neuronal analysis and allowed to refine the classification of distinct neuronal subsets of the peripheral nervous system. Delineating the sensory and immunoregulatory capacity of different neuronal subsets could inform a better understanding of the response happening in tissues that coordinate physiologic functions, tissue homeostasis and immunity. Here, we summarize current subsets of peripheral neurons and discuss neuronal regulation of immune responses, focusing on neuro-immune interactions in the gastrointestinal tract. The nervous system as a central coordinator of immune reactions and tissue homeostasis may predispose for novel promising therapeutic approaches for a large variety of diseases including but not limited to chronic inflammation.

An Integrated View on Neuronal Subsets in the Peripheral Nervous System and Their Role in Immunoregulation.

Frontiers in immunology

immunology

The dynamic nature of the SIV population during disease progression in the SIV/macaque model of AIDS and the factors responsible for its behavior have not been documented, largely owing to the lack of sufficient spatial and temporal sampling of both viral and host data from SIV-infected animals. In this study, we detail Bayesian coalescent inference of the changing collective intra-host viral effective population size ( Ne ) from various tissues over the course of infection and its relationship with what we demonstrate is a continuously changing immune cell repertoire within the blood. Although the relative contribution of these factors varied among hosts and time points, the adaptive immune response best explained the overall periodic dynamic behavior of the effective virus population. Data exposing the nature of the relationship between the virus and immune cell populations revealed the plausibility of an eco-evolutionary mathematical model, which was able to mimic the large-scale oscillations in Ne through virus escape from relatively few, early immunodominant responses, followed by slower escape from several subdominant and weakened immune populations. The results of this study suggest that SIV diversity within the untreated host is governed by a predator-prey relationship, wherein differing phases of infection are the result of adaptation in response to varying immune responses. Previous investigations into viral population dynamics using sequence data have focused on single estimates of the effective viral population size ( Ne ) or point estimates over sparse sampling data to provide insight into the precise impact of immune selection on virus adaptive behavior. Herein, we describe the use of the coalescent phylogenetic frame- work to estimate the relative changes in Ne over time in order to quantify the relationship with empirical data on the dynamic immune composition of the host. This relationship has allowed us to expand on earlier simulations to build a predator-prey model that explains the deterministic behavior of the virus over the course of disease progression. We show that sequential viral adaptation can occur in response to phases of varying immune pressure, providing a broader picture of the viral response throughout the entire course of progression to AIDS.

Predator-Prey Dynamics of Intra-Host Simian Immunodeficiency Virus Evolution Within the Untreated Host.

Frontiers in immunology

immunology

Although various immunotherapies have exerted promising effects on cancer treatment, many patients with cancer continue to exhibit poor responses. Because of its negative regulatory effects on T cells and its biological functions related to immune and inflammatory responses, there has been considerable emphasis on a protein-coding gene named lymphocyte-activation gene 3 (LAG3). Recently, evidence demonstrated marked synergy in its targeted therapy with programmed death-1 and programmed death-1 ligand-1 (PD-1/PD-L1) blockade, and a variety of LAG3 targeted agents are in clinical trials, indicating the important role of LAG3 in immunotherapy. This mini-review discusses preclinical and clinical studies investigating PD-1 pathway blockade in combination with LAG3 inhibition as a potentially more effective immunotherapy strategy for further development in the clinic. This strategy might provide a new approach for the design of more effective and precise cancer immune checkpoint therapies.

Research Progress Concerning Dual Blockade of Lymphocyte-Activation Gene 3 and Programmed Death-1/Programmed Death-1 Ligand-1 Blockade in Cancer Immunotherapy: Preclinical and Clinical Evidence of This Potentially More Effective Immunotherapy Strategy.

Frontiers in immunology

immunology

Maternal RhD alloimmunization is an inflammatory response against protein antigens in fetal red blood cells (RBC). However, not all women become alloimmunized when exposed to RhD + fetal RBC. Thus, this study aimed to evaluate levels of inflammatory chemokines in RhD - pregnant women with erythrocyte alloimmunization. CXCL8, CXCL9, CCL5, and CXCL10 levels were determined from cell culture supernatants by flow cytometry in 46 (30 non-alloimmunized RhD - and 16 previously alloimmunized RhD - ) pregnant women. CXCL8 levels were significantly higher ( P < 0.004), and CXCL9 ( P < 0.008) and CXCL10 ( P < 0.003) levels were significantly lower in alloimmunized pregnant women. No significant difference in CCL5 levels was detected between the groups. Fetal RHD genotyping was performed in the alloimmunized RhD - group by real-time PCR. Anti-D alloantibody was detected in 10 mothers and anti-D and -C in six mothers. Twelve fetuses were RHD positive and four were RHD negative. Further studies of serum chemokines and placenta tissue could provide a better understanding of the cells involved in the pathogenesis of maternal erythrocyte alloimmunization.

High Levels of CXCL8 and Low Levels of CXCL9 and CXCL10 in Women with Maternal RhD Alloimmunization.

Frontiers in immunology

immunology

Clinically, immune cell function is correlated with pathogenesis of endometrial polyp (EP) and infertility of women of reproductive-age. However, the underlying immune cell hallmark in EP patients remains unclear. Here, we focused on analyzing circulating immune cells, and attempted to reveal the correlation between peripheral immune cell functional phenotypes and fertility in EP patients. Through comparison of circulating CD4 + /CD8 + T cells, NK cells, and γδ T cells between 64 EP patients and 68 healthy females, we found that γδ T cells, but not CD4 + /CD8 + T cells and NK cells, were immunologically correlated with conception rate and conception interval time. Specifically, total γδ T cells and the Vδ1 + PD1 + γδ T subpopulation decreased whereas the Vδ1/Vδ2 ratio increased in EP patients compared to healthy controls. Moreover, the patients with the higher Vδ1/Vδ2 ratio (median value equals 1.04) had a poorer fertility and longer interval time of conception (210 days versus 158 days for control). Meanwhile, higher Vδ1 + PD1 + γδ T cell proportion (median equals 15.7) was positively correlative with both higher conception rate and shortened median conception interval time (130 days for Vδ1 + PD1 high group versus 194 days for Vδ1 + PD1 low group). Notably, in healthy controls, both Vδ1/Vδ2 ratio and Vδ1 + PD1 + γδ T cell proportion correlated with pregnancy rate oppositely, comparing to EP patients. Together, our results suggested that imbalanced γδ T cell population occurred in EP patients, and that Vδ1/Vδ2 ratio and PD-1 expression of Vδ1 + γδ T cells could be potentially developed into valuable predictors for fertility in EP patients.

Circulating PD1+Vδ1+γδ T Cell Predicts Fertility in Endometrial Polyp Patients of Reproductive-Age.

Frontiers in immunology

immunology

Periodontitis increases the risk of nonalcoholic fatty liver disease (NAFLD); however, the underlying mechanisms are unclear. Here, we show that gut dysbiosis induced by oral administration of Porphyromonas gingivalis , a representative periodontopathic bacterium, is involved in the aggravation of NAFLD pathology. C57BL/6N mice were administered either vehicle, P. gingivalis , or Prevotella intermedia , another periodontopathic bacterium with weaker periodontal pathogenicity, followed by feeding on a choline-deficient, l-amino acid-defined, high-fat diet with 60 kcal% fat and 0.1% methionine (CDAHFD60). The gut microbial communities were analyzed by pyrosequencing the 16S ribosomal RNA genes. Metagenomic analysis was used to determine the relative abundance of the Kyoto Encyclopedia of Genes and Genomes pathways encoded in the gut microbiota. Serum metabolites were analyzed using nuclear magnetic resonance-based metabolomics coupled with multivariate statistical analyses. Hepatic gene expression profiles were analyzed via DNA microarray and quantitative polymerase chain reaction. CDAHFD60 feeding induced hepatic steatosis, and in combination with bacterial administration, it further aggravated NAFLD pathology, thereby increasing fibrosis. Gene expression analysis of liver samples revealed that genes involved in NAFLD pathology were perturbed, and the two bacteria induced distinct expression profiles. This might be due to quantitative and qualitative differences in the influx of bacterial products in the gut because the serum endotoxin levels, compositions of the gut microbiota, and serum metabolite profiles induced by the ingested P. intermedia and P. gingivalis were different. Swallowed periodontopathic bacteria aggravate NAFLD pathology, likely due to dysregulation of gene expression by inducing gut dysbiosis and subsequent influx of gut bacteria and/or bacterial products.

Oral Pathobiont-Induced Changes in Gut Microbiota Aggravate the Pathology of Nonalcoholic Fatty Liver Disease in Mice.

Frontiers in immunology

immunology

Cytotoxic T lymphocytes (CTLs) have been suggested to play an important role in controlling human immunodeficiency virus (HIV-1 or simply HIV) infection. HIV, due to its high mutation rate, can evade recognition of T cell responses by generating escape variants that cannot be recognized by HIV-specific CTLs. Although HIV escape from CTL responses has been well documented, factors contributing to the timing and the rate of viral escape from T cells have not been fully elucidated. Fitness costs associated with escape and magnitude of the epitope-specific T cell response are generally considered to be the key in determining timing of HIV escape. Several previous analyses generally ignored the kinetics of T cell responses in predicting viral escape by either considering constant or maximal T cell response; several studies also considered escape from different T cell responses to be independent. Here, we focus our analysis on data from two patients from a recent study with relatively frequent measurements of both virus sequences and HIV-specific T cell response to determine impact of CTL kinetics on viral escape. In contrast with our expectation, we found that including temporal dynamics of epitope-specific T cell response did not improve the quality of fit of different models to escape data. We also found that for well-sampled escape data, the estimates of the model parameters including T cell killing efficacy did not strongly depend on the underlying model for escapes: models assuming independent, sequential, or concurrent escapes from multiple CTL responses gave similar estimates for CTL killing efficacy. Interestingly, the model assuming sequential escapes (i.e., escapes occurring along a defined pathway) was unable to accurately describe data on escapes occurring rapidly within a short-time window, suggesting that some of model assumptions must be violated for such escapes. Our results thus suggest that the current sparse measurements of temporal CTL dynamics in blood bear little quantitative information to improve predictions of HIV escape kinetics. More frequent measurements using more sensitive techniques and sampling in secondary lymphoid tissues may allow to better understand whether and how CTL kinetics impacts viral escape.

Kinetics of HIV-Specific CTL Responses Plays a Minimal Role in Determining HIV Escape Dynamics.

Frontiers in immunology

immunology

The anti-inflammatory effect of an α7nAChR agonist, PNU-282987, has previously been explored in the context of inflammatory disease. However, the effects of PNU-282987 on type 2 innate lymphoid cells (ILC2s)-mediated allergic airway inflammation has not yet been established. To determine the effects of PNU-282987 on the function of ILC2s in the context of IL-33- or Alternaria Alternata (AA)- induced airway inflammation. PNU-282987 was administered to mice that received recombinant IL-33 or AA intranasal challenges. Lung histological analysis and flow cytometry were performed to determine airway inflammation and the infiltration and activation of ILC2s. The previously published α7nAChR agonist GTS-21 was employed as a comparable reagent. ILC2s were isolated from murine lung tissue and cultured in vitro in the presence of IL-33, IL-2, and IL-7 with/without either PNU-282987 or GTS-21. The expression of the transcription factors GATA3, IKK, and NF-κB were also determined. PNU-282987 and GTS-21 significantly reduced goblet cell hyperplasia in the airway, eosinophil infiltration, and ILC2s numbers in BALF, following IL-33 or AA challenge. In vitro IL-33 stimulation of isolated lung ILC2s showed a reduction of GATA3 and Ki67 in response to PNU-282987 or GTS-21 treatments. There was a significant reduction in IKK and NF-κB phosphorylation in the PNU-282987-treated group when compared to the GTS-21-treated ILC2s. PNU-282987 inhibits ILC2-associated airway inflammation, where its effects were comparable to that of GTS-21.

A Selective α7 Nicotinic Acetylcholine Receptor Agonist, PNU-282987, Attenuates ILC2s Activation and Alternaria-Induced Airway Inflammation.

Frontiers in immunology

immunology

Bacterial cells in their natural environments encounter rapid and large changes in external osmolality. For instance, enteric bacteria such as Escherichia coli experience a rapid decrease when they exit from host intestines. Changes in osmolality alter the mechanical load on the cell envelope, and previous studies have shown that large osmotic shocks can slow down bacterial growth and impact cytoplasmic diffusion. However, it remains unclear how cells maintain envelope integrity and regulate envelope synthesis in response to osmotic shocks. In this study, we developed an agarose pad-based protocol to assay envelope stiffness by measuring population-averaged cell length before and after a hyperosmotic shock. Pad-based measurements exhibited an apparently larger length change compared with single-cell dynamics in a microfluidic device, which we found was quantitatively explained by a transient increase in division rate after the shock. Inhibiting cell division led to consistent measurements between agarose pad-based and microfluidic measurements. Directly after hyperosmotic shock, FtsZ concentration and Z-ring intensity increased, and the rate of septum constriction increased. These findings establish an agarose pad-based protocol for quantifying cell envelope stiffness, and demonstrate that mechanical perturbations can have profound effects on bacterial physiology.

Hyperosmotic Shock Transiently Accelerates Constriction Rate in Escherichia coli.

Frontiers in microbiology

microbiology

Iris yellow spot, caused by Iris yellow spot orthotospovirus (IYSV) (Genus: Orthotospovirus , Family: Tospoviridae ), is an important disease of Allium spp. The complete N gene sequences of 142 IYSV isolates of curated sequence data from GenBank were used to determine the genetic diversity and evolutionary pattern. In silico restriction fragment length polymorphism (RFLP) analysis, codon-based maximum likelihood studies, genetic differentiation and gene flow within the populations of IYSV genotypes were investigated. Bayesian phylogenetic analysis was carried out to estimate the evolutionary rate. In silico RFLP analysis of N gene sequences categorized IYSV isolates into two major genotypes viz ., IYSV Netherlands (IYSV NL ; 55.63%), IYSV Brazil (IYSV BR ; 38.73%) and the rest fell in neither group [IYSV other (IYSV other ; 5.63%)]. Phylogenetic tree largely corroborated the results of RFLP analysis and the IYSV genotypes clustered into IYSV NL and IYSV BR genotypes. Genetic diversity test revealed IYSV other to be more diverse than IYSV NL and IYSV BR . IYSV NL and IYSV BR genotypes are under purifying selection and population expansion, whereas IYSV other showed decreasing population size and hence appear to be under balancing selection. IYSV BR is least differentiated from IYSV other compared to IYSV NL genotype based on nucleotide diversity. Three putative recombinant events were found in the N gene of IYSV isolates based on RDP analysis, however, RAT substantiated two among them. The marginal likelihood mean substitution rate was 5.08 × 10 -5 subs/site/year and 95% highest posterior density (HPD) substitution rate between 5.11 × 10 -5 and 5.06 × 10 -5 . Findings suggest that IYSV continues to evolve using population expansion strategies. The substitution rates identified are similar to other plant RNA viruses.

Viruses Without Borders: Global Analysis of the Population Structure, Haplotype Distribution, and Evolutionary Pattern of Iris Yellow Spot Orthotospovirus (Family Tospoviridae, Genus Orthotospovirus).

Frontiers in microbiology

microbiology

Antimicrobial resistance (AMR) is one of the most significant threats to global public health. As antibiotic failure is increasing, phages are gradually becoming important agents in the post-antibiotic era. In this study, the therapeutic effects and safety of kpssk3, a previously isolated phage infecting carbapenem-resistant hypermucoviscous Klebsiella pneumoniae (CR-HMKP), were evaluated in a mouse model of systemic CR-HMKP infection. The therapeutic efficacy experiment showed that intraperitoneal injection with a single dose of phage kpssk3 (1 × 10 7 PFU/mouse) 3 h post infection protected 100% of BALB/c mice against bacteremia induced by intraperitoneal challenge with a 2 × LD 100 dose of NY03, a CR-HMKP clinical isolate. In addition, mice were treated with antibiotics from three classes (polymyxin B, tigecycline, and ceftazidime/avibactam plus aztreonam), and the 7 days survival rates of the treated mice were 20, 20, and 90%, respectively. The safety test consisted of 2 parts: determining the cytotoxicity of kpssk3 and evaluating the short- and long-term impacts of phage therapy on the mouse gut microbiota. Phage kpssk3 was shown to not be cytotoxic to mammalian cells in vitro or in vivo . Fecal samples were collected from the phage-treated mice at 3 time points before (0 day) and after (3 and 10 days) phage therapy to study the change in the gut microbiome via high-throughput 16S rDNA sequence analysis, which revealed no notable alterations in the gut microbiota except for decreases in the Chao1 and ACE indexes.

Safety and Efficacy of a Phage, kpssk3, in an in vivo Model of Carbapenem-Resistant Hypermucoviscous Klebsiella pneumoniae Bacteremia.

Frontiers in microbiology

microbiology

While modern developments in agriculture have allowed for increases in crop yields and rapid human population growth, they have also drastically altered biogeochemical cycles, including the biotransformation of nitrogen. Denitrification is a critical process performed by bacteria and fungi that removes nitrate in surface waters, thereby serving as a potential natural remediation strategy. We previously reported that constant inundation resulted in a coupling of denitrification gene abundances with denitrification rates in sediments, but these relationships were not maintained in periodically-inundated or non-inundated environments. In this study, we utilized Illumina next-generation sequencing to further evaluate how the microbial community responds to these hydrologic regimes and how this community is related to denitrification rates at three sites along a creek in an agricultural watershed over 2 years. The hydrologic connectivity of the sampling location had a significantly greater influence on the denitrification rate ( P = 0.010), denitrification gene abundances ( P < 0.001), and the prokaryotic community ( P < 0.001), than did other spatiotemporal factors (e.g., creek sample site or sample month) within the same year. However, annual variability among denitrification rates was also observed ( P < 0.001). Furthermore, the denitrification rate was significantly positively correlated with water nitrate concentration (Spearman's ρ = 0.56, P < 0.0001), denitrification gene abundances (ρ = 0.23-0.47, P ≤ 0.006), and the abundances of members of the families Burkholderiaceae, Anaerolinaceae, Microbacteriaceae, Acidimicrobineae incertae sedis, Cytophagaceae , and Hyphomicrobiaceae (ρ = 0.17-0.25, P ≤ 0.041). Prokaryotic community composition accounted for the least amount of variation in denitrification rates (22%), while the collective influence of spatiotemporal factors and gene abundances accounted for 37%, with 40% of the variation related to interactions among all parameters. Results of this study suggest that the hydrologic connectivity at each location had a greater effect on the prokaryotic community than did spatiotemporal differences, where inundation is associated with shifts favoring increased denitrification potential. We further establish that while complex interactions among the prokaryotic community influence denitrification, the link between hydrologic connectivity, microbial community composition, and genetic potential for biogeochemical cycling is a promising avenue to explore hydrologic remediation strategies such as periodic flooding.

Increased Denitrification Rates Associated with Shifts in Prokaryotic Community Composition Caused by Varying Hydrologic Connectivity.

Frontiers in microbiology

microbiology

The time-to-result for culture-based microorganism recovery and phenotypic antimicrobial susceptibility testing necessitates initial use of empiric (frequently broad-spectrum) antimicrobial therapy. If the empiric therapy is not optimal, this can lead to adverse patient outcomes and contribute to increasing antibiotic resistance in pathogens. New, more rapid technologies are emerging to meet this need. Many of these are based on identifying resistance genes, rather than directly assaying resistance phenotypes, and thus require interpretation to translate the genotype into treatment recommendations. These interpretations, like other parts of clinical diagnostic workflows, are likely to be increasingly automated in the future. We set out to evaluate the two major approaches that could be amenable to automation pipelines: rules-based methods and machine learning methods. The rules-based algorithm makes predictions based upon current, curated knowledge of Enterobacteriaceae resistance genes. The machine-learning algorithm predicts resistance and susceptibility based on a model built from a training set of variably resistant isolates. As our test set, we used whole genome sequence data from 78 clinical Enterobacteriaceae isolates, previously identified to represent a variety of phenotypes, from fully-susceptible to pan-resistant strains for the antibiotics tested. We tested three antibiotic resistance determinant databases for their utility in identifying the complete resistome for each isolate. The predictions of the rules-based and machine learning algorithms for these isolates were compared to results of phenotype-based diagnostics. The rules based and machine-learning predictions achieved agreement with standard-of-care phenotypic diagnostics of 89.0 and 90.3%, respectively, across twelve antibiotic agents from six major antibiotic classes. Several sources of disagreement between the algorithms were identified. Novel variants of known resistance factors and incomplete genome assembly confounded the rules-based algorithm, resulting in predictions based on gene family, rather than on knowledge of the specific variant found. Low-frequency resistance caused errors in the machine-learning algorithm because those genes were not seen or seen infrequently in the test set. We also identified an example of variability in the phenotype-based results that led to disagreement with both genotype-based methods. Genotype-based antimicrobial susceptibility testing shows great promise as a diagnostic tool, and we outline specific research goals to further refine this methodology.

Evaluation of Machine Learning and Rules-Based Approaches for Predicting Antimicrobial Resistance Profiles in Gram-negative Bacilli from Whole Genome Sequence Data.

Frontiers in microbiology

microbiology

While vegetation has intensively been surveyed on mountain summits, limited knowledge exists about the diversity and community structure of soil biota. Here, we study how climatic variables, vegetation, parent material, soil properties, and slope aspect affect the soil microbiome on 10 GLORIA (Global Observation Research Initiative in Alpine environments) mountain summits ranging from the lower alpine to the nival zone in Switzerland. At these summits we sampled soils from all four aspects and examined how the bacterial and fungal communities vary by using Illumina MiSeq sequencing. We found that mountain summit soils contain highly diverse microbial communities with a total of 10,406 bacterial and 6,291 fungal taxa. Bacterial α-diversity increased with increasing soil pH and decreased with increasing elevation, whereas fungal α-diversity did not change significantly. Soil pH was the strongest predictor for microbial β-diversity. Bacterial and fungal community structures exhibited a significant positive relationship with plant communities, indicating that summits with a more distinct plant composition also revealed more distinct microbial communities. The influence of elevation was stronger than aspect on the soil microbiome. Several microbial taxa responded to elevation and soil pH. Chloroflexi and Mucoromycota were significantly more abundant on summits at higher elevations, whereas the relative abundance of Basidiomycota and Agaricomycetes decreased with elevation. Most bacterial OTUs belonging to the phylum Acidobacteria were indicators for siliceous parent material and several OTUs belonging to the phylum Planctomycetes were associated with calcareous soils. The trends for fungi were less clear. Indicator OTUs belonging to the genera Mortierella and Naganishia showed a mixed response to parent material, demonstrating their ubiquitous and opportunistic behaviour in soils. Overall, fungal communities responded weakly to abiotic and biotic factors. In contrast, bacterial communities were strongly influenced by environmental changes suggesting they will be strongly affected by future climate change and associated temperature increase and an upward migration of vegetation. Our results provide the first insights into the soil microbiome of mountain summits in the European Alps that are shaped as a result of highly variable local environmental conditions and may help to predict responses of the soil biota to global climate change.

The Soil Microbiome of GLORIA Mountain Summits in the Swiss Alps.

Frontiers in microbiology

microbiology

Sub-acute ruminal acidosis (SARA) is a gastrointestinal functional disorder in livestock characterized by low rumen pH, which reduces rumen function, microbial diversity, host performance, and host immune function. Dietary management is used to prevent SARA, often with yeast supplementation as a pH buffer. Almost nothing is known about the effect of SARA or yeast supplementation on ruminal protozoal and fungal diversity, despite their roles in fiber degradation. Dairy cows were switched from a high-fiber to high-grain diet abruptly to induce SARA, with and without active dry yeast (ADY, Saccharomyces cerevisiae ) supplementation, and sampled from the rumen fluid, solids, and epimural fractions to determine microbial diversity using the protozoal 18S rRNA and the fungal ITS1 genes via Illumina MiSeq sequencing. Diet-induced SARA dramatically increased the number and abundance of rare fungal taxa, even in fluid fractions where total reads were very low, and reduced protozoal diversity. SARA selected for more lactic-acid utilizing taxa, and fewer fiber-degrading taxa. ADY treatment increased fungal richness (OTUs) but not diversity (Inverse Simpson, Shannon), but increased protozoal richness and diversity in some fractions. ADY treatment itself significantly ( P < 0.05) affected the abundance of numerous fungal genera as seen in the high-fiber diet: Lewia, Neocallimastix , and Phoma were increased, while Alternaria, Candida Orpinomyces , and Piromyces spp. were decreased. Likewise, for protozoa, ADY itself increased Isotricha intestinalis but decreased Entodinium furca spp. Multivariate analyses showed diet type was most significant in driving diversity, followed by yeast treatment, for AMOVA, ANOSIM, and weighted UniFrac. Diet, ADY, and location were all significant factors for fungi (PERMANOVA, P = 0.0001, P = 0.0452, P = 0.0068, Monte Carlo correction, respectively, and location was a significant factor ( P = 0.001, Monte Carlo correction) for protozoa. Diet-induced SARA shifts diversity of rumen fungi and protozoa and selects against fiber-degrading species. Supplementation with ADY mitigated this reduction in protozoa, presumptively by triggering microbial diversity shifts (as seen even in the high-fiber diet) that resulted in pH stabilization. ADY did not recover the initial community structure that was seen in pre-SARA conditions.

An Investigation into Rumen Fungal and Protozoal Diversity in Three Rumen Fractions, during High-Fiber or Grain-Induced Sub-Acute Ruminal Acidosis Conditions, with or without Active Dry Yeast Supplementation.

Frontiers in microbiology

microbiology

Intercropping influences the soil microbiota via litter and root exudate inputs, but the mechanisms by which root exudates mediate the soil microbial community and soil organic matter (SOM) are still unclear. In this study, we selected three aromatic plants ( Ocimum basilicum , Tr1; Satureja hortensis , Tr2; Ageratum houstonianum , Tr3) as intercrops that separately grew between rows of pear trees, and no plants were grown as the control in a pear orchard during the spring-summer season for 3 years. The soil from each plot was collected using a stainless-steel corer by five-point sampling between rows of pear trees. The bacterial and fungal communities of the different aromatic intercrops were analyzed by 16S and ITS rRNA gene amplicon sequencing; their functional profiles were predicted by PICRUSt and FUNGuild analyses. The root exudates of the aromatic plants were analyzed by a liquid chromatography-tandem mass spectrometry (LC-MS) system. Compared with the control treatment, all intercropping treatments with aromatic plants significantly increased SOM and soil water content and decreased pH values. The contents of total nitrogen and alkali-hydrolyzable nitrogen in Tr1 and Tr2 were higher than those in Tr3. In Tr3 soil, the relative content of saccharides increased little, whereas the changes in amine (increases) and alcohols (decreases) were rapid. Ageratum houstonianum intercropping decreased the microbial community diversity and significantly influenced the relative abundances of the dominant microbiota (Actinobacteria, Verrucomicrobia, Gemmatimonadetes, Cyanobacteria, Ascomycota, and Basidiomycota) at the phylum, class, and order levels, which increased the assemblage of functional groups (nitrite ammonification, nitrate ammonification, and ureolysis groups). Our study suggested that the main root exudates from aromatic plants shaped the microbial diversity, structure, and functional groups related to the N cycle during SOM mineralization and that intercropping with aromatic plants (especially basil and summer savory) increased N release in the orchard soil.

Intercropping With Aromatic Plants Increased the Soil Organic Matter Content and Changed the Microbial Community in a Pear Orchard.

Frontiers in microbiology

microbiology

The influence of water and nitrogen (N) management on wheat have been investigated, but studies on the impact of long-term interactive water and N management on microbial structure and function are limited. Soil chemical properties and plants determine the soil microbial communities whose functions involved in nutrient cycling may affect plant productivity. There is an urgent need to elucidate the underlying mechanisms to optimize these microbial communities for agricultural sustainability in the winter wheat production area of the North China Plain. We performed high-throughput sequencing and quantitative PCR of the 16S rRNA gene on soil from a 7-year-old stationary field experiment to investigate the response of bacterial communities and function to water and N management. It was observed that water and N management significantly influenced wheat growth, soil properties and bacterial diversity. N application caused a significant decrease in the number of operational taxonomic units (OTUs), and both Richness and Shannon diversity indices, in the absence of irrigation. Irrigation led to an increase in the relative abundance of Planctomycetes, Latescibacteria, Anaerolineae, and Chloroflexia. In addition, most bacterial taxa were correlated with soil and plant properties. Some functions related to carbohydrate transport, transcription, inorganic ion transport and lipid transport were enriched in irrigation treatment, while N enriched predicted functions related to amino acid transport and metabolism, signal transduction, and cell wall/membrane/envelope biogenesis. Understanding the impact of N application and irrigation on the structure and function of soil bacteria is important for developing strategies for sustainable wheat production. Therefore, concurrent irrigation and N application may improve wheat yield and help to maintain those ecosystem functions that are driven by the soil microbial community.

Bacterial Community Structure and Predicted Function in Wheat Soil From the North China Plain Are Closely Linked With Soil and Plant Characteristics After Seven Years of Irrigation and Nitrogen Application.

Frontiers in microbiology

microbiology

Bacterial brain abscesses (BAs) are difficult to treat with conventional antibiotics. Thus, the development of alternative therapeutic strategies for BAs is of high priority. Identifying the virulence determinants that contribute to BA formation induced by Staphylococcus aureus would improve the effectiveness of interventions for this disease. In this study, RT-qPCR was performed to compare the expression levels of 42 putative virulence determinants of S. aureus strains Newman and XQ during murine BA formation, ear colonization, and bacteremia. The alterations in the expression levels of 23 genes were further confirmed through specific TaqMan RT-qPCR. Eleven S. aureus genes that persistently upregulated expression levels during BA infection were identified, and their functions in BA formation were confirmed through isogenic mutant experiments. Bacterial loads and BA volumes in mice infected with isdA, isdC, lgt, hla , or spa deletion mutants and the hla / spa double mutant strain were lower than those in mice infected with the wild-type Newman strain. The therapeutic application of monoclonal antibodies against Hla and SpA decreased bacterial loads and BA volume in mice infected with Newman. This study provides insights into the virulence determinants that contribute to staphylococcal BA formation and a paradigm for antivirulence factor therapy against S. aureus infections.

Virulence Determinants Are Required for Brain Abscess Formation Through Staphylococcus aureus Infection and Are Potential Targets of Antivirulence Factor Therapy.

Frontiers in microbiology

microbiology

Chlamydia trachomatis is the major cause of infectious blindness and represents the most common bacterial sexually transmitted infection worldwide. Considering the potential side effects of antibiotic therapy and increasing threat of antibiotic resistance, alternative therapeutic strategies are needed. Previous studies showed that water filtered infrared A alone (wIRA) or in combination with visible light (wIRA/VIS) reduced C. trachomatis infectivity. Furthermore, wIRA/VIS irradiation led to secretion of pro-inflammatory cytokines similar to that observed upon C. trachomatis infection. We confirmed the results of previous studies, namely that cytokine secretion (IL-6, IL-8, and RANTES/CCL5) upon wIRA/VIS treatment, and the subsequent reduction of chlamydial infectivity, are independent of the addition of cycloheximide, a host protein synthesis inhibitor. Reproducible cytokine release upon irradiation indicated that cytokines might be involved in the anti-chlamydial mechanism of wIRA/VIS. This hypothesis was tested by inhibiting IL-6, IL-8, and RANTES secretion in C. trachomatis or mock-infected cells by gene silencing or pharmaceutical inhibition. Celastrol, a substance derived from Trypterygium wilfordii , used in traditional Chinese medicine and known for anti-cancer and anti-inflammatory effects, was used for IL-6 and IL-8 inhibition, while Maraviroc, a competitive CCR5 antagonist and anti-HIV drug, served as a RANTES/CCL5 inhibitor. HeLa cell cytotoxicity and impact on chlamydial morphology, size and inclusion number was evaluated upon increasing inhibitor concentration, and concentrations of 0.1 and 1 μM Celastrol and 10 and 20 μM Maraviroc were subsequently selected for irradiation experiments. Celastrol at any concentration reduced chlamydial infectivity, an effect only observed for 20 μM Maraviroc. Triple dose irradiation (24, 36, 40 hpi) significantly reduced chlamydial infectivity regardless of IL-6, IL-8, or RANTES/CCL5 gene silencing, Celastrol or Maraviroc treatment. Neither gene silencing nor pharmaceutical cytokine inhibition provoked the chlamydial stress response. The anti-chlamydial effect of wIRA/VIS is independent of cytokine inhibition under all conditions evaluated. Thus, factors other than host cell cytokines must be involved in the working mechanism of wIRA/VIS. This study gives a first insight into the working mechanism of wIRA/VIS in relation to an integral component of the host immune system and supports the potential of wIRA/VIS as a promising new tool for treatment in trachoma.

Water Filtered Infrared A and Visible Light (wIRA/VIS) Irradiation Reduces Chlamydia trachomatis Infectivity Independent of Targeted Cytokine Inhibition.

Frontiers in microbiology

microbiology

The use of bacteriophages as therapeutic agents is hindered by their narrow and specific host range, and by a lack of the knowledge concerning the molecular mechanism of receptor recognition. Two P2-like coliphages, named P88 and pro147, were induced from Escherichia coli strains K88 and DE147, respectively. A comparison of the genomes of these two and other P2-like coliphages obtained from GenBank showed that the tail fiber protein genes, which are the key genes for receptor recognition in other myoviridae phages, showed more diversity than the conserved lysin, replicase, and terminase genes. Firstly, replacing hypervariable region 2 (HR2: amino acids 716-746) of the tail fiber protein of P88 with that of pro147 changed the host range of P88. Then, replacing six amino acids in HR2 with the corresponding residues from pro147 altered the host range only in these mutants with changes at position 730 (leucine) and 744 (glutamic acid). Thus, we predicted that these amino acids are vital to establish the host range of P88. This study provided a vector of lysogenic bacteria that could be used to change or expand the phage host range of P88. These results illustrated that, in P2-like phage P88, the tail fiber protein determined the receptor recognition. Amino acids 716-746 and the amino acids at positions 730 and 744 were important for receptor recognition.

Inducible Prophage Mutant of Escherichia coli Can Lyse New Host and the Key Sites of Receptor Recognition Identification.

Frontiers in microbiology

microbiology

Microalgal species are potential resource of both biofuels and high-value metabolites, and their production is growth dependent. Growth parameters can be screened for the selection of novel microalgal species that produce molecules of interest. In this context our review confirms that, autotrophic and heterotrophic organisms have demonstrated a dual potential, namely the ability to produce lipids as well as value-added products (particularly carotenoids) under influence of various physico-chemical stresses on microalgae. Some species of microalgae can synthesize, besides some pigments, very-long-chain polyunsaturated fatty acids (VL-PUFA,>20C) such as docosahexaenoic acid and eicosapentaenoic acid, those have significant applications in food and health. Producing value-added by-products in addition to biofuels, fatty acid methyl esters (FAME), and lipids has the potential to improve microalgae-based biorefineries by employing either the autotrophic or the heterotrophic mode, which could be an offshoot of biotechnology. The review considers the potential of microalgae to produce a range of products and indicates future directions for developing suitable criteria for choosing novel isolates through bioprospecting large gene pool of microalga obtained from various habitats and climatic conditions.

A Review on the Assessment of Stress Conditions for Simultaneous Production of Microalgal Lipids and Carotenoids.

Frontiers in microbiology

microbiology

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is an urgent public health problem worldwide, and its rapid evolution in the clinical environment has been a major concern. A total of 99 CRKP isolates spreading in the intensive care unit (ICU) setting were included and subjected to whole-genome sequencing, and their sequence types (STs), serotype loci, and virulence determinants were screened based on genome data. The phylogenetic structure was reconstructed based on the core genome multilocus sequence typing method. Regions of recombination were assessed. Biofilm formation, serum resistance assays, and a Galleria mellonella infection model were used to evaluate strain virulence. A novel ST, designated ST4496, emerged in the ICU and spread for 6 months before its disappearance. ST4496 was closely related to ST11, with only a single-allele variant, and ST11 is the most dominant clinical clone in China. Recombination events occurred at capsule biosynthesis loci and divided the strains of ST11 and its derivative ST4496 into three clusters, including ST11-KL47, ST11-KL64, and ST4496-KL47. The phylogenetic structure indicated that ST11-KL47 was probably the origin of ST11-related strain evolution and presented more diversity in terms of both sequence similarity and phenotypes. ST4496-KL47 cluster strains presented less virulence than ST11-KL64, which was probably one of the factors preventing the former from spreading widely. In conclusion, ST4496-KL47 was probably derived from ST11-KL47 via intraspecies shifting but was less competitive than ST11-KL64, which also evolved from ST11-KL47 and developed increased virulence via capsule biosynthesis locus recombination. ST11-KL64 has the potential to be the predominant CRKP clone in China.

The Emergence of Novel Sequence Type Strains Reveals an Evolutionary Process of Intraspecies Clone Shifting in ICU-Spreading Carbapenem-Resistant Klebsiella pneumoniae.

Frontiers in microbiology

microbiology

The obligate intracellular pathogen Chlamydia trachomatis (Ctr) is the causative agent of the most common form of sexually transmitted disease in the United States. Genital infections with C. trachomatis can lead to inflammatory tissue damage followed by scarring and tissue remodeling during wound healing. Extensive scarring can lead to ectopic pregnancy or infertility. Classically activated macrophages (CA mϕ), with their anti-microbial effector mechanisms, are known to be involved in acute inflammatory processes during the course of infection. In contrast, alternatively activated macrophages (AA mϕ) contribute to tissue repair at sites of wound healing, and have reduced bactericidal functions. They are present during infection, and thus potentially can provide a growth niche for C. trachomatis during a course of infection. To address this question, macrophages derived from CD14-positive monocytes magnetically isolated from peripheral blood mononuclear cells (PBMC) were treated with interferon-γ or interleukin-4 to produce CA mϕ or AA mϕ, respectively. Confocal microscopy of chlamydial inclusions and quantification of infectious yields revealed better pathogen growth and development in AA mϕ than CA mϕ, which correlated with the reduced expression of indoleamine 2,3-dioxygenase, a known anti-chlamydial effector of the host. Furthermore, AA mϕ stained strongly for transferrin receptor and secreted higher amounts of anti-inflammatory interleukin-10 compared to CA mϕ, characteristics that indicate its suitability as host to C. trachomatis . CA, AA, and resting mϕ were infected with Ctr serovar L2. The data suggest that IL-10 produced by infected AA mϕ attenuated the anti-chlamydial function of CA mϕ with growth recovery observed in infected CA mϕ in the presence of infected, but not mock-infected AA mϕ. This could be related to our observation that IL-10 treatment of infected CA mϕ promoted better chlamydial growth. Thus, in addition to serving as an additional niche, AA mϕ might also serve as a means to modulate the immediate environment by attenuating the anti-chlamydial functions of nearby CA mϕ in a manner that could involve IL-10 produced by infected AA mϕ.

Alternatively Activated Macrophages Are Host Cells for Chlamydia trachomatis and Reverse Anti-chlamydial Classically Activated Macrophages.

Frontiers in microbiology

microbiology

Recent development in genome editing technologies has enabled site-directed deprivation of a nucleotide sequence in the chromosome in mammalian cells. Human immunodeficiency (HIV) infection causes integration of proviral DNA into the chromosome, which potentially leads to re-emergence of the virus, but conventional treatment cannot delete the proviral DNA sequence from the cells infected with HIV. In the present study, the transcription activator-like effector nucleases (TALENs) specific for the HIV p17 gene were constructed, and their activities to destroy the target sequence were evaluated. SSA assay showed a high activity of a pair of p17-specific TALENs. A human T lymphoid cell line, Jurkat, was infected with a lentivirus vector followed by transfection with the TALEN-HIV by electroporation. The target sequence was destructed in approximately 10-95% of the p17 polymerase chain reaction clones, and the efficiencies depended on the Jurkat-HIV clones. Because p17 plays essential roles for assembly and budding of HIV, and this gene has relatively low nucleotide sequence diversity, genome editing procedures targeting p17 may provide a therapeutic benefit for HIV infection.

Specific Destruction of HIV Proviral p17 Gene in T Lymphoid Cells Achieved by the Genome Editing Technology.

Frontiers in microbiology

microbiology

Similar to what has been described in other Gram-negative bacteria, Brucella melitensis releases outer membrane vesicles (OMVs). OMVs from B. melitensis 16M and the rough-mutant B. melitensis VTRM1 were able to induce a protective immune response against virulent B. melitensis in mice models. The presence of some proteins which had previously been reported to induce protection against Brucella were found in the proteome of OMVs from B. melitensis 16M. However, the proteome of OMVs from B. melitensis VTRM1 had not previously been determined. In order to be better understand the role of OMVs in host-cell interactions, the aim of this work was to compare the proteomes of OMVs from B. melitensis 16M and the derived rough-mutant B. melitensis VTRM1, as well as to characterize the immune response induced by vesicles on host cells. Additionally, the effect of SDS and proteinase K on the stability of OMVs was analyzed. OMVs from B. melitensis 16M (smooth strain) and the B. melitensis VTRM1 rough mutant (lacking the O -polysaccharide side chain) were analyzed through liquid chromatography-mass spectrometry (LC-MS/MS). OMVs were treated with proteinase K, sodium deoxycholate, and SDS, and then their protein profile was determined using SDS-PAGE. Furthermore, PBMCs were treated with OMVs in order to measure their effect on cytoskeleton, surface molecules, apoptosis, DNA damage, proliferation, and cytokine-induction. A total of 131 proteins were identified in OMVs from B. melitensis 16M, and 43 in OMVs from B. melitensis VTRM1. Proteome comparison showed that 22 orthologous proteins were common in vesicles from both strains, and their core proteome contained Omp31, Omp25, GroL, and Omp16. After a subsequent detergent and enzyme treatment, OMVs from B. melitensis VTRM1 exhibited higher sensitive compared to OMVs from the B. melitensis 16M strain. Neither OMVs induced IL-17, proliferation, apoptosis or DNA damage. Nonetheless, OMVs from the smooth and rough strains induced overproduction of TNFα and IL-6, as well as actin and tubulin rearrangements in the cytoskeleton. Moreover, OMVs from both strains inhibited PD-L1 expression in T-cells. These data revealed significant differences in OMVs derived from the rough and smooth Brucella strains, among which, the presence or absence of complete LPS appeared to be crucial to protect proteins contained within vesicles and to drive the immune response.

Outer Membrane Vesicles From Brucella melitensis Modulate Immune Response and Induce Cytoskeleton Rearrangement in Peripheral Blood Mononuclear Cells.

Frontiers in microbiology

microbiology

Helicobacter pylori is a pathogen involved in gastric diseases such as ulcers and carcinomas. H. pylori's urease is an important virulence factor produced in large amounts by this bacterium. In previous studies, we have shown that this protein is able to activate several cell types like neutrophils, monocytes, platelets, endothelial cells, and gastric epithelial cells. Angiogenesis is a physiological process implicated in growth, invasion and metastization of tumors. Here, we have analyzed the angiogenic potential of H. pylori urease (HPU) in gastric epithelial cells. No cytotoxicity was observed in AGS, Kato-III, and MKN28 gastric cell lines treated with 300 nM HPU, as evaluated by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As we previously reported in neutrophils, treatment with 300 nM HPU also had an anti-apoptotic effect in gastric epithelial cells leading to a 2.2-fold increase in the levels of Bcl-X L after 6 h, and a decrease of 80% in the content of BAD, after 48 h, two mitochondrial proteins involved in regulation of apoptosis. Within 10 min of exposure, HPU is rapidly internalized by gastric epithelial cells. Treatment of the gastric cells with methyl-β-cyclodextrin abolished HPU internalization suggesting a cholesterol-dependent process. HPU induces the expression of pro-angiogenic factors and the decrease of expression of anti-angiogenic factors by AGS cells. The angiogenic activity of HPU was analyzed using in vitro and in vivo models. HPU induced formation of tube-like structures by human umbilical vascular endothelial cells in a 9 h experiment. In the chicken embryo chorioallantoic membrane model, HPU induced intense neo-vascularization after 3 days. In conclusion, our results indicate that besides allowing bacterial colonization of the gastric mucosa, H. pylori 's urease triggers processes that initiate pro-angiogenic responses in different cellular models. Thus, this bacterial urease, a major virulence factor, may also play a role in gastric carcinoma development.

A New Role for Helicobacter pylori Urease: Contributions to Angiogenesis.

Frontiers in microbiology

microbiology

The cell surface of Gram-negative bacteria usually exhibits a net negative charge mostly conferred by lipopolysaccharides (LPS). This property sensitizes bacterial cells to cationic antimicrobial peptides, such as polymyxin B, by favoring their binding to the cell surface. Gram-negative bacteria can modify their surface to counteract these compounds such as the decoration of their LPS by positively charged groups. For example, in Escherichia coli and Salmonella , EptA and ArnT add amine-containing groups to the lipid A moiety. In contrast, LpxT enhances the net negative charge by catalyzing the synthesis of tri-phosphorylated lipid A, whose function is yet unknown. Here, we report that E. coli has the intrinsic ability to resist polymyxin B upon the simultaneous activation of the two component regulatory systems PhoPQ and PmrAB by intricate environmental cues. Among many LPS modifications, only EptA- and ArnT-dependent decorations were required for polymyxin B resistance. Conversely, the acquisition of polymyxin B resistance compromised the innate resistance of E. coli to deoxycholate, a major component of bile. The inhibition of LpxT by PmrR, under PmrAB-inducing conditions, specifically accounted for the acquired susceptibility to deoxycholate. We also report that the kinetics of intestinal colonization by the E. coli lpxT mutant was impaired as compared to wild-type in a mouse model of infection and that lpxT was upregulated at the temperature of the host. Together, these findings highlight an important function of LpxT and suggest that a tight equilibrium between EptA- and LpxT-dependent decorations, which occur at the same position of lipid A, is critical for the life style of E. coli .

LpxT-Dependent Phosphorylation of Lipid A in Escherichia coli Increases Resistance to Deoxycholate and Enhances Gut Colonization.

Frontiers in microbiology

microbiology

Near infrared (NIR) spectroscopy combined with Monte Carlo uninformative variable elimination (MC-UVE) and nonlinear calibration methods employed to determine gossypol content in cottonseeds were investigated. The reference method was performed by high performance liquid chromatography coupled to an ultraviolet detector (HPLC-UV). MC-UVE was employed to extract the effective information from the full NIR spectra. Nonlinear calibration methods were applied to establish the models compared with the linear method. The optimal model for gossypol content was obtained by MC-UVE-WLS-SVM, with root mean squares error of prediction (RMSEP) of 0.0422, coefficient of determination (R 2 ) of 0.9331, and residual predictive deviation (RPD) of 3.8374, respectively, which was accurate and robust enough to substitute for traditional gossypol measurements. The nonlinear methods performed more reliable than linear method during the development of calibration models. Furthermore, MC-UVE could provide better and simpler calibration models than full spectra.

Determination of gossypol content in cottonseeds by near infrared spectroscopy based on Monte Carlo uninformative variable elimination and nonlinear calibration methods.

Food chemistry

chemistry

Among the existing multiplex genetically modified organism (GMO) detection methods, significant problems are highlighted, including amplification asymmetry of different targets, and the low detection throughput, which limits their capacity to meet the requirements of high-throughput analysis. To mitigate these challenges, a 'turn-on' ultra-sensitive multiplex real-time fluorescent quantitative biosensor is developed. In this system, the multiplex ligation-dependent amplification (MLPA), universal primer and universal probe are innovatively combined, which can enhanced the amplification specificity, overcome asymmetric amplification and guarantee the homogeneity of amplification efficiency simultaneously. Furthermore, both single and multiplex detection results can be output by the fluorescent group labeled on universal TaqMan probes for different targets in real-time. After optimization, the quantitative detection limit was 5 pg. In conclusion, this strategy could serve as an important tool for GMO detection in processed and commercially available products, even in the fields that require reliable and sensitive detection of DNA targets.

A 'turn-on' ultra-sensitive multiplex real-time fluorescent quantitative biosensor mediated by a universal primer and probe for the detection of genetically modified organisms.

Food chemistry

chemistry

The δ 2 H and δ 18 O of 105 salmonids cultured in freshwater and seawater and from different regions were combined with linear discriminant analysis (LDA), k-nearest neighbor (KNN), and random forest (RF) to create discrimination models. To assess the stability of the discrimination models, seasonal variation in δ 2 H and δ 18 O in salmonids cultured in different systems was studied. δ 2 H and δ 18 O were significantly different between salmonids cultured in freshwater and seawater and from different geographical origins. δ 2 H and δ 18 O of salmonids cultured in an open system were vulnerable to seasonal effects. The KNN model had 100% accuracy for identifying the production methods of salmonids and was less affected by seasonal variation. The RF model had the highest accuracy for identifying the geographical origins of salmonids with an accuracy of over 80%. Thus, δ 2 H and δ 18 O were more effective for identifying the production methods of salmonids than their geographical origins.

Efficacy of using stable isotopes coupled with chemometrics to differentiate the production method and geographical origin of farmed salmonids.

Food chemistry

chemistry

The effects of honeys from different floral origins on alcohol metabolism were compared, and the correlation between their chemical compositions and antialcholic effects was analyzed. The results demonstrated that the five types of investigated honeys from different floral origins had different effects on alcohol metabolism, and the blood alcohol removal rate by these honeys ranged from 18.01% to 49.17%. Ziziphus jujuba honey exhibited the best blood alcohol removal effect, and meanwhile significantly enhanced the activity of alcohol-metabolizing enzymes including alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). Chemical composition analysis also showed that honeys from different floral origins were considerably different in the contents of sugars, minerals, ascorbic acid and phenolics. Ziziphus jujuba honey had the highest fructose/glucose ratio, ascorbic acid and phenolics contents, and higher contents of minerals, especially K, Ca, Mg, Fe, Cu, Zn and Mn. This chemical composition might contribute to its better anti-alcoholic effect.

Anti-alcoholic effects of honeys from different floral origins and their correlation with honey chemical compositions.

Food chemistry

chemistry

The purpose of this study was to construct a fusion model using probe-based and non-probe-based fluorescence spectroscopy and low-field nuclear magnetic resonance spectroscopy (Low-field NMR) for rapid quality evaluation of frying oil. Iron tetraphenylporphyrin (FeTPP) was selected as the probe to detect polar compounds in frying oil samples. Non-probe-based fluorescence spectroscopy and low-field NMR were employed to determine the fluorescence changes of antioxidants, triglycerides and fatty acids in frying oil samples. Compared to the models constructed using non-fusion data, the fusion-data models achieved a better regression prediction performance and correlation coefficients with values of 0.9837 and 0.9823 for the training and test sets, respectively. This study suggested that the multiple data fusion method was capable to construct better regression models to rapidly evaluate the quality of frying oil and other food with high oil contents.

Intelligent evaluation of total polar compounds (TPC) content of frying oil based on fluorescence spectroscopy and low-field NMR.

Food chemistry

chemistry

The potential of pulsed electric field (PEF) of different intensities (8, 18, and 28 kV/cm) on the conformation and gelation properties of myofibrillar proteins (MPs) extracted from pale, soft, and exudative-like (PSE-like) chicken meat was investigated. The results showed a positive correlation between gelation properties and PEF intensities in the range of 8-18 kV/cm; however, a further increase in intensity had a negative impact. Optimized PEF treatment (18 kV/cm) was capable of inducing MPs with a relatively small particle size, thus contributing to the production of a more homogeneous gel structure. The water distribution and mobility in the gel system significantly changed with increasing PEF intensities, the proportion of immobilized water (P 21 ) increased, and that of free water (P 22 ) decreased. Based on molecular dynamics simulations (MDS), an increasing trend in the number of hydrogen bonds and a reduction in the radius of gyration (Rg) after PEF treatment.

Effects of pulsed electric fields on the conformation and gelation properties of myofibrillar proteins isolated from pale, soft, exudative (PSE)-like chicken breast meat: A molecular dynamics study.

Food chemistry

chemistry

Clinical trials show an inverse relationship between the consumption of antioxidant-rich tree nuts and the development of chronic diseases. This study examined antioxidant efficacy of U.S. pecans using a modified cellular antioxidant activity (CAA) assay with comparisons to data from in vitro antioxidant assays (hydrophilic-oxygen radical absorbance capacity {H-ORAC FL } and ferric reducing antioxidant power {FRAP}). Crude phenolic extracts from both raw and roasted pecans were analyzed. In the CAA assay, pecan phenolics were taken up by human colorectal adenocarcinoma (Caco-2) cells and bestowed CAA, determined by monitoring the fluorescence of 2',7'-dichlorofluorescein. Phenolics (25-100 μg/mL) demonstrated a reduction in fluorescence by 37-69% for raw and 26-68% for roasted pecans. The primary active phenolic constituents were determined by high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) to be epi(catechin) dimers and trimers. These oligomeric procyanidins, ranging in size from 560 to 840 g/mol appear to be small enough for cellular uptake, showing pecans are an effective antioxidant in biological systems, regardless of roasting.

Cellular evaluation of the antioxidant activity of U.S. Pecans [Carya illinoinensis (Wangenh.) K. Koch].

Food chemistry

chemistry

Durum wheat (Triticum turgidum ssp. durum) is widely grown in the Mediterranean area. The semolina obtained by this grain is used to prepare pasta, couscous, and baked products all over the world. The growing area affects the characteristics of Durum wheat; consequently, it is relevant to trace this product. The present study aims at developing an analytical methodology which would allow tracing durum semolina harvested in 7 different Italian macro-areas. In order to achieve this goal, 597 samples of semolina have been analysed by Near Infrared Spectroscopy, and by measuring alveographic parameters. Eventually, the information collected have been handled by a multi-block classifier (SO-PLS-LDA) in order to predict the origin of samples. The proposed approach provided extremely satisfactory results (in external validation, on a test set of 140 objects), correctly classifying all samples according to their growing area, confirming it represents a suitable solution for tracing durum wheat semolina.

Multi-block classification of Italian semolina based on Near Infrared Spectroscopy (NIR) analysis and alveographic indices.

Food chemistry

chemistry

Dry-cured loins elaborated from frozen (-20 °C/20 weeks)/thawed longissimus dorsi muscles (F) were compared with counterparts elaborated from fresh (unfrozen) muscles (UF) for the extent of protein oxidation (carbonylation and Schiff base formation) and their sensory profile (quantitative-descriptive analysis). All samples had similar moisture, fat and protein contents (p>0.05). In accordance with previous studies, freezing meat prior to processing affected the oxidative stability of meat proteins. This chemical change occurred concomitantly with modifications of the sensory profile of the loins as F-samples received significantly (p<0.05) higher scores for rancid and salty flavor, hardness and fibrousness than UF-counterparts. The formation of cross-links (assessed as Schiff bases) during freezing and the subsequent processing may have contributed to strengthening the meat structure and hence, impairing the texture properties of dry-cured loins.

Effect of protein oxidation on the impaired quality of dry-cured loins produced from frozen pork meat.

Food chemistry

chemistry

Rabbit is a healthy meat, with low allergenicity and excellent nutritional properties. The global popularity of rabbit meat makes it a target for food fraud. We present a LC-QTOF-MS/MS approach for detecting and identifying rabbit-specific peptide-markers from thermally processed meat products to differentiate rabbit from other commonly-consumed animal species. We identified 49 heat-stable specific peptides. We selected the most stable markers for testing complex meat matrices by analysing pâtés-type products with a rabbit meat content ranging from 5% to 85%. Of the 49 heat-stable peptides detected in pure cooked rabbit meat, three were consistently detected in all investigated pâté samples i.e., SSVFVADPK, AFFGHYLYEVAR and PHSHPALTPEQK. Monitoring meat species other than rabbit in the examined pâtés using pork-, lamb- and chicken-specific peptides identified the presence of undeclared chicken in two samples. The results confirm that LC-QTOF-MS/MS is a suitable tool for multi-species detection in processed meat products, particularly for authentication purposes.

LC-QTOF-MS identification of rabbit-specific peptides for authenticating the species composition of meat products.

Food chemistry

chemistry

Analytical chromatographic techniques for mycotoxins control are well established, but they often depend on costly immunoaffinity sample clean-up. Serum albumins, particularly that from bovine origin (BSA), have stable binding affinity towards some mycotoxins, and can be cheaper alternative receptors for sample clean-up due to their wide availability. Thus, this work used BSA immobilized in agarose beads as a novel solid-phase extraction method for quantification of ochratoxin A (OTA) in wine. Constructed BSA-agarose columns could extract OTA efficiently from red wine after its dilution (4-fold) in 0.1 M Tris pH 8.0. The method was linear (R 2 = 0.9999) in the OTA concentration range studied (0.05 to 3.0 μg L -1 ), with recovery rates above 98%. It also showed low detection (0.017 μg L -1 ) and quantification (0.051 μg L -1 ) limits. The efficacy of the BSA-based method was further validated by direct comparison with commercial immunoaffinity columns. Portuguese wines analyzed by both methods had agreeing results.

BSA-based sample clean-up columns for ochratoxin A determination in wine: Method development and validation.

Food chemistry

chemistry

Bovine milk lipids can be replaced with cheaper indigenous vegetable oils to produce milk alternatives with healthier saturated/unsaturated fat balance for those in areas where milk supply is poor or even absent. A wide range of vegetable oils can be used, but their impacts when blended with skimmed milk powder to formulate filled milk powder (FMP) are still unknown. We investigate the baseline variances in 12 types of FMP produced onsite with 3 proportions (10%, 20% and 30%) of 4 different vegetable oils (i.e., coconut, palm, soya-bean and sunflower) using fatty acid- and near infrared spectra profiles. Chemometric analyses revealed 8 significant overlapping clusters of FMP types but 100% classification efficiency was achieved. Sunflower oil, and particularly soya-bean FMP types had statistically the lowest indices of atherogenicity and thrombogenicity. This appears to be the first chemometrics study of FMP; the spectral analytical models used may be effective for product monitoring.

Chemometric studies of the effects of milk fat replacement with different proportions of vegetable oils in the formulation of fat-filled milk powders: Implications for quality assurance.

Food chemistry

chemistry

The occurrence of deoxynivalenol, 3- and 15-deoxynivalenol and deoxynivalenol-3-glucoside in 84 durum wheat samples, from the Argentinean main growing area, was investigated during 2012/13 and 2013/14 using LC-MS/MS. Deoxynivalenol was found in all samples at concentrations varying between <LOQ (50μg/kg) and 9480μg/kg. Deoxynivalenol-3-glucoside was detected in 94% of the samples at concentrations ranging from <LOQ (50μg/kg) to 850μg/kg. Moreover, the acetylated derivatives were also detected but at lower frequency (49%). To the best of our knowledge, this is the first report of deoxynivalenol-3-glucoside in wheat in Argentina. All the commercial cultivars transformed deoxynivalenol to its glucosylated form at conversion rates between 6 and 22%. The results obtained alert of the potential risk present in durum wheat for Argentinean consumers but also show that some of the commercial cultivars currently on used could be promising candidates for breeding programs intended to obtained Fusarium head blight resistance.

Occurrence of deoxynivalenol and deoxynivalenol-3-glucoside in durum wheat from Argentina.

Food chemistry

chemistry

Plasma is a medium of unbound negative and positive particles with the overall electrical charge being roughly zero. Non-thermal plasma processing is an emerging green technology with great potential to improve the quality and microbial safety of various food materials. Starch is a major component of many food products and is an important ingredient for food and other industries. There has been increasing interest in utilizing plasma to modify the functionalities of starch through interactions with reactive species. This mini-review summarises the impact of plasma on composition, chemical and granular structures, physicochemical properties, and uses of starch. Structure-function relationships of starch components as affected by plasma modifications are discussed. Effect of plasma on the properties of wheat flour, which is a typical example of starch based complex food systems, is also reviewed. Future research directions on how to better utilise plasma to improve the functionalities of starch are suggested.

Plasma modification of starch.

Food chemistry

chemistry

In this work, to improve the availability of the recognition sites of molecularly imprinted polymers (MIPs), ordered macroporous molecularly imprinted polymers (OMMIPs) were facilely prepared by grafting a quercetin-MIPs layer on the pore walls of the ordered macroporous thiol group functionalized silica. The pore structures were characterized by FTIR, Raman, SEM, BET and TGA measurements. The results indicated that OMMIPs possessed a nanoscale polymer layer, a more regular macroporous structure and a greater porosity compared with the traditional bulk MIPs (TBMIPs). The polymer content of OMMIPs was about 49.7%. Kinetic and isothermal adsorption experiments indicated that OMMIPs exhibited higher affinity and selectivity towards quercetin than its structural analogues. Moreover, OMMIPs could improve the intra-particle adsorption and thus provide a significant improvement in recognition sites availability over TBMIPs. Using the quercetin-OMMIPs as SPE sorbent, quercetin was directly extracted from the crude Gingko leaves extract with a satisfying selectivity and elution recovery.

Ordered macroporous molecularly imprinted polymers prepared by a surface imprinting method and their applications to the direct extraction of flavonoids from Gingko leaves.

Food chemistry

chemistry

A highly specific competitive enzyme-linked immunosorbent assay (ELISA) protocol has been developed to identify and classify almond products based on differential proteomic analysis. We applied two-dimensional electrophoresis to compare the differences between almond and apricot kernels to search for almond-specific proteins. The amino acid of apricot Pru-1 was sequenced and aligned to almond Pru-1. One peptide, RQGRQQGRQQQEEGR, which exists in almond but not in apricot, was used as hapten to prepare monoclonal antibody against almond Pru-1. An optimized ELISA method was established using this antibody. The assay did not exhibit cross-reactivity with the tested apricot kernels and other edible plant seeds. The limit of detection (LOD) was 2.5-100μg/g based on different food samples. The recoveries of fortified samples at levels of twofold and eightfold LOD ranged from 82% to 96%. The coefficients of variation were less than 13.0%. Using 7M urea as extracting solution, the heat-treated protein loss ratios were 2%, 5% and 15% under pasteurization (65°C for 30min), baking (150°C for 30min) and autoclaved sterilization (120°C for 15min), respectively.

High-specificity quantification method for almond-by-products, based on differential proteomic analysis.

Food chemistry

chemistry

Chelation of iron and zinc in wheat as phytates lowers their bio-accessibility. Steeping and germination (15 °C, 120 h) lowered phytate content from 0.96% to only 0.81% of initial dry matter. A multifactorial experiment in which (steeped/germinated) wheat was subjected to different time (2-24 h), temperature (20-80 °C) and pH (2.0-8.0) conditions showed that hydrothermal processing of germinated (15 °C, 120 h) wheat at 50 °C and pH 3.8 for 24 h reduced phytate content by 95%. X-ray absorption near-edge structure imaging showed that it indeed abolished chelation of iron to phytate. It also proved that iron was oxidized during steeping, germination and hydrothermal processing. It was further shown that zinc and iron bio-accessibility were respectively 3 and 5% in wheat and 27 and 37% in hydrothermally processed wheat. Thus, hydrothermal processing of (germinated) wheat paves the way for increasing elemental bio-accessibility in whole grain-based products.

The impact of steeping, germination and hydrothermal processing of wheat (Triticum aestivum L.) grains on phytate hydrolysis and the distribution, speciation and bio-accessibility of iron and zinc elements.

Food chemistry

chemistry

Rice bran wax (RBW) is a traditional plant based natural wax and an increasingly popular component in textiles, fruit coatings and cosmetics. Properties of RBW can be modified by acyglycerols, and the resulting products can possess features with great potential in different applications. In this study, RBW was interesterified with palm olein (POL) catalyzed by Lipozyme TL IM, and the effects of RBW on the crystallization rate, solid fat content (SFC) and thermodynamic properties were investigated. The crystallization rates of RBW-based enzymatically interesterified (EIE) products were significantly higher than both the starting mixture and fully hydrogenated rapeseed oil (FHRSO). The EIE RBW-based samples were predominantly crystallized in β' form, and presented a much smoother SFC profile as compared to physically blended raw materials. The SFC values were significantly decreased, conversely increased, and remained constant, and at 10 °C, 20-30 °C, and 35-40 °C as the wax ester and acylglycerols compositions changes. Overall, RBW-based samples after EIE showed an increased hardness and good surface properties, which make it a potential plastic fats substitute.

Interesterification of rice bran wax and palm olein catalyzed by lipase: Crystallization behaviours and characterization.

Food chemistry

chemistry

A new multiresidue method was developed for determination of 25 pesticide residues in red wine by gas chromatography coupled to mass spectrometry with a single run of 23.63 min. Samples were extracted from wine with solid phase extraction using Oasis HLB. Mixture of methanol and water was used for rinsing, while acetonitrile and n-hexane were used as elution solvents. Method was validated according to SANCO/12571/2013 criteria in wide linearity range (limit of quantification - 400 μg L(-1)). Limits of quantification (LOQ) were well below 10 μg L(-1) for most pesticides and recoveries at 2×LOQ and 10×LOQ concentration levels were in range 70-120%. Precision, expressed as a relative standard deviation, was always under 14%. The method was applied to 32 red wine samples from Croatia. Pesticides were detected in 30 samples with a total of 15 pesticides found, 7 of which were at a high concentration.

Novel multiresidue method for determination of pesticides in red wine using gas chromatography-mass spectrometry and solid phase extraction.

Food chemistry

chemistry

The antioxidant activities of polysaccharide from bitter gourd were studied. It was found that bitter gourd polysaccharide could significantly increase SOD and CAT contents in serum, liver and brain of mice, and reduce MDA levels in serum, liver and brain to a certain extent in vivo. So, bitter gourd polysaccharide should be a potential antioxidant.

The antioxidant activities in vivo of bitter gourd polysaccharide.

International journal of biological macromolecules

economics

Fusarium head blight (FHB) disease caused by Fusarium graminearum is one of the most important diseases of wheat in humid and warm areas. This disease significantly reduces yield as well as seed quality. The aim of this work was to evaluate the possibility of control of FHB by chitosan (CS) and chitosan nanoparticles (CS/NPs). In vitro, the application of various concentrations of CS and CS/NPs showed significant inhibition of both radial mycelial growth and number of colonies formed against F. graminearum. The application of 1000 and 5000ppm concentration of CS and CS/NPs produced maximum inhibition of radial mycelial growth in comparison to the control, respectively. The microscopic examination, of treated F. graminearum with the CS and CS/NPs, showed dehydration and deformation in mycelial growth and some hyphae were collapsed. The maximum percentage reduction number of colonies was observed in 5000ppm concentration of both CS and CS/NPs. To test the effect of CS and CS/NPs on spore germination, four concentrations were used for 4 and 24h incubation. The 24h incubation of F. graminearum spores with a 5000ppm solution of CS greatly reduced the number of germinating spores. In greenhouse trials, the disease severity percentage was low when CS and CS/NPs were applied before fungus inoculation on the plants and 1000ppm concentration. The spores of F. graminearum germinated on the anther, hyphae penetrated into anther and colonized the palea, lemma and glume after 24 and 72 hpi, respectively. Wherease, the spikelets treated with CS and CS/NPs were infected slowly. Light microscopy and TEM observations indicated that mycelium penetrated into the cells through stoma and transited to other cells by cell wall or plasmodesmata. Mycelial growth caused conidia into cells but CS and CS/NPs prevented of it's growth. Results showed that CS and CS/NPs could be a useful biological pesticide for controlling FHB.

Application of chitosan and chitosan nanoparticles for the control of Fusarium head blight of wheat (Fusarium graminearum) in vitro and greenhouse.

International journal of biological macromolecules

economics

The crustacean shells of crabs and shrimps produces quantities of by-products, leading to seriously environmental pollution and human health problems during industrial processing, yet they turned into high-value useful products, such as chitin and chitosan. To prepare them under large-scale submerged fermentation level, shrimp shell powders (SSPs) was fermented by successive three-step fermentation of Serratia marcescens B742, Lactobacillus plantarum ATCC 8014 and Rhizopus japonicus M193 to extract chitin and chitosan based on previously optimal conditions. Moreover, the key parameters was investigated to monitor the changes of resulted products during fermentation process. The results showed that the yield of prepared chitin and chitosan reached 21.35 and 13.11% with the recovery rate of 74.67 and 63.42%, respectively. The degree of deacetylation (DDA) and molecular mass (MM) of produced chitosan were 81.23% and 512.06kDa, respectively. The obtained chitin and chitosan was characterized using Fourier transform infrared spectrometer (FT-IR) and X-ray diffraction (XRD) analysis. The established microbial fermentation method can be applied for the industrial large-scale production of chitin and chitosan, while the use of chemical reagents was significantly reduced.

The preparation and characterization of chitin and chitosan under large-scale submerged fermentation level using shrimp by-products as substrate.

International journal of biological macromolecules

economics

The large water holding capacity of Basil Seed (Ocimum basilicum L.) Mucilage (BSM) gives it potential to produce a valuable polymer for water holding applications such as wound dressing. The objective of this research was to prepare a natural-based antibacterial wound dressing from BSM by freeze-drying. Various contents of zinc oxide nanoparticles (ZnO-NP) were incorporated as an antibacterial agent. BSM hydrogel sponge showed considerable porosity and degree of swelling. From FTIR analysis, hydrogen bond and electrostatic interaction between BSM molecules and ZnO-NP were confirmed. SEM images revealed an interconnecting open-cell structure of pores in the BSM hydrogel sponge with a good distribution of ZnO-NP. Moreover, increase in ZnO-NP content improved the mechanical properties (stress at maximum load 8.9 MPa, Young's modulus 151 MPa and strain at maximum load 51%), thermal properties, water retention capacity and antibacterial activity. Cytotoxicity and cell adhesion studies of BSM hydrogel sponge indicated non-cytotoxicity and non-adherent nature of the sponge.

Characterization of an antibacterial wound dressing from basil seed (Ocimum basilicum L.) mucilage-ZnO nanocomposite.

International journal of biological macromolecules

economics

The objective of this study was to evaluate the effects of drying temperature (30, 50, 70, and 90 °C) and genotype (yellow floury corn, white floury corn, and yellow flint corn) on the morphology and technological and thermal properties of corn starch. The white and yellow genotypes with floury endosperm (soft) had spherical starch granules, while the granules of the yellow flint genotype were polyhedral. White floury corn showed higher extraction yield, higher resistance during granules' swelling before the physical collapse, as it was not affected by the increase in drying temperature, and at 90 °C it presented higher breakdown and energy necessary for gelatinization. There were peak viscosity and gelatinization enthalpy reduction on starch from yellow floury corn and yellow flint corn according to the increase in drying temperature. Drying temperatures below 50 °C are indicated for all genotypes studied. White floury corn is indicated due to the higher extraction yield presented. Yellow floury corn is indicated for application in chilled and frozen products due to lower starch retrogradation. The yellow flint corn is indicated for application as a thickening agent due to higher gel hardness.

Effects of drying temperature and genotype on morphology and technological, thermal, and pasting properties of corn starch.

International journal of biological macromolecules

economics

Nanoparticles have been investigated as flagging tests for the sensitive DNA recognition that can be utilized as a part of field applications to defeat restrictions. Gold nanoparticles (AuNPs) have been widely utilized due to its optical property and capacity to get functionalized with a mixed bag of biomolecules. This study exhibits the utilization of AuNPs functionalized with single-stranded oligonucleotide (AuNP-oligo test) for fast the identification of Human Papillomavirus (HPV). This test is displayed on interdigitated electrode sensor and supported by colorimetric assay. DNA conjugated AuNP has optical property that can be controlled for the applications in diagnostics. With its identification abilities, this methodology incorporates minimal effort, strong reagents and basic identification of HPV.

A direct detection of human papillomavirus 16 genomic DNA using gold nanoprobes.

International journal of biological macromolecules

economics

Dioscorea bulbifera or air potato has been used as a folk remedy to treat cancer. A mannose binding lectin from bulbils of D. bulbifera was purified in a single step by affinity chromatography on mucin coupled Sepharose 4B column, determined by its fine sugar specificity by glycan array analysis and studied for its clinical potential in cancer and HIV research. SDS-PAGE showed that lectin is a monomer of M r 24kDa. DBL agglutinated only rabbit erythrocytes and was inhibited by mucin, asialomucin, fetuin, asialofetuin and transferrin but not by any monosaccharides. Glycan array analysis of DBL revealed its affinity toward high mannose N-linked glycans with enhanced affinity for terminal mannose including N-linked glycans of HIV envelope glycoprotein gp120 and has strong anti-reverse transcriptase activity. DBL showed strong binding to non-metastatic human colon epithelial cancer HT 29, metastatic SW 620 and hepatocellular HepG2 cell lines. DBL showed dose and time dependent growth inhibitory effects on all the three cell lines HT 29, SW 620 and HepG2 with IC 50 of 110μg, 9.8μg, 40μg respectively at 72h. Inhibitory effect of DBL was effectively blocked in presence of competing glycans like mucin. DBL has promising clinical potential both in cancer and HIV research.

Purification, characterization and biological significance of mannose binding lectin from Dioscorea bulbifera bulbils.

International journal of biological macromolecules

economics

We present a facile synthesis of bovine serum albumin (BSA) conjugated low-dimensional ZnS nanocrystals. The experimental parameters such as effects of BSA concentration and precursor vol ratios of Zn:S on the formation of ZnS nanoparticles in BSA matrix were investigated. The ZnS crystalline sizes of 1.9, 1.8 and 1.6nm were obtained by using the BSA concentrations of 1×10 -4 , 5×10 -4 and 10×10 -4 g/mL, respectively, with a fixed Zn:S vol ratio of 1:1. The ZnS samples prepared from 1:10 and 10:1 vol ratios of Zn:S at BSA concentration of 5×10 -4 g/mL shows the crystalline sizes of ZnS are 2.1 and 1.5nm, respectively. FT-IR analysis suggests that the prepared ZnS nanoparticles might be conjugated through the interactions of hydroxyl and amine groups present in BSA. We evaluate the cytotoxicity of the prepared ZnS nanoparticles, the THP-1 cells showed a good viability (>88%) for all the prepared ZnS samples. The plausible mechanism for the formation of ZnS-BSA composite has also been discussed.

Facile synthesis of bovine serum albumin conjugated low-dimensional ZnS nanocrystals.

International journal of biological macromolecules

economics

Exopolysaccharides (EPS) are important bioproducts produced by some genera of lactic acid bacteria. EPS are famous for their shelf-life improving properties, techno-functional enhancing abilities in food and dairy industries, besides their beneficial health effects. Furthermore, exopolysaccharides have many prospective and well-established contributions in the field of drugs and diagnostic industry. In this review, classification of EPS produced by LAB was presented. Moreover, current and potential applications of EPS in food, dairy, baking industries, cereal-based, and functional products were described. Also, some clinical and pharmaceutical applications of EPS such as intelligent drug delivery systems (microsystems and nanosystems for sustained delivery), interpenetrating polymer networks (IPNs), anticancer drug-targeting, recombinant macromolecular biopharmaceuticals, gene delivery, tissue engineering, and role of EPS in diagnostics were highlighted. Finally, future prospects concerning enhancing EPS production, minimizing costs of their production, and exploring their contribution in further applications were discussed.

Contributions of exopolysaccharides from lactic acid bacteria as biotechnological tools in food, pharmaceutical, and medical applications.

International journal of biological macromolecules

economics

To selectively target telomeric G-quadruplex (G4) DNA, monomeric and dimeric naphthalene diimides (NDIs) were investigated as binders of multimeric G4 structures able to discriminate duplex DNA. These NDIs were analysed by the affinity chromatography-based screening G4-CPG (G-quadruplex on Controlled Pore Glass), using the sequence d[AGGG(TTAGGG) 7 ] (tel46), folding into two consecutive G4s, as model of the human telomeric G4 multimer. In parallel, a telomeric G4 monomer (tel26) and a duplex structure (ds27) were used as controls. According to G4-CPG screening, NDI-5 proved to be the best ligand in terms of dimeric G4 vs. duplex DNA selectivity and was analysed by circular dichroism (CD), gel electrophoresis, isothermal titration calorimetry (ITC) and fluorescence spectroscopy in its interactions with tel46. NDI-5 strongly binds and stabilizes tel46 G4, favouring a hybrid folding in K + -containing buffer. Under these conditions, the binding process comprises a first event involving three molecules of NDI-5 and a second one in which other six molecules bind to the DNA. In a metal cation-free system, NDI-5 induces tel46 G4 folding, as indicated by CD and PAGE, favouring an antiparallel structuring. Docking simulations showed that NDI-5 can effectively bind to the pocket between two G4 units, representing a promising ligand for multimeric G4s.

On the binding of naphthalene diimides to a human telomeric G-quadruplex multimer model.

International journal of biological macromolecules

economics

Epidermal growth factor (EGF) is a local growth factor that stimulates cell growth, proliferation, and differentiation by binding to its receptor EGFR. EGF and EGFR are involved in many aspects of the development of carcinomas. Because EGFR has been found to be over-expressed in many tumors of epithelial origin, it is a potential target for antitumor therapy. In this study we designed a mutated form of hEGF (mEGF) with a deletion of four amino acids residues (Gln 43 , Tyr 44 , Arg 45 and Asp 46 ) in order to show importance of Leu spatial location for EGFR binding/activation. Expression vector pET32a + and E. Coli, strain Rosetta-gami B (DE3) were used to enhance solubility of the recombinant protein with yielding approximately 10mg/l of cell culture. The purified cleaved hEGF as well as non-cleaved fusion protein were biologically active, which was confirmed by their equal ability to stimulate proliferation of MCF7 cells. The mEGF showed specificity and high affinity for EGFR binding, however binding affinity of mEGF for EGFR was reduced about 11.5 fold compared with that of hEGF. The mEGF effect on the MCF7 cell proliferation had a relatively different outcome; mEGF simulated differential cell growth in a dose dependent manner. On the other hand, in MDA-MB468 cells, hEGF and mEGF induced growth inhibition, which was much more severe for hEGF than that of mEGF. Also, hEGF strongly induced the phosphorylation of EGF receptor in MDA-MB468 cells while mEGF induced poor EGFR phosphorylation. The same observations were also made for migration of cancer cells, especially induction of MDA-MB468 migration by mEGF was significantly lower than that of hEGF, suggesting a connection between tyrosine phosphorylation of EGFR and cell migration. Docking analysis revealed that the binding affinity and the buried surface area of mEGF to EGFR complex are lower than those of hEGF/EGFR. Although theoretical studies confirmed reduction in mEGF-EGFR binding affinity, the data of the present study indicate that mEGF is a potential EGFR blocker but may highlight it as excellent delivery agent of protein/non-protein toxins as well as for α-, β-, γ-emitting radio-immunotherapy.

Development of a human epidermal growth factor derivative with EGFR-blocking and depleted biological activities: A comparative in vitro study using EGFR-positive breast cancer cells.

International journal of biological macromolecules

economics

This study investigated the optimization of thermal, functional and rheological properties of Ethyl Cellulose (EC)-based oleogel considering different concentrations of Behenic Acid (BA) and stability of water in oleogel (w/og) emulsions. The results showed that the combination of EC and BA improved the oleogel properties at specific ratios (2:4 and 1:5 wt%). High strength (G' > 1000 mPa) with good thermo-responsive and viscoelastic behavior in the range of 45-60 °C and low loss of oil (<0.2%) were observed in these oleogel formulations. Polarized light microscopy images and XRD results showed the presence of crystals and high proportion of crystalline regions in the mentioned formulations. There were no significant differences among solid fat content (SFC) of EC contained oleogels. The FTIR results indicated new hydrogen bonds formation. The w/og stabilized emulsions with EC: BA (1:5 wt%) oleogel showed high physical stability even at high ratios of disperse phase (5 to 45 wt% of water). The particle size and polydispersity index (PDI) of emulsions were reduced significantly to 250 nm and 0.19, respectively by increasing the ratio of water phase to 45:55 w/og. The oleogel and developed Pickering w/og emulsion has good potential in the formulation of low calorie food products.

Development of behenic acid-ethyl cellulose oleogel stabilized Pickering emulsions as low calorie fat replacer.

International journal of biological macromolecules

economics

Advanced melanoma patients that are not included in common genetic classificatory groups lack effective and safe therapeutic options. Chemotherapy and immunotherapy show unsatisfactory results and devastating adverse effects for these called triple wild-type patients. New approaches exploring the intrinsic antitumor properties of gold nanoparticles might reverse this scenario as a safer and more effective alternative. Therefore, we investigated the efficacy and safety of a composite made of gum arabic-functionalized gold nanorods (GA-AuNRs) against triple wild-type melanoma. The natural polymer gum arabic successfully stabilized the nanorods in the biological environment and was essential to improve their biocompatibility. In vivo results obtained from treating triple wild-type melanoma-bearing mice showed that GA-AuNRs remarkably reduced primary tumor growth by 45%. Furthermore, GA-AuNRs induced tumor histological features associated with better prognosis while also reducing superficial lung metastasis depth and the incidence of intrapulmonary metastasis. GA-AuNRs' efficacy comes from their capacity to reduce melanoma cells ability to invade the extracellular matrix and grow into colonies, in addition to a likely immunomodulatory effect induced by gum arabic. Additionally, a broad safety investigation found no evidence of adverse effects after GA-AuNRs treatment. Therefore, this study unprecedentedly reports GA-AuNRs as a potential nanomedicine for advanced triple wild-type melanomas.

Biocompatible gum arabic-gold nanorod composite as an effective therapy for mistreated melanomas.

International journal of biological macromolecules

economics

A novel chitinase (P1724) was discovered from a Qinghai-Tibetan plateau microbial metagenome. P1724 contains two GH18 family catalytic domains and is phylogenetically distant from any of the chitinases studied. P1724 and its truncated versions, P1724(∆cGH18) and P1724(∆nGH18), were produced in Escherichia coli and characterized. Using colloidal chitin as substrate, the three recombinant proteins showed maximum hydrolytic activities at 40 °C, pH 5.0-6.0 and 0-0.5 M NaCl, and were cold adaptive, as they remained active at 4 °C; their chitinase activities were decreased with the presence of Cu 2+ and EDTA, but increased with Ba 2+ and Ca 2+ ; they all showed both chitobiosidase and endochitinase activities. Compared to P1724(∆nGH18), P1724 and P1724(∆cGH18) shared more similarities in temperature and pH stabilities, NaCl tolerance, and substrate affinity, suggesting the N-terminal GH18 domain contributed more than the C-terminal GH18 did in biochemical characteristics of P1724. k cat /K m value of P1724 was significantly higher than the sum values of P1724(∆cGH18) and P1724(∆nGH18), which indicated that two GH18 domains of P1724 worked cooperatively in degrading chitin. This study has not only broadened the understanding of unknown chitinases in nature but also discussed the strategy of adding additional catalytic domains in enzyme engineering.

The discovery and characterization of a novel chitinase with dual catalytic domains from a Qinghai-Tibetan Plateau wetland soil metagenome.

International journal of biological macromolecules

economics

The exposed hydrophobic patches of protein are widely detected through the binding by the fluorescent probes such as 1-anilino-8-naphthalene sulfonate (ANS), Nile Red (NR) and 1-(N-phenylamino) naphthalene, N-(1-Naphthyl) aniline (1NPN). Interestingly, at pH4, where the Toxoplasma gondii Ferredoxin-NADP(+) reductase (TgFNR) is stable, an exclusive binding and fluorescence emission was observed for ANS. To understand the underlying difference in the binding of ANS, NR and 1NPN; their effect on the protein structure was studied in detail. ANS was found to interact with TgFNR via electrostatic as well as hydrophobic interactions at pH4. NR and 1NPN did not show any such binding to TgFNR in the similar conditions, however showed strong hydrophobic interaction in the presence of NaCl or DSS (2, 2-dimethyl-2-silapentane-5-sulfonate). The subsequent structural studies suggest that ANS, NaCl and DSS induced partial unfolding of TgFNR by modulating ionic interactions of the enzyme, leading to the exposure of buried hydrophobic patches amicable for the binding by NR and 1NPN. The induced unfolding of TgFNR by ANS is unique and thus cautions to use the fluorescent dye as simple indicator to probe the exposed hydrophobic patches of the protein or its folding intermediates.

New insight on 8-anilino-1-naphthalene sulfonic acid interaction with TgFNR for hydrophobic exposure analysis.

International journal of biological macromolecules

economics

We develop a robust micro-patterned double-layer film that can adhere firmly to the tissue and provide a sustained release of ascorbic acid (AA) for corneal regeneration. This double-layer film consists of a AA reservoir sodium alginate (SA) adhesive and an anisotropic layer made of micro-patterned silk nanofibrils (SNF) incorporated gelatin methacrylate (GelMA) (S/G). The S/G layer facilitates the adhesion and orientation of corneal stroma cells, depending on the pattern sizes (50 μm (P1) and 100 (P2) μm). Results reveal that more than 90% and 80% of the cells are located at angles close to the vertical axis (0-20°) in the sample with the smaller and larger pattern size, respectively. The mechanical robustness and 90% light transmission of this hybrid film originate from the micro-patterned S/G layer. However, the micro-pattern size does not show a significant role in the mechanical properties of hybrid films (tensile strength of S/G-SA, S/G-SA(P 1 ), and S/G-SA(P 2 ) is 3.4 ± 0.1 MPa, 3.6 ± 0.6 MPa and 3.3 ± 0.2 MPa, respectively). In addition, the strong adhesion to the tissue of this double-layer film is related to the alginate layer. AA can release in a controlled manner, which can significantly promote corneal stroma cells' attachment, alignment, and proliferation compared to the control (AA-free micro-patterned film). Our results reveal that this innovative multifunctional S/G-SA + AA film can be a proper candidate for use in stroma tissue engineering of the human cornea.

Robust and double-layer micro-patterned bioadhesive based on silk nanofibril/GelMA-alginate for stroma tissue engineering.

International journal of biological macromolecules

economics

We report the preparation of both control chitosan and magnetic chitosan beads as biosorbents using chitosan as matrix and magnetite (Fe 3 O 4 ) nanoparticles as reinforcement followed by detailed advanced characterization. The batch trials were performed to study the adsorption kinetics of biosorbents by removing As (III) and As (V) species from water systems. The experimental data was inserted into Langmuir and Freundlich's isotherms to undertake the mechanism and adsorption capacity of the test biosorbents. Under Langmuir's isotherm, maximum monolayer adsorption capacity (q max ) of the biosorbent was observed to be 73.69 and 79.49 mg/g for As (III) and As (V) species, respectively, under specified conditions. The optimum doses of 1.5 and 2 g/L of MCBB at pH 6.7 showed 99.5 and 99% removal of As (V) and As (III) , respectively. The analysis demonstrated that the biosorption process obeyed pseudo 2nd order kinetics with linear regression coefficient (R 2 ) of >0.999. The regeneration and reusability of biosorbents were also assessed.

Development of sustainable magnetic chitosan biosorbent beads for kinetic remediation of arsenic contaminated water.

International journal of biological macromolecules

economics

Dendritic cells (DCs) are the key regulators of immune responses. In this study, the effect of an exopolysaccharide (EPS) from the culture broth of Trichoderma pseudokoningii on the phenotypic and functional maturation of murine DCs and its underlying molecular mechanisms were investigated. It showed that EPS induced the morphological changes of DCs and the enhanced expression of DCs featured surface molecules CD11c, CD86, CD80 and major histocompatibility complex II (MHC-II). Flow cytometry analysis showed that the treatment with EPS could reduce FITC-dextran uptake by DCs. Sequentially, the results of ELISA indicated that EPS could increase the production of interleukin-12p70 (IL-12p70) in culture supernatant of DCs. Immunofluorescence staining and western blot analysis further revealed that EPS significantly prompted nuclear factor (NF)-κB subunit p65 translocation, IκB-α protein degradation, and p38 mitogen-activated protein kinase (MAPK) phosphorylation. And the production of IL-12p70 was significantly decreased in condition of the inhibition of p38 or NF-κB signaling pathway. These findings suggested that EPS could induce DCs maturation through both p38 MAPK and NF-κB signaling pathways.

Exopolysaccharide from Trichoderma pseudokoningii promotes maturation of murine dendritic cells.

International journal of biological macromolecules

economics

Fibronectin type-II (FnII) family proteins are the major proteins in many mammalian species including bull, horse and pig. In the present study, a major FnII protein has been identified and isolated from donkey (Equus hemionus) seminal plasma, which we refer to as Donkey Seminal Plasma protein-1 (DSP-1). The amino acid sequence determined by mass spectrometry and computational modeling studies revealed that DSP-1 is homologous to other mammalian seminal plasma proteins, including bovine PDC-109 (also known as BSP-A1/A2) and equine HSP-1/2. High-resolution LC-MS analysis indicated that the protein is heterogeneously glycosylated and also contains multiple acetylations, occurring in the attached glycans. Structural and thermal stability studies on DSP-1 employing CD spectroscopy and differential scanning calorimetry showed that the protein unfolds at ~43 °C and binding to phosphorylcholine (PrC) - the head group moiety of choline phospholipids - increases its thermal stability. Intrinsic fluorescence titrations revealed that DSP-1 recognizes lyso-phosphatidylcholine with over 100-fold higher affinity than PrC. Further, interaction of DSP-1 with erythrocytes, a model cell membrane, revealed that DSP-1 binding is mediated by a specific interaction with choline phospholipids and results in membrane perturbation, suggesting that binding of this protein to sperm plasma membrane could be physiologically significant.

Purification, molecular characterization and ligand binding properties of the major donkey seminal plasma protein DSP-1.

International journal of biological macromolecules

economics

Polymer electrolyte membrane fuel cells represent a next-generation power supply technology that may be used in a diverse range of applications. Towards this end, the rational design and engineering of functional nanomaterials as low-cost, high-performance catalysts is of critical significance in the wide-spread commercialization of fuel cell technology. One major bottleneck is the oxygen reduction reaction (ORR) at the cathode. Whereas platinum-based nanoparticles have been used as the catalysts of choice, further engineering of the nanoparticles is urgently needed to enhance the catalytic performance and concurrently reduce the costs. Extensive research has also been extended to non-platinum metals or even metal-free nanocatalysts that may be viable alternatives to platinum. In this review article, we will summarize recent progress in these areas of research within the context of interfacial electron transfer: (a) interactions between metal elements in alloy nanoparticles, (b) metal-ligand interfacial bonding interactions, (c) metal-carbon substrate interactions, and (d) heteroatom doping of graphitic carbons. Results have shown that ready manipulation of the electronic interactions between the catalyst surface and oxygen species may serve as a fundamental mechanism for the optimization of the catalytic performance.

Impacts of interfacial charge transfer on nanoparticle electrocatalytic activity towards oxygen reduction.

Physical chemistry chemical physics : PCCP

chemistry

Interfacial reaction and transport processes contribute crucially to the overall performance and impedance of electrochemical systems. The influence of the electrochemical double layer and the interfacial reactions on the impedance of lithium intercalation batteries is investigated with a modeling approach. Our generic theory for charge and electron transfer reactions at electrified interfaces and its simplified adaptation, a reduced interface model, are compared with the standard for electrochemical interface modeling, the Butler-Volmer ansatz, in terms of numerically simulated impedance spectra. Both of our interface models inherently provide significant impedance characteristics that go beyond the standard approach due to their theoretically consistent derivation. We discuss resistant and capacitive contributions of the double layer to impedance spectra and analyze the effect of strongly correlated interfacial dynamics.

The electrochemical double layer and its impedance behavior in lithium-ion batteries.

Physical chemistry chemical physics : PCCP

chemistry

A model of phosphorylated and ATP-containing B-Raf protein kinase is needed as a tool for the structure-based design of new allosteric inhibitors, since no crystal structure of such a system has been resolved. Here, we present the development of such a model as well as a thorough analysis of its structural features. This model was prepared using a systematic molecular dynamics approach considering the presence or absence of both the phosphate group at the Thr599 site and the ATP molecule. Then, different structural features (i.e. DFG motif, Mg 2+ binding loop, activation loop, phosphorylation site and αC-helix region) were analysed for each trajectory to validate the aimed 2pBRAF_ATP model. Moreover, the structure and activating interactions of this 2pBRAF_ATP model were found to be in agreement with previously reported information. Finally, the model was further validated by means of a molecular docking study with our previously developed lead compound I confirming that this ATP-containing, phosphorylated protein model is suitable for further structure-based design studies.

Development of the first model of a phosphorylated, ATP/Mg2+-containing B-Raf monomer by molecular dynamics simulations: a tool for structure-based design.

Physical chemistry chemical physics : PCCP

chemistry

Cubane-type Mo 3 S 4 cluster hydrides decorated with phosphine ligands are active catalysts for the transfer hydrogenation of nitroarenes to aniline derivatives in the presence of formic acid (HCOOH) and triethylamine (Et 3 N). The process is highly selective and most of the cluster species involved in the catalytic cycle have been identified through reaction monitoring. Formation of a dihydrogen cluster intermediate has also been postulated based on previous kinetic and theoretical studies. However, the different steps involved in the transfer hydrogenation from the cluster to the nitroarene to finally produce aniline remain unclear. Herein, we report an in-depth computational investigation into this mechanism. Et 3 N reduces the activation barrier associated with the formation of Mo-HHOOCH dihydrogen species. The global catalytic process is highly exergonic and occurs in three consecutive steps with nitrosobenzene and N-phenylhydroxylamine as reaction intermediates. Our computational findings explain how hydrogen is transferred from these Mo-HHOOCH dihydrogen adducts to nitrobenzene with the concomitant formation of nitrosobenzene and the formate substituted cluster. Then, a β-hydride elimination reaction accompanied by CO 2 release regenerates the cluster hydride. Two additional steps are needed for hydrogen transfer from the dihydrogen cluster to nitrosobenzene and N-phenylhydroxylamine to finally produce aniline. Our results show that the three metal centres in the Mo 3 S 4 unit act independently, so the cluster can exist in up to ten different forms that are capable of opening a wide range of reaction paths. This behaviour reveals the outstanding catalytic possibilities of this kind of cluster complexes, which work as highly efficient catalytic machines.

On the catalytic transfer hydrogenation of nitroarenes by a cubane-type Mo3S4 cluster hydride: disentangling the nature of the reaction mechanism.

Physical chemistry chemical physics : PCCP

chemistry

Proton conduction in alkali metal ion-exchanged porous ionic crystals A 2 [Cr 3 O(OOCH) 6 (etpy) 3 ] 2 [α-SiW 12 O 40 ]·nH 2 O [I-A + ] (A = Li, Na, K, Cs, etpy = 4-ethylpyridine) is investigated. Single crystal and powder X-ray diffraction measurements show that I-A + possesses analogous one-dimensional channels where alkali metal ions (A + ) and water of crystallization exist. Impedance spectroscopy and water diffusion measurements of I-A + show that proton conductivities are low (10 -7 -10 -6 S cm -1 ) under low relative humidity (RH), and protons mostly migrate as H 3 O + with H 2 O as vehicles (vehicle mechanism). The proton conductivity of I-A + increases with the increase in RH and is largely dependent on the types of alkali metal ions. I-Li + shows a high proton conductivity of 1.9 × 10 -3 S cm -1 (323 K) and a low activation energy of 0.23 eV under RH 95%. Under high RH, alkali metal ions with high ionic potentials (e.g., Li + ) form a dense and extensive hydrogen-bonding network of water molecules with mobile protons at the periphery, which leads to high proton conductivities and low activation energies via rearrangement of the hydrogen-bonding network (Grotthuss mechanism).

Proton conduction in alkali metal ion-exchanged porous ionic crystals.

Physical chemistry chemical physics : PCCP

chemistry

X-ray reflectivity and transmission electron microscopy (TEM) were used to characterize the morphological changes in thin film electrodes with alternating Ni and NiO layers during lithiation as a function of the Ni buffer layer thickness. Complete lithiation of the active NiO layers occurs only when the thickness of the Ni/NiO bilayers are less than 75 Å - a threshold value that is determined by the sum of the Ni quantity in the Ni/NiO bilayer of the multilayer stack. Thicker Ni/NiO bilayers present a kinetic barrier for lithium ion diffusion inside the stack resulting in partial lithiation of the multilayer electrodes in which only the top NiO layer lithiates. Lithiation of NiO layers in a multilayer stack also leads to an interface-specific reaction that is observed to increase the thicknesses of adjacent Ni layers by 3-4 Å and is associated with the formation of a low-density Li 2 O layer, corresponding to an interfacially-driven phase separation of the NiO. Rate dependent cyclic voltammetry studies reveal a linear relation between the peak current and scan rate suggesting that the lithiation kinetics are controlled by charge transfer resistance at the liquid-solid interface.

Lithiation of multilayer Ni/NiO electrodes: criticality of nickel layer thicknesses on conversion reaction kinetics.

Physical chemistry chemical physics : PCCP

chemistry

Graphene is widely used in numerous scientific fields including physics, chemistry and materials science due to its exceptional electrical, thermal, optical and mechanical properties. However, the poor solubility/dispersibility strongly limits the practical applications of graphene. In this work, hydroxypropyl hydrazine (HPH) was synthesized to reduce graphene oxide (GO) under mild conditions. The as-produced graphene sheets with a 3D-porous structure show admirable dispersion stability in N,N-dimethylacetamide (DMAc) and the graphene sheets are more effective absorbents for Cu 2+ removal than those reduced by hydrazine hydrate. A mechanism for removal of epoxides and carboxides from GO by HPH has been proposed. This one-step reducing and dispersing process of GO is more efficient, environmentally benign and safer for the bulk-scale production of 3D porous graphene.

One-step reducing and dispersing graphene oxide via hydroxypropyl hydrazine and its applications in Cu2+ removal.

Physical chemistry chemical physics : PCCP

chemistry

Graphitic carbon nitride (g-C 3 N 4 ) synthesised from a urea precursor is an excellent CO 2 reduction photocatalyst using [Co(bpy) n ] 2+ as a co-catalyst. A five-fold increase in activity for the highly polymerised urea derived g-C 3 N 4 is achieved compared to alternative precursors. Transient absorption, time-resolved and steady-state emission studies indicate that the enhanced activity is related to both an increased driving force for photoelectron transfer and a greater availability of photogenerated charges.

Photochemical CO2 reduction using structurally controlled g-C3N4.

Physical chemistry chemical physics : PCCP

chemistry

The indirect method for the construction of quantum mechanics (QM)/molecular mechanics (MM) free energy landscapes provides a cheaper alternative for free energy simulations at the QM level. The indirect method features a direct calculation of the free energy profile with a computationally efficient but less accurate Hamiltonian (i.e. low-level Hamiltonian) and a low-level-to-high-level correction. In the thermodynamic cycle, the direct low-level calculation along the physically meaningful reaction coordinate is corrected via the alchemical method, which is often achieved with perturbation-based techniques. In our previous work, a multi-dimensional nonequilibrium pulling framework is proposed for the indirect construction of QM/MM free energy landscapes. Previously, we focused on obtaining semi-empirical QM (SQM) results indirectly from direct MM simulations and MM to SQM corrections. In this work, we apply this method to obtain results under ab initio QM Hamiltonians by combining direct SQM results and SQM to QM corrections. A series of SQM and QM Hamiltonians are benchmarked. It is observed that PM6 achieves the best performance among the low-level Hamiltonians. Therefore, we recommend using PM6 as the low-level theory in the indirect free energy simulation. Considering its higher similarity to the high-level Hamiltonians, PM6 corrected with the bond charge correction could be more accurate than the existing AM1-BCC model. Another central result in the current work is a basic protocol of choosing the strength of restraints and an appropriate time step in nonequilibrium free energy simulation at the stiff spring limit. We provide theoretical derivations to emphasize the importance of using a sufficiently large force constant and choosing an appropriate time step. It is worth noting that a general rule of thumb for choosing the time step, according to our derivation, is that a time step of 1 fs or smaller should be used, as long as the stiff spring approximation is employed, even in simulations with constraints on bonds involving hydrogen atoms.

BAR-based multi-dimensional nonequilibrium pulling for indirect construction of QM/MM free energy landscapes: from semi-empirical to ab initio.

Physical chemistry chemical physics : PCCP

chemistry

The OH initiated oxidation of HNO3 in the UT/LS plays an important role in controlling the O3 budget, removing HOx radicals whilst driving NOx/y partitioning chemistry by yielding NO3 radicals: OH + HNO3 → H2O + NO3. In this paper, k1(T, P) was measured using OH (A ← X) Laser Induced Fluorescence (LIF) and the data was modelled over the 223-298 K temperature and 25-750 Torr pressure ranges, using the modified Lindemann-Hinshelwood expression , where k0 = 5.2 × 10-14 exp(200/T) cm3 s-1, k2 = 8.4 × 10-17 exp(1900/T) cm3 s-1 and k3 = 1.6 × 10-34 exp(1745/T) cm3 s-1. A significant source of experimental uncertainty derives from accurate determination of HNO3 concentration, which is impacted by heterogeneous uptake of the low volatility HNO3 onto cold surfaces of the reactors. Our results represent the determination of k1(T, P) using two different in situ [HNO3] measurements: VUV absorption and a new two photon Photolysis Induced Fluoresence (PIF). Experimental results are discussed along with a computational master equation calculation (MESMER), which highlight the need for further theoretical study into the OH + HNO3 mechanism and potential energy surface. The atmospheric impact of these new rate constants were modelled using the STOCHEM-CRI chemistry transport global model, which have shown a small reduction in global budgets of key atmospheric species, with more significant changes in the NOx/HNO3 ratio, peaking in the tropical upper troposphere regions.

Reaction kinetics of OH + HNO3 under conditions relevant to the upper troposphere/lower stratosphere.

Physical chemistry chemical physics : PCCP

chemistry

Aluminum monochloride (AlCl) has been proposed as a promising candidate for laser cooling to ultracold temperatures, and recent spectroscopy results support this prediction. It is challenging to produce large numbers of AlCl molecules because it is a highly reactive open-shell molecule and must be generated in situ . Here we show that pulsed-laser ablation of stable, non-toxic mixtures of Al with alkali or alkaline earth chlorides, denoted XCl n , can provide a robust and reliable source of cold AlCl molecules. Both the chemical identity of XCl n and the Al : XCl n molar ratio are varied, and the yield of AlCl is monitored using absorption spectroscopy in a cryogenic gas. For KCl, the production of Al and K atoms was also monitored. We model the AlCl production in the limits of nonequilibrium recombination dominated by first-encounter events. The non-equilibrium model is in agreement with the data and also reproduces the observed trend with different XCl n precursors. We find that AlCl production is limited by the solid-state densities of Al and Cl atoms and the recondensation of Al atoms in the ablation plume. We suggest future directions for optimizing the production of cold AlCl molecules using laser ablation.

Optimizing pulsed-laser ablation production of AlCl molecules for laser cooling.

Physical chemistry chemical physics : PCCP

chemistry

The dynamics of the magnetic moment reversal is studied for ErFeO3 and NdFeO3 single crystals. The reversal occurs at 41 and 5.1 K for ErFeO3 and NdFeO3, respectively, at a field of 300 Oe. The dynamics of the magnetization reversal process depends on the temperature at which the reversal occurs. The reversal is abrupt if the thermal energy is far higher than the energy of Zeeman splitting of the rare earth ion levels by internal fields, as observed for ErFeO3. A gradual magnetization reversal occurs for NdFeO3 over 64 s, when the thermal energy at the temperature of the reversal is well below the Zeeman splitting energy of Nd3+ spins. A mechanism for this gradual magnetization reversal is proposed in terms of the thermal re-population of Zeeman doublets of Nd3+ ions, the splitting energy of which continuously changes during the magnetization reversal.

Magnetization reversal on different time-scales for ErFeO3 and NdFeO3 single crystals.

Physical chemistry chemical physics : PCCP

chemistry

From the analysis of UV/Vis absorption spectra, emission and excitation emission spectra of solutions of all-trans-1,6-diphenyl-1,3,5-hexatriene (DPH) in carbon disulfide (CS 2 ) within a wide range of temperatures, we can conclude that these spectra do not support the inversion of excited electronic states in DPH by an increased polarizability of the solvent as proposed by Kohler and Itoh. The proposal by Kohler and Itoh is widely accepted as an elegant demonstration of the inversion of the 1 1 Bu and 2 1 Ag states in the polyene DPH by a mere increase in the solvent polarizability. The evidence provided in this paper confirms, as has recently been proposed, that a ghost state as the first electronic state, 2 1 Ag, is not necessary to explain the photophysics of DPH. The photophysics of DPH is plausibly explicable with the 1 1 Bu state, which is directly excitable by photonic absorption from the fundamental electronic state of the compound. In this paper, we provide evidence that the UV/Vis spectra of DPH in CS 2 at 77 K are most likely generated from a molecular structure of DPH constrained by solid CS 2 , which is clearly distinct from that present in liquid CS 2 .

Inversion of the 11Bu and 21Ag electronic states of all-trans-1,6-diphenyl-1,3,5-hexatriene in carbon disulfide.

Physical chemistry chemical physics : PCCP

chemistry

The consideration of polar interactions is of vital importance for the development of predictive and accurate thermodynamic models for polar fluids, as they govern most of their thermodynamic properties, making them highly non-ideal fluids. We present here for the first time the extension of the soft-SAFT equation of state (EoS), named polar soft-SAFT, to explicitly model intermolecular polar interactions (dipolar and quadrupolar), using the approach of Jog and Chapman (P. K. Jog and W. G. Chapman, Mol. Phys., 1999, 97(3), 307-319). The theory is first validated using molecular simulation data for a wide range of polar model systems including Stockmayer fluids, LJ dimers with dipole, and quadrupolar LJ fluids, for a wide range of thermophysical properties such as liquid density, vapour pressure, surface tension and heat capacities. Excellent agreement between polar soft-SAFT and simulation data has been obtained for all examined fluids and properties for systems exhibiting low to intermediate polar strength, while the agreement deteriorates at very high polar strengths. Once validated with simulations, the equation has been applied to calculate vapour-liquid equilibria (VLE), surface tension and second-order derivative properties of systems such as 2-ketone and methane chloride families as showcases for dipolar fluids and the benzene family for quadrupolar fluids, finding very good agreement with experimental data. In order to preserve the robustness of the model, the experimental value of the dipole or quadrupole was used in these calculations, while the additional parameter for the polar fluids was set a priori rather than included in the fitting procedure. The excellent agreement found with simulations and experiments empowers the soft-SAFT equation with new capabilities for the development of robust and accurate molecular models of polar fluids of industrial relevance.

Polar soft-SAFT: theory and comparison with molecular simulations and experimental data of pure polar fluids.

Physical chemistry chemical physics : PCCP

chemistry

Although general porous materials have a low dielectric constant, their uncontrollable opened porous structure results in high dielectric loss and poor barrier properties, thus limiting their application as interconnect dielectrics. In this study, polymeric nanoporous materials with well-controlled closed pores were prepared by incorporating polystyrene (PS) hollow nanoparticles into polyethylene (PE/HoPS). SEM images suggested a closed porous structure for PE/HoPS. In order to show the effect of the porous structure on dielectric properties, nanoporous materials with an opened or uncontrollable porous structure were prepared by etching SiO2/PE or PE/PS@SiO2 composites. PE/HoPSs composites showed an apparently lower dielectric constant and loss compared with the opened porous PE, demonstrating the advantages of a closed porous structure upon enhancing low-dielectric performance. The low dielectric performance of the PE/HoPS composites is linked with high water resistance owing to their closed porous characteristics. When incorporating 15.3 wt% HoPS (porosity: ∼6.9%), the dielectric constant reached 2.08. This value is lower than that calculated from the serial model. Our work revealed that the incorporation of HoPS not only reduces the porosity, but also alters the intrinsic properties of PE, as a result, leading to a greatly reduced dielectric constant.

Preparation and unique dielectric properties of nanoporous materials with well-controlled closed-nanopores.

Physical chemistry chemical physics : PCCP

chemistry