Determinants of CRISPR Cas12a nuclease activation by DNA and RNA targets.

The RNA-guided CRISPR-associated (Cas) enzyme Cas12a cleaves specific double-stranded (ds-) or single-stranded (ss-) DNA targets (in cis), unleashing non-specific ssDNA cleavage (in trans). Though this trans-activity is widely coopted for diagnostics, little is known about target determinants promoting optimal enzyme performance. Using quantitative kinetics, we show formation of activated nuclease proceeds via two steps whereby rapid binding of Cas12a ribonucleoprotein to target is followed by a slower allosteric transition. Activation does not require a canonical protospacer-adjacent motif (PAM), nor is utilization of such PAMs predictive of high trans-activity. We identify several target determinants that can profoundly impact activation times, including bases within the PAM (for ds- but not ssDNA targets) and sequences within and outside those complementary to the spacer, DNA topology, target length, presence of non-specific DNA, and ribose backbone itself, uncovering previously uncharacterized cleavage of and activation by RNA targets. The results provide insight into the mechanism of Cas12a activation, with direct implications on the role of Cas12a in bacterial immunity and for Cas-based diagnostics.

Diversification of refugia types needed to secure the future of coral reefs subject to climate change.

Identifying locations of refugia from the thermal stresses of climate change for coral reefs and better managing them is one of the key recommendations for climate change adaptation. We review and summarize approximately 30 years of applied research focused on identifying climate refugia to prioritize the conservation actions for coral reefs under rapid climate change. We found that currently proposed climate refugia and the locations predicted to avoid future coral losses are highly reliant on  excess heat metrics, such as degree heating weeks. However, many existing alternative environmental, ecological, and life-history variables could be used to identify other types of refugia that lead to the desired diversified portfolio for coral reef conservation. To improve conservation priorities for coral reefs, there is a need to evaluate and validate the predictions of climate refugia with long-term field data on coral abundance, diversity, and functioning. There is also the need to identify and safeguard locations displaying resistance toprolonged exposure to heat waves and the ability to recover quickly after thermal exposure. We recommend using more metrics to identify a portfolio of potential refugia sites for coral reefs that can avoid, resist, and recover from exposure to high ocean temperatures and the consequences of climate change, thereby shifting past efforts focused on avoidance to a diversified risk-spreading portfolio that can be used to improve strategic coral reef conservation in a rapidly warming climate.

Diversificación de los tipos de refugio necesarios para asegurar el futuro de los arrecifes de coral sujetos al cambio climático Resumen Una de las principales recomendaciones para la adaptación al cambio climático es identificar los refugios de los arrecifes de coral frente al estrés térmico del cambio climático y mejorar su gestión. Revisamos y resumimos ∼30 años de investigación aplicada centrada en la identificación de refugios climáticos para priorizar las acciones de conservación de los arrecifes de coral bajo un rápido cambio climático. Descubrimos que los refugios climáticos propuestos actualmente y las ubicaciones que pueden evitarlos dependen en gran medida de métricas de exceso de calor, como las semanas de calentamiento en grados (SCG). Sin embargo, existen muchas variables alternativas de historia vital, ambientales y ecológicas que podrían utilizarse para identificar otros tipos de refugios que resulten en el acervo diversificado que se desea para la conservación de los arrecifes de coral. Para mejorar las prioridades de conservación de los arrecifes de coral, es necesario evaluar y validar las predicciones sobre refugios climáticos con datos de campo a largo plazo sobre abundancia, diversidad y funcionamiento de los corales. También es necesario identificar y salvaguardar lugares que muestren resistencia a la exposición climática prolongada a olas de calor y la capacidad de recuperarse rápidamente tras la exposición térmica. Recomendamos utilizar más métricas para identificar un acervo de posibles lugares de refugio para los arrecifes de coral que puedan evitar, resistir y recuperarse de la exposición a las altas temperaturas oceánicas y las consecuencias del cambio climático, para así desplazar los esfuerzos pasados centrados en la evitación hacia un acervo diversificado de riesgos que pueda utilizarse para mejorar la conservación estratégica de los arrecifes de coral en un clima que se calienta rápidamente.

CaCO3 Nanoparticles Delivering MicroRNA-200c Suppress Oral Squamous Cell Carcinoma.

MicroRNA (miR)-200c suppresses the initiation and progression of oral squamous cell carcinoma (OSCC), the most prevalent head and neck cancer with high recurrence, metastasis, and mortality rates. However, miR-200c-based gene therapy to inhibit OSCC growth has yet to be reported. To develop an miR-based gene therapy to improve the outcomes of OSCC treatment, this study investigates the feasibility of plasmid DNA (pDNA) encoding miR-200c delivered via nonviral CaCO3-based nanoparticles to inhibit OSCC tumor growth. CaCO3-based nanoparticles with various ratios of CaCO3 and protamine sulfate (PS) were used to transfect pDNA encoding miR-200c into OSCC cells, and the efficiency of these nanoparticles was evaluated. The proliferation, migration, and associated oncogene production, as well as in vivo tumor growth for OSCC cells overexpressing miR-200c, were also quantified. It was observed that, while CaCO3-based nanoparticles improve transfection efficiencies of pDNA miR-200c, the ratio of CaCO3 to PS significantly influences the transfection efficiency. Overexpression of miR-200c significantly reduced proliferation, migration, and oncogene expression of OSCC cells, as well as the tumor size of cell line-derived xenografts (CDX) in mice. In addition, a local administration of pDNA miR-200c using CaCO3 delivery significantly enhanced miR-200c transfection and suppressed tumor growth of CDX in mice. These results strongly indicate that the nanocomplexes of CaCO3/pDNA miR-200c may potentially be used to reduce oral cancer recurrence and improve clinical outcomes in OSCC treatment, while more comprehensive examinations to confirm the safety and efficacy of the CaCO3/pDNA miR-200c system using various preclinical models are needed.

CD5L is a canonical component of circulatory IgM.

Immunoglobulin M (IgM) is an evolutionary conserved key component of humoral immunity, and the first antibody isotype to emerge during an immune response. IgM is a large (1 MDa), multimeric protein, for which both hexameric and pentameric structures have been described, the latter additionally containing a joining (J) chain. Using a combination of single-particle mass spectrometry and mass photometry, proteomics, and immunochemical assays, we here demonstrate that circulatory (serum) IgM exclusively exists as a complex of J-chain-containing pentamers covalently bound to the small (36 kDa) protein CD5 antigen-like (CD5L, also called apoptosis inhibitor of macrophage). In sharp contrast, secretory IgM in saliva and milk is principally devoid of CD5L. Unlike IgM itself, CD5L is not produced by B cells, implying that it associates with IgM in the extracellular space. We demonstrate that CD5L integration has functional implications, i.e., it diminishes IgM binding to two of its receptors, the FcαµR and the polymeric Immunoglobulin receptor. On the other hand, binding to FcµR as well as complement activation via C1q seem unaffected by CD5L integration. Taken together, we redefine the composition of circulatory IgM as a J-chain containing pentamer, always in complex with CD5L.

Artificial surface labelling of Escherichia coli with StrepTagII antigen to study how monoclonal antibodies drive complement-mediated killing.

Antibodies play a key role in the immune defence against Gram-negative bacteria. After binding to bacterial surface antigens, IgG and IgM can activate the complement system and trigger formation of lytic membrane attack complex (MAC) pores. Molecular studies to compare functional activity of antibodies on bacteria are hampered by the limited availability of well-defined antibodies against bacterial surface antigens. Therefore, we genetically engineered E. coli by expressing the StrepTagII antigen into outer membrane protein X (OmpX) and validated that these engineered bacteria were recognised by anti-StrepTagII antibodies. We then combined this antigen-antibody system with a purified complement assay to avoid interference of serum components and directly compare MAC-mediated bacterial killing via IgG1 and pentameric IgM. While both IgG1 and IgM could induce MAC-mediated killing, we show that IgM has an increased capacity to induce complement-mediated killing of E. coli compared to IgG1. While Fc mutations that enhance IgG clustering after target binding could not improve MAC formation, mutations that cause formation of pre-assembled IgG hexamers enhanced the complement activating capacity of IgG1. Altogether, we here present a system to study antibody-dependent complement activation on E. coli and show IgM's enhanced capacity over IgG to induce complement-mediated lysis of E. coli.

MEMO1 Is Required for Ameloblast Maturation and Functional Enamel Formation.

Coordinated mineralization of soft tissue is central to organismal form and function, while dysregulated mineralization underlies several human pathologies. Oral epithelial-derived ameloblasts are polarized, secretory cells responsible for generating enamel, the most mineralized substance in the human body. Defects in ameloblast development result in enamel anomalies, including amelogenesis imperfecta. Identifying proteins critical in ameloblast development can provide insight into specific pathologies associated with enamel-related disorders or, more broadly, mechanisms of mineralization. Previous studies identified a role for MEMO1 in bone mineralization; however, whether MEMO1 functions in the generation of additional mineralized structures remains unknown. Here, we identify a critical role for MEMO1 in enamel mineralization. First, we show that Memo1 is expressed in ameloblasts and, second, that its conditional deletion from ameloblasts results in enamel defects, characterized by a decline in mineral density and tooth integrity. Histology revealed that the mineralization defects in Memo1 mutant ameloblasts correlated with a disruption in ameloblast morphology. Finally, molecular profiling of ameloblasts and their progenitors in Memo1 oral epithelial mutants revealed a disruption to cytoskeletal-associated genes and a reduction in late-stage ameloblast markers, relative to controls. Collectively, our findings integrate MEMO1 into an emerging network of molecules important for ameloblast development and provide a system to further interrogate the relationship of cytoskeletal and amelogenesis-related defects.

Purified complement C3b triggers phagocytosis and activation of human neutrophils via complement receptor 1.

The complement system provides vital immune protection against infectious agents by labeling them with complement fragments that enhance phagocytosis by immune cells. Many details of complement-mediated phagocytosis remain elusive, partly because it is difficult to study the role of individual complement proteins on target surfaces. Here, we employ serum-free methods to couple purified complement C3b onto E. coli bacteria and beads and then expose human neutrophils to these C3b-coated targets. We examine the neutrophil response using a combination of flow cytometry, confocal microscopy, luminometry, single-live-cell/single-target manipulation, and dynamic analysis of neutrophil spreading on opsonin-coated surfaces. We show that purified C3b can potently trigger phagocytosis and killing of bacterial cells via Complement receptor 1. Comparison of neutrophil phagocytosis of C3b- versus antibody-coated beads with single-bead/single-target analysis exposes a similar cell morphology during engulfment. However, bulk phagocytosis assays of C3b-beads combined with DNA-based quenching reveal that these are poorly internalized compared to their IgG1 counterparts. Similarly, neutrophils spread slower on C3b-coated compared to IgG-coated surfaces. These observations support the requirement of multiple stimulations for efficient C3b-mediated uptake. Together, our results establish the existence of a direct pathway of phagocytic uptake of C3b-coated targets and present methodologies to study this process.

Does Walking Help to Generate a Differential Diagnosis?

TheoryIn Medicine, arriving at the correct diagnosis is of paramount importance for patient health and safety, yet is a difficult task especially when a patient presents with symptoms that do not fit typical patterns of disease. This task can be further complicated by errors of judgment, with the failure to consider all possible diagnoses being the most common of such errors. In this study, we investigated the process of differential diagnosis generation within the growing evidence that diagnostic performance can be increased by activities such as walking as was previously shown in Oppezzo and Schwartz's 2014 study. Hypotheses: It was hypothesized that an increase in performance, as expressed by a greater number of plausible differential diagnoses, would be seen in the walking group. Method: Eighteen medical students in their last two months of pre-clerkship training and eighteen second year family medicine residents were shown four different lists of a constellation of signs and symptoms. Participants were asked to generate differential diagnoses over five minutes per each list. All participants sat when completing the first two lists (pretest phase), and then were equally and randomly assigned to sitting versus walking on a treadmill when completing the last two lists (post-test phase). The number of total and unique differential diagnoses generated was determined, before being submitted to a three-member expert panel who identified appropriate unique differential diagnoses. Results: Two-way mixed ANOVAs were conducted to investigate the impact of exercise on the number of total, unique, and appropriate unique ideas generated and compared between pretest and post-test phases. Conclusions: We conclude that there is neither an increase nor a decrease in the number or quality of differential diagnoses generated by the sitting and walking groups within a population that has acquired some level of expertise.

Relationship quality in higher education and the interplay with student engagement and loyalty.

BACKGROUND: To date, studies that have investigated the bonds between students and their institution have emphasized the importance of student-staff relationships. Measuring the quality of those relationships (i.e., relationship quality) appears to help with investigating the relational ties students have with their higher education institutions. Growing interest has arisen in further investigating relationship quality in higher education, as it might predict students' involvement with the institution (e.g., student engagement and student loyalty). So far, most studies have used a cross-sectional design, so that causality could not be determined. AIMS: The aim of this longitudinal study was twofold. First, we investigated the temporal ordering of the relation between the relationship quality dimensions of trust (in benevolence and honesty) and affect (satisfaction, affective commitment, and affective conflict). Second, we examined the ordering of the paths between relationship quality, student engagement, and student loyalty. Our objectives were to gain a deeper understanding of the relationship quality construct in higher education and its later outcomes. SAMPLE: Participants (N = 1649) were students from three Dutch higher education institutions who were studying in a technology economics or social sciences program. METHODS: Longitudinal data from two time points were used to evaluate two types of cross-lagged panel models. In the first analysis, we could not assume measurement invariance for affective conflict over time. Therefore, we tested an alternative model without affective conflict, using the latent variables of trust and affect, the student engagement dimensions and student loyalty. In the second type of model, we investigated the manifest variables of relationship quality, student engagement, and student loyalty. The hypotheses were tested by evaluating simultaneous comparisons between estimates. RESULTS: Results indicated that the relation between relationship quality at Time 1 with student engagement and loyalty at Time 2 was stronger than the reverse ordering in the first model. In the second model, results indicated that cross-lagged relations between trust in benevolence and trust in honesty at Time 1 and affective commitment, affective conflict, and satisfaction at Time 2 were more likely than the reverse ordering. Furthermore, cross-lagged relations from relationship quality at Time 1 to student engagement and student loyalty at Time 2 also supported our hypothesis. CONCLUSIONS: This study contributes to the existing higher education literature, indicating that students' trust in the quality of their relationship with faculty/staff is essential for developing students' affective commitment and satisfaction and for avoiding conflict over time. Second, relationship quality factors positively influence students' engagement in their studies and their loyalty towards the institution. A relational approach to establishing (long-lasting) bonds with students appears to be fruitful as an approach for educational psychologists and for practitioners' guidance and strategies. Recommendations are made for future research to further examine relationship quality in higher education in Europe and beyond.

Assessing Concerns and Care Needs of Expectant Parents: Development and Feasibility of a Structured Interview.

Many adverse situations for parenting and healthy child development can be detected before a child's birth. The aim of this project was to develop and test an instrument to use in prenatal home visits, to improve the identification of adverse situations and care needs during pregnancy. The preSPARK is based on a valid and reliable broad-scope structured interview called SPARK (Structured Problem Analysis of Raising Kids). The preSPARK focuses on 12 topics ranging from aspects of the period before pregnancy to future parents' expectations. The preSPARK was tested in daily practice for feasibility and discriminative capacity. User experience was assessed from the perspective of the professional. In total, 64 home visits using the preSPARK were carried out by 21 nurses. About 24% of the expectant parents needed intensive help or immediate action on one or more topics. The risk assessment showed 29% of the participants were at high risk, 40% at increased risk, and 31% at low risk for future parenting and child developmental problems. The nurses indicated that the preSPARK provides a good structure for home visits and gives insight in interrelated factors. The preSPARK is feasible in daily practice and clarifies risks and care needs of expectant parents.

Kinetic analysis of Cas12a and Cas13a RNA-Guided nucleases for development of improved CRISPR-Based diagnostics.

Bacterial CRISPR systems provide acquired immunity against invading nucleic acids by activating RNA-programmable RNases and DNases. Cas13a and Cas12a enzymes bound to CRISPR RNA (crRNA) recognize specific nucleic acid targets, initiating cleavage of the targets as well as non-target (trans) nucleic acids. Here, we examine the kinetics of single-turnover target and multi-turnover trans-nuclease activities of both enzymes. High-turnover, non-specific Cas13a trans-RNase activity is coupled to rapid binding of target RNA. By contrast, low-turnover Cas12a trans-nuclease activity is coupled to relatively slow cleavage of target DNA, selective for DNA over RNA, indifferent to base identity, and preferential for single-stranded substrates. Combining multiple crRNA increases detection sensitivity of targets, an approach we use to quantify pathogen DNA in samples from patients suspected of Buruli ulcer disease. Results reveal that these enzymes are kinetically adapted to play distinct roles in bacterial adaptive immunity and show how kinetic analysis can be applied to CRISPR-based diagnostics.

Outer membrane permeabilization by the membrane attack complex sensitizes Gram-negative bacteria to antimicrobial proteins in serum and phagocytes.

Infections with Gram-negative bacteria form an increasing risk for human health due to antibiotic resistance. Our immune system contains various antimicrobial proteins that can degrade the bacterial cell envelope. However, many of these proteins do not function on Gram-negative bacteria, because the impermeable outer membrane of these bacteria prevents such components from reaching their targets. Here we show that complement-dependent formation of Membrane Attack Complex (MAC) pores permeabilizes this barrier, allowing antimicrobial proteins to cross the outer membrane and exert their antimicrobial function. Specifically, we demonstrate that MAC-dependent outer membrane damage enables human lysozyme to degrade the cell wall of E. coli. Using flow cytometry and confocal microscopy, we show that the combination of MAC pores and lysozyme triggers effective E. coli cell wall degradation in human serum, thereby altering the bacterial cell morphology from rod-shaped to spherical. Completely assembled MAC pores are required to sensitize E. coli to the antimicrobial actions of lysozyme and other immune factors, such as Human Group IIA-secreted Phospholipase A2. Next to these effects in a serum environment, we observed that the MAC also sensitizes E. coli to more efficient degradation and killing inside human neutrophils. Altogether, this study serves as a proof of principle on how different players of the human immune system can work together to degrade the complex cell envelope of Gram-negative bacteria. This knowledge may facilitate the development of new antimicrobials that could stimulate or work synergistically with the immune system.

Does walking improve diagnosis of skin conditions at varying levels of medical expertise?

The use of walking workstations in educational and work settings has been shown to improve cognitive abilities. At the same time, it has been repeatedly shown that medical residents around the world do not meet exercise guidelines, mainly due to a scarcity of available free time. Our study investigates the boundaries of the previously observed phenomenon of improved cognitive performance with physical activity using materials that represent real life tasks. Participants had different level of expertise and involved second year psychology students, medical students, and family medicine residents. We examined the effect of being physically inactive (i.e., sitting) or active (i.e., walking) while diagnosing multiple complex presentations of four skin conditions. We assumed that being physically active, irrespective of the level of expertise, will bolster diagnostic performance. Our findings show, however, that being physically active does not change the performance level of participants with different levels of medical expertise. Implications for medical education and suggestions for further research will be discussed.

New injectable self-assembled hydrogels that promote angiogenesis through a bioactive degradation product.

Hydrogels used in regenerative medicine are often designed to allow cellular infiltration, degradation, and neovascularization. Low molecular weight hydrogels (LMWHs), formed by self-assembly via non-covalent interactions, are gaining significant interest because they are soft, easy to use and injectable. We propose LMWHs as suitable body implant materials that can stimulate tissue regeneration. We produced four new LMWHs with molecular entities containing nucleic acid and lipid building blocks and analyzed the foreign body response upon subcutaneous implantation into mice. Despite being infiltrated with macrophages, none of the hydrogels triggered detrimental inflammatory responses. Most macrophages present in the hydrogel-surrounding tissue acquired an immuno-modulatory rather than inflammatory phenotype. Concomitantly, no fibrotic capsule was formed after three weeks. Our glyconucleolipid LMWHs exhibited different degradation kinetics in vivo and in vitro. LMWHs with high angiogenic properties in vivo, were found to release glyconucleoside (glucose covalently linked to thymidine via a triazole moiety) as a common by-product of in vitro LMWH degradation. Chemically synthesized glyconucleoside exhibited angiogenic properties in vitro in scratch assays with monolayers of human endothelial cells and in vivo using the chick chorioallantoic membrane assay. Collectively, LMWHs hold promise as efficient scaffolds for various regenerative applications by displaying good biointegration without causing fibrosis, and by promoting angiogenesis through the release of a pro-angiogenic degradation product. STATEMENT OF SIGNIFICANCE: The main limitations of biomaterials developed in the field of tissue engineering remains their biocompatibility and vascularisation properties. In this context, we developed injectable Low Molecular Weight Hydrogels (LMWH) exhibiting thixotropic (reversible gelation) and thermal reversible properties. LMWH having injectability is of great advantage since it allows for their delivery without wounding the surrounding tissues. The resulting gels aim at forming scaffolds that the host cells colonize without major inflammation, and that won't be insulated by a strong fibrosis reaction. Importantly, their molecular degradation releases a product (a glycosyl-nucleoside conjugate) promoting angiogenesis. In this sense, these LMWH represent an important advance in the development of biomaterials promoting tissue regeneration.

Self-reported adverse childhood experiences and quality of life among children in the two last grades of Dutch elementary education.

BACKGROUND: Adverse Childhood Experiences (ACEs) may have a life-long impact on mental health and are related to physical disease, such as diabetes and cardiovascular diseases in adulthood. Research on ACEs suffers from recall bias when performed with adults. OBJECTIVE: To estimate the prevalence of ACEs and the interrelationships between ACEs as reported by children, and to determine the impact on their self-reported quality of life (QoL). Children's opinions on the ACE-Questionnaire were also obtained. METHOD: A cross-sectional study was conducted with a child version of the ACE-Questionnaire. This questionnaire assesses parental separation or divorce, physical and emotional child abuse and neglect, sexual violence, domestic violence, household substance abuse, psychological issues or suicide, and incarceration of a household member. QoL was measured with the Kidscreen-10. PARTICIPANTS AND SETTING: The questionnaire was completed by 644 children at a mean age of 11 years (range 9-13 years), in the two last grades of regular elementary schools, recruited throughout the Netherlands. RESULTS: Data were weighted by ethnicity to obtain a representative sample of children in Dutch elementary education. Of all children, 45.3% had one or more out of ten ACEs. Child maltreatment was experienced by 26.4%. ACEs often co-occurred. A higher number of ACEs correlated with a lower mean level of QoL (p <  0.001). Mean QoL was 8.5 points lower (Cohen's d = 0.8) in children who experienced child maltreatment. Children's opinions on the questionnaire were positive in 82.4%. CONCLUSION: Prevention of ACEs, professional training and trauma-focus in schools are urgently needed.

Effective chemical virus inactivation of patient serum compatible with accurate serodiagnosis of infections.

OBJECTIVES: Highly pathogenic viruses such as EBOV are a threat to routine laboratory workers. Inactivation procedures with Triton X-100 0.1% and/or heat are currently recommended, but have unknown effects on the accuracy of serological testing. Furthermore, virus inactivation by Triton X-100 0.1% was shown to be ineffective in serum. This study aimed to demonstrate virus inactivation in serum by Triton X-100 1% and maintained accuracy of serological testing. METHODS: A panel of 19 serological tests was run on patient serum samples after treatment with Triton X-100 1%, 0.1%, and 0.1% + heat inactivation at 60°C for 1 h. Mean differences between measurements (bias) were calculated applying the Bland-Altman method. To determine effectiveness of virus inactivation, herpes simplex virus 1 (HSV-1) was spiked into medium containing 90% or 1% serum, and treated with Triton X-100 0.1% or 1%. Infectious titres were then determined on Vero cells. RESULTS: Serological measurements showed good agreement between controls and samples treated with Triton X-100 0.1% and 1%, with an estimated bias of 0.6 ± 9.2% (n = 258) and -0.1 ± 18.6% (n = 174), respectively. Discordant qualitative results were rare. Conversely, heat inactivation alone and combined with Triton X-100 0.1% triggered a bias of 17.5 ± 66.4% (n = 200) and 37.9 ± 79.8% (n = 160), respectively. Triton X-100 1% completely inactivated HSV-1 in 1% and 90% serum while Triton X-100 0.1% failed to do so in 90% serum. CONCLUSIONS: Unlike heat inactivation, Triton X-100 1% enabled accurate serological testing and completely inactivated HSV-1 in serum. This simple method could allow safe routine serological diagnostics in high-risk patients.

Association between self-reported sexually transmitted infection treatment and mental health symptoms in conflict-affected eastern Democratic Republic of the Congo.

Although poor mental health has been associated with sexual risk behavior, few studies have examined the association between mental health and sexually transmitted infections (STIs) in conflict-affected settings. With elevated symptoms of poor mental health in conflict-affected settings, it is important to consider if and how mental health may be a risk factor for STIs in these settings. We used cross-sectional logistic regression to examine the association between symptoms of depression, anxiety, and post-traumatic stress disorder (PTSD) with having been treated for an STI in rural South Kivu, Democratic Republic of the Congo. Among 753 adults, those with elevated self-reported symptoms of depression and anxiety (adjusted OR = 2.73, 95% CI 1.68, 4.44) and PTSD (adjusted OR = 1.89, 95% CI 1.17, 3.06) had higher odds of reporting ever being treated for an STI than those who were not symptomatic. Our findings suggest that future studies are needed to more rigorously examine the relationship between mental health and STIs.

Cerebrospinal fluid CXCL13 as a diagnostic marker of neuroborreliosis in children: a retrospective case-control study.

BACKGROUND: Lyme neuroborreliosis (LNB) is a frequent manifestation of Lyme disease in children and its current diagnosis has limitations. The elevation of the chemokine CXCL13 in the cerebrospinal fluid (CSF) of adult patients with LNB has been demonstrated and suggested as a new diagnostic marker. Our aim was to evaluate this marker in the CSF of children with suspected LNB and to determine a CXCL13 cut-off concentration that would discriminate between LNB and other central nervous system (CNS) infections. METHODS: For this single-center retrospective case-control study we used a diagnostic-approved ELISA to measure CXCL13 concentrations in the CSF of 185 children with LNB suspicion at presentation. Patients were classified into definite LNB (cases), non-LNB (controls with other CNS affections), and possible LNB. A receiver-operating characteristic curve was generated by comparison of cases and controls. RESULTS: CXCL13 was significantly elevated in the CSF of 53 children with definite LNB (median 774.7 pg/ml) compared to 91 control patients (median 4.5 pg/ml, p < 0.001). A cut-off of 55 pg/ml resulted in a sensitivity of 96.7% and a specificity of 98.1% for the diagnosis of definite LNB and the test exhibited a diagnostic odds ratio of 1525.3. Elevated CSF CXCL13 levels were also detected in three controls with viral meningitis (enterovirus n = 1, varicella-zoster virus n = 2) while other CNS affections such as idiopathic facial palsy did not lead to CXCL13 elevation. Of the 41 patients with possible LNB, 27% had CXCL13 values above the cut-off of 55 pg/ml (median 16.7 pg/ml). CONCLUSIONS: CSF CXCL13 is highly elevated in children during early LNB as previously shown in adults. CXCL13 is a highly sensitive and specific marker that helps to differentiate LNB from other CNS affections in children.

Assessment of engineered cells using CellNet and RNA-seq.

CellNet is a computational platform designed to assess cell populations engineered by either directed differentiation of pluripotent stem cells (PSCs) or direct conversion, and to suggest specific hypotheses to improve cell fate engineering protocols. CellNet takes as input gene expression data and compares them with large data sets of normal expression profiles compiled from public sources, in regard to the extent to which cell- and tissue-specific gene regulatory networks are established. CellNet was originally designed to work with human or mouse microarray expression data for 21 cell or tissue (C/T) types. Here we describe how to apply CellNet to RNA-seq data and how to build a completely new CellNet platform applicable to, for example, other species or additional cell and tissue types. Once the raw data have been preprocessed, running CellNet takes only several minutes, whereas the time required to create a completely new CellNet is several hours.

In vitro assessment of a collagen/alginate composite scaffold for regenerative endodontics.

AIM: To develop a biological scaffold that could be moulded to reproduce the geometry of a gutta-percha point with precision and allow the differentiation of mesenchymal stem cells into osteoblasts to be used as a regenerative endodontic material. METHODOLOGY: A collagen/alginate composite scaffold was cast into a sodium alginate mould to produce a gutta-percha point-like cone. Prior to gelation, the cone was seeded with human stem cells from the apical papilla (SCAPs) to evaluate cell/scaffold interactions. The reconstructed tissue was characterized after 8 days in culture. Elastic modulus, tissue compaction and cell differentiation were assessed. Student t-tests and the Mann-Whitney U test were performed. RESULTS: The fabrication method developed enabled the shape of a gutta-percha point to be mimicked with great accuracy and reproducibility (P = 0.31). Stem cells seeded into this composite scaffold were able to spread, survive and proliferate (P < 0.001). Moreover, they were able to differentiate into osteoblasts and produce calcified osseous extracellular matrix (P < 0.001). The construct showed no significant contraction after 8 days, preserving its shape and tip diameter (P = 0.58). CONCLUSIONS: The composite scaffold could present substantial benefits compared to synthetic materials. It could provide a favourable healing environment in the root canal conducive for regenerative endodontics and is therefore appropriate to be evaluated in vivo in further studies.