Miracle-dz/UniFAR-FTUs huggingface dataset:
High-Content Analysis Provides Mechanistic Insights into the Testicular Toxicity of Bisphenol A and Selected Analogues in Mouse Spermatogonial Cells

Bisphenol A (BPA), an endocrine-disrupting compound, was found to be a testicular toxicant in animal models. Bisphenol S (BPS), bisphenol AF (BPAF), and tetrabromobisphenol A (TBBPA) were recently introduced to the market as alternatives to BPA. However, toxicological data of these compounds in the male reproductive system are still limited so far. This study developed and validated an automated multi-parametric high-content analysis (HCA) using the C18-4 spermatogonial cell line as a model. We applied these validated HCA, including nuclear morphology, DNA content, cell cycle progression, DNA synthesis, cytoskeleton integrity, and DNA damage responses, to characterize and compare the testicular toxicities of BPA and 3 selected commercial available BPA analogues, BPS, BPAF, and TBBPA. HCA revealed BPAF and TBBPA exhibited higher spermatogonial toxicities as compared with BPA and BPS, including dose- and time-dependent alterations in nuclear morphology, cell cycle, DNA damage responses, and perturbation of the cytoskeleton. Our results demonstrated that this specific culture model together with HCA can be utilized for quantitative screening and discriminating of chemical-specific testicular toxicity in spermatogonial cells. It also provides a fast and cost-effective approach for the identification of environmental chemicals that could have detrimental effects on reproduction.
Actin Monomers Activate Inverted Formin 2 by Competing with Its Autoinhibitory Interaction*

Background: INF2 is not regulated by typical formin autoinhibition. Results: INF2 is autoinhibited in cells and is constitutively active in biochemical actin polymerization assays containing only actin monomers but is inhibited by proteins that bind actin monomers. Conclusion: INF2 can be activated by actin monomers. Significance: A component of INF2 regulation might be the ability to sense free actin monomer levels. INF2 is an unusual formin protein in that it accelerates both actin polymerization and depolymerization, the latter through an actin filament-severing activity. Similar to other formins, INF2 possesses a dimeric formin homology 2 (FH2) domain that binds filament barbed ends and is critical for polymerization and depolymerization activities. In addition, INF2 binds actin monomers through its diaphanous autoregulatory domain (DAD) that resembles a Wiskott-Aldrich syndrome protein homology 2 (WH2) sequence C-terminal to the FH2 that participates in both polymerization and depolymerization. INF2-DAD is also predicted to participate in an autoinhibitory interaction with the N-terminal diaphanous inhibitory domain (DID). In this work, we show that actin monomer binding to the DAD of INF2 competes with the DID/DAD interaction, thereby activating actin polymerization. INF2 is autoinhibited in cells because mutation of a key DID residue results in constitutive INF2 activity. In contrast, purified full-length INF2 is constitutively active in biochemical actin polymerization assays containing only INF2 and actin monomers. Addition of proteins that compete with INF2-DAD for actin binding (profilin or the WH2 from Wiskott-Aldrich syndrome protein) decrease full-length INF2 activity while not significantly decreasing activity of an INF2 construct lacking the DID sequence. Profilin-mediated INF2 inhibition is relieved by an anti-N-terminal antibody for INF2 that blocks the DID/DAD interaction. These results suggest that free actin monomers can serve as INF2 activators by competing with the DID/DAD interaction. We also find that, in contrast to past results, the DID-containing N terminus of INF2 does not directly bind the Rho GTPase Cdc42.
A Mechanistic Basis for the Co-evolution of Chicken Tapasin and Major Histocompatibility Complex Class I (MHC I) Proteins*

Background: Tapasin edits the MHC I peptide repertoire and is highly polymorphic in birds but not mammals. Results: Two chicken MHC I alleles differ in peptide binding properties and participate in an allele-specific interaction with tapasin. Conclusion: Tapasin-MHC alleles have co-evolved by balancing interaction characteristics against MHC peptide-binding ability. Significance: Variations in the functional attributes of tapasin and MHC I alleles determine effective antigen presentation. MHC class I molecules display peptides at the cell surface to cytotoxic T cells. The co-factor tapasin functions to ensure that MHC I becomes loaded with high affinity peptides. In most mammals, the tapasin gene appears to have little sequence diversity and few alleles and is located distal to several classical MHC I loci, so tapasin appears to function in a universal way to assist MHC I peptide loading. In contrast, the chicken tapasin gene is tightly linked to the single dominantly expressed MHC I locus and is highly polymorphic and moderately diverse in sequence. Therefore, tapasin-assisted loading of MHC I in chickens may occur in a haplotype-specific way, via the co-evolution of chicken tapasin and MHC I. Here we demonstrate a mechanistic basis for this co-evolution, revealing differences in the ability of two chicken MHC I alleles to bind and release peptides in the presence or absence of tapasin, where, as in mammals, efficient self-loading is negatively correlated with tapasin-assisted loading. We found that a polymorphic residue in the MHC I α3 domain thought to bind tapasin influenced both tapasin function and intrinsic peptide binding properties. Differences were also evident between the MHC alleles in their interactions with tapasin. Last, we show that a mismatched combination of tapasin and MHC alleles exhibit significantly impaired MHC I maturation in vivo and that polymorphic MHC residues thought to contact tapasin influence maturation efficiency. Collectively, this supports the possibility that tapasin and BF2 proteins have co-evolved, resulting in allele-specific peptide loading in vivo.
Constitutive Activation of Signal Transducer and Activator of Transcription 3 (STAT3) and Nuclear Factor κB Signaling in Glioblastoma Cancer Stem Cells Regulates the Notch Pathway*

Background: Glioma cancer stem cells (CSCs) are believed to drive tumorigenesis. Results: Glioma CSCs show constitutive activation of the STAT3/NF-κB signaling pathway and the Notch pathway. Conclusion: A novel relationship between glioma CSCs and the Notch pathway is defined, involving the constitutive activation of STAT3 and NF-κB signaling. Significance: The STAT3, NF-κB, and Notch pathways provide novel therapeutic targets to treat glioma. Malignant gliomas are locally aggressive, highly vascular tumors that have a dismal prognosis, and present therapies provide little improvement in the disease course and outcome. Many types of malignancies, including glioblastoma, originate from a population of cancer stem cells (CSCs) that are able to initiate and maintain tumors. Although CSCs only represent a small fraction of cells within a tumor, their high tumor-initiating capacity and therapeutic resistance drives tumorigenesis. Therefore, it is imperative to identify pathways associated with CSCs to devise strategies to selectively target them. In this study, we describe a novel relationship between glioblastoma CSCs and the Notch pathway, which involves the constitutive activation of STAT3 and NF-κB signaling. Glioma CSCs were isolated and maintained in vitro using an adherent culture system, and the biological properties were compared with the traditional cultures of CSCs grown as multicellular spheres under nonadherent culture conditions. Interestingly, both adherent and spheroid glioma CSCs show constitutive activation of the STAT3/NF-κB signaling pathway and up-regulation of STAT3- and NF-κB-dependent genes. Gene expression profiling also identified components of the Notch pathway as being deregulated in glioma CSCs, and the deregulated expression of these genes was sensitive to treatment with STAT3 and NF-κB inhibitors. This finding is particularly important because Notch signaling appears to play a key role in CSCs in a variety of cancers and controls cell fate determination, survival, proliferation, and the maintenance of stem cells. The constitutive activation of STAT3 and NF-κB signaling pathways that leads to the regulation of Notch pathway genes in glioma CSCs identifies novel therapeutic targets for the treatment of glioma.
High school students' experiences of learning through research on the Internet

One of the challenges that school educators face today is the need to integrate learning technologies into the learning experience in a meaningful and sustainable way. The current research focuses on how students’ experience learning through new technologies in high schools across New South Wales, Australia, specifically when they learn by researching through the Internet. Open-ended questionnaires were administered to over 300 high school students to explore their conceptions of learning through research on the Internet, approaches to learning through research on the Internet, and approaches to integrating various sources of knowledge. Students’ descriptions of their learning experiences show qualitative variation in their conceptions and approaches to using the Internet in their learning, variation that is reflected in their academic performance. Our analysis suggests that the experience of learning through research on the Internet is highly relational, a finding that emphasizes the importance of integration of various sources of knowledge both for student approaches to learning and for teacher approaches to designing learning tasks.
Crystal Structure of 3-Hydroxybenzoate 6-Hydroxylase Uncovers Lipid-assisted Flavoprotein Strategy for Regioselective Aromatic Hydroxylation*

Background: 3-Hydroxybenzoate 6-hydroxylase (3HB6H) is a flavoprotein monooxygenase involved in the catabolism of aromatic compounds in soil microorganisms. Results: The enzyme crystal structure features natively bound phospholipids and a Tyr-His pair for substrate binding and catalysis. Conclusion: 3HB6H has a peculiar substrate-binding site that uses a bound lipid to help to discriminate between ortho- and para-hydroxylation. Significance: 3HB6H structure uncovers new flavoprotein strategy for regioselective aromatic hydroxylation. 3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is a dimeric flavoprotein that catalyzes the NADH- and oxygen-dependent para-hydroxylation of 3-hydroxybenzoate to 2,5-dihydroxybenzoate. In this study, we report the crystal structure of 3HB6H as expressed in Escherichia coli. The overall fold of 3HB6H is similar to that of p-hydroxybenzoate hydroxylase and other flavoprotein aromatic hydroxylases. Unexpectedly, a lipid ligand is bound to each 3HB6H monomer. Mass spectral analysis identified the ligand as a mixture of phosphatidylglycerol and phosphatidylethanolamine. The fatty acid chains occupy hydrophobic channels that deeply penetrate into the interior of the substrate-binding domain of each subunit, whereas the hydrophilic part is exposed on the protein surface, connecting the dimerization domains via a few interactions. Most remarkably, the terminal part of a phospholipid acyl chain is directly involved in the substrate-binding site. Co-crystallized chloride ion and the crystal structure of the H213S variant with bound 3-hydroxybenzoate provide hints about oxygen activation and substrate hydroxylation. Essential roles are played by His-213 in catalysis and Tyr-105 in substrate binding. This phospholipid-assisted strategy to control regioselective aromatic hydroxylation is of relevance for optimization of flavin-dependent biocatalysts.
The impact of extrinsic and intrinsic rewards on referral strength in a professional service context

The purpose of this paper is to investigate the relationship between referring clients of professional services firms and rewards. An analytical model of referral strength is proposed and includes extrinsic and intrinsic rewards, importance of the reward and referral behaviour (talkers). The findings indicate that extrinsic rewards that are not important to a client can have a significantly negative impact on referral strength. Extrinsic rewards that are valued have a positive and significant indirect impact on talkers. Intrinsic rewards are significantly associated with positive word-of-mouth but do not influence the strength of referrals. The paper finishes with a discussion of the theoretical and practical implications of the findings.
The Natural Compound Cantharidin Induces Cancer Cell Death through Inhibition of Heat Shock Protein 70 (HSP70) and Bcl-2-associated Athanogene Domain 3 (BAG3) Expression by Blocking Heat Shock Factor 1 (HSF1) Binding to Promoters*

Background: HSF1 is a transcription factor that enhances cancer formation and progression. Results: Cantharidin inhibited the binding of HSF1 to the HSP70 promoter and subsequently blocked HSF1-dependent HSP70 expression. Conclusion: The HSF1-dependent expression of HSP70 and BAG3 is inhibited by cantharidin, causing the down-regulation of antiapoptotic BCL-2 family proteins, especially MCL-1. Significance: This information provides a new target molecule and pathway of cantharidin. Heat shock factor 1 (HSF1) enhances the survival of cancer cells under various stresses. The knock-out of HSF1 impairs cancer formation and progression, suggesting that HSF1 is a promising therapeutic target. To identify inhibitors of HSF1 activity, we performed cell-based screening with a library of marketed and experimental drugs and identified cantharidin as an HSF1 inhibitor. Cantharidin is a potent antitumor agent from traditional Chinese medicine. Cantharidin inhibited heat shock-induced luciferase activity with an IC50 of 4.2 μm. In contrast, cantharidin did not inhibit NF-κB luciferase reporter activity, demonstrating that cantharidin is not a general transcription inhibitor. When the HCT-116 colorectal cancer cells were exposed to heat shock in the presence of cantharidin, the induction of HSF1 downstream target proteins, such as HSP70 and BAG3 (Bcl-2-associated athanogene domain 3), was suppressed. HSP70 and its co-chaperone BAG3 have been reported to protect cells from apoptosis by stabilizing anti-apoptotic Bcl-2 family proteins. As expected, treating HCT-116 cancer cells with cantharidin significantly decreased the amounts of BCL-2, BCL-xL, and MCL-1 protein and induced apoptotic cell death. Chromatin immunoprecipitation analysis showed that cantharidin inhibited the binding of HSF1 to the HSP70 promoter and subsequently blocked HSF1-dependent p-TEFb recruitment. Therefore, the p-TEFb-dependent phosphorylation of the C-terminal domain of RNA polymerase II was blocked, arresting transcription at the elongation step. Protein phosphatase 2A inhibition with PP2CA siRNA or okadaic acid did not block HSF1 activity, suggesting that cantharidin inhibits HSF1 in a protein phosphatase 2A-independent manner. We show for the first time that cantharidin inhibits HSF1 transcriptional activity.
Effect of applying a treatment threshold in a population. An example of pulmonary tuberculosis in Rwanda.

PURPOSE Clinicians often think treatment thresholds should be adapted to the setting. We intended to explore the effect in terms of harm because of false negatives and true and false positives of the application of a treatment threshold for pulmonary tuberculosis from a patient's perspective at different prevalence levels in a developing country. METHODS In a cohort of 300 patients with chronic cough, we estimated the prevalence of pulmonary tuberculosis, and the sensitivity and specificity of key predictors with latent class analysis (LCA). We computed the post-test probability of individual patients based on these data. With disease- and treatment-related mortality and morbidity, and without cost or regret, we calculated the break-even point of disease probability where treating versus not treating resulted in similar total harm from the patient's perspective. We estimated the total harm of applying this threshold to the cohort, and to hypothetical settings with different disease prevalence. RESULTS The threshold was computed at 0.026, suggesting treatment for all patients of the cohort. Hypothetically lowering the prevalence showed that the lowest total harm in the cohort always coincides with this threshold, but that numbers of treated patients drop considerably. CONCLUSION For pulmonary tuberculosis a decision threshold solely based on utilities without cost or regret leads to a very low threshold. The lowest total harm is found always at this disease probability, irrespective of the distribution of the patients. Although these findings might suggest an excess prescription at reference level, this is not the case in settings with lower prevalence.
The impact of diabetes education on blood glucose self-monitoring among older adults.

OBJECTIVES To determine whether attending diabetes education is associated with blood glucose self-monitoring among unselected older adults in routine clinical care. METHOD A cross-sectional population-based study was carried out on 15,190 people with diabetes aged 65-79 years. Subjects were identified using a registry of doctor-diagnosed diabetes derived from administrative data, and attendance at diabetes education centres (DECs) was determined from a separate registry of DEC utilization for 2006. Outcomes were derived using administrative data. The primary outcome was prescriptions filled for glucose self-monitoring supplies. The secondary outcomes were prescriptions for antihypertensive drugs, prescriptions for lipid-lowering drugs and eye examinations. RESULTS DEC attendance was associated with glucose self-monitoring, after adjusting for baseline differences between attendees and non-attendees (adjusted odds ratio 6.45, 95% confidence interval 5.61 to 7.42). All of the secondary outcomes were also independently associated with DEC attendance. CONCLUSIONS This study suggests that diabetes education is associated with self-management behaviour in real-world clinical care. These findings support the effectiveness of self-management education programmes to increase self-care behaviours.
Peptidoglycan ld-Carboxypeptidase Pgp2 Influences Campylobacter jejuni Helical Cell Shape and Pathogenic Properties and Provides the Substrate for the dl-Carboxypeptidase Pgp1*

Background: C. jejuni helical shape is important to pathogenesis. Results: Deletion of pgp2 results in loss of C. jejuni helical shape and change in peptidoglycan structure and pathogenic properties. Conclusion: Pgp2 is a ld-carboxypeptidase cleaving peptidoglycan tetrapeptides to tripeptides. Significance: Characterization of enzymes involved in C. jejuni peptidoglycan and cell shape maintenance is crucial to the understanding of fundamental properties of this organism. Despite the importance of Campylobacter jejuni as a pathogen, little is known about the fundamental aspects of its peptidoglycan (PG) structure and factors modulating its helical morphology. A PG dl-carboxypeptidase Pgp1 essential for maintenance of C. jejuni helical shape was recently identified. Bioinformatic analysis revealed the CJJ81176_0915 gene product as co-occurring with Pgp1 in several organisms. Deletion of cjj81176_0915 (renamed pgp2) resulted in straight morphology, representing the second C. jejuni gene affecting cell shape. The PG structure of a Δpgp2 mutant showed an increase in tetrapeptide-containing muropeptides and a complete absence of tripeptides, consistent with ld-carboxypeptidase activity, which was confirmed biochemically. PG analysis of a Δpgp1Δpgp2 double mutant demonstrated that Pgp2 activity is required to generate the tripeptide substrate for Pgp1. Loss of pgp2 affected several pathogenic properties; the deletion strain was defective for motility in semisolid agar, biofilm formation, and fluorescence on calcofluor white. Δpgp2 PG also caused decreased stimulation of the human nucleotide-binding oligomerization domain 1 (Nod1) proinflammatory mediator in comparison with wild type, as expected from the reduction in muropeptide tripeptides (the primary Nod1 agonist) in the mutant; however, these changes did not alter the ability of the Δpgp2 mutant strain to survive within human epithelial cells or to elicit secretion of IL-8 from epithelial cells after infection. The pgp2 mutant also showed significantly reduced fitness in a chick colonization model. Collectively, these analyses enhance our understanding of C. jejuni PG maturation and help to clarify how PG structure and cell shape impact pathogenic attributes.
The pathogenic white-rot fungus Heterobasidion parviporum triggers non-specific defence responses in the bark of Norway spruce.

Norway spruce [Picea abies (L.) Karst. ] is one of the economically most important conifer species in Europe. The major pathogen on Norway spruce is Heterobasidion parviporum (Fr.) Niemelä & Korhonen. To achieve a better understanding of Norway spruce's defence mechanisms, transcriptional responses in bark to H. parviporum infection were compared with the response to wounding using cDNA-amplified fragment length polymorphism. The majority of the recovered transcript-derived fragments (TDFs) showed a similar expression pattern for infection and wounding treatment, although inoculated samples showed an enhanced reaction. Genes related to systemic acquired resistance, e.g., PR1, accumulated after H. parviporum infection. Simultaneously, several transcripts involved in various aspects of jasmonic acid (JA)- and ethylene (ET)-mediated signalling accumulated. Genes involved in the ubiquitin/proteasome system were also regulated. Expression patterns have been confirmed by quantitative polymerase chain reaction. The expression patterns of the isolated TDFs suggest that infection with H. parviporum in Norway spruce induces a broad defence, with many similarities to non-specific defence responses in angiosperms. The parallel induction of salicylic acid- and JA/ET-mediated pathways implies spatially separated responses in different cell layers, with and without hyphal contact. A set of TDFs were analysed in an independent experiment with unrelated material treated with wounding or with inoculation with H. parviporum or Phlebiopsis gigantea, verifying the original observations and underlining the non-specific defence responses. In addition, our data suggest that rerouting of carbon in secondary metabolism is an integral part of Norway spruce induced defence. We report the sequences of three 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase genes (PaDAHP1, PaDAHP2 and PaDAHP3) and their relative expression in response to wounding and infection with H. parviporum and P. gigantea. The results clearly indicate differential regulation of the three DAHPs in the induced defence responses in Norway spruce. This study gives insights into the central mechanisms in the induced defences in Norway spruce.