Effectiveness of an ultrasound basic cancer training program through on-site training and virtual case discussions in rural Tanzania: a proof-of-concept study.

Introduction: Cancer rates are rising in low- and middle-income countries. While point-of-care ultrasound is now available globally and could serve to mitigate against this rise, its use in diagnosing cancers is inconsistent in lower-resourced healthcare contexts. This proof-of-concept study investigates the feasibility of an ultrasound training concept in a low-resource setting. It evaluates whether this educational concept led to improved knowledge and application of ultrasound diagnostics, cancer screening and staging and patient care. Material and methods: The curriculum was developed through expert exchange and is based on the World Health Organisation's Manual of Diagnostic Ultrasound. It consisted of two didactic components: an on-site training phase across 5 days for a total of 24 hours, and a digital follow-up phase involving the meeting of a bi-weekly tumor board online. The learning objectives of the on-site training were normal imaging and recognition of common pathologies of the abdominal organs, vessels, lymph nodes, female breasts and lungs. The virtual tumour boards met to discuss cases and ultrasound findings, thus aiding continuing professional development after the training sessions had concluded. The face-to-face course component was accompanied by tests given before and after training as well as an evaluation sheet (Likert-scale with 1 = 'completely/very good' and 7 'not at all/very poor'). Results: Of 20 participants from a rural hospital in Tanzania, a total of 16 were included in the analysis (clinical officers n = 6; medical officers n = 10). A significant increase in knowledge (p < 0.01) was measured both in the subjective self-assessment and in the theoretical competence tests. In multivariate linear regression, the status 'medical officers yes' (ß = 5.4; p = 0.04) had a significant influence on theory test results at T2. During the 24 virtual tumour board meetings, 28 cases were discussed and a continuous improvement in image acquisition quality was observed. Conclusion: The ultrasound education concept comes with a sustainable increase in clinical competence and improved oncological ultrasound screening locally. There is potential for the transfer of the concept to other locations, which can be explored in the future.

Transforming Growth Factor ß1 and Gap Junction Protein Alpha 4 Gene Heterogeneity in Relation to the Severity of Clinical Disease in Cystic Fibrosis.

BACKGROUND: High TGFß1-producing variants cause severe clinical disease in F508del homozygous patients. Lately, we showed that a single nucleotide polymorphism (SNP), rs41266431, in the GJA4 gene modifies the disease severity of cystic fibrosis (CF). Our aim was to investigate whether the clinical phenotype associated with GJA4 variants was independent of TGFß1 variants. METHODS: Homozygous F508del patients (n = 115, mean age 27.2 years, m/f (65/50)) were included in this study. A deep sequence analysis was performed for GJA4 and TGBß1, and disease severity was assessed over 3 years using lung function tests (LFTs), body mass index, diabetes mellitus, colonization with Pseudomonas aeruginosa, survival to end-stage lung disease (ESLD), as well as distinct inflammatory biomarkers. RESULTS: The analyses revealed that one SNP (rs41266431) in GJA4 may be clinically relevant. Carriers homozygous for the G variant (n = 84; 73%) presented with worse LFTs (forced vital capacity (FVC) % predicted: mean 80/86.6, p < 0.035) and a lower survival to ESLD (p < 0.029). For the TGBß1 variant: 509 carriers of the C variant (CT + CC genotype, n = 105, 91.3%) had better LFTs (Forced expiratory flow at 75% of the FVC (FEF75% predicted: median 40/29.5, p < 0.015), although a similar outcome to ESLD. A gene-gene interaction was not observed between TGBß1 and GJA4 variants for any clinical measure. CONCLUSIONS: GJA4 variants are independent of TGBß1 variants. Both variants had an impact on the LFTs, although only GJA4 variants were associated with an improved outcome for ESLD. CLINICAL TRIAL REGISTRATION: The study was registered with ClinicalTrials.gov, number NCT04242420, retrospectively on January 24th, 2020.

Proteasome Inhibition Sensitizes Liposarcoma to MDM2 Inhibition with Nutlin-3 by Activating the ATF4/CHOP Stress Response Pathway.

Liposarcoma is the most commonly occurring soft-tissue sarcoma and is frequently characterized by amplification of chromosome region 12q13-15 harboring the oncogenes MDM2 and CDK4. This unique genetic profile makes liposarcoma an attractive candidate for targeted therapeutics. While CDK4/6 inhibitors are currently employed for treatment of several cancers, MDM2 inhibitors have yet to attain clinical approval. Here, we report the molecular characterization of the response of liposarcoma to the MDM2 inhibitor nutlin-3. Treatment with nutlin-3 led to upregulation of two nodes of the proteostasis network: the ribosome and the proteasome. CRISPR/Cas9 was used to perform a genome-wide loss of function screen that identified PSMD9, which encodes a proteasome subunit, as a regulator of response to nutlin-3. Accordingly, pharmacologic studies with a panel of proteasome inhibitors revealed strong combinatorial induction of apoptosis with nutlin-3. Mechanistic studies identified activation of the ATF4/CHOP stress response axis as a potential node of interaction between nutlin-3 and the proteasome inhibitor carfilzomib. CRISPR/Cas9 gene editing experiments confirmed that ATF4, CHOP, and the BH3-only protein, NOXA, are all required for nutlin-3 and carfilzomib-induced apoptosis. Furthermore, activation of the unfolded protein response using tunicamycin and thapsigargin was sufficient to activate the ATF4/CHOP stress response axis and sensitize to nutlin-3. Finally, cell line and patient-derived xenograft models demonstrated combinatorial effects of treatment with idasanutlin and carfilzomib on liposarcoma growth in vivo. Together, these data indicate that targeting of the proteasome could improve the efficacy of MDM2 inhibitors in liposarcoma. SIGNIFICANCE: Targeting the proteasome in combination with MDM2 inhibition activates the ATF4/CHOP stress response axis to induce apoptosis in liposarcoma, providing a potential therapeutic approach for the most common soft-tissue sarcoma.

A genome-wide association study with tissue transcriptomics identifies genetic drivers for classic bladder exstrophy.

Classic bladder exstrophy represents the most severe end of all human congenital anomalies of the kidney and urinary tract and is associated with bladder cancer susceptibility. Previous genetic studies identified one locus to be involved in classic bladder exstrophy, but were limited to a restrict number of cohort. Here we show the largest classic bladder exstrophy genome-wide association analysis to date where we identify eight genome-wide significant loci, seven of which are novel. In these regions reside ten coding and four non-coding genes. Among the coding genes is EFNA1, strongly expressed in mouse embryonic genital tubercle, urethra, and primitive bladder. Re-sequence of EFNA1 in the investigated classic bladder exstrophy cohort of our study displays an enrichment of rare protein altering variants. We show that all coding genes are expressed and/or significantly regulated in both mouse and human embryonic developmental bladder stages. Furthermore, nine of the coding genes residing in the regions of genome-wide significance are differentially expressed in bladder cancers. Our data suggest genetic drivers for classic bladder exstrophy, as well as a possible role for these drivers to relevant bladder cancer susceptibility.

Application of Poly-L-Lysine for Tailoring Graphene Oxide Mediated Contact Formation between Lithium Titanium Oxide LTO Surfaces for Batteries.

When producing stable electrodes, polymeric binders are highly functional materials that are effective in dispersing lithium-based oxides such as Li4Ti5O12 (LTO) and carbon-based materials and establishing the conductivity of the multiphase composites. Nowadays, binders such as polyvinylidene fluoride (PVDF) are used, requiring dedicated recycling strategies due to their low biodegradability and use of toxic solvents to dissolve it. Better structuring of the carbon layers and a low amount of binder could reduce the number of inactive materials in the electrode. In this study, we use computational and experimental methods to explore the use of the poly amino acid poly-L-lysine (PLL) as a novel biodegradable binder that is placed directly between nanostructured LTO and reduced graphene oxide. Density functional theory (DFT) calculations allowed us to determine that the (111) surface is the most stable LTO surface exposed to lysine. We performed Kubo-Greenwood electrical conductivity (KGEC) calculations to determine the electrical conductivity values for the hybrid LTO-lysine-rGO system. We found that the presence of the lysine-based binder at the interface increased the conductivity of the interface by four-fold relative to LTO-rGO in a lysine monolayer configuration, while two-stack lysine molecules resulted in 0.3-fold (in the plane orientation) and 0.26-fold (out of plane orientation) increases. These outcomes suggest that monolayers of lysine would specifically favor the conductivity. Experimentally, the assembly of graphene oxide on poly-L-lysine-TiO2 with sputter-deposited titania as a smooth and hydrophilic model substrate was investigated using a layer-by-layer (LBL) approach to realize the required composite morphology. Characterization techniques such as X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), Kelvin probe force microscopy (KPFM), scanning electron microscopy (SEM) were used to characterize the formed layers. Our experimental results show that thin layers of rGO were assembled on the TiO2 using PLL. Furthermore, the PLL adsorbates decrease the work function difference between the rGO- and the non-rGO-coated surface and increased the specific discharge capacity of the LTO-rGO composite material. Further experimental studies are necessary to determine the influence of the PLL for aspects such as the solid electrolyte interface, dendrite formation, and crack formation.

Mannose-Binding Lectin (MBL) and Gap Junction Protein Alpha 4 (GJA4) Gene Heterogeneity in Relation to Severity of Clinical Disease in Cystic Fibrosis.

BACKGROUND: Recently, we provided evidence that a single nucleotide polymorphism (SNP), rs41266431, on the gap junction protein alpha 4 (GJA4) gene, acts as a modifier for clinical disease severity in patients with cystic fibrosis (CF). These features are very similar to those of variants of the mannose-binding lectin (MBL). This study aimed to clarify whether the clinical disease phenotype associated with GJA4 variants is independent of MBL variants. METHODS: One hundred and twelve patients with homozygous F508del (mean age, 27.6 years; m/f, 61/51) were recruited from the CF centers of Bonn, Frankfurt, and Amsterdam. A sequence analysis was performed for GJA4 and MBL. The clinical phenotype was assessed over three years using pulmonary function tests, body mass index, Pseudomonas aeruginosa colonization, diabetes mellitus, survival to end-stage lung disease, and inflammatory markers. RESULTS: A clinically relevant SNP of GJA4 was identified by sequence analysis. Pulmonary function (FVC% pred, mean 78/85; p < 0.055) and survival to end-stage lung disease were lower (p < 0.043) for this variant (rs41266431) in carriers homozygous for the G variant (n = 82/112; 73%) than in other carriers. Serum MBL (820/372 ng/mL, p < 0.001) was significantly higher in "MBL-sufficient" genotypes (n = 79/112; 71%) than in "MBL-insufficient" genotypes, and a trend for a significant difference in BMI percentiles (35.2/23.8; p < 0.059) was observed. For the MBL-sufficient genotype (median age at death, 38/26 years), there was a trend for better survival (p < 0.076). There was no augmentation by gene-gene interaction between MBL and GJA4 variants for any outcome parameter. CONCLUSIONS: The clinical disease phenotype associated with GJA4 variants is independent of MBL variants. MBL-sufficient variants were associated with superior BMI and a trend for better survival than MBL insufficient variants.

Diagnostic Performance of Point Shear Wave Elastography Using Acoustic Radiation Force Impulse Technology in Peripheral Pulmonary Consolidations: A Feasibility Study.

The aim of the study described here was to evaluate the diagnostic performance of lung ultrasound point shear wave elastography using acoustic radiation force impulse (ARFI) in peripheral pulmonary consolidations (PPCs). A total of 87 patients with PPCs diagnosed from April to December 2020 were included retrospectively in the study. The inclusion criteria were (i) a PPC >1 cm; (ii) valid ARFI measurements; and (iii) confirmation of the diagnosis of a PPC by histocytological examination and/or clinical and radiological follow-up. The presence of pleural effusions and mean ARFI velocities (MAVs) of PPCs were evaluated. To examine the MAV for potential cutoff values between benign and malignant PPCs, a receiver operating characteristic analysis was implemented. In total, 48 of 87 PPCs (55.2%) were accompanied by pleural effusions. Benign PPCs had significantly lower MAVs than malignant PPCs (1.82 ± 0.97 m/s vs. 3.05 ± 0.73 m/s, p < 0.001). Selecting 2.21 m/s as a cutoff value yielded a sensitivity and specificity of 89.7% and 75.9%, respectively, in diagnosing malignant PPCs (area under the curve = 0.852, 95% confidence interval: 0.773-0.931). In summary, ARFI elastography may be an additional non-invasive tool for differentiating benign from malignant PPCs. Furthermore, the feasibility of using ARFI elastography in PPCs associated with pleural effusions was proved. However, there is some degree of overlap between different disease entities, and diagnosis should always take into account the clinical background.

PPAR-Responsive Elements Enriched with Alu Repeats May Contribute to Distinctive PPARγ-DNMT1 Interactions in the Genome.

BACKGROUND: PPARγ (peroxisome proliferator-activated receptor gamma) is involved in the pathology of numerous diseases, including UM and other types of cancer. Emerging evidence suggests that an interaction between PPARγ and DNMTs (DNA methyltransferase) plays a role in cancer that is yet to be defined. METHODS: The configuration of the repeating elements was performed with CAP3 and MAFFT, and the structural modelling was conducted with HDOCK. An evolutionary action scores algorithm was used to identify oncogenic variants. A systematic bioinformatic appraisal of PPARγ and DNMT1 was performed across 29 tumor types and UM available in The Cancer Genome Atlas (TCGA). RESULTS: PPAR-responsive elements (PPREs) enriched with Alu repeats are associated with different genomic regions, particularly the promotor region of DNMT1. PPARγ-DNMT1 co-expression is significantly associated with several cancers. C-terminals of PPARγ and DNMT1 appear to be the potential protein-protein interaction sites where disease-specific mutations may directly impair the respective protein functions. Furthermore, PPARγ expression could be identified as an additional prognostic marker for UM. CONCLUSIONS: We hypothesize that the function of PPARγ requires an additional contribution of Alu repeats which may directly influence the DNMT1 network. Regarding UM, PPARγ appears to be an additional discriminatory prognostic marker, in particular in disomy 3 tumors.

Impact of a Gap Junction Protein Alpha 4 Variant on Clinical Disease Phenotype in F508del Homozygous Patients With Cystic Fibrosis.

BACKGROUND: Lung disease phenotype varies widely even in the F508del (homozygous) genotype. Leukocyte-driven inflammation is important for pulmonary disease pathogenesis in cystic fibrosis (CF). Blood cytokines correlate negatively with pulmonary function in F508del homozygous patients, and gap junction proteins (GJA) might be related to the influx of blood cells into the lung and influence disease course. We aimed to assess the relationship between GJA1/GJA4 genotypes and the clinical disease phenotype. METHODS: One-hundred-and-sixteen homozygous F508del patients (mean age 27 years, m/f 66/50) were recruited from the CF centers of Bonn, Frankfurt, and Amsterdam. Sequence analysis was performed for GJA1 and GJA4. The clinical disease course was assessed over 3 years using pulmonary function tests, body mass index, Pseudomonas aeruginosa colonization, diabetes mellitus, survival to end-stage lung disease, blood and sputum inflammatory markers. RESULTS: Sequence analysis revealed one clinically relevant single nucleotide polymorphism. In this GJA4 variant (rs41266431), homozygous G variant carriers (n = 84/116; 72.4%) had poorer pulmonary function (FVC% pred: mean 78/86, p < 0.040) and survival to end-stage lung disease was lower (p < 0.029). The frequency of P. aeruginosa colonization was not influenced by the genotype, but in those chronically colonized, those with the G/G genotype had reduced pulmonary function (FVC% pred: mean 67/80, p < 0.049). Serum interleukin-8 (median: 12.4/6.7 pg/ml, p < 0.052) and sputum leukocytes (2305/437.5 pg/ml, p < 0.025) were higher for the G/G genotype. CONCLUSIONS: In carriers of the A allele (27.6%) the GJA4 variant is associated with significantly better protection against end-stage lung disease and superior pulmonary function test results in F508del homozygous patients. This SNP has the potential of a modifier gene for phenotyping severity of CF lung disease, in addition to the CFTR genotype. CLINICAL TRIAL REGISTRATION: The study was registered with ClinicalTrials.gov, number NCT04242420, retrospectively on January 24th, 2020.

A context-dependent bifurcation in the Pointed transcriptional effector network contributes specificity and robustness to retinal cell fate acquisition.

Spatiotemporally precise and robust cell fate transitions, which depend on specific signaling cues, are fundamental to the development of appropriately patterned tissues. The fidelity and precision with which photoreceptor fates are recruited in the Drosophila eye exemplifies these principles. The fly eye consists of a highly ordered array of ~750 ommatidia, each of which contains eight distinct photoreceptors, R1-R8, specified sequentially in a precise spatial pattern. Recruitment of R1-R7 fates requires reiterative receptor tyrosine kinase / mitogen activated protein kinase (MAPK) signaling mediated by the transcriptional effector Pointed (Pnt). However the overall signaling levels experienced by R2-R5 cells are distinct from those experienced by R1, R6 and R7. A relay mechanism between two Pnt isoforms initiated by MAPK activation directs the universal transcriptional response. Here we ask how the generic Pnt response is tailored to these two rounds of photoreceptor fate transitions. We find that during R2-R5 specification PntP2 is coexpressed with a closely related but previously uncharacterized isoform, PntP3. Using CRISPR/Cas9-generated isoform specific null alleles we show that under otherwise wild type conditions, R2-R5 fate specification is robust to loss of either PntP2 or PntP3, and that the two activate pntP1 redundantly; however under conditions of reduced MAPK activity, both are required. Mechanistically, our data suggest that intrinsic activity differences between PntP2 and PntP3, combined with positive and unexpected negative transcriptional auto- and cross-regulation, buffer first-round fates against conditions of compromised RTK signaling. In contrast, in a mechanism that may be adaptive to the stronger signaling environment used to specify R1, R6 and R7 fates, the Pnt network resets to a simpler topology in which PntP2 uniquely activates pntP1 and auto-activates its own transcription. We propose that differences in expression patterns, transcriptional activities and regulatory interactions between Pnt isoforms together facilitate context-appropriate cell fate specification in different signaling environments.

SLC20A1 Is Involved in Urinary Tract and Urorectal Development.

Previous studies in developing Xenopus and zebrafish reported that the phosphate transporter slc20a1a is expressed in pronephric kidneys. The recent identification of SLC20A1 as a monoallelic candidate gene for cloacal exstrophy further suggests its involvement in the urinary tract and urorectal development. However, little is known of the functional role of SLC20A1 in urinary tract development. Here, we investigated this using morpholino oligonucleotide knockdown of the zebrafish ortholog slc20a1a. This caused kidney cysts and malformations of the cloaca. Moreover, in morphants we demonstrated dysfunctional voiding and hindgut opening defects mimicking imperforate anus in human cloacal exstrophy. Furthermore, we performed immunohistochemistry of an unaffected 6-week-old human embryo and detected SLC20A1 in the urinary tract and the abdominal midline, structures implicated in the pathogenesis of cloacal exstrophy. Additionally, we resequenced SLC20A1 in 690 individuals with bladder exstrophy-epispadias complex (BEEC) including 84 individuals with cloacal exstrophy. We identified two additional monoallelic de novo variants. One was identified in a case-parent trio with classic bladder exstrophy, and one additional novel de novo variant was detected in an affected mother who transmitted this variant to her affected son. To study the potential cellular impact of SLC20A1 variants, we expressed them in HEK293 cells. Here, phosphate transport was not compromised, suggesting that it is not a disease mechanism. However, there was a tendency for lower levels of cleaved caspase-3, perhaps implicating apoptosis pathways in the disease. Our results suggest SLC20A1 is involved in urinary tract and urorectal development and implicate SLC20A1 as a disease-gene for BEEC.

A Metabolomics Approach to Screening for Autism Risk in the Children's Autism Metabolome Project.

Autism spectrum disorder (ASD) is biologically and behaviorally heterogeneous. Delayed diagnosis of ASD is common and problematic. The complexity of ASD and the low sensitivity of available screening tools are key factors in delayed diagnosis. Identification of biomarkers that reduce complexity through stratification into reliable subpopulations can assist in earlier diagnosis, provide insight into the biology of ASD, and potentially suggest targeted interventions. Quantitative metabolomic analysis was performed on plasma samples from 708 fasting children, aged 18 to 48 months, enrolled in the Children's Autism Metabolome Project (CAMP). The primary goal was to identify alterations in metabolism helpful in stratifying ASD subjects into subpopulations with shared metabolic phenotypes (i.e., metabotypes). Metabotypes associated with ASD were identified in a discovery set of 357 subjects. The reproducibility of the metabotypes was validated in an independent replication set of 351 CAMP subjects. Thirty-four candidate metabotypes that differentiated subsets of ASD from typically developing participants were identified with sensitivity of at least 5% and specificity greater than 95%. The 34 metabotypes formed six metabolic clusters based on ratios of either lactate or pyruvate, succinate, glycine, ornithine, 4-hydroxyproline, or α-ketoglutarate with other metabolites. Optimization of a subset of new and previously defined metabotypes into a screening battery resulted in 53% sensitivity (95% confidence interval [CI], 48%-57%) and 91% specificity (95% CI, 86%-94%). Thus, our metabolomic screening tool detects more than 50% of the autistic participants in the CAMP study. Further development of this metabolomic screening approach may facilitate earlier referral and diagnosis of ASD and, ultimately, more targeted treatments. LAY SUMMARY: Analysis of a selected set of metabolites in blood samples from children with autism and typically developing children identified reproducible differences in the metabolism of about half of the children with autism. Testing for these differences in blood samples can be used to help screen children as young as 18 months for risk of autism that, in turn, can facilitate earlier diagnoses. In addition, differences may lead to biological insights that produce more precise treatment options. We are exploring other blood-based molecules to determine if still a higher percentage of children with autism can be detected using this strategy. Autism Res 2020, 13: 1270-1285. © 2020 The Authors. Autism Research published by International Society for Autism Research published by Wiley Periodicals LLC.

Ubiquitin Carboxyl-Terminal Hydrolases (UCHs): Potential Mediators for Cancer and Neurodegeneration.

Emerging evidence suggests an inverse association between cancer and neurodegenerative diseases (NDD). Although phenotypically different, both diseases display a significant imbalance in the ubiquitination/deubiquitination processes. Therefore, we particularly investigated the expression of ubiquitin C-terminal hydrolases (UCHs: UCH-L1, UCH-L3, UCH-L5 and BAP1), a subfamily of deubiquitinating enzymes (DUBs), using publically available datasets (GTEx, TCGA) and observed altered expression of UCH-L1, UCH-L3, UCH-L5 in 17 cancer types. Interestingly, UCH-L1 (known to be enriched in neurons and interacting with the Parkinson's disease-associated protein α-synuclein) appeared to be a prognostic indicator of unfavorable outcome in endometrial and urothelial cancer, while increased expression of UCH-L3 and UCH-L5 was associated with poor survival in liver and thyroid cancer, respectively. In normal tissues, UCH-L1 was found to be strongly expressed in the cerebral cortex and hypothalamus, while UCH-L3 expression was somewhat higher in the testis. The occurrence of mutation rates in UCHs also suggests that BAP1 and UCH-L5 may play a more dominant role in cancers than UCH-L1 and UCH-L3. We also characterized the functional context and configuration of the repeat elements in the promoter of DUBs genes and found that UCHs are highly discriminatory for catabolic function and are mainly enriched with LINE/CR1 repeats. Regarding the thesis of an inverse association between cancer and NDD, we observed that among all DUBs, UCHs are the one most involved in both entities. Considering a putative therapeutic potential based on presumed common mechanisms, it will be useful to determine whether other DUBs can compensate for the loss of UCH activity under physiological conditions. However, experimental evidence is required to substantiate this argument.

A case of Type 1 Dent disease presenting with isolated persistent proteinuria.

Dent disease is a rare X-linked recessive tubular disorder, characterized by the triad of low molecular-weight proteinuria, hypercalciuria, nephrocalcinosis and/or nephrolithiasis. It is caused by mutations in the CLCN5 gene or OCRL gene. Thirty to 80% of affected males develop end-stage kidney disease between the ages of 30 and 50 years. Some children were reported to present with isolated persistent proteinuria and a part of these patients were diagnosed as having focal segmental glomerulosclerosis with kidney biopsy. Although there is no specific treatment, treatment of proteinuria and hypercalciuria is thought to delay the progression of the disease. For this reason, awareness of the disease findings and early diagnosis are important. In this case report, we present a boy followed-up with isolated persistent proteinuria and then diagnosed as having Dent disease with mutation analysis that showed c.328_330delT (p.Phe110Trpfs27*) in the CLCN5 gene. The importance of researching low-molecular- weight proteinuria and considering Dent disease in the differential diagnosis of children presenting with isolated persistent proteinuria has been emphasized.

Mutational Landscape of the BAP1 Locus Reveals an Intrinsic Control to Regulate the miRNA Network and the Binding of Protein Complexes in Uveal Melanoma.

The BAP1 (BRCA1-associated protein 1) gene is associated with a variety of human cancers. With its gene product being a nuclear ubiquitin carboxy-terminal hydrolase with deubiquitinase activity, BAP1 acts as a tumor suppressor gene with potential pleiotropic effects in multiple tumor types. Herein, we focused specifically on uveal melanoma (UM) in which BAP1 mutations are associated with a metastasizing phenotype and decreased survival rates. We identified the ubiquitin carboxyl hydrolase (UCH) domain as a major hotspot region for the pathogenic mutations with a high evolutionary action (EA) score. This also includes the mutations at conserved catalytic sites and the ones overlapping with the phosphorylation residues. Computational protein interaction studies revealed that distant BAP1-associated protein complexes (FOXK2, ASXL1, BARD1, BRCA1) could be directly impacted by this mutation paradigm. We also described the conformational transition related to BAP1-BRCA-BARD1 complex, which may pose critical implications for mutations, especially at the docking interfaces of these three proteins. The mutations affect - independent of being somatic or germline - the binding affinity of miRNAs embedded within the BAP1 locus, thereby altering the unique regulatory network. Apart from UM, BAP1 gene expression and survival associations were found to be predictive for the prognosis in several (n = 29) other cancer types. Herein, we suggest that although BAP1 is conceptually a driver gene in UM, it might contribute through its interaction partners and its regulatory miRNA network to various aspects of cancer. Taken together, these findings will pave the way to evaluate BAP1 in a variety of other human cancers with a shared mutational spectrum.

Frequent detection of Saffold cardiovirus in adenoids.

Saffold virus (SAFV) is classified into the Cardiovirus genus of the Picornaviridae family. Up to now, eleven genotypes have been identified however, their clinical significance remains unclear. Here, we investigated the presence of SAFV in asymptomatic patients admitted for adenoidectomy. A total of 70 adenoid tissue samples were collected from children with clinical symptoms caused by hypertrophy of adenoids but without symptoms of airway infection. Samples were investigated for SAFV by RT-nested PCR and sequence analysis. Eleven of 70 (15.7%) samples were positive for SAFV. Nasopharyngeal swabs were available from 45 children just before surgery. SAFV was rarely found and only in children with SAFV-positive adenoids 2/8. Our findings indicate that the presence of SAFV seems to be more frequent in adenoid tissue than expected. This could support the notion of a longer than previously anticipated persistence of SAFV nucleic acids in the respiratory tract and possibly a chronic infection. Further investigations are necessary to establish the role of SAFV infection in humans.

Towards a Central Role of ISL1 in the Bladder Exstrophy⁻Epispadias Complex (BEEC): Computational Characterization of Genetic Variants and Structural Modelling.

Genetic factors play a critical role in the development of human diseases. Recently, several molecular genetic studies have provided multiple lines of evidence for a critical role of genetic factors in the expression of human bladder exstrophy-epispadias complex (BEEC). At this point, ISL1 (ISL LIM homeobox 1) has emerged as the major susceptibility gene for classic bladder exstrophy (CBE), in a multifactorial disease model. Here, GWAS (Genome wide association studies) discovery and replication studies, as well as the re-sequencing of ISL1, identified sequence variants (rs9291768, rs6874700, c.137C > G (p.Ala46Gly)) associated with CBE. Here, we aimed to determine the molecular and functional consequences of these sequence variants and estimate the dependence of ISL1 protein on other predicted candidates. We used: (i) computational analysis of conserved sequence motifs to perform an evolutionary conservation analysis, based on a Bayesian algorithm, and (ii) computational 3D structural modeling. Furthermore, we looked into long non-coding RNAs (lncRNAs) residing within the ISL1 region, aiming to predict their targets. Our analysis suggests that the ISL1 protein specific N-terminal LIM domain (which harbors the variant c.137C>G), limits its transcriptional ability, and might interfere with ISL1-estrogen receptor α interactions. In conclusion, our analysis provides further useful insights about the ISL1 gene, which is involved in the formation of the BEEC, and in the development of the urinary bladder.

ΔNp63α Suppresses TGFB2 Expression and RHOA Activity to Drive Cell Proliferation in Squamous Cell Carcinomas.

The transcriptional repressor ΔNp63α is a potent oncogene widely overexpressed in squamous cell carcinomas (SCCs) of diverse tissue origins, where it promotes malignant cell proliferation and survival. We report here the results of a genome-wide CRISPR screen to identify pathways controlling ΔNp63α-dependent cell proliferation, which revealed that the small GTPase RHOA blocks cell division upon ΔNp63α knockdown. After ΔNp63α depletion, RHOA activity is increased, and cells undergo RHOA-dependent proliferation arrest along with transcriptome changes indicative of increased TGF-ß signaling. Mechanistically, ΔNp63α represses transcription of TGFB2, which induces a cell cycle arrest that is partially dependent on RHOA. Ectopic TGFB2 activates RHOA and impairs SCC proliferation, and TGFB2 neutralization restores cell proliferation during ΔNp63α depletion. Genomic data from tumors demonstrate inactivation of RHOA and the TGFBR2 receptor and ΔNp63α overexpression in more than 80% of lung SCCs. These results reveal a signaling pathway controlling SCC proliferation that is potentially amenable to pharmacological intervention.

Autophagy Inhibition Mediates Apoptosis Sensitization in Cancer Therapy by Relieving FOXO3a Turnover.

Macroautophagy (autophagy) is intimately linked with cell death and allows cells to evade apoptosis. This has prompted clinical trials to combine autophagy inhibitors with other drugs with the aim of increasing the likelihood of cancer cells dying. However, the molecular basis for such effects is unknown. Here, we describe a transcriptional mechanism that connects autophagy to apoptosis. The autophagy-regulating transcription factor, FOXO3a, is itself turned over by basal autophagy creating a potential feedback loop. Increased FOXO3a upon autophagy inhibition stimulates transcription of the pro-apoptotic BBC3/PUMA gene to cause apoptosis sensitization. This mechanism explains how autophagy inhibition can sensitize tumor cells to chemotherapy drugs and allows an autophagy inhibitor to change the action of an MDM2-targeted drug from growth inhibition to apoptosis, reducing tumor burden in vivo. Thus, a link between two processes mediated via a single transcription factor binding site in the genome can be leveraged to improve anti-cancer therapies.

Featured Article: Modulation of the OGF-OGFr pathway alters cytokine profiles in experimental autoimmune encephalomyelitis and multiple sclerosis.

The endogenous neuropeptide opioid growth factor, chemically termed [Met5]-enkephalin, has growth inhibitory and immunomodulatory properties. Opioid growth factor is distributed widely throughout most tissues, is autocrine and paracrine produced, and interacts at the nuclear-associated receptor, OGFr. Serum levels of opioid growth factor are decreased in patients with multiple sclerosis and in animals with experimental autoimmune encephalomyelitis suggesting that the OGF-OGFr pathway becomes dysregulated in this disease. This study begins to assess other cytokines that are altered following opioid growth factor or low-dose naltrexone modulation of the OGF-OGFr axis in mice with experimental autoimmune encephalomyelitis using serum samples collected in mice treated for 10 or 20 days and assayed by a multiplex cytokine assay for inflammatory markers. Cytokines of interest were validated in mice at six days following immunization for experimental autoimmune encephalomyelitis. In addition, selected cytokines were validated with serum from MS patients treated with low-dose naltrexone alone or low-dose naltrexone in combination with glatiramer acetate (Copaxone®). Experimental autoimmune encephalomyelitis mice had elevated levels of 7 of 10 cytokines. Treatment with opioid growth factor or low-dose naltrexone resulted in elevated expression levels of the IL-6 cytokine, and significantly reduced IL-10 values, relative to saline-treated experimental autoimmune encephalomyelitis mice. TNF-γ values were increased in experimental autoimmune encephalomyelitis mice relative to normal, but were not altered by opioid growth factor or low-dose naltrexone. IFN-γ levels were reduced in opioid growth factor- or low-dose naltrexone-treated experimental autoimmune encephalomyelitis mice relative to saline-treated mice at 10 days, and elevated relative to normal values at 20 days. Validation studies revealed that within six days of immunization, opioid growth factor or low-dose naltrexone modulated IL-6 and IL-10 cytokine expression. Validation in human serum revealed markedly reduced IL-6 cytokine levels in MS patients taking low-dose naltrexone relative to standard care. In summary, modulation of the OGF-OGFr pathway regulates some inflammatory cytokines, and together with opioid growth factor serum levels, may begin to form a panel of valid biomarkers to monitor progression of multiple sclerosis and response to therapy. Impact statement Modulation of the opioid growth factor (OGF)-OGF receptor (OGFr) alters inflammatory cytokine expression in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). Multiplex cytokine assays demonstrated that mice with chronic EAE and treated with either OGF or low-dose naltrexone (LDN) had decreased expression of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and the anti-inflammatory cytokine IL-10 within 10 days or treatment, as well as increased serum expression of the pro-inflammatory cytokine IL-6, relative to immunized mice receiving saline. Multiplex data were validated using ELISA kits and serum from MS patients treated with LDN and revealed decreased in IL-6 levels in patients taking LDN relative to standard care alone. These data, along with serum levels of OGF, begin to formulate a selective biomarker profile for MS that is easily measured and effective at monitoring disease progression and response to therapy.