Molecular methods for the identification of Aspergillus species

Invasive aspergillosis (IA) is a leading cause of morbidity and mortality in immunocompromised hosts. In some institutions, species of Aspergillus less susceptible to amphotericin B than Aspergillus fumigatus are becoming more common, making an accurate identification of species important. However, species identification has traditionally relied on macroscopic colony characteristics and microscopic morphology, which may require several days of culture. Additional sub-culturing on specialized media may be required to induce conidia formation; in some cases conidia may never form, confounding identification. Therefore, rapid, nucleic acid-based methods that identify species of Aspergillus independent of morphology are now being developed to augment or replace phenotypic identification methods. The most successful methods to date have employed polymerase chain reaction (PCR) amplification of target sequences within the ribosomal RNA gene complex, including the 28S ribosomal subunit (D1-D2 region) and the internal transcribed spacers 1 and 2 (ITS1 and ITS2 regions). We therefore developed a PCR-based assay to differentiate medically important species of Aspergillus from one another, and from other opportunistic moulds and yeasts, by employing universal, pan-fungal primers directed to conserved ribosomal genes and species-specific DNA probes directed to the highly variable ITS2 region. Amplicons were then detected in a simple, colorimetric enzyme immunoassay format (PCR-EIA). DNA sequencing of the ITS1 and ITS2 regions and of the D1-D2 region was also conducted for the differentiation of species by comparative GenBank sequence analysis. The PCR-EIA method was found to be rapid, sensitive, and specific for the identification and differentiation of the most medically important species of Aspergillus. In addition, methods to identify species of Aspergillus by comparative GenBank sequence analysis were found to be more reliable using the ITS1 and ITS2 regions than the D1-D2 regio

Search for the exotic Ξ−−(1860)\Xi^{--}(1860) Resonance in 340GeV/c Σ−\Sigma^--Nucleus Interactions

We report on a high statistics search for the $\Xi^{--}(1860)$ resonance in $\Sigma^-$-nucleus collisions at 340GeV/c. No evidence for this resonance is found in our data sample which contains 676000 $\Xi^-$ candidates above background. For the decay channel $\Xi^{--}(1860) \to \Xi^-\pi^-$ and the kinematic range 0.15$<x_F<$0.9 we find a 3$\sigma$ upper limit for the production cross section of 3.1 and 3.5 $\mu$b per nucleon for reactions with carbon and copper, respectively.Comment: 5 pages, 4 figures, modification of ref. 43 and 4

Tumor Heterogeneity of Fibroblast Growth Factor Receptor 3 (FGFR3) Mutations in Invasive Bladder Cancer: Implications for Peri-Operative anti-FGFR3 Treatment

Background: Fibroblast growth factor receptor 3 (FGFR3) is an actionable target in bladder cancer. Preclinical studies show that anti-FGFR3 treatment slows down tumor growth, suggesting that this tyrosine kinase receptor is a candidate for personalized bladder cancer treatment, particularly in patients with mutated FGFR3. We addressed tumor heterogeneity in a large multicenter, multi-laboratory study, as this may have significant impact on therapeutic response. Patients: and methods We evaluated possible FGFR3 heterogeneity by the PCR-SNaPshot method in the superficial and deep compartments of tumors obtained by transurethral resection (TUR, n = 61) and in radical cystectomy (RC, n = 614) specimens and corresponding cancer-positive lymph nodes (LN+, n = 201).Results: We found FGFR3 mutations in 13/34 (38%) T1 and 8/27 (30%) ≥T2-TUR samples, with 100% concordance between superficial and deeper parts in T1-TUR samples. Of eight FGFR3 mutant ≥T2-TUR samples, only 4 (50%) displayed the mutation in the deeper part. We found 67/614 (11%) FGFR3 mutations in RC specimens. FGFR3 mutation was associated with pN0 (P < 0.001) at RC. In 10/201 (5%) LN+, an FGFR3 mutation was found, all concordant with the corresponding RC specimen. In the remaining 191 cases, RC and LN+ were both wild type.Conclusions: FGFR3 mutation status seems promising to guide decision-making on adjuvant anti-FGFR3 therapy as it appeared homogeneous in RC and LN+. Based on the results of TUR, the deep part of the tumor needs to be assessed if neoadjuvant anti-FGFR3 treatment is considered. We conclude that studies on the heterogeneity of actionable molecular targets should precede clinical trials with these drugs in the perioperative setting

DLG4-related synaptopathy: a new rare brain disorder

PURPOSE: Postsynaptic density protein-95 (PSD-95), encoded by DLG4, regulates excitatory synaptic function in the brain. Here we present the clinical and genetic features of 53 patients (42 previously unpublished) with DLG4 variants.METHODS: The clinical and genetic information were collected through GeneMatcher collaboration. All the individuals were investigated by local clinicians and the gene variants were identified by clinical exome/genome sequencing.RESULTS: The clinical picture was predominated by early onset global developmental delay, intellectual disability, autism spectrum disorder, and attention deficit-hyperactivity disorder, all of which point to a brain disorder. Marfanoid habitus, which was previously suggested to be a characteristic feature of DLG4-related phenotypes, was found in only nine individuals and despite some overlapping features, a distinct facial dysmorphism could not be established. Of the 45 different DLG4 variants, 39 were predicted to lead to loss of protein function and the majority occurred de novo (four with unknown origin). The six missense variants identified were suggested to lead to structural or functional changes by protein modeling studies.CONCLUSION: The present study shows that clinical manifestations associated with DLG4 overlap with those found in other neurodevelopmental disorders of synaptic dysfunction; thus, we designate this group of disorders as DLG4-related synaptopathy.Genetics of disease, diagnosis and treatmen

Histone H3.3 beyond cancer: Germline mutations in Histone 3 Family 3A and 3B cause a previously unidentified neurodegenerative disorder in 46 patients

Although somatic mutations in Histone 3.3 (H3.3) are well-studied drivers of oncogenesis, the role of germline mutations remains unreported. We analyze 46 patients bearing de novo germline mutations in histone 3 family 3A (H3F3A) or H3F3B with progressive neurologic dysfunction and congenital anomalies without malignancies. Molecular modeling of all 37 variants demonstrated clear disruptions in interactions with DNA, other histones, and histone chaperone proteins. Patient histone posttranslational modifications (PTMs) analysis revealed notably aberrant local PTM patterns distinct from the somatic lysine mutations that cause global PTM dysregulation. RNA sequencing on patient cells demonstrated up-regulated gene expression related to mitosis and cell division, and cellular assays confirmed an increased proliferative capacity. A zebrafish model showed craniofacial anomalies and a defect in Foxd3-derived glia. These data suggest that the mechanism of germline mutations are distinct from cancer-associated somatic histone mutations but may converge on control of cell proliferation