Single-cell tumor-immune microenvironment of BRCA1/2 mutated high-grade serous ovarian cancer.

The majority of high-grade serous ovarian cancers (HGSCs) are deficient in homologous recombination (HR) DNA repair, most commonly due to mutations or hypermethylation of the BRCA1/2 genes. We aimed to discover how BRCA1/2 mutations shape the cellular phenotypes and spatial interactions of the tumor microenvironment. Using a highly multiplex immunofluorescence and image analysis we generate spatial proteomic data for 21 markers in 124,623 single cells from 112 tumor cores originating from 31 tumors with BRCA1/2 mutation (BRCA1/2mut), and from 13 tumors without alterations in HR genes. We identify a phenotypically distinct tumor microenvironment in the BRCA1/2mut tumors with evidence of increased immunosurveillance. Importantly, we report a prognostic role of a proliferative tumor-cell subpopulation, which associates with enhanced spatial tumor-immune interactions by CD8+ and CD4 + T-cells in the BRCA1/2mut tumors. The single-cell spatial landscapes indicate distinct patterns of spatial immunosurveillance with the potential to improve immunotherapeutic strategies and patient stratification in HGSC.

Development of highly selective SH3 binding peptides for Crk and CRKL which disrupt Crk-complexes with DOCK180, SoS and C3G.

Many Src Homology 3 (SH3) domains function as molecular adhesives in intracellular signal transduction. Based on previous ultrastructural studies, short motifs which bind to the first SH3 domains of the adapters Crk and CRKL were selectively mutagenised to generate Crk/CRKL SH3-binding peptides of very high affinity and selectivity. Affinities were increased up to 20-fold compared to the best wildtype sequences, while the selectivity against a similar SH3 domain [Grb2SH3(N)] was not only retained, but sometimes increased. Blot techniques with GST-fusion peptides and in solution precipitation assays with biotinylated high affinity Crk binding peptides (HACBPs) were subsequently used to analyse the binding of these sequences to a large panel of SH3 domain-containing fusion proteins. Only those proteins which contained the CrkSH3(1) or CRKLSH3(1) domains bound efficiently to the HACBPs. A GST-HACBP fusion protein precipitated Crk and CRKL proteins out of 35S-labelled and unlabelled cell lysates. Very little binding of other cellular proteins to HACBP was detectable, indicative of a great preference for Crk and CRKL when compared to the wide variety of other endogenous cellular proteins. Moreover, HACBP disrupted in vitro preexisting Crk-complexes with DOCK180 and the exchange factors SoS and C3G, which are known targets of Crk adapters, in a concentration dependent manner. HACBP-based molecules should therefore be useful as highly selective inhibitors of intracellular signalling processes involving Crk and CRKL.

The effects of noise in transient EOAE newborn hearing screening.

The use of transient evoked otoacoustic emissions (TEOAEs) has been advocated as the first stage entry level technique for universal newborn hearing screening. To date, the majority of TEOAE infant testing has been conducted under controlled noise conditions; i.e., acoustically treated sound suites. As a result, previously reported TEOAE evaluations may not realistically represent test outcomes in actual hospital screening settings. The purpose of this study was to compare the results of TEOAEs with auditory brainstem response (ABR) hearing screening in a hospital environment where noise conditions do not meet the same ambient noise specifications as those found in sound rooms. A total of 119 stable newborns (67 high risk, 52 normal) ranging in post-conceptual age (PCA) from 33 to 41 weeks received both the ABR and TEOAE screening protocols. Testing was conducted at crib side in either the well baby nursery or the neonatal special care unit (NSCU). Newborn ABR screening failed 8 (3.8%) of 224 ears, whereas TEOAE testing failed 85 (38.4%) and could not test another 22 (9.8%) ears. That is, only 117 (52.2%) of the 224 ears passed the TEOAE test. Using the ABR as the reference test the specificity and sensitivity for TEOAE was 52% and 50%, respectively. Noise levels measured by the probe microphone within the ear canal exceeded those levels (30 dBA SPL) recommended for TEOAE newborn hearing screening. Results of this study suggest that under realistic hearing screening test conditions, TEOAE results may be influenced by the level of noise in the testing environment. Whereas significant advances have been attained in TEOAE measurement during the past decade, clinical evidence supports the need for continued research aimed at solving problems before this technique can be used efficiently for newborn screening.

Follow-up services in newborn hearing screening programs.

Newborn hearing screening programs have gained wide acceptance as a means of identifying infants at risk for hearing loss. For the most part, the auditory brainstem response (ABR) technique has been the measurement tool universally adopted in the evaluation of high-risk infants. Over the years, the ABR has been used successfully with a negligible false-negative rate. Unfortunately, program follow-up services have not received similar attention, and there is a lack in program development. This article describes a series of follow-up measures that include the use of a questionnaire sent to the parents/caregivers of 401 infants who pass either the initial or retest ABR screen. A total of 262 (65%) response questionnaires were returned. The results of the questionnaire and recommendations regarding follow-up services are discussed.

Automated and conventional ABR screening techniques in high-risk infants.

Test validity is determined by the proportion of results that are diagnostically confirmed and predicted on the measures used to identify the disease process. This article summarizes the results of a series of 224 stable high-risk infants who were screened by automated (ALGO-1) and conventional (Bio-logics LT) ABR instrumentation. Failure criteria was defined as the absence or prolongation of a replicable wave V response (conventional) or Refer by the automated system. The overall failure rates at a 35 dB screening level were comparable between devices. Sensitivity and specificity measures for the ALGO-1 unit were 100 and 96 percent, respectively. Permanent hearing loss was demonstrated in 5 percent of the newborns screened in this study. Advantages of the automated system include a dual artifact rejection system, attenuating ear couplers, and a battery operated design. These findings suggest that the automated ABR screener is a viable alternative to conventional ABR instrumentation for the limited purpose of neonatal auditory screening.

Hearing loss in prison inmates.

The incidence of hearing disorders in 34 State Penitentiary prison inmates all with a previous history of drug abuse were investigated. Subjects were evaluated using routine pure-tone air conduction audiometry, immittance measures, and short-latency auditory brain stem responses. Of the 34 inmates, 20 (58.8%) demonstrated normal bilateral peripheral hearing sensitivity, whereas 10 (29.4%) inmates presented with some degree of hearing impairment. In addition, the conflicting results of elevated pure-tone thresholds with normal immittance measures and normal ABR findings suggested that four (11.8%) subjects exhibited functional hearing loss. The results of this study support the reported high incidence of hearing loss in the prison population. The synergistic effects of drug abuse, noise exposure, and head trauma as possible contributing factors are discussed.