Monitoring of the primary drying of a lyophilization process in vials

An innovative and modular system (LyoMonitor) for monitoring the primary drying of a lyophilization process in vials is illustrated: it integrates some commercial devices (pressure gauges, moisture sensor and mass spectrometer), an innovative balance and a manometric temperature measurement system based on an improved algorithm (DPE) to estimate sublimating interface temperature and position, product temperature profile, heat and mass transfer coefficients. A soft-sensor using a multipoint wireless thermometer can also estimate the previous parameters in a large number of vials. The performances of the previous devices for the determination of the end of the primary drying are compared. Finally, all these sensors can be used for control purposes and for the optimization of the process recipe; the use of DPE in a control loop will be shown as an exampl

Expert consensus guidelines for the genetic diagnosis of Alport syndrome

Recent expert guidelines recommend genetic testing for the diagnosis of Alport syndrome. Here, we describe current best practice and likely future developments. In individuals with suspected Alport syndrome, all three COL4A5, COL4A3 and COL4A4 genes should be examined for pathogenic variants, probably by high throughput-targeted next generation sequencing (NGS) technologies, with a customised panel for simultaneous testing of the three Alport genes. These techniques identify up to 95% of pathogenic COL4A variants. Where causative pathogenic variants cannot be demonstrated, the DNA should be examined for deletions or insertions by re-examining the NGS sequencing data or with multiplex ligation-dependent probe amplification (MLPA). These techniques identify a further 5% of variants, and the remaining few changes include deep intronic splicing variants or cases of somatic mosaicism. Where no pathogenic variants are found, the basis for the clinical diagnosis should be reviewed. Genes in which mutations produce similar clinical features to Alport syndrome (resulting in focal and segmental glomerulosclerosis, complement pathway disorders, MYH9-related disorders, etc.) should be examined. NGS approaches have identified novel combinations of pathogenic variants in Alport syndrome. Two variants, with one in COL4A3 and another in COL4A4, produce a more severe phenotype than an uncomplicated heterozygous change. NGS may also identify further coincidental pathogenic variants in genes for podocyte-expressed proteins that also modify the phenotype. Our understanding of the genetics of Alport syndrome is evolving rapidly, and both genetic and non-genetic factors are likely to contribute to the observed phenotypic variability

Clinical and molecular characterization of COVID-19 hospitalized patients

Clinical and molecular characterization by Whole Exome Sequencing (WES) is reported in 35 COVID-19 patients attending the University Hospital in Siena, Italy, from April 7 to May 7, 2020. Eighty percent of patients required respiratory assistance, half of them being on mechanical ventilation. Fiftyone percent had hepatic involvement and hyposmia was ascertained in 3 patients. Searching for common genes by collapsing methods against 150 WES of controls of the Italian population failed to give straightforward statistically significant results with the exception of two genes. This result is not unexpected since we are facing the most challenging common disorder triggered by environmental factors with a strong underlying heritability (50%). The lesson learned from Autism-Spectrum-Disorders prompted us to re-analyse the cohort treating each patient as an independent case, following a Mendelian-like model. We identified for each patient an average of 2.5 pathogenic mutations involved in virus infection susceptibility and pinpointing to one or more rare disorder(s). To our knowledge, this is the first report on WES and COVID-19. Our results suggest a combined model for COVID-19 susceptibility with a number of common susceptibility genes which represent the favorite background in which additional host private mutations may determine disease progression

Array comparative genomic hybridization in retinoma and retinoblastoma tissues

In retinoblastoma, two RB1 mutations are necessary for tumor development. Recurrent genomic rearrangements may represent subsequent events required for retinoblastoma progression. Array-comparative genomic hybridization was carried out in 18 eye samples, 10 from bilateral and eight from unilateral retinoblastoma patients. Two unilateral cases also showed areas of retinoma. The most frequent imbalance in retinoblastomas was 6p gain (40%), followed by gains at 1q12-q25.3, 2p24.3-p24.2, 9q22.2, and 9q33.1 and losses at 11q24.3, 13q13.2-q22.3, and 16q12.1-q21. Bilateral cases showed a lower number of imbalances than unilateral cases (P = 0.002). Unilateral cases were divided into low-level (≤4) and high-level (÷7) chromosomal instability groups. The first group presented with younger age at diagnosis (mean 511 days) compared with the second group (mean 1606 days). In one retinoma case ophthalmoscopically diagnosed as a benign lesion no rearrangements were detected, whereas the adjacent retinoblastoma displayed seven aberrations. The other retinoma case identified by retrospective histopathological examination shared three rearrangements with the adjacent retinoblastoma. Two other gene-free rearrangements were retinoma specific. One rearrangement, dup5p, was retinoblastoma specific and included the SKP2 gene. Genomic profiling indicated that the first retinoma was a pretumoral lesion, whereas the other represents a subclone of cells bearing 'benign' rearrangements overwhelmed by another subclone presenting aberrations with higher 'oncogenic' potential. In summary, the present study shows that bilateral and unilateral retinoblastoma have different chromosomal instability that correlates with the age of tumor onset in unilateral cases. This is the first report of genomic profiling in retinoma tissue, shedding light on the different nature of lesions named 'retinoma'. (Cancer Sci 2009; 100: 465–471

Immunological fingerprint of 4CMenB recombinant antigens via protein microarray reveals key immunosignatures correlating with bactericidal activity

Serogroup B meningococcus (MenB) is a leading cause of meningitis and sepsis across the world and vaccination is the most effective way to protect against this disease. 4CMenB is a multi-component vaccine against MenB, which is now licensed for use in subjects >2 months of age in several countries. In this study, we describe the development and use of an ad hoc protein microarray to study the immune response induced by the three major 4CMenB antigenic components (fHbp, NHBA and NadA) in individual sera from vaccinated infants, adolescents and adults. The resulting 4CMenB protein antigen fingerprinting allowed the identification of specific human antibody repertoire correlating with the bactericidal response elicited in each subject. This work represents an example of epitope mapping of the immune response induced by a multicomponent vaccine in different age groups with the identification of protective signatures. It shows the high flexibility of this microarray based methodology in terms of high-throughput information and minimal volume of biological samples needed

Germline mosaicism in Rett syndrome identified by prenatal diagnosis

Rett syndrome is an X-linked neurodevelopmental dominant disorder that affects almost exclusively girls. The vast majority of cases are sporadic and are caused by de novo mutations in the MECP2 gene, located in Xq28. Only few familial cases have been reported: in four cases, the mother was an asymptomatic carrier and in other four cases, the germline mosaicism in the mother was postulated. Owing to the above reported cases of germline mosaicism, we decided to offer prenatal diagnosis to all expectant mothers with a Rett daughter despite the absence of the causative mutation in parents' blood. We describe here the outcome of the first nine cases of prenatal diagnosis followed by our center. In eight cases, the fetus did not carry the mutation. In one case, the female fetus did carry the same mutation of the affected sister. The couple decided to interrupt the pregnancy and to devolve fetal tissues for research purposes. Our results indicate that prenatal diagnosis should be proposed to all couples with a Rett daughter, even when the mutation is apparently de novo. Moreover, one positive prenatal test among the first nine cases indicates that germline mosaicism may be seriously considered for the assessment of recurrence risk during genetic counseling

Clinical and molecular characterization of COVID-19 hospitalized patients

Clinical and molecular characterization by Whole Exome Sequencing (WES) is reported in 35 COVID-19 patients attending the University Hospital in Siena, Italy, from April 7 to May 7, 2020. Eighty percent of patients required respiratory assistance, half of them being on mechanical ventilation. Fiftyone percent had hepatic involvement and hyposmia was ascertained in 3 patients. Searching for common genes by collapsing methods against 150 WES of controls of the Italian population failed to give straightforward statistically significant results with the exception of two genes. This result is not unexpected since we are facing the most challenging common disorder triggered by environmental factors with a strong underlying heritability (50%). The lesson learned from Autism-Spectrum-Disorders prompted us to re-analyse the cohort treating each patient as an independent case, following a Mendelian-like model. We identified for each patient an average of 2.5 pathogenic mutations involved in virus infection susceptibility and pinpointing to one or more rare disorder(s). To our knowledge, this is the first report on WES and COVID-19. Our results suggest a combined model for COVID-19 susceptibility with a number of common susceptibility genes which represent the favorite background in which additional host private mutations may determine disease progression