Fluorescent reporter proteins (FP) have become an indispensable tool for the optimization of microbial cell factories and in synthetic biology per se. The applicability of the currently available FPs is, however, constrained by species-dependent performance and misfolding at elevated temperatures. To obtain functional reporters for thermophilic, biotechnologically important bacteria such as Parageobacillus thermoglucosidasius, an in vivo screening approach based on a mutational library of superfolder GFP was applied. Flow cytometry-based benchmarking of a set of GFPs, sfGFPs and species-specific codon-optimized variants revealed that none of the proteins was satisfyingly detectable in P. thermoglucosidasius at its optimal growth temperature of 60 °C. An undirected mutagenesis approach coupled to fluorescence-activated cell sorting allowed the isolation of sfGFP variants that were extremely well expressed in the chassis background at 60 °C. Notably, a few nucleotide substitutions, including silent mutations, significantly improved the functionality and brightness. The best mutant sfGFP(N39D/A179A) showed an 885-fold enhanced mean fluorescence intensity (MFI) at 60 °C and is the most reliable reporter protein with respect to cell-to-cell variation and signal intensity reported so far. The in vitro spectral and thermostability properties were unaltered as compared to the parental sfGFP protein, strongly indicating that the combination of the amino acid exchange and an altered translation or folding speed, or protection from degradation, contribute to the strongly improved in vivo performance. Furthermore, sfGFP(N39D/A179A) and the newly developed cyan and yellow derivatives were successfully used for labeling several industrially relevant thermophilic bacilli, thus proving their broad applicability. This study illustrates the power of in vivo isolation of thermostable proteins to obtain reporters for highly efficient fluorescence labeling. Successful expression in a variety of thermophilic bacteria proved that the novel FPs are highly suitable for imaging and flow cytometry-based studies. This enables a reliable cell tracking and single-cell-based real-time monitoring of biological processes that are of industrial and biotechnological interest.

Angiotensin-converting enzyme 2 (ACE2) is the main entry point in the airways for SARS-CoV-2. ACE2 binding to SARS-CoV-2 protein Spike triggers viral fusion with the cell membrane, resulting in viral RNA genome delivery into the host. Despite ACE2’s critical role in SARS-CoV-2 infection, an understanding of ACE2 expression, including in response to viral infection, remains unclear. Until now ACE2 was thought to encode five transcripts and one 805 amino acid protein. Here we identify a novel short isoform of ACE2. Short ACE2 is expressed in the airway epithelium, the main site of SARS-CoV-2 infection; it is substantially upregulated in response to interferon stimulation and RV infection, but not in response to SARS-CoV-2 infection, and it shows differential regulation in asthma patients. This short isoform lacks SARS-CoV-2 spike glycoprotein high-affinity binding sites and altogether, our data are consistent with a model where short ACE2 may influence host susceptibility to SARS-CoV-2 infection.

It is well known that modifiers play a role in ameliorating or exacerbating disease phenotypes in patients and carriers of recessively inherited disorders such as sickle cell disease and thalassemia. Here, we give an overview of the literature concerning a recently described association in carriers of SUPT5H Loss-of-Function variants with a beta-thalassemia-like phenotype including the characteristic elevated levels of HbA₂. That SUPT5H acts as modifier in beta-thalassemia carriers became evident from three reported cases in whom combined heterozygosity of SUPT5H and HBB gene variants was observed to resemble a mild beta-thalassemia intermedia phenotype. The different SUPT5H variants and hematologic parameters reported are collected and reviewed to provide insight into the possible effects on hematologic expression, as well as potential disease mechanisms in carriers and patients.

Rare genetic respiratory disease has an incidence rate of more than 1:2500 live births in Northern Europe and carries significant disease burden. Early diagnosis improves outcomes, but many individuals remain without a confident genetic diagnosis. Improved and expanded molecular testing methods are required to improve genetic diagnosis rates and thereby improve clinical outcomes. Using primary ciliary dyskinesia (PCD) as an exemplar rare genetic respiratory disease, we developed a standardized method to identify pathogenic variants using whole transcriptome RNA-sequencing (RNA-seq) of nasal epithelial cells cultured at air-liquid interface (ALI). The method was optimized using cells from healthy volunteers, and people with rhino-pulmonary disease but no diagnostic indication of PCD. We validated the method using nasal epithelial cells from PCD patients with known genetic cause. We then assessed the ability of RNA-seq to identify pathogenic variants and the disease mechanism in PCD likely patients but in whom DNA genetic testing was inconclusive. The majority of 49 targeted PCD genes were optimally identified in RNA-seq data from nasal epithelial cells grown for 21 days at ALI culture. Four PCD-likely patients without a previous genetic diagnosis received a confirmed genetic diagnosis from the findings of the RNA-seq data. We demonstrate the clinical potential of RNA-seq of nasal epithelial cells to identify variants in individuals with genetically unsolved PCD. This uplifted genetic diagnosis should improve genetic counselling, enables family cascade screening, opens the door to potential personalised treatment and care approaches. This methodology could be implemented in other rare lung diseases such as cystic fibrosis.

Hypophosphatemic rickets is a rare bone disease characterized by short stature, bone deformities, impaired bone mineralization, and dental problems. Most commonly, hypophosphatemic rickets is caused by pathogenic variants in the X-chromosomal PHEX gene, but autosomal dominant and recessive forms also exist. We investigated a Finnish family in which the son (index, 29 yr) and mother (56 yr) had hypophosphatemia since childhood. Both patients had typical clinical, radiographic, and biochemical features of hypophosphatemic rickets, including a pathological fracture in the son. Gene panels and whole-exome sequencing did not reveal any pathogenic variants in the known hypophosphatemia genes. Therefore, we performed whole genome sequencing and identified a deep intronic variant (c.2147 + 1197A > G) in PHEX. Both the affected individuals, but none of the unaffected family members, had the same variant, as confirmed by Sanger sequencing. According to RT-PCR, whole transcriptomic data, and in silico analyses, the variant led to a new splice donor site in intron 21 and an 84 basepair pseudoexon between exons 21 and 22, likely leading to the synthesis of abnormal PHEX protein. Our study underscores the importance of intronic PHEX variants in X-linked hypophosphatemia (XLH). In patients with features of XLH but negative gene panel or whole-exome sequencing results, the combination of whole-genome sequencing and whole transcriptomics should be considered to detect possible deep intronic variants. The methodologies presented have the potential to be used more widely in other rare diseases.