Comparative Analysis of Five Multiplex RT-PCR Assays in the Screening of SARS-CoV-2 Variants.

The rapid and presumptive detection of SARS-CoV-2 variants may be performed using multiplex RT-PCR assays. The aim of this study was to evaluate the diagnostic performance of five qualitative RT-PCR tests as compared with next-generation sequencing (NGS). We retrospectively examined a multi-variant panel (n = 72) of SARS-CoV-2-positive nasopharyngeal swabs categorized as variants of concern (Alpha, Beta, Gamma and Delta), variants under monitoring (Iota and Kappa) and wild-type strains circulating in Liguria (Italy) from January to August 2021. First, NGS libraries of study samples were prepared and mapped to the reference genome. Then, specimens were screened for the detection of L452R, W152C, K417T, K417N, E484Q, E484K and N501Y mutations using the SARS-CoV-2 Variants II Assay Allplex, UltraGene Assay SARS-CoV-2 452R & 484K & 484Q Mutations V1, COVID-19 Ultra Variant Catcher, SARS-CoV-2 Extended ELITe MGB and Simplexa SARS-CoV-2 Variants Direct. The overall accuracy of these assays ranged from 96.9% to 100%. Specificity and sensitivity were 100% and 96-100%, respectively. We highly recommend the use of these assays as second-level tests in the routine workflow of SARS-CoV-2 laboratory diagnostics, as they are accurate, user friendly, low cost, may identify specific mutations in about 2-3 h and, therefore, optimize the surveillance of SARS-CoV-2 variants.

Two Novel CAPN5 Variants Associated with Mild and Severe Autosomal Dominant Neovascular Inflammatory Vitreoretinopathy Phenotypes.

Purpose: We report two new CAPN5 mutations associated with a phenotype of Autosomal Dominant Neovascular Inflammatory Vitreoretinopathy. Methods: We performed next generation sequencing in two patients with ADNIV phenotype; the variants identified were explored further. Results: Patient 1 was heterozygous for CAPN5 c.799G>A, p.(Gly267Ser). Patient 2 was heterozygous for CAPN5 c.1126G>A, p.(Gly376Ser). Both amino acids are highly conserved across species. Patient 1 had a severe phenotype and his mutation lies within the protein's catalytic domain. Patient 2 had a mild phenotype and her mutation is the first ADNIV-causing mutation to be described in the regulatory domain of Calpain-5. Conclusions: Our findings potentially add two new ADNIV-causing CAPN5 mutations to the three previously described. We recommend CAPN5 genetic testing in all patients with a possible ADNIV phenotype, to develop our understanding of Calpain-5; a protein which could potentially provide therapeutically accessible targets for the treatment of many leading causes of blindness.

Proliferative arrest and activation of apoptosis related genes in Rolly Protein-silenced cells.

Here we describe a novel small polypeptide expressed in chick embryo and mouse adult tissues referred to as Rolly Protein (Rolp), expressed at the highest levels in tibial cartilage and lung respectively. Investigating its putative role in cartilage differentiation we found that its expression is restricted to proliferative stages consistently with a decreased proliferation rate observed in Rolp-silenced cells. Additional functional studies demonstrate that inhibition of Rolp expression causes a transcription modulation of genes involved in apoptosis. The results here provided strongly suggest an active role of Rolp in the control of cell proliferation and apoptosis.

CALbeta, a novel lipocalin associated with chondrogenesis and inflammation.

We have previously demonstrated the association of the chicken lipocalin Ex-FABP with cartilage formation and inflammatory responses as a marker of these processes (Descalzi Cancedda et al., Biochim. Biophys. Acta 1482, 127-135, 2000). Here we report the isolation and characterisation of a new lipocalin gene laying upstream the Ex-FABP, thus representing the second member of a possible genomic cluster. This gene contains an open reading frame coding for a polypeptide of about 19 kDa. The amino-acid sequence revealed a conserved lipocalin secondary structure. Tissue distribution of the protein in developing embryos showed a preferential expression in the heart although mRNA transcripts could be detected also in muscle, lung and liver. The lowest expression was observed in the stomach, brain and skin. During endochondral formation of long bones, the protein is differentially distributed, as the transcripts, evidenced in the tibia by in situ hybridisation, are present in the hypertrophic cone of the cartilage and mostly absent in the area of the proliferating chondrocytes. Such developmental regulation was observed also in vitro in cultured chondrocytes where the transcripts were barely detectable in dedifferentiated cells but highly expressed in hypertrophic chondrocytes. The protein was also significantly induced by lipopolysaccharide stimulation of chondrocytes, indicating a possible involvement in acute phase response. Raising specific antibodies in a rabbit allowed validating, at the protein level, all the transcriptional data. Moreover, we gained evidence that the protein is actively secreted in the extracellular matrix surrounding the chondrocytes. Because of its peculiar expression in cartilage, this new protein was named chondrogenesis-associated lipocalin beta (thereafter referred to as CAL beta). The close similarity between Ex-FABP and CAL beta expression patterns supports the hypothesis of a genomic organisation in a cluster where both genes could be co-ordinately regulated.

A chondrogenesis-related lipocalin cluster includes a third new gene, CALgamma.

We have previously reported the modulation, during chondrogenesis and/or inflammation, of two chicken genes laying in the same genomic locus and coding for two polypeptides of the lipocalin protein family, the extracellular fatty acid binding protein (ExFABP) and the chondrogenesis associated lipocalin beta (CALbeta). A third gene, located within the same cluster and coding for a new lipocalin, CALgamma, has been identified and is here characterized. Tissue distribution analyzed by real-time quantitative reverse transcriptase-polymerase chain reaction in chicken embryos shows a ubiquitous expression with predominant levels of mRNA transcripts in the liver and the brain. In the developing tibia, a high expression of CALgamma mRNA was evidenced by in situ hybridization within the pre-hypertrophic and the hypertrophic zones of the bone-forming cartilage. In agreement, dedifferentiated chondrocytes in vitro express the transcripts to the highest level when they re-differentiate reaching hypertrophy. Such peculiar developmental pattern of expression that is analogous to those already described for Ex-FABP and CALbeta suggests that all three proteins may act synergistically in the process of endochondral bone formation. Moreover, like Ex-FABP and CALbeta, CALgamma is also highly induced in dedifferentiated chondrocytes upon stimulation with lypopolysaccharides, indicating that the whole cluster quite possibly is transcriptionally activated not only in physiological morphogenic differentiation but also in pathological acute phase response.

Phylogeny and regulation of four lipocalin genes clustered in the chicken genome: evidence of a functional diversification after gene duplication.

A novel lipocalin gene is here reported that represents the fourth member of a cluster we have identified in the chicken genome. This cluster also includes Chondrogenesis-Associated Lipocalins beta and gamma (CAL beta, CAL gamma) and Extracellular Fatty Acid Binding Protein (Ex-FABP). The new gene codes for a 22-kDa secreted protein with three cysteine residues and a series of sequence features well conserved in the lipocalin family. All the genes in the cluster are structurally similar presenting comparable exon/intron boundary positions and exon sizes. A phylogenetic analysis indicates the monophyletic grouping of these genes, and their relationship with the lipocalins alpha-1-microglobulin (A1mg), complement factor 8 gamma chain (C8GC), prostaglandin D synthase (PGDS), and neutrophil-gelatinase-associated lipocalin (NGAL). The new cluster gene appears to be the ortholog of the mammalian C8GC and was thus named Ggal-C8GC. This orthology also suggests that this lipocalin was present in the ancestor common to reptiles and mammals. In addition to other expressing tissues, Ex-FABP, CAL beta and CAL gamma genes are highly transcribed in chondrocytes at late stages of chondrogenesis during endochondral bone formation and/or upon inflammatory stimulation. Here, we show that they are also transcriptionally induced when chondrocytes are subjected to various biological events as cell quiescence, cell shape transition, and hormonal stimulation. By contrast, Ggal-C8GC transcripts are only barely detectable in chondrocytes, but are more abundant in liver, kidney, brain, heart, skeletal muscle and particularly in skin. Moreover, no expression induction was observed neither during chondrocyte differentiation, nor upon any of the stimulations mentioned above. This indicates that the Ggal-C8GC gene was co-opted for a novel function after the duplication events that gave rise to the cluster. The peculiar coordinated regulation of Ex-FABP, CAL beta and CAL gamma, and the apparent divergent role of Ggal-C8GC suggest that these gene duplications may have been maintained during evolution by a sub-functionalization mechanism where some common function(s) are shared by several members of the cluster and some other specialized function(s) are unique to other members.

Serum-free growth medium sustains commitment of human articular chondrocyte through maintenance of Sox9 expression.

Human articular cartilage heals poorly in adults and current surgical procedures do not provide long-term repair. Cell therapy and tissue engineering could become the treatment of choice, but suffer a major limitation as chondrocytes in vitro lose the differentiated phenotype. In vivo, the chondrogenic lineage is specified by transcription factor Sox9. Thus, cell-based therapy could be successful if Sox9 expression and chondrogenic commitment of the expanded cells were preserved. To achieve this goal, we developed a serum-free medium that supports cell proliferation and preserves the differentiation potential. Indeed, expression of Sox9 is maintained when the conventionally used serum is substituted for by this defined supplement. Spontaneous cartilage formation after expansion in serum-free medium is obtained in vitro in a high-density pellet culture and confirmed in vivo in a functional assay in immunodeficient mice. By contrast, cells grown in serum lose the expression of Sox9 and fail to reform cartilage both in vitro and in vivo unless they are rescued by chondrogenic inducers such as transforming growth factor beta(1) and dexamethasone. Our data emphasize the importance of the microenvironment in modulating commitment, plasticity, and phenotype of chondrocytes, and provide an experimental system to study their physiological or pathological metabolism in a controlled context.

Descemet membrane air-bubble separation in donor corneas.

We describe a technique to obtain Descemet-endothelium disks from donors. To detach Descemet membrane, an air bubble was introduced in the deep stroma of human donor corneas mounted on an artificial chamber. In Group A (n = 5), the bubble was left inflated. In Group B (n = 4), the bubble was deflated immediately after the membrane was detached. In Group C (n = 7), the Descemet-endothelium disk was trephined and separated from the stroma after the bubble was deflated. All tissues were stored at 4°C. Descemet detachment was achieved in 89% of the tissues. After 48 hours, the mean endothelial loss was 83% ± 10% (SD), 15% ± 11%, and 3% ± 3% in the 3 groups, respectively. With this technique, Descemet-endothelium disks were obtained without significant alterations in the endothelial layer.