Diagnostic Performance and Tolerability of Saliva and Nasopharyngeal Swab Specimens in the Detection of SARS-CoV-2 by RT-PCR.

Saliva is a promising alternative for a nasopharyngeal swab (NPS) in specimen collection to detect SARS-CoV-2. We compared the diagnostic performance and tolerability of saliva collection versus NPS in a clinical setting. Paired NPS and saliva specimens were collected sequentially from participants (n = 250) at the Turku University Hospital drive-in coronavirus testing station in the spring of 2022, with Omicron BA.2 as the dominant SARS-CoV-2 variant. Discomfort and preference for the sampling method were assessed. The specimens were analyzed for SARS-CoV-2 using real-time multiplex reverse transcriptase PCR (RT-PCR) with a laboratory-developed test (LDT) and two commercial kits (PerkinElmer SARS-CoV-2 and PerkinElmer SARS-CoV-2 Plus) for several target genes. Among the 250 participants, 246 had respiratory symptoms. With LDT, SARS-CoV-2 was detected in 135 and 134 participants from NPS and saliva, respectively. Of the 250 specimens, 11 gave a discordant outcome, resulting in excellent agreement between the specimen types (Cohen's kappa coefficient of 0.911; P = 0.763). The cycle threshold (CT) values of LDT and commercial kit target genes were significantly lower from NPS than from saliva. A total of 172 (69%) participants assessed saliva sampling as more tolerable than NPS (P < 0.0001). Our findings present saliva as an applicable alternative for SARS-CoV-2 diagnostics. However, the lower CT values obtained from NPS indicate that NPS may be a slightly more sensitive specimen type. Participants preferred saliva sampling, although delivering an adequate volume of saliva was challenging for some participants. IMPORTANCE The extensive testing of SARS-CoV-2 is vital in controlling the spread of COVID-19. The reference standard for specimen collection is a nasopharyngeal swab (NPS). However, the discomfort of NPS sampling, the risk of nosocomial infections, and global material shortages have accelerated the development of alternative testing methods. Our study demonstrates that patients tolerate saliva sampling better than NPS. Of importance, although the RT-PCR qualitative test results seem to correspond between NPS and saliva, we show significantly lower CT values for NPS, compared to saliva, thus contradicting the suggested superiority of the saliva specimen over NPS in the detection of the Omicron variants of SARS-CoV-2. Future research is still required to enable individual planning for specimen collection and to determine the effects of different SARS-CoV-2 variants on the sensitivity of the saliva matrix.

Erbb4 regulates the oocyte microenvironment during folliculogenesis.

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders leading to infertility in women affecting reproductive, endocrine and metabolic systems. Recent genomewide association studies on PCOS cohorts revealed a single nucleotide polymorphism (SNP) in the ERBB4 receptor tyrosine kinase 4 gene, but its role in ovary development or during folliculogenesis remains poorly understood. Since no genetic animal models mimicking all PCOS reproductive features are available, we conditionally deleted Erbb4 in murine granulosa cells (GCs) under the control of Amh promoter. While we have demonstrated that Erbb4 deletion displayed aberrant ovarian function by affecting the reproductive function (asynchronous oestrous cycle leading to few ovulations and subfertility) and metabolic function (obesity), their ovaries also present severe structural and functional abnormalities (impaired oocyte development). Hormone analysis revealed an up-regulation of serum luteinizing hormone, hyperandrogenism, increased production of ovarian and circulating anti-Müllerian hormone. Our data implicate that Erbb4 deletion in GCs leads to defective intercellular junctions between the GCs and oocytes, causing changes in the expression of genes regulating the local microenvironment of the follicles. In vitro culture assays reducing the level of Erbb4 via shRNAs confirm that Erbb4 is essential for regulating Amh level. In conclusion, our results indicate a functional role for Erbb4 in the ovary, especially during folliculogenesis and its reduced expression plays an important role in reproductive pathophysiology, such as PCOS development.

Sequence environment of BMP-dependent activating elements controls transcriptional responses to Dpp signaling in Drosophila.

Intercellular signaling pathways activate transcription factors, which, along with tissue-specific co-factors, regulate expression of target genes. Responses to TGFß/BMP signals are mediated by Smad proteins, which form complexes and accumulate in the nucleus to directly bind and regulate enhancers of BMP targets upon signaling. In Drosophila, gene activation by BMP signaling often requires, in addition to direct input by Smads, the signal-dependent removal of the transcriptional repressor Brk. Previous studies on enhancers of BMP-activated genes have defined a BMP-responsive motif, the AE, which integrates activatory and repressive input by the Smad complex and Brk, respectively. Here, we address whether sequence variations within the core AE sequences might endow the motif with additional properties accounting for qualitative and quantitative differences in BMP responses, including tissue specificity of transcriptional activation and differential sensitivity to Smad and Brk inputs. By analyzing and cross-comparing three distinct BMP-responsive enhancers from the genes wit and Dad in two different epithelia, the wing imaginal disc and the follicular epithelium, we demonstrate that differences in the AEs contribute neither to the observed tissue-restriction of BMP responses nor to differences in the utilization of the Smad and Brk branches for transcriptional activation. Rather, our results suggest that the cis-environment of the BMP-response elements not only dictates tissue specificity but also differential sensitivity to the two BMP mediators.

ErbB4 tyrosine kinase inhibition impairs neuromuscular development in zebrafish embryos.

Tyrosine kinase inhibitors are widely used in the clinic, but limited information is available about their toxicity in developing organisms. Here, we tested the effect of tyrosine kinase inhibitors targeting the ErbB receptors for their effects on developing zebrafish ( Danio rerio) embryos. Embryos treated with wide-spectrum pan-ErbB inhibitors or erbb4a-targeting antisense oligonucleotides demonstrated reduced locomotion, reduced diameter of skeletal muscle fibers, and reduced expression of muscle-specific genes, as well as reduced motoneuron length. The phenotypes in the skeletal muscle, as well as the defect in motility, were rescued both by microinjection of human ERBB4 mRNA and by transposon-mediated muscle-specific ERBB4 overexpression. The role of ErbB4 in regulating motility was further controlled by targeted mutation of the endogenous erbb4a locus in the zebrafish genome by CRISPR/Cas9. These observations demonstrate a potential for the ErbB tyrosine kinase inhibitors to induce neuromuscular toxicity in a developing organism via a mechanism involving inhibition of ErbB4 function.

Gene regulation during Drosophila eggshell patterning.

A common path to the formation of complex 3D structures starts with an epithelial sheet that is patterned by inductive cues that control the spatiotemporal activities of transcription factors. These activities are then interpreted by the cis-regulatory regions of the genes involved in cell differentiation and tissue morphogenesis. Although this general strategy has been documented in multiple developmental contexts, the range of experimental models in which each of the steps can be examined in detail and evaluated in its effect on the final structure remains very limited. Studies of the Drosophila eggshell patterning provide unique insights into the multiscale mechanisms that connect gene regulation and 3D epithelial morphogenesis. Here we review the current understanding of this system, emphasizing how the recent identification of cis-regulatory regions of genes within the eggshell patterning network enables mechanistic analysis of its spatiotemporal dynamics and evolutionary diversification. It appears that cis-regulatory changes can account for only some aspects of the morphological diversity of Drosophila eggshells, such as the prominent differences in the number of the respiratory dorsal appendages. Other changes, such as the appearance of the respiratory eggshell ridges, are caused by changes in the spatial distribution of inductive signals. Both types of mechanisms are at play in this rapidly evolving system, which provides an excellent model of developmental patterning and morphogenesis.

Molecular Detection of Candidatus Bartonella hemsundetiensis in Bats.

Although bats have been implicated as reservoir hosts for a number of zoonotic and life-threatening viruses, the bat bacterial flora and its zoonotic threat remain elusive. However, members of the vector-borne bacterial genera Bartonella causing various human as well as animal diseases have recently been isolated or detected from bats and their ectoparasites. In this study, we sampled 124 insectivorous microbats (Daubenton's bat, Myotis daubentonii) for peripheral blood in southwestern Finland in 2010. A Bartonella-specific PCR targeting rpoB (RNA polymerase ß-subunit) was positive with blood samples from 46 bats (prevalence 37%). Scaled mass indexes of the infected and noninfected bats did not differ (p = 0.057). One rpoB sequence was identical with the rpoB sequence of B. naantaliensis strain 2574/1, previously isolated from bats in Finland. The rest of the sequences were highly similar to each other with nucleotide identity scores of 96% or higher. Nucleotide identity scores to the previously described type strain sequences of Bartonella or other database entries were no higher than 87%. Sequence analyses of another gene, gltA (citrate synthase), gave no higher than 90% nucleotide identity scores. On the basis of the conventional 95% sequence similarity cutoff in bacterial species delineation, a novel species of Bartonella was detected. We propose a species name Candidatus B. hemsundetiensis. Phylogenetic analyses based on rpoB and gltA sequences indicate that Candidatus B. hemsundetiensis clusters in a deep-branching position close to the ancestral species B. tamiae and B. bacilliformis. Our study reinforces the importance of bats as reservoirs of Bartonella.

BMP-dependent gene repression cascade in Drosophila eggshell patterning.

Bone Morphogenetic Proteins (BMPs) signal by activating Smad transcription factors to control a number of decisions during animal development. In Drosophila, signaling by the BMP ligand Decapentaplegic (Dpp) involves the activity of brinker (brk) which, in most contexts, is repressed by Dpp. Brk encodes a transcription factor which represses BMP signaling output by antagonizing Smad-dependent target gene activation. Here, we study BMP-dependent gene regulation during Drosophila oogenesis by following the signal transmission from Dpp to its target broad (br), a gene with a crucial function in eggshell patterning. We identify regulatory sequences that account for expression of both brk and br, and connect these to the transcription factors of the pathway. We show that Dpp directly regulates brk transcription through Smad- and Schnurri (Shn)-dependent repression. Brk is epistatic to Dpp in br expression and activates br indirectly, through removal of a repressor, which is yet to be identified. Our work provides first cis-regulatory insights into transcriptional interpretation of BMP signaling in eggshell morphogenesis and defines a transcriptional cascade that connects Dpp to target gene regulation.

ErbB4, a receptor tyrosine kinase, coordinates organization of the seminiferous tubules in the developing testis.

Although close to every fifth couple nowadays has difficulty conceiving, the molecular mechanisms behind the decline in human reproduction remain poorly understood. We report here that the receptor tyrosine kinase Erbb4 is a candidate causal gene, because it is expressed in a sexually dimorphic manner and is abundant in the developing and adult testes in the mouse. Sertoli cell-specific Erbb4-knockout mice have a compromised 3-dimensional organization of the testicular seminiferous tubules that affects their fertility. More specifically, adhesion defects are observed in the absence of Erbb4, which are characterized by changes in the expression of laminin-1, N-cadherin, claudin-3, and certain cell-cell junction components between the Sertoli and germ cells. Interestingly, Erbb4 knockout also had an effect on the Leydig cells, which suggests a paracrine influence of Sertoli cells expressing ErbB4. Many of the defects observed in Erbb4-knockout mice are rescued in targeted ERBB4 gain-of-function mice, pointing to a coordination role for ErbB4 in the developing testis. Thus, the ErbB4 receptor tyrosine kinase promotes seminiferous tubule development by controlling Sertoli cell and germ cell adhesion.

Bats as reservoir hosts of human bacterial pathogen, Bartonella mayotimonensis.

A plethora of pathogenic viruses colonize bats. However, bat bacterial flora and its zoonotic threat remain ill defined. In a study initially conducted as a quantitative metagenomic analysis of the fecal bacterial flora of the Daubenton's bat in Finland, we unexpectedly detected DNA of several hemotrophic and ectoparasite-transmitted bacterial genera, including Bartonella. Bartonella spp. also were either detected or isolated from the peripheral blood of Daubenton's, northern, and whiskered bats and were detected in the ectoparasites of Daubenton's, northern, and Brandt's bats. The blood isolates belong to the Candidatus-status species B. mayotimonensis, a recently identified etiologic agent of endocarditis in humans, and a new Bartonella species (B. naantaliensis sp. nov.). Phylogenetic analysis of bat-colonizing Bartonella spp. throughout the world demonstrates a distinct B. mayotimonensis cluster in the Northern Hemisphere. The findings of this field study highlight bats as potent reservoirs of human bacterial pathogens.

Cat scratch disease caused by Bartonella grahamii in an immunocompromised patient.

Bartonella grahamii colonizes rodents worldwide and has been detected in questing Ixodes ricinus ticks. Here, the first human B. grahamii infection confirmed by multilocus sequence typing is reported. The route of transmission and clinical picture of the patient are similar to those seen in patients with cat scratch disease, which is typically diagnosed as a Bartonella henselae infection.

ErbB4 modulates tubular cell polarity and lumen diameter during kidney development.

ErbB4 receptor tyrosine kinase contributes to the development of the heart, the central nervous system, and the lactating mammary gland, but whether it has a role in the development of the kidney epithelium is unknown. Here, we found that expression of Erbb4 isoforms JM-a CYT-1 and JM-a CYT-2 was first detectable around embryonic day 13 in the mouse, mainly in the collecting ducts and both the proximal and distal tubules. In vitro, overexpression of a relevant ErbB4 isoform promoted proliferation and disturbed polarization of kidney epithelial cells when cultured as three-dimensional structures. We examined ErbB4 function in developing kidney tubules in vivo with Pax8-Cre-mediated conditional overexpression of Rosa26 locus-targeted ERBB4 and with conditional Erbb4 knock-out mice. The Pax8-Cre-driven ERBB4 overexpression enhanced proliferation in the collecting ducts, reduced the size of epithelial duct lumens, and promoted formation of cortical tubular cysts. These defects were associated with changes in the subcellular distribution of markers of epithelial cell polarity. Similarly, the Pax8-Cre-mediated Erbb4 knock-out mice manifested dysfunctional kidneys with larger duct lumens and epithelial cell mispolarization. Taken together, these data suggest that ErbB4 signaling modulates proliferation and polarization, cellular functions critical for the development of epithelial ducts in the kidney.

Function of ERBB4 is determined by alternative splicing.

Alternative splicing is a central tool  of evolution that significantly increases the size of transcriptomes and generates functional specification. Within the human ERBB receptor gene family, only ERBB4 is known to produce functionally distinct isoforms as a result of alternative splicing. While ErbB4 signaling has been demonstrated to regulate cellular processes involved in embryogenesis, carcinogenesis and cardiovascular and psychiatric diseases, relatively little is known about the contribution of the individual isoforms in the different biological contexts. Here, we review recent findings as well as provide novel data about the distribution and functions of the ERBB4 splice variants. These observations represent an example of how minor alterations in the transcripts of a single gene can result in even antagonistic cellular responses. The observations also underline the significance of understanding the unique functions of isoforms of a potential drug target gene.

Cell death or survival promoted by alternative isoforms of ErbB4.

The significance of ErbB4 in tumor biology is poorly understood. The ERBB4 gene is alternatively spliced producing juxtamembrane (JM-a and JM-b) and cytoplasmic (CYT-1 and CYT-2) isoforms. Here, signaling via the two alternative ErbB4 JM isoforms (JM-a CYT-2 and JM-b CYT-2) was compared. Fibroblasts expressing ErbB4 JM-a demonstrated enhanced ErbB4 autophosphorylation, growth, and survival. In contrast, cells overexpressing ErbB4 JM-b underwent starvation-induced death. Both pro- and antisurvival responses to the two ErbB4 isoforms were sensitive to an ErbB kinase inhibitor. Platelet-derived growth factor receptor-alpha (PDGFRA) was identified as an ErbB4 target gene that was differentially regulated by the two ErbB4 isoforms. The soluble intracellular domain of ErbB4, released from the JM-a but not from the JM-b isoform, associated with the transcription factor AP-2 and promoted its potential to enhance PDGFRA transcription. Survival of cells expressing JM-a was suppressed by targeting either PDGFR-α or AP-2, whereas cells expressing JM-b were rescued from cell death by the PDGFR-α agonist, PDGF-BB. These findings indicate that two alternative ErbB4 isoforms may promote antagonistic cellular responses and suggest that pharmacological inhibition of ErbB4 kinase activity may lead to either suppression or promotion of cellular growth.