Reverse-Transcription Loop-Mediated Isothermal Amplification Has High Accuracy for Detecting Severe Acute Respiratory Syndrome Coronavirus 2 in Saliva and Nasopharyngeal/Oropharyngeal Swabs from Asymptomatic and Symptomatic Individuals.

Previous studies have described reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for the rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal/oropharyngeal swab and saliva samples. This multisite clinical evaluation describes the validation of an improved sample preparation method for extraction-free RT-LAMP and reports clinical performance of four RT-LAMP assay formats for SARS-CoV-2 detection. Direct RT-LAMP was performed on 559 swabs and 86,760 saliva samples and RNA RT-LAMP on extracted RNA from 12,619 swabs and 12,521 saliva samples from asymptomatic and symptomatic individuals across health care and community settings. For direct RT-LAMP, overall diagnostic sensitivity (DSe) was 70.35% (95% CI, 63.48%-76.60%) on swabs and 84.62% (95% CI, 79.50%-88.88%) on saliva, with diagnostic specificity of 100% (95% CI, 98.98%-100.00%) on swabs and 100% (95% CI, 99.72%-100.00%) on saliva, compared with quantitative RT-PCR (RT-qPCR); analyzing samples with RT-qPCR ORF1ab CT values of ≤25 and ≤33, DSe values were 100% (95% CI, 96.34%-100%) and 77.78% (95% CI, 70.99%-83.62%) for swabs, and 99.01% (95% CI, 94.61%-99.97%) and 87.61% (95% CI, 82.69%-91.54%) for saliva, respectively. For RNA RT-LAMP, overall DSe and diagnostic specificity were 96.06% (95% CI, 92.88%-98.12%) and 99.99% (95% CI, 99.95%-100%) for swabs, and 80.65% (95% CI, 73.54%-86.54%) and 99.99% (95% CI, 99.95%-100%) for saliva, respectively. These findings demonstrate that RT-LAMP is applicable to a variety of use cases, including frequent, interval-based direct RT-LAMP of saliva from asymptomatic individuals who may otherwise be missed using symptomatic testing alone.

A highly effective reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of SARS-CoV-2 infection.

The COVID-19 pandemic has illustrated the importance of simple, rapid and accurate diagnostic testing. This study describes the validation of a new rapid SARS-CoV-2 RT-LAMP assay for use on extracted RNA or directly from swab offering an alternative diagnostic pathway that does not rely on traditional reagents that are often in short supply during a pandemic. Analytical specificity (ASp) of this new RT-LAMP assay was 100% and analytical sensitivity (ASe) was between 1 × 101 and 1 × 102 copies per reaction when using a synthetic DNA target. The overall diagnostic sensitivity (DSe) and specificity (DSp) of RNA RT-LAMP was 97% and 99% respectively, relative to the standard of care rRT-PCR. When a CT cut-off of 33 was employed, above which increasingly evidence suggests there is a low risk of patients shedding infectious virus, the diagnostic sensitivity was 100%. The DSe and DSp of Direct RT-LAMP (that does not require RNA extraction) was 67% and 97%, respectively. When setting CT cut-offs of ≤33 and ≤25, the DSe increased to 75% and 100%, respectively, time from swab-to-result, CT < 25, was < 15 min. We propose that RNA RT-LAMP could replace rRT-PCR where there is a need for increased sample throughput and Direct RT-LAMP as a near-patient screening tool to rapidly identify highly contagious individuals within emergency departments and care homes during times of increased disease prevalence ensuring negative results still get laboratory confirmation.

The Hippo pathway integrates PI3K-Akt signals with mechanical and polarity cues to control tissue growth.

The Hippo signalling pathway restricts cell proliferation in animal tissues by inhibiting Yes-associated protein (YAP or YAP1) and Transcriptional Activator with a PDZ domain (TAZ or WW-domain-containing transcriptional activator [WWTR1]), coactivators of the Scalloped (Sd or TEAD) DNA-binding transcription factor. Drosophila has a single YAP/TAZ homolog named Yorkie (Yki) that is regulated by Hippo pathway signalling in response to epithelial polarity and tissue mechanics during development. Here, we show that Yki translocates to the nucleus to drive Sd-mediated cell proliferation in the ovarian follicle cell epithelium in response to mechanical stretching caused by the growth of the germline. Importantly, mechanically induced Yki nuclear localisation also requires nutritionally induced insulin/insulin-like growth factor 1 (IGF-1) signalling (IIS) via phosphatidyl inositol-3-kinase (PI3K), phosphoinositide-dependent kinase 1 (PDK1 or PDPK1), and protein kinase B (Akt or PKB) in the follicular epithelium. We find similar results in the developing Drosophila wing, where Yki becomes nuclear in the mechanically stretched cells of the wing pouch during larval feeding, which induces IIS, but translocates to the cytoplasm upon cessation of feeding in the third instar stage. Inactivating Akt prevents nuclear Yki localisation in the wing disc, while ectopic activation of the insulin receptor, PI3K, or Akt/PKB is sufficient to maintain nuclear Yki in mechanically stimulated cells of the wing pouch even after feeding ceases. Finally, IIS also promotes YAP nuclear localisation in response to mechanical cues in mammalian skin epithelia. Thus, the Hippo pathway has a physiological function as an integrator of epithelial cell polarity, tissue mechanics, and nutritional cues to control cell proliferation and tissue growth in both Drosophila and mammals.

YAP drives cutaneous squamous cell carcinoma formation and progression.

Squamous cell carcinoma (SCC) can progress to malignant metastatic cancer, including an aggressive subtype known as spindle cell carcinoma (spSCC). spSCC formation involves epithelial-to-mesenchymal transition (EMT), yet the molecular basis of this event remains unknown. The transcriptional co-activator YAP undergoes recurrent amplification in human SCC and overexpression of YAP drives SCC formation in mice. Here, we show that human spSCC tumours also feature strong nuclear localisation of YAP and overexpression of activated YAP (NLS-YAP-5SA) with Keratin-5 (K5-CreERt) is sufficient to induce rapid formation of both SCC and spSCC in mice. spSCC tumours arise at sites of epithelial scratch wounding, where tumour-initiating epithelial cells undergo EMT to generate spSCC. Expression of the EMT transcription factor ZEB1 arises upon wounding and is a defining characteristic of spSCC in mice and humans. Thus, the wound healing response synergises with YAP to drive metaplastic transformation of SCC to spSCC.

YAP and TAZ in epithelial stem cells: A sensor for cell polarity, mechanical forces and tissue damage.

The YAP/TAZ family of transcriptional co-activators drives cell proliferation in epithelial tissues and cancers. Yet, how YAP and TAZ are physiologically regulated remains unclear. Here we review recent reports that YAP and TAZ act primarily as sensors of epithelial cell polarity, being inhibited when cells differentiate an apical membrane domain, and being activated when cells contact the extracellular matrix via their basal membrane domain. Apical signalling occurs via the canonical Crumbs/CRB-Hippo/MST-Warts/LATS kinase cascade to phosphorylate and inhibit YAP/TAZ. Basal signalling occurs via Integrins and Src family kinases to phosphorylate and activate YAP/TAZ. Thus, YAP/TAZ is localised to the nucleus in basal stem/progenitor cells and cytoplasm in differentiated squamous cells or columnar cells. In addition, other signals such as mechanical forces, tissue damage and possibly receptor tyrosine kinases (RTKs) can influence MST-LATS or Src family kinase activity to modulate YAP/TAZ activity.

Integrin signalling regulates YAP and TAZ to control skin homeostasis.

The skin is a squamous epithelium that is continuously renewed by a population of basal layer stem/progenitor cells and can heal wounds. Here, we show that the transcription regulators YAP and TAZ localise to the nucleus in the basal layer of skin and are elevated upon wound healing. Skin-specific deletion of both YAP and TAZ in adult mice slows proliferation of basal layer cells, leads to hair loss and impairs regeneration after wounding. Contact with the basal extracellular matrix and consequent integrin-Src signalling is a key determinant of the nuclear localisation of YAP/TAZ in basal layer cells and in skin tumours. Contact with the basement membrane is lost in differentiating daughter cells, where YAP and TAZ become mostly cytoplasmic. In other types of squamous epithelia and squamous cell carcinomas, a similar control mechanism is present. By contrast, columnar epithelia differentiate an apical domain that recruits CRB3, Merlin (also known as NF2), KIBRA (also known as WWC1) and SAV1 to induce Hippo signalling and retain YAP/TAZ in the cytoplasm despite contact with the basal layer extracellular matrix. When columnar epithelial tumours lose their apical domain and become invasive, YAP/TAZ becomes nuclear and tumour growth becomes sensitive to the Src inhibitor Dasatinib.

Adeno-associated virus Rep represses the human integration site promoter by two pathways that are similar to those required for the regulation of the viral p5 promoter.

UNLABELLED: Adeno-associated virus serotype 2 (AAV2) can efficiently replicate in cells that have been infected with helper viruses, such as adenovirus or herpesvirus. However, in the absence of helper virus infection, AAV2 establishes latency by integrating its genome site specifically into PPP1R12C, a gene located on chromosome 19. This integration target site falls into one of the most gene-dense regions of the human genome, thus inviting the question as to whether the virus has evolved mechanisms to control this complex transcriptional environment in order to facilitate integration, maintain an apparently innocuous latency, and/or establish conditions that are conducive to the rescue of the integrated viral genome. The viral replication (Rep) proteins control and direct every known aspect of the viral life cycle and have been shown to tightly control all AAV2 promoters. In addition, a number of heterologous promoters are repressed by the AAV2 Rep proteins. Here, we demonstrate that Rep proteins efficiently repress expression from the target site PPP1R12C promoter. We find evidence that this repression employs mechanisms similar to those described for Rep-mediated AAV2 p5 promoter regulation. Furthermore, we show that the repression of the cellular target site promoter is based on two distinct mechanisms, one relying on the presence of a functional Rep binding motif within the 5' untranslated region (UTR) of PPP1R12C, whereas the second pathway requires only an intact nucleoside triphosphate (NTP) binding site within the Rep proteins, indicating the possible reliance of this pathway on interactions of the Rep proteins with cellular proteins that mediate or regulate cellular transcription. IMPORTANCE: The observation that repression of transcription from the adeno-associated virus serotype 2 (AAV2) p5 and integration target site promoters is mediated by shared mechanisms highlights the possible coevolution of virus and host and could lead to the identification of host factors that the virus exploits to navigate its life cycle.