Zyxin regulates migration of renal epithelial cells through activation of hepatocyte nuclear factor-1ß.

Hepatocyte nuclear factor-1ß (HNF-1ß) is an epithelial tissue-specific transcription factor that regulates gene expression in the kidney, liver, pancreas, intestine, and other organs. Mutations of HNF-1ß in humans produce renal cysts and congenital kidney anomalies. Here, we identify the LIM-domain protein zyxin as a novel binding partner of HNF-1ß in renal epithelial cells. Zyxin shuttles to the nucleus where it colocalizes with HNF-1ß. Immunoprecipitation of zyxin in leptomycin B-treated cells results in coprecipitation of HNF-1ß. The protein interaction requires the second LIM domain of zyxin and two distinct domains of HNF-1ß. Overexpression of zyxin stimulates the transcriptional activity of HNF-1ß, whereas small interfering RNA silencing of zyxin inhibits HNF-1ß-dependent transcription. Epidermal growth factor (EGF) induces translocation of zyxin into the nucleus and stimulates HNF-1ß-dependent promoter activity. The EGF-mediated nuclear translocation of zyxin requires activation of Akt. Expression of dominant-negative mutant HNF-1ß, knockdown of zyxin, or inhibition of Akt inhibits EGF-stimulated cell migration. These findings reveal a novel pathway by which extracellular signals are transmitted to the nucleus to regulate the activity of a transcription factor that is essential for renal epithelial differentiation.

Agency factors associated with first response systems that improve out-of-hospital cardiac arrest outcomes.

BACKGROUND: Data are conflicting regarding the association between first responder (FR) intervention and improved outcomes after out-of-hospital cardiac arrest (OHCA). We evaluated characteristics of agencies that have positive associations between FR interventions and outcomes. METHODS: We analyzed the 2016-2021 national Cardiac Arrest Registry to Enhance Survival (CARES). We defined the exposures as FR CPR and AED. The outcome was survival with favorable neurologic status. We used logistic regression models to evaluate the association between FR interventions with OHCA outcome for each agency, stratifying agencies into positive association (95% confidence interval above 1) and no/inverse association (95% confidence below or including 1). We compared characteristics between cohorts. RESULTS: For the association between FR CPR and outcomes, 21 agencies caring for 42,856 OHCAs had a positive association; 371 agencies caring for 449,824 OHCAs had no association. For FR AED, 47 agencies caring for 103,120 OHCAs had a positive association; 262 agencies caring for 327,761 OHCAs had no association. Comparing agency characteristics for FR CPR, agencies with a positive association had more annual OHCAs (+300), lower FR CPR rate (-11.3%), and lower FR AED rate (-10.8%). Comparing FR AED, agencies with a positive association had more OHCAs per year (+150.5), lower FR CPR rate (-6.8%), lower FR AED rate (-13.3%), lower response time (-0.6 minutes), and more OHCAs from high-income neighborhoods (+3.7%). CONCLUSION: FR AED more commonly had a positive association with outcomes than FR CPR. Agencies with better outcomes from FR interventions treated more OHCAs and had lower rates of FR intervention.

Ultrafast RNA extraction-free SARS-CoV-2 detection by direct RT-PCR using a rapid thermal cycling approach.

The COVID19 pandemic has underlined the need for quick and high-throughput SARS-CoV-2 detection assays. Here we report the development of a direct RT-PCR detection method that can reliably detect SARS-CoV-2 gRNA in nasopharyngeal swab samples in under 27 minutes without needing nucleic acid extraction. Fluorescence readouts were highly linear, robust, and sensitive with a LoD95% of determined at 1.46 copies/µL as determined by RT-PCR on a surrogate sample panel containing clinical samples with varying SARS-CoV-2 viral load. We benchmarked our direct RT-PCR method against a reference qPCR method in 368 nasopharyngeal swab samples, confirming a sensitivity score of 99.4% and a specificity score of 98.5% as compared to the reference method. In summary, we here describe a novel rapid direct RT-PCR method to detect SARS-CoV-2 gRNA in clinical specimens, which can be completed in significantly less time compared to conventional PCR methods making it ideal for large-scale screening applications.

Mutation of hepatocyte nuclear factor-1beta inhibits Pkhd1 gene expression and produces renal cysts in mice.

Hepatocyte nuclear factor-1beta (HNF-1beta) is a Pit-1, Oct-1/2, UNC-86 (POU)/homeodomain-containing transcription factor that regulates tissue-specific gene expression in the liver, kidney, and other organs. Humans with autosomal dominant mutations of HNF-1beta develop maturity-onset diabetes of the young type 5 (MODY5) and congenital cystic abnormalities of the kidney. Autosomal recessive polycystic kidney disease (ARPKD) is an inherited cystic disorder that produces renal failure in infants and children and is caused by mutations of PKHD1. The proximal promoter of the mouse Pkhd1 gene contains an evolutionarily conserved HNF-1-binding site that is located near a region of deoxyribonuclease hypersensitivity. HNF-1beta and the structurally related HNF-1alpha bind specifically to the Pkhd1 promoter and stimulate gene transcription. Mutations of the HNF-1 site or expression of a dominant-negative HNF-1beta mutant inhibit Pkhd1 promoter activity in transfected cells. Transgenic mice expressing a dominant-negative HNF-1beta mutant under the control of a kidney-specific promoter develop renal cysts, similarly to humans with MODY5. Pkhd1 transcripts are absent in the cells lining the cysts but are present in morphologically normal surrounding tubules. These studies identify a link between two cystic disease genes, HNF1beta (MODY5) and PKHD1 (ARPKD). HNF-1beta directly regulates the transcription of Pkhd1, and inhibition of PKHD1 gene expression may contribute to the formation of renal cysts in humans with MODY5.

Squamous cell carcinoma arising in a dentigerous cyst in a 16-month-old girl.

Squamous cell carcinoma arising in a dentigerous cyst is a rare lesion with distinct histopathology and stringent criteria for diagnosis.(1,2) During the past century, <60 cases have been reported.(3) Of the previously documented cases, all have occurred in adults. We present the case of a squamous cell carcinoma arising in a dentigerous cyst in a 16-month-old girl. This case poses interesting questions regarding the pathophysiology of these tumors and highlights the importance of maintaining a high index of suspicion regardless of age when evaluating cystic lesions of the mandible and maxilla.

Roles of HNF-1beta in kidney development and congenital cystic diseases.

Hepatocyte nuclear factor-1beta (HNF-1beta) is a Pit-1/Oct-1/Unc-86 (POU)/homeodomain-containing transcription factor that regulates tissue-specific gene expression in the kidney, liver, pancreas, and other epithelial organs. Mutations of HNF-1beta produce maturity-onset diabetes of the young type 5 (MODY5) and are associated with congenital cystic abnormalities of the kidney. Transgenic mice expressing mutant HNF-1beta under the control of a kidney-specific promoter develop kidney cysts and renal failure, which is similar to the phenotype of humans with MODY5. Similarly, kidney-specific deletion of HNF-1beta using Cre/loxP recombination results in renal cyst formation. HNF-1beta directly regulates the Pkhd1 promoter. HNF-1beta mutant mice show decreased expression of Pkhd1, the gene that is mutated in humans with autosomal-recessive polycystic kidney disease (ARPKD). These studies demonstrate that HNF-1beta is required for the development of the mammalian kidney. They establish a previously unrecognized link between two renal cystic diseases, MODY5 and ARPKD, and suggest that the mechanism of cyst formation in humans with mutations of HNF-1beta involves down-regulation of PKHD1 gene transcription.