Incursion of SARS-CoV-2 BA.2.86.1 variant into Israel: National-scale wastewater surveillance using a novel quantitative real-time PCR assay.

The emergence of the SARS-CoV-2 variant BA.2.86.1 raised a considerable concern, due to the large number of potentially virulent mutations. In this study, we developed a novel assay that specifically detects variant BA.2.86.1, and used it to screen environmental samples from wastewater treatment sites across Israel. By using a multiplex assay that included a general SARS-CoV-2 reaction, together with the BA.2.86.1-specific reaction and a control reaction, we quantified the absolute number of viral copies in each sample and its relative abundance, compared with the total copy number of circulating SARS-CoV-2. Evaluation of the new reactions showed that they are both sensitive and specific, detecting down to four copies per reaction, and maintain specificity in the presence of Omicron variants BA.1, 2 and 4 RNA. Examination of 279 samples from 30 wastewater collection sites during August-September 2023 showed that 35 samples (12.5 %) were positive, from 18 sites. Quantitative analysis of the samples showed that the relative abundance of variant BA.2.86.1 with respect to the total viral load of SARS-CoV-2 was very low and consisted between 0.01 % and 0.6 % of the total SARS-CoV-2 circulation. This study demonstrates the importance of combining wastewater surveillance with the development of specialized diagnostic assays, when clinical testing is insufficient. This approach may be useful for timely response by public health authorities in future outbreaks.

Fast, accurate, and cost-effective poultry sex genotyping using real-time polymerase chain reaction.

According to The Organization for Economic Co-operation and Development (OECD), demand for poultry meat and eggs consumption is growing consistently since poultry meat and eggs are readily available and cheap source for nutritional protein. As such, there is pressing demand from industry for improved protocols to determine chicken sex, especially in layer industry since only females can lay eggs. Extensive efforts are being dedicated to avoiding male chicks culling by developing in-ovo sexing detection methods. Any established in-ovo detection method will need to be validated by embryo genotyping. Therefore, there is a growing demand for fast, inexpensive, and precise method for proper discrimination between males and females in the poultry science community. Our aim with this study was to develop an accurate, high-throughput protocol for sex determination using small volumes of blood. We designed primers targeting the Hint-W gene within the W chromosome clearly distinguishing between males and females. In the interest of establishing an efficient protocol without the need for gel electrophoresis, crude DNA extraction without further purification was coupled with qPCR. We validated the accuracy of our method using established protocols and gonad phenotyping and tested our protocol with four different chicken breeds, day-nine embryos, day-old chicks and adult chicken. In summary, we developed a fast, cost-effective, and accurate method for the genotyping of sex chromosomes in chicken.

Characterization and biological function of milk-derived miRNAs.

SCOPE: Breastfeeding is associated with reduced risk of infection, immune-mediated disorders, obesity, and even cancer. Recently it was found that breast milk contains a variety of microRNAs (miRNAs) in the skim and fat layer that can be transferred to infants, and appear to play important roles in those biological functions. METHODS AND RESULTS: This study applied next generation sequencing and quantitative real-time PCR analysis to determine the miRNA expression profile of the skim and fat fraction of human, goat, and bovine milk as well as infant formulas. Human and mammalian milk were found to contain known advantageous miRNAs in exosomes and also in the fat layer. These miRNAs are highly conserved in human, bovine and goat milk. However, they were not detected in several infant formulas. Further, miRNAs present in milk were able to enter normal and tumor cells and affect their biological functions. Following incubation of milk derived human miRNA with normal and cancer cells, the expression of miRNA-148a was upregulated and the expression of the DNA methyltransferase1 target gene of miRNA-148a was down regulated. CONCLUSION: These results reinforce previous findings on the importance of miRNA in breast milk. Future studies should concentrate on the addition of miRNA to infant formulas.

Feasibility of Unbiased RNA Profiling of Colorectal Tumors: A Proof of Principle.

Despite recent advances in molecular profiling of colorectal cancer (CRC), as of yet this has not translated into an unbiased molecular liquid biopsy profile which can accurately screen for early CRC. In this study we depict the profile of early stage CRC as well as for advanced adenomas (AA) by combination of current molecular knowledge with microarray technology, using efficient circulating free plasma RNA purification from blood and RNA amplification technologies. We joined literature search with Affymetrix gene chip experimental procedure to draw the circulating free plasma RNA profile of colorectal cancer disease reflected in blood. The RNA panel was tested by two datasets comparing patients with CRC with healthy subjects and patients with AA to healthy subjects. For the CRC patient cohort (28 CRC cases vs. 41 healthy controls), the ROC analysis of the selected biomarker panel generated a sensitivity of 75% and a specificity of 93% for the detection of CRC using 8-gene classification model. For the AA patient cohort (28 subjects vs. 46 healthy controls), a sensitivity of 60% and a specificity of 87% were calculated using a 2-gene classification model. We have identified a panel of 8 plasma RNA markers as a preliminary panel for CRC detection and subset markers suitable for AA detection. Subjected to extensive clinical validation we suggest that this panel represents a feasible approach and a potential strategy for noninvasive early diagnosis, as a first-line screening test for asymptomatic, average-risk population before colonoscopy.

PTH increases FGF23 gene expression and mediates the high-FGF23 levels of experimental kidney failure: a bone parathyroid feedback loop.

Parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23) target the kidney to cause a phosphaturia. FGF23 also acts on the parathyroid to decrease PTH expression, but in chronic kidney disease (CKD) there are high-serum PTH and FGF23 levels and resistance of the parathyroid to FGF23. We now report that PTH acts on bone to increase FGF23 expression and characterize the signal transduction pathway whereby PTH increases FGF23 expression. Remarkably, we show that PTH is necessary for the high-FGF23 levels of early kidney failure due to an adenine high-phosphorus diet. Parathyroidectomy before the diet totally prevented the fivefold increase in FGF23 levels in kidney failure rats. Moreover, parathyroidectomy of early kidney failure rats corrected their high-FGF23 levels. Therefore, in early kidney failure, the high-FGF23 levels are dependent on the high-PTH levels. PTH infusion for 3 days to mice with normal renal function increased serum FGF23 and calvaria FGF23 mRNA levels. To demonstrate a direct effect of PTH on FGF23, we added PTH to rat osteoblast-like UMR106 cells. PTH increased FGF23 mRNA levels (4-fold) and this effect was mimicked by a PKA activator, forskolin. PTH also decreased SOST mRNA levels (3-fold). SOST codes for sclerostin, a Wnt pathway inhibitor, which is a PTH receptor (PTH1R) target. The effect of PTH was prevented by added sclerostin. Therefore, PTH increases FGF23 expression which involves the PKA and Wnt pathways. The effect of PTH on FGF23 completes a bone-parathyroid endocrine feedback loop. Importantly, secondary hyperparathyroidism is essential for the high-FGF23 levels in early CKD.

Human PTH gene regulation in vivo using transgenic mice.

To study the regulation of the human PTH (hPTH) gene in vivo, we generated transgenic mice with the hPTH gene expressed in the mouse parathyroid using a bacterial artificial chromosome (BAC) containing the hPTH gene within its 144-kb chromosomal region. The BAC construct maintains the native hPTH gene surrounding sequences and isolates it from positional effects. The transgenic mice had normal levels of serum mouse PTH (mPTH) in addition to both intact and bioactive hPTH. Despite the presence of both mPTH and hPTH, serum calcium and 1,25(OH)(2) vitamin D levels were normal. The lack of response to hPTH may be due to tachyphylaxis of the mPTH receptor (PTH1R) and/or impaired recognition of the mPTH1R. In contrast, the regulation of hPTH levels in the mouse was intact. A calcium-depleted diet increased serum mPTH and both intact and bioactive hPTH. mPTH and hPTH mRNA levels were also markedly increased. The calcimimetic R-568 dramatically decreased mPTH and hPTH serum levels. Administered recombinant fibroblast growth factor (FGF)23 decreased hPTH. Therefore, the regulation of hPTH gene expression and serum hPTH levels is intact in the transgenic mice, indicating preservation of the signal transduction of the parathyroid calcium receptor and the Klotho-FGF receptor between mouse and man.

The calcium-sensing receptor regulates parathyroid hormone gene expression in transfected HEK293 cells.

BACKGROUND: The parathyroid calcium receptor determines parathyroid hormone secretion and the response of parathyroid hormone gene expression to serum Ca2+ in the parathyroid gland. Serum Ca2+ regulates parathyroid hormone gene expression in vivo post-transcriptionally affecting parathyroid hormone mRNA stability through the interaction of trans-acting proteins to a defined cis element in the parathyroid hormone mRNA 3'-untranslated region. These parathyroid hormone mRNA binding proteins include AUF1 which stabilizes and KSRP which destabilizes the parathyroid hormone mRNA. There is no parathyroid cell line; therefore, we developed a parathyroid engineered cell using expression vectors for the full-length human parathyroid hormone gene and the human calcium receptor. RESULTS: Co-transfection of the human calcium receptor and the human parathyroid hormone plasmid into HEK293 cells decreased parathyroid hormone mRNA levels and secreted parathyroid hormone compared with cells that do not express the calcium receptor. The decreased parathyroid hormone mRNA correlated with decreased parathyroid hormone mRNA stability in vitro, which was dependent upon the 3'-UTR cis element. Moreover, parathyroid hormone gene expression was regulated by Ca2+ and the calcimimetic R568, in cells co-transfected with the calcium receptor but not in cells without the calcium receptor. RNA immunoprecipitation analysis in calcium receptor-transfected cells showed increased KSRP-parathyroid hormone mRNA binding and decreased binding to AUF1. The calcium receptor led to post-translational modifications in AUF1 as occurs in the parathyroid in vivo after activation of the calcium receptor. CONCLUSION: The expression of the calcium receptor is sufficient to confer the regulation of parathyroid hormone gene expression to these heterologous cells. The calcium receptor decreases parathyroid hormone gene expression in these engineered cells through the parathyroid hormone mRNA 3'-UTR cis element and the balanced interactions of the trans-acting factors KSRP and AUF1 with parathyroid hormone mRNA, as in vivo in the parathyroid. This is the first demonstration that the calcium receptor can regulate parathyroid hormone gene expression in heterologous cells.

Fibroblast growth factor 23 acts on the parathyroid to decrease parathyroid hormone secretion.

PURPOSE OF REVIEW: The aim of this article is to describe the intriguing action of fibroblast growth factor 23 on the parathyroid. RECENT FINDINGS: Fibroblast growth factor 23 inhibits renal phosphate reabsorption and calcitriol production. It is the principal phosphaturic factor in a bone-kidney axis coordinating systemic phosphate homeostasis and bone mineralization. Fibroblast growth factor 23 acts at its target tissues by binding to the Klotho-FGFR1c complex and it has recently been confirmed that the fibroblast growth factor 23 receptor is present not only in renal tissue but also in the parathyroid. Fibroblast growth factor 23 leads to a decrease in parathyroid hormone mRNA and serum parathyroid hormone levels by the mitogen-activated protein kinase pathway both in vivo and in vitro. SUMMARY: Fibroblast growth factor 23 is secreted by osteocytes and acts through its receptor the heterodimer of Klotho-FGFR1c in the kidney and parathyroid. In the kidney it leads to phosphaturia and decreased calcitriol synthesis, and in the parathyroid it activates the mitogen-activated protein kinase pathway to decrease parathyroid hormone gene expression and secretion. The decreased parathyroid hormone levels would then also contribute to a decrease in calcitriol synthesis. A bone-kidney-parathyroid hormonal network is now apparent which regulates phosphate, calcium and calcitriol homeostasis. Fibroblast growth factor 23 is the major factor regulating phosphate, and parathyroid hormone the major factor for calcium and calcitriol balances between these factors.

The parathyroid is a target organ for FGF23 in rats.

Phosphate homeostasis is maintained by a counterbalance between efflux from the kidney and influx from intestine and bone. FGF23 is a bone-derived phosphaturic hormone that acts on the kidney to increase phosphate excretion and suppress biosynthesis of vitamin D. FGF23 signals with highest efficacy through several FGF receptors (FGFRs) bound by the transmembrane protein Klotho as a coreceptor. Since most tissues express FGFR, expression of Klotho determines FGF23 target organs. Here we identify the parathyroid as a target organ for FGF23 in rats. We show that the parathyroid gland expressed Klotho and 2 FGFRs. The administration of recombinant FGF23 led to an increase in parathyroid Klotho levels. In addition, FGF23 activated the MAPK pathway in the parathyroid through ERK1/2 phosphorylation and increased early growth response 1 mRNA levels. Using both rats and in vitro rat parathyroid cultures, we show that FGF23 suppressed both parathyroid hormone (PTH) secretion and PTH gene expression. The FGF23-induced decrease in PTH secretion was prevented by a MAPK inhibitor. These data indicate that FGF23 acts directly on the parathyroid through the MAPK pathway to decrease serum PTH. This bone-parathyroid endocrine axis adds a new dimension to the understanding of mineral homeostasis.

Increased parathyroid hormone gene expression in secondary hyperparathyroidism of experimental uremia is reversed by calcimimetics: correlation with posttranslational modification of the trans acting factor AUF1.

Most patients with chronic kidney disease develop secondary hyperparathyroidism with disabling systemic complications. Calcimimetic agents are effective tools in the management of secondary hyperparathyroidism, acting through allosteric modification of the calcium-sensing receptor (CaR) on the parathyroid gland (PT) to decrease parathyroid hormone (PTH) secretion and PT cell proliferation. This study showed that rats that were fed an adenine high-phosphorus diet had increased serum PTH and PTH mRNA levels at 7 and 21 d. For studying the effect of activation of the CaR by the calcimimetics R-568 on PTH gene expression, R-568 was given by gavage to uremic rats for the last 4 d of a 7-d adenine high-phosphorus diet. R-568 decreased both PTH mRNA and serum PTH levels. The effect of the calcimimetic on PTH gene expression was posttranscriptional and correlated with differences in protein-RNA binding and posttranslational modifications of the trans acting factor AUF1 in the PT. The AUF1 modifications as a result of uremia were reversed by treatment with R-568 to those of normal rats. Therefore, uremia and activation of the CaR mediated by calcimimetics modify AUF1 posttranslationally. These modifications in AUF1 correlate with changes in protein-PTH mRNA binding and PTH mRNA levels.

Calcineurin Abeta is central to the expression of the renal type II Na/Pi co-transporter gene and to the regulation of renal phosphate transport.

The sensing and response to extracellular phosphate (Pi) concentration is preserved from prokaryotes to mammals and ensures an adequate supply of Pi in the face of large differences in its availability. In mammals, the kidneys are central to Pi homeostasis. Renal Pi reabsorption is mediated by a Na/Pi co-transporter that is regulated by a renal Pi sensing system and humoral factors. The signal transduction by which Pi regulates type II Na/Pi activity is largely unknown. It is shown that calcineurin inhibitors specifically and dramatically decrease type II Na/Pi gene expression in a proximal tubule cell line and in vivo. Mice with genetic deletion of the calcineurin Abeta gene had a marked decrease in type II Na/Pi mRNA levels and remarkably did not show the expected increase in type II Na/Pi mRNA levels after the challenge of a low-Pi diet. In contrast, the regulation of renal 25(OH)-vitamin D 1alpha-hydroxylase gene expression by Pi was intact. This is the first demonstration that calcineurin has a crucial role in the signal transduction pathway regulating renal Pi homeostasis both in vitro and in vivo. These results suggest that the use of calcineurin inhibitors contributes to the renal Pi wasting seen in renal transplant patients.