Genetic diversity and evolutionary convergence of cryptic SARS- CoV-2 lineages detected via wastewater sequencing.

Wastewater-based epidemiology (WBE) is an effective way of tracking the appearance and spread of SARS-COV-2 lineages through communities. Beginning in early 2021, we implemented a targeted approach to amplify and sequence the receptor binding domain (RBD) of SARS-COV-2 to characterize viral lineages present in sewersheds. Over the course of 2021, we reproducibly detected multiple SARS-COV-2 RBD lineages that have never been observed in patient samples in 9 sewersheds located in 3 states in the USA. These cryptic lineages contained between 4 to 24 amino acid substitutions in the RBD and were observed intermittently in the sewersheds in which they were found for as long as 14 months. Many of the amino acid substitutions in these lineages occurred at residues also mutated in the Omicron variant of concern (VOC), often with the same substitutions. One of the sewersheds contained a lineage that appeared to be derived from the Alpha VOC, but the majority of the lineages appeared to be derived from pre-VOC SARS-COV-2 lineages. Specifically, several of the cryptic lineages from New York City appeared to be derived from a common ancestor that most likely diverged in early 2020. While the source of these cryptic lineages has not been resolved, it seems increasingly likely that they were derived from long-term patient infections or animal reservoirs. Our findings demonstrate that SARS-COV-2 genetic diversity is greater than what is commonly observed through routine SARS-CoV-2 surveillance. Wastewater sampling may more fully capture SARS-CoV-2 genetic diversity than patient sampling and could reveal new VOCs before they emerge in the wider human population.

Genetic Diversity and Evolutionary Convergence of Cryptic SARS-CoV-2 Lineages Detected Via Wastewater Sequencing.

Wastewater-based epidemiology (WBE) is an effective way of tracking the appearance and spread of SARS-COV-2 lineages through communities. Beginning in early 2021, we implemented a targeted approach to amplify and sequence the receptor binding domain (RBD) of SARS-COV-2 to characterize viral lineages present in sewersheds. Over the course of 2021, we reproducibly detected multiple SARS-COV-2 RBD lineages that have never been observed in patient samples in 9 sewersheds located in 3 states in the USA. These cryptic lineages contained between 4 to 24 amino acid substitutions in the RBD and were observed intermittently in the sewersheds in which they were found for as long as 14 months. Many of the amino acid substitutions in these lineages occurred at residues also mutated in the Omicron variant of concern (VOC), often with the same substitution. One of the sewersheds contained a lineage that appeared to be derived from the Alpha VOC, but the majority of the lineages appeared to be derived from pre-VOC SARS-COV-2 lineages. Specifically, several of the cryptic lineages from New York City appeared to be derived from a common ancestor that most likely diverged in early 2020. While the source of these cryptic lineages has not been resolved, it seems increasingly likely that they were derived from immunocompromised patients or animal reservoirs. Our findings demonstrate that SARS-COV-2 genetic diversity is greater than what is commonly observed through routine SARS-CoV-2 surveillance. Wastewater sampling may more fully capture SARS-CoV-2 genetic diversity than patient sampling and could reveal new VOCs before they emerge in the wider human population. Author Summary: During the COVID-19 pandemic, wastewater-based epidemiology has become an effective public health tool. Because many infected individuals shed SARS-CoV-2 in feces, wastewater has been monitored to reveal infection trends in the sewersheds from which the samples were derived. Here we report novel SARS-CoV-2 lineages in wastewater samples obtained from 3 different states in the USA. These lineages appeared in specific sewersheds intermittently over periods of up to 14 months, but generally have not been detected beyond the sewersheds in which they were initially found. Many of these lineages may have diverged in early 2020. Although these lineages share considerable overlap with each other, they have never been observed in patients anywhere in the world. While the wastewater lineages have similarities with lineages observed in long-term infections of immunocompromised patients, animal reservoirs cannot be ruled out as a potential source.

Salinomycin induces selective cytotoxicity to MCF-7 mammosphere cells through targeting the Hedgehog signaling pathway.

Breast cancer stem cells (BCSCs) are believed to be responsible for tumor chemoresistance, recurrence, and metastasis formation. Salinomycin (SAL), a carboxylic polyether ionophore, has been reported to act as a selective breast CSC inhibitor. However, the molecular mechanisms underlying SAL-induced cytotoxicity on BCSCs remain unclear. The Hedgehog (Hh) signaling pathway plays an important role in CSC maintenance and carcinogenesis. Here, we investigated whether SAL induces cytotoxicity on BCSCs through targeting Hh pathway. In the present study, we cultured breast cancer MCF-7 cells in suspension in serum-free medium to obtain breast CSC-enriched MCF-7 mammospheres (MCF-7 MS). MCF-7 MS cells possessed typical BCSC properties, such as CD44+CD24-/low phenotype, high expression of OCT4 (a stem cell marker), increased colony-forming ability, strong migration and invasion capabilities, differentiation potential, and strong tumorigenicity in xenografted mice. SAL exhibited selective cytotoxicity to MCF-7 MS cells relative to MCF-7 cells. The Hh pathway was highly activated in BCSC-enriched MCF-7 MS cells and SAL inhibited Hh signaling activation by downregulating the expression of critical components of the Hh pathway such as PTCH, SMO, Gli1, and Gli2, and subsequently repressing the expression of their essential downstream targets including C-myc, Bcl-2, and Snail (but not cyclin D1). Conversely, Shh-induced Hh signaling activation could largely reverse SAL-mediated inhibitory effects. These findings suggest that SAL-induced selective cytotoxicity against MCF-7 MS cells is associated with the inhibition of Hh signaling activation and the expression of downstream targets and the Hh pathway is an important player and a possible drug target in the pathogenesis of BCSCs.

[Relief of urinary retention after radical hysterectomy for cervical cancer patients by acupressure: a randomized controlled trial].

OBJECTIVE: To study whether acupressure could relieve urinary retention after radical hysterectomy in cervical cancer patients. METHODS: A randomized controlled prospective double-blinded trial was carried out in 107 urinary retention patients undergoing grade III radical hysterectomy. They were assigned to Group A (positive acupoints, 40 cases), Group B (negative acupoints, 32 cases) , and Group C (with no acupoints, 35 cases). All patients received protective 115 000 potassium permanganate sitz bath, 15 - 20 min each time, 3 times per day. Patients in Group A received acupressure at positive points [liniao point and Qihai (RN6)] combined points by syndrome typing [Guanyuan (RN4) , Zhongji (RN3) , Shenshu (BL23) , Zusanli (ST36), Sanyinjiao (SP6), and Taixi (K13)]. Patients in Group B received negative acupressure at sham-acupoints (for adjusting gastrointestinal functions). Patients in Group C only received conventional sitz bath. All medication was performed 3 times per day, 7 days as one therapeutic course, 21 days in total. The residual urine volume was detected. The recovery time for bladder function was recorded. The average residual urine volume was also recorded at day 7, 14, and 21. RESULTS: Compared with Group B and C, the time for ureter retention was shortened for mild and severe CKD patients in Group A (P <0. 01). The residual urine volume was also lessened for mild and severe CKD patients in Group A at day 7, 14, and 21 (P <0.01). CONCLUSION: Cervical cancer patients could relieve urinary retention by self-acupressure after radical hysterectomy.

Genome Sequences of Pseudoalteromonas Strains ATCC BAA-314, ATCC 70018, and ATCC 70019.

The assembly and annotation of the draft genome sequences for Pseudoalteromonas strains ATCC BAA314, ATCC 700518, and ATCC 700519 reveal candidates for promoting symbiosis between Pseudoalteromonas strains and eukaryotes. Groups of genes generally associated with virulence are present in all three strains, suggesting that these bacteria may be pathogenic under specific circumstances.

Gene expression profile in rat adrenal zona glomerulosa cells stimulated with aldosterone secretagogues.

The mineralocorticoid aldosterone, mainly produced by the adrenal gland, is essential for life, but an abnormally excessive secretion causes severe pathological effects including hypertension and target organ injury in the heart and kidney. The aim of this study was to determine the gene regulatory network triggered by aldosterone secretagogues in a nontransformed cell system. Freshly isolated rat adrenal zona glomerulosa cells were stimulated with the two main aldosterone secretagogues, angiotensin II and potassium, for 2 h and subjected to whole genome expression studies using multiple biological and bioinformatics tools. Several genes were differentially expressed by ANG II (n = 133) or potassium (n = 216). Genes belonging to the nucleic acid binding and transcription factor activity categories were significantly enriched. A subset of the most regulated genes was confirmed by real-time RT-PCR, and then their expression was analyzed in time curve studies. Differentially expressed genes were grouped according to their time response expression pattern, and their promoter regions were analyzed for common regulatory transcription factor binding sites. Finally, data mining with gene promoters, transcription factors, and literature databases was performed to generate gene interaction networks for either ANG II or potassium. This paper provides for the first time a complete study of the genes that are regulated, and the interaction between them, by aldosterone secretagogues in rat adrenal cells. Increasing our knowledge of adrenal physiology and gene regulation in nontransformed cell systems could lead us to a better approach for the discovery of candidate genes involved in pathological conditions of the adrenal cortex.

Regulators of G-protein signaling 4 in adrenal gland: localization, regulation, and role in aldosterone secretion.

Regulators of G-protein signaling (RGS proteins) interact with Galpha subunits of heterotrimeric G-proteins, accelerating the rate of GTP hydrolysis and finalizing the intracellular signaling triggered by the G-protein-coupled receptor (GPCR)-ligand interaction. Angiotensin II (Ang II) interacts with its GPCR in adrenal zona glomerulosa cells and triggers a cascade of intracellular signals that regulates steroidogenesis and proliferation. On screening for adrenal zona glomerulosa-specific genes, we found that RGS4 was exclusively localized in the zona glomerulosa of the rat adrenal cortex. We studied RGS4 expression and regulation in the rat adrenal gland, including the signaling pathways involved, as well as the role of RGS4 in steroidogenesis in human adrenocortical H295R cells. We reported that RGS4 mRNA expression in the rat adrenal gland was restricted to the adrenal zonal glomerulosa and upregulated by low-salt diet and Ang II infusion in rat adrenal glands in vivo. In H295R cells, Ang II caused a rapid and transient increase in RGS4 mRNA levels mediated by the calcium/calmodulin/calmodulin-dependent protein kinase and protein kinase C pathways. RGS4 overexpression by retroviral infection in H295R cells decreased Ang II-stimulated aldosterone secretion. In reporter assays, RGS4 decreased Ang II-mediated aldosterone synthase upregulation. In summary, RGS4 is an adrenal gland zona glomerulosa-specific gene that is upregulated by aldosterone secretagogues, in vivo and in vitro, and functions as a negative feedback of Ang II-triggered intracellular signaling. Alterations in RGS4 expression levels or functions may be involved in deregulations of Ang II signaling and abnormal aldosterone secretion.

Adrenal transcription regulatory genes modulated by angiotensin II and their role in steroidogenesis.

Transcription regulatory genes are crucial modulators of cell physiology and metabolism whose intracellular levels are tightly controlled to respond to extracellular stimuli. We studied transcription regulatory genes modulated by angiotensin II, one of the most important regulators of adrenal cortical cell function, and their role in adrenal steroidogenesis in H295R human adrenocortical cells. Angiotensin II-modulated transcription regulatory genes were identified with high-density oligonucleotide microarrays and the results validated by real-time RT-PCR. Cotransfection reporter assays were performed in H295R cells to analyze the role of these transcription regulatory genes in the control of the expression of 11beta-hydroxylase and aldosterone synthase, the last and unique enzymes of the glucocorticoid and mineralocorticoid biosynthetic pathways, respectively. We selected a subset of the most regulated genes for reporter plasmid studies to determine the effect on these enzymes. BHLHB2, BTG2, and SALL1 decreased expression of both enzymes, whereas CITED2, EGR2, ELL2, FOS, FOSB, HDAC5, MAFF, MITF, NFIL3, NR4A1, NR4A2, NR4A3, PER1, and VDR increased expression for both enzymes. By the ratio of aldosterone synthase to 11beta-hydroxylase expression, NFIL3, NR4A1, NR4A2, and NR4A3 show the greatest selectivity toward upregulating expression of the mineralocorticoid biosynthetic pathway preferentially. In summary, this study reports for the first time a set of transcription regulatory genes that are modulated by angiotensin II and their role in adrenal gland steroidogenesis. Abnormal regulation of the mineralocorticoid or glucocorticoid biosynthesis pathways is involved in several pathophysiological conditions; hence the modulated transcription regulatory genes described may correlate with adrenal steroidogenesis pathologies.

Regulation of 11 beta-hydroxysteroid dehydrogenase enzymes in the rat kidney by estradiol.

The 11beta-hydroxysteroid dehydrogenase (11betaHSD) type 1 (11betaHSD1) enzyme is an NADP+-dependent oxidoreductase, usually reductase, of major glucocorticoids. The NAD+-dependent type 2 (11betaHSD2) enzyme is an oxidase that inactivates cortisol and corticosterone, conferring extrinsic specificity of the mineralocorticoid receptor for aldosterone. We reported that addition of a reducing agent to renal homogenates results in the monomerization of 11betaHSD2 dimers and a significant increase in NAD+-dependent corticosterone conversion. Estrogenic effects on expression, dimerization, and activity of the kidney 11betaHSD1 and -2 enzymes are described herein. Renal 11betaHSD1 mRNA and protein expressions were decreased to very low levels by estradiol (E2) treatment of both intact and castrated male rats; testosterone had no effect. NADP+-dependent enzymatic activity of renal homogenates from E2-treated rats measured under nonreducing conditions was less than that of homogenates from intact animals. Addition of 10 mM DTT to aliquots from these same homogenates abrogated the difference in NADP+-dependent activity between E2-treated and control rats. In contrast, 11betaHSD2 mRNA and protein expressions were significantly increased by E2 treatment. There was a marked increase in the number of juxtamedullary proximal tubules stained by the antibody against 11betaHSD2 after the administration of E2. Notwithstanding, neither the total corticosterone and 11-dehydrocorticosterone excreted in the urine nor their ratio differed between E2- and vehicle-treated rats. NAD+-dependent enzymatic activity in the absence or presence of a reducing agent demonstrated that the increase in 11betaHSD2 protein was not associated with an increase in in vitro activity unless the dimers were reduced to monomers.

Interferon-inducible genes in the rat adrenal gland and vascular smooth muscle cells.

Chronic stimulation of the renin-angiotensin system results in increased zona glomerulosa cells and in cells expressing the final enzyme in the synthesis of aldosterone, the cytochrome P-450 aldosterone synthase. The genes activated during adrenal remodeling are not well defined. We have reported that the expression of interferon-inducible genes, 9-27, 1-8D and 1-8U in H295R cells is stimulated by A-II. The 9-27 gene is expressed mainly in leukocytes and is associated with cell proliferation. In this study, we searched for similar genes in a rat zona glomerulosa cDNA library, and examined the regulation of the expression of these genes. We found the Rat8 gene, which has been reported to be similar to human interferon-inducible genes, as well as two similar genes, No. 10 (1096 bp), and No. 16 (630 bp). Rat8 gene and No. 16 were mainly expressed in zona glomerulosa. The product of No. 10 is thought to be a secreted protein, unlike those of 8 and 16, and its expression in the adrenal was weak in comparison. The control of the expression of rat8 or No. 16 genes differs depending on the tissue. Expression in A10 cells (derived from rat embryo thoracic aorta) was not stimulated by A-II, nor was it influenced by salt intake in the adrenal gland, but it was reduced in vascular smooth muscle cells (VSMC) of rats on a low sodium diet. These results show that genes similar to the human 1-8 gene family are expressed in rat adrenal glomerulosa cells and VSMC, but their expression is not regulated by A-II. The function of these genes in VSMC and adrenal is unknown.