A rapid assessment of wastewater for genomic surveillance of SARS-CoV-2 variants at sewershed scale in Louisville, KY.

In this communication, we report on the genomic surveillance of SARS-CoV-2 using wastewater samples in Jefferson County, KY. In February 2021, we analyzed seven wastewater samples for SARS-CoV-2 genomic surveillance. Variants observed in smaller catchment areas, such as neighborhood manhole locations, were not necessarily consistent when compared to associated variant results in downstream treatment plants, suggesting catchment size or population could impact the ability to detect diversity.

Ubiquilin1 represses migration and epithelial-to-mesenchymal transition of human non-small cell lung cancer cells.

Ubiquilin1 (UBQLN1) is a ubiquitin-like domain and a ubiquitin-associated domain containing protein that has been reported to be involved in shuttling proteins to the proteasome, especially during endoplasmic reticulum-associated protein degradation. Thus, UBQLN1 function has been shown to be critical for combating a number of neurological disorders caused by protein aggregation, such as amyotrophic lateral sclerosis, Alzheimer's disease and Huntington's disease. A role for UBQLN1 in regulating processes involved in tumorigenesis has not been demonstrated. Herein, we show that loss of UBQLN1 causes increased cell migration and invasion, actin cytoskeleton reorganization and induction of epithelial-to-mesenchymal transition (EMT). Loss of UBQLN1 results in a significant decrease in the expression of epithelial markers including E-cadherin and claudin1, whereas expression of mesenchymal markers including Vimentin, Snail and ZEB1 are significantly elevated. Interestingly, we found that ZEB1 is required for induction of mesenchymal-like properties following loss of UBQLN1 and ZEB1 is capable of repressing expression of UBQLN1, suggesting a physiological, reciprocal regulation of EMT by UBQLN1 and ZEB1. Further, we find evidence for a role for UBQLN2 in also regulating EMT and cell migration. These observations have potential clinical relevance because the UBQLN1 gene is lost and underexpressed in a large percentage of human cancer cell lines, and primary human lung cancer samples and recurrent mutations in all five UBQLN family members have been identified in human lung cancers. Taken together, our results suggest for the first time a role for UBQLN family members in cancer biology.

Targeting cathepsin L (CL) by specific ribozymes decreases CL protein synthesis and cartilage destruction in rheumatoid arthritis.

The present study was undertaken to examine whether ribozymes cleaving specifically cathepsin L (CL) mRNA are able to decrease the synthesis of CL protease in rheumatoid arthritis synovial fibroblasts (RA-SF) and thereby reduce the invasiveness into cartilage both in vitro and in the SCID mouse coimplantation model of RA. Two different ribozymes that cleave CL mRNA specifically at positions 533 (RzCL533) and 790 (RzCL790) were generated. Using retroviral gene transfer, RA-SF were transduced with the ribozyme constructs or the empty vector. To examine the effect of the ribozymes on the mRNA level, quantitative analysis for CL mRNA was performed using real-time PCR. For evaluation on the protein level, ELISA using specific anti-CL antibodies was performed. In addition, transduced RA-SF were examined in vitro in a three-dimensional destruction assay evaluating their ability to degrade extracellular matrix produced by human chondrocytes. Matrix destruction was monitored by the release of soluble glycosaminoglycans (sGAG). Using the in vivo SCID mouse coimplantation model of RA, RzCL533-transduced RA-SF and control cells were coimplanted with human cartilage for 60 days. After being killed, invasion of RA-SF into the cartilage was evaluated by using a semiquantitative score. Transduction of RA-SF with RzCL533 and RzCL790 ribozymes decreased significantly the expression of CL mRNA to 44% (range 25-62%) and 20% (range 1-43%), respectively, when compared to mock-transduced cells. The protein concentration of CL in the cell culture supernatants of transduced RA-SF was decreased from 16.0 ng/ml in the mock constructs to 4.1 and 8.2 ng/ml (mean), respectively. Using the in vitro cartilage destruction assay, the release of sGAG decreased to 46 and 60%, respectively, after 14 days when compared to mock-transduced cells. In the SCID mouse coimplantation model of RA, RzCL533-transduced RA-SF revealed a significant lower cartilage invasion when compared to mock and untransduced cells. Using retroviral gene transfer, ribozymes cleaving CL mRNA inhibit specifically the synthesis of this matrix-degrading enzyme and reduce cartilage destruction in in vitro and in vivo models. Our study therefore suggests that ribozymes targeting CL could be a novel and efficient tool to inhibit joint destruction in RA.

Intra- and intermolecular triplex DNA formation in the murine c-myb proto-oncogene promoter are inhibited by mithramycin.

Mithramycin inhibits transcription by binding to G/C-rich sequences, thereby preventing regulatory protein binding. However, it is also possible that mithramycin inhibits gene expression by preventing intramolecular triplex DNA assembly. We tested this hypothesis using the DNA triplex adopted by the murine c-myb proto-oncogene. The 5'-regulatory region of c-myb contains two polypurine:polypyrimidine tracts with imperfect mirror symmetry, which are highly conserved in the murine and human c-myb sequences. The DNA binding drugs mithramycin and distamycin bind to one of these regions as determined by DNase I protection assay. Gel mobility shift assays, nuclease and chemical hypersensitivity and 2D-gel topological analyses as well as triplex-specific antibody binding studies confirmed the formation of purine*purine:pyrimidine inter- and pyrimidine*purine:pyrimidine intra-molecular triplex structures in this sequence. Mithramycin binding within the triplex target site displaces the major groove-bound oligonucleotide, and also abrogates the supercoil-dependent H-DNA formation, whereas distamycin binding had no such effects. Molecular modeling studies further support these observations. Triplex-specific antibody staining of cells pretreated with mithramycin demonstrates a reversal of chromosomal triplex structures compared to the non-treated and distamycin-treated cells. These observations suggest that DNA minor groove-binding drugs interfere with gene expression by precluding intramolecular triplex formation, as well as by physically preventing regulatory protein binding.

Variable expression of cathepsin B and D correlates with highly invasive and metastatic phenotype of oral cancer.

The expression levels of cathepsins B, D, and L in oral cancer surgical specimens were determined using immunocytochemical analysis. Cathepsins B and D are frequently overexpressed in squamous cell carcinomas, whereas their overexpression was less frequent in verrucous carcinoma and basaloid squamous cell carcinomas. Elevated level of cathepsin B in oral carcinomas was significantly associated with advanced tumor stage (P < .05) and poor histologic malignancy grade (P < .001). Increased expression of cathepsin D correlated significantly with the presence of metastasis (P < .05), poor histologic malignancy grade (P < .001), and high proliferation rate (P < .05). Cathepsin L was less frequently overexpressed in oral cancers than cathepsin B and D. These findings indicate that there is a strong cause/effect relationship between the expression levels of cathepsin B and D in oral cancers and their local invasive and metastatic growth patterns. Thus, cathepsins B and D are useful prognostic markers as well as promising gene therapy targets for oral cancer.

The integral divalent cation within the intermolecular purine*purine. pyrimidine structure: a variable determinant of the potential for and characteristics of the triple helical association.

In vitro assembly of an intermolecular purine*purine.pyrimidine triple helix requires the presence of a divalent cation. The relationships between cation coordination and triplex assembly were investigated, and we have obtained new evidence for at least three functionally distinct potential modes of divalent cation coordination. (i) The positive influence of the divalent cation on the affinity of the third strand for its specific target correlates with affinity of the cation for coordination to phosphate. (ii) Once assembled, the integrity of the triple helical structure remains dependent upon its divalent cation component. A mode of heterocyclic coordination/chelation is favorable to triplex formation by decreasing the relative tendency for efflux of integral cations from within the triple helical structure. (iii) There is also a detrimental mode of base coordination through which a divalent cation may actively antagonize triplex assembly, even in the presence of other supportive divalent cations. These results demonstrate the considerable impact of the cationic component, and suggest ways in which the triple helical association might be positively or negatively modulated.

RN first assistants expand their perioperative role.

At the Cleveland Clinic Foundation, the RN first assistant (RNFA) role has expanded to include radial artery (RA) harvesting for coronary artery bypass surgery. This new role encompasses preoperatively assessing the RA with the Allen's test and perfusion index; intraoperatively removing the RA, dissecting the RA as a pedicle, and maintaining hemostasis and wound closure; and postoperatively collaborating with the postoperative muse clinician during patient care and management in the intensive care unit. This article outlines the RNFA's expanded role in this detailed surgical procedure.

Divalent transition metal cations counteract potassium-induced quadruplex assembly of oligo(dG) sequences.

Nucleic acids containing tracts of contiguous guanines tend to self-associate into four-stranded (quadruplex) structures, based on reciprocal non-Watson-Crick (G*G*G*G) hydrogen bonds. The quadruplex structure is induced/stabilized by monovalent cations, particularly potassium. Using circular dichroism, we have determined that the induction/stabilization of quadruplex structure by K+is specifically counteracted by low concentrations of Mn2+(4-10 mM), Co2+(0.3-2 mM) or Ni2+(0.3-0.8 mM). G-Tract-containing single strands are also capable of sequence-specific non-Watson-Crick interaction with d(G. C)-tract-containing (target) sequences within double-stranded DNA. The assembly of these G*G.C-based triple helical structures is supported by magnesium, but is potently inhibited by potassium due to sequestration of the G-tract single strand into quadruplex structure. We have used DNase I protection assays to demonstrate that competition between quadruplex self-association and triplex assembly is altered in the presence of Mn2+, Co2+or Ni2+. By specifically counteracting the induction/stabilization of quadruplex structure by potassium, these divalent transition metal cations allow triplex formation in the presence of K+and shift the position of equilibrium so that a very high proportion of triplex target sites are bound. Thus, variation of the cation environment can differentially promote the assembly of multistranded nucleic acid structural alternatives.

The RN first assistant's role during inferior epigastric artery harvesting.

At the Cleveland Clinic Foundation, RN first assistants (RNFAs) have replaced inexperienced interns and rotating general surgery residents as surgical first assistants. Their new role includes harvesting inferior epigastric arteries (IEAs) for coronary artery bypass procedures. The RNFAs remove the IEAs through paramedian incisions that extend from below the umbilicus to the groin, dissect the IEAs as pedicles, and complete wound closure. This is one example of the expanded role of RNFAs during complex surgical procedures.

What is the basis of sequence-directed curvature in DNAs containing A tracts?

A variety of solution and gel experiments show that DNAs containing tracts of 4-8 A's repeated in phase with the helix repeat are curved. Several independent analyses of these experiments argue that curvature resides in the A tracts themselves. In x-ray crystallographic studies of several DNAs containing A tracts, however, the A tracts are uncurved, leading to models in which curvature resides in the non-A tracts. This "curved general sequence model" has several problems, in our view. We review those, and we describe recent experiments that show that the dehydrating agents commonly used in x-ray crystallography markedly reduce curvature in gels and in solution, calling into question the ability of crystallography to determine the structural basis of DNA curvature. Finally, we discuss the critical role of hydration in curved DNAs and suggest new experiments that we hope could finally determine exactly which sequences are responsible for curvature.

The role of the registered nurse-first assistant in the implantable left ventricular assist device program.

Successful support of patients using the implantable left ventricular assist device requires sustained and coordinated efforts by physicians and medical personnel. The authors describe the role of their registered nurse-first assistant (RNFA) as it has evolved through caring for 43 implantable pneumatic left ventricular assist device patients and 8 vented-electric left ventricular assist system patients during a 3 year period. Intraoperatively, the RNFA is responsible for pump assembly, including pre sealing all grafts and connecting areas of the pump using a combination of cryoprecipitate and thrombin. The RNFA assists with pump insertion during surgery. At device explantation, the RNFA dismantles the pump according to the FDA protocol for disassembly. Post operatively, the RNFA assesses and maintains patient hemodynamic stability and intervenes to manage hemodynamic and mechanical problems. Of the 51 patients, 13 are still on support, 9 died before transplantation (17.6%), and post transplant survival is 96.0%. In conclusion, an active left ventricular assist device program requires skilled personnel to manage complex problems and contributes to a successful patient outcome.

Dehydrating agents sharply reduce curvature in DNAs containing A tracts.

The structural basis of DNA curvature remains elusive, because models for curvature based on crystallographic structures of molecules containing A tracts do not agree with any of the models for sequence-directed curvature based on solution studies. Here we demonstrate that the difference is probably due to MPD (2-methyl-2,4-pentanediol), the dehydrating agent commonly used in crystallography. One characteristic signature of curved DNA molecules is that they run anomalously slowly on polyacrylamide gels, appearing to be larger than they actually are. The gel anomalies of three curved DNAs from trypanosome kinetoplast minicircles drop monotonically with increasing MPD concentration, indicating that MPD straightens molecules that are curved in aqueous solution. This is not due to some non-specific effect of MPD on poly(dA) or polypurine tracts, because control molecules containing dA70 and dG43 run normally over the full range of MPD concentrations. Circular dichroism spectra are not affected by MPD, ruling out a conformational change to a structure outside the B-DNA family. The effect is not due to MPD-induced changes in phasing of the curved sequences, because MPD has virtually no effect on the linking numbers of relaxed plasmids containing either curved sequences or dA70. At the concentrations of MPD used in X-ray crystallography, the curvature of DNAs containing A tracts is substantially lower than in solution, which probably explains the ongoing discrepancies between the crystallographic results and models based on solution studies.

Rapid method for the fractionation of nuclear proteins and their complexes by batch elution from hydroxyapatite.

A new procedure for the separation and purification of nuclear proteins and their complexes by batch elution from hydroxyapatite is presented. This method allows to isolate such proteins with different basic character faster and more efficiently than procedures using column chromatography, while showing high selectivity, sensitivity, simplicity, mild conditions of purification, reproducibility and protein stability.

Potassium permanganate as an in situ probe for B-Z and Z-Z junctions.

The availability of DNA structural probes that can be applied to living cells is essential for the analysis of biological functions of unusual DNA structures adopted in vivo. We have developed a chemical probe assay to detect and quantitate left-handed Z-DNA structures in recombinant plasmids in growing E. coli cells. Potassium permanganate selectively reacts with B-Z or Z-Z junction regions in supercoiled plasmids harbored in the cells. Restriction enzyme recognition sites located at these junctions are not cleaved by the corresponding endonuclease after modification with KMnO4. This inhibition of cleavage allows the determination of the relative amounts of B- and Z-forms of the cloned inserts inside the cell. We have successfully applied this method to monitor the extent of Z-DNA formation in E. coli as a function of the growth phase and mutated topoisomerase or gyrase activities. The assay can in principle be used for any unusual DNA structure that contains a restriction recognition site inside or near the structural alteration. It can be a useful tool to analyze in vivo correlations between DNA structure and gene regulatory events.

Topoisomerase mutants and physiological conditions control supercoiling and Z-DNA formation in vivo.

The influence of topoisomerase I and gyrase mutations in Escherichia coli on the supercoiled density of recombinant plasmids and the stability of left-handed Z-DNA was investigated. The formation of Z-DNA in vivo by dC-dG sequences of different lengths was used to determine the effective plasmid supercoil densities in the mutant strains. The presence of Z-DNA in the cells was detected by linking number and EcoRI methylase inhibition assays. A change in the unrestrained superhelical tension in vivo directly effects the B- to Z-DNA transition. Alterations in the internal or external environment of the cells, such as the inactivation of gyrase or topoisomerase I, a gyrase temperature-sensitive mutant, or starvation of cells, have a dramatic influence on the topology of plasmids. Also, E. coli has significantly more superhelical strain than Klebsiella, Morganella, or Enterobacter. These studies indicate that linking deficiency and effective supercoil density are mutually independent variables of plasmid tertiary structure. A variety of factors, such as protein-DNA interactions, activity of topoisomerases, and the resulting supercoil density, contribute to the B to Z transition inside living cells.

Cytosine methylation enhances Z-DNA formation in vivo.

The influence of cytosine methylation on the supercoil-stabilized B-Z equilibrium in Escherichia coli was analyzed by two independent assays. Both the M.EcoRI inhibition assay and the linking-number assay have been used previously to establish that dC-dG segments of sufficient lengths can exist as left-handed helices in vivo. A series of dC-dG plasmid inserts with Z-form potential, ranging in length from 14 to 74 base pairs, was investigated. Complete methylation of cytosine at all HhaI sites, including the inserts, was obtained by coexpression of the HhaI methyltransferase (M.HhaI) in cells also carrying a dC-dG-containing plasmid. Both assays showed that for all lengths of dC-dG inserts, the relative amounts of B and Z helices were shifted to more Z-DNA in the presence of M.HhaI than in the absence of M.HhaI. These results indicate that cytosine methylation enhances the formation of Z-DNA helices at the superhelix density present in E. coli. The B-Z equilibrium, in combination with site-specific base methylation, may constitute a concerted mechanism for the modulation of DNA topology and DNA-protein interactions.

Lupus-inducing drugs alter the structure of supercoiled circular DNA domains.

We analyzed the effects of procainamide (PROC), hydralazine (HYD), N-acetylprocainamide (NAPA), and L-canavanine (CAN) on circular supercoiled plasmids as models for chromosomal loop domains. The supercoil-dependent B-Z equilibrium in recombinant plasmids was used as an indicator of structural changes induced in circular DNA. Two-dimensional gel electrophoresis showed that PROC and HYD strongly inhibited supercoil-induced Z-DNA formation, whereas NAPA caused less pronounced changes in the B-Z equilibrium, and CAN had no effect. Gel retardation assays showed that the binding of a Z-DNA-specific autoimmune antibody to a Z-DNA-containing plasmid was strongly perturbed by HYD, but not influenced by CAN. Both PROC and NAPA showed moderate inhibition of antibody binding. Our results demonstrate the different potentials of these 4 drugs to interact with DNA and to alter the tertiary topology of DNA domains. It is conceivable that the in vivo capacity of PROC and HYD to induce antinuclear antibodies may be related to their ability to influence structural features in chromosomal DNA domains or nucleosomes, thus liberating antigenic structural epitopes in DNA and/or DNA-associated proteins.

Probing salt-induced and supercoiling-induced B-Z junctions in DNA by bisulfitemethoxyamine.

Salt-induced and supercoiling-induced B-Z junctions in pWR756, a plasmid containing (GC)16, were probed with bisulfite-methoxyamine, a modification reagent specific for single-stranded nucleic acids. The modification sites were analyzed with S1 nuclease and the modified cytosines were determined from termination sites of DNA chain elongation by DNA polymerase. The results showed that most accessible cytosines are the same for both types of B-Z junctions.