Urine osmolality assessment through the integration of urea hydrolysis and impedance measurement.

We present the development and validation of an impedance-based urine osmometer for accurate and portable measurement of urine osmolality. The urine osmolality of a urine sample can be estimated by determining the concentrations of the conductive solutes and urea, which make up approximately 94% of the urine composition. Our method utilizes impedance measurements to determine the conductive solutes and urea after hydrolysis with urease enzyme. We built an impedance model using sodium chloride (NaCl) and urea at various known concentrations. In this work, we validated the accuracy of the impedance-based urine osmometer by developing a proof-of-concept first prototype and an integrated urine dipstick second prototype, where both prototypes exhibit an average accuracy of 95.5 ± 2.4% and 89.9 ± 9.1%, respectively in comparison to a clinical freezing point osmometer in the hospital laboratory. While the integrated dipstick design exhibited a slightly lower accuracy than the first prototype, it eliminated the need for pre-mixing or manual pipetting. Impedance calibration curves for conductive and non-conductive solutes consistently yielded results for NaCl but underscored challenges in achieving uniform urease enzyme coating on the dipstick. We also investigated the impact of storing urine at room temperature for 24 hours, demonstrating negligible differences in osmolality values. Overall, our impedance-based urine osmometer presents a promising tool for point-of-care urine osmolality measurements, addressing the demand for a portable, accurate, and user-friendly device with potential applications in clinical and home settings.

Feasibility and utility of facemask sampling in the detection of SARS-CoV-2 during an ongoing pandemic.

Easy access to screening for timely identification and isolation of infectious COVID-19 patients remains crucial in sustaining the international efforts to control COVID-19 spread. A major barrier limiting broad-based screening is the lack of a simple, rapid, and cost-effective COVID-19 testing method. We evaluated the feasibility and utility of facemask sampling in a cohort of 42 COVID-19-positive and 36 COVID-19-negative patients. We used a prototype of Steri-Strips™ (3 M) applied to the inner surface of looped surgical facemasks (Assure), which was worn by patients for a minimum wear time of 3 h, then removed and sent for SARS-CoV-2 PCR testing. Baseline demographics and symptomatology were also collected. Facemask sampling positivity was highest within the first 5 days of symptomatic presentation. Patients with nasopharyngeal and/or oropharyngeal swab SARS-CoV-2 PCR Ct values < 25.09 had SARS-CoV-2 detected on facemask sampling, while patients with Ct values ≥ 25.2 had no SARS-CoV-2 detected on facemask sampling. Facemask sampling can identify patients with COVID-19 during the early symptomatic phase or those with high viral loads, hence allowing timely identification and isolation of those with the highest transmission risk. Given the widespread use of facemasks, this method can potentially be easily applied to achieve broad-based, or even continuous, population screening.

Anti-survivin effect of the small molecule inhibitor YM155 in RCC cells is mediated by time-dependent inhibition of the NF-κB pathway.

Constitutive activation of the NF-κB signaling cascade is associated with tumourigenesis and poor prognosis in many human cancers including RCC. YM155, a small molecule inhibitor of survivin, was previously shown to potently inhibit the viability of immortalized and patient derived renal cell carcinoma (RCC) cell lines. Here we investigated the role of NF-κB signaling to the anti-cancer properties of YM155 in RCC786.0 cells. YM155 diminished nuclear levels of p65 and phosphorylated p65 and attenuated the transcriptional competencies of the p65/p50 heterodimers. Accordingly, we found that YM155 diminished the transcription of NF-κB target gene survivin. Events that led to the interception of the nuclear translocation of p65/p50 were the activation of the deubiquinating enzyme CYLD by YM155, which led to the inhibition of IKKß, stabilization of IκBα and retention of NF-κB heterodimers in the cytosol. Importantly, the suppressive effects of YM155 were time-dependent and observed at the 24 h time point, and not earlier. TNF-α, a stimulator of NF-κB signaling did not affect its inhibitory properties. The ability of YM155 to intercept a major transcriptional pathway like NF-κB, would have important ramifications on the pharmacodynamics effects elicited by this unusual molecule.

The mechanistic effects of the dioxonaphthoimidazolium analog YM155 in renal cell carcinoma cell cycling and apoptosis.

AIM: To investigate the effect of dioxonaphthoimidazolium analog YM155 on cell cycle progression of the clear-cell variant of renal cell carcinoma (ccRCC). MAIN METHODS: Cell cycle analysis was performed using bromodeoxyuridine (BrdU) and PI, apoptosis initiation was monitored using Annexin V and proteins expression was determined using western immunoblotting. KEY FINDINGS: Here, we showed that YM155 activated stress-related molecules (histone H2AX, checkpoint kinases Chk1 and Chk2, p53) that mediate DNA damage checkpoint responses. The coordinated activation of these effector molecules disrupts progression of the cell cycle at the S phase as deduced from BrdU pulsing experiments and the ensuing changes in the levels of proteins (cyclins, CDKs, CDK inhibitors, phosphatases) that control cell cycle progression. Notably, we found increases in cyclin E and Cdc2 which regulate transition of cells from G1 to S, even as losses were observed for other CDKs and their cyclin partners. Furthermore, by inducing a loss in total pRb possibly by promoting its degradation, YM155 promoted the E2F transcription of genes that regulate entry into the S phase. After 24 h, cell cycle arrest to repair YM155-inflicted DNA damage was overtaken by p53-mediated apoptosis. YM155 induced increases in pro-apoptotic proteins (Bax and Bad), diminished anti-apoptotic proteins (Mcl-1, Bcl-xl, XIAP, survivin) and initiated cleavage of apoptotic marker proteins caspase 3 and PARP. SIGNIFICANCE: Taken together, the added insight provided on the cell cycle perturbative effects of YM155 may assist clinicians in framing rational choices for combining YM155 with other anti-cancer drugs or treatment modalities in ccRCC.

Action of YM155 on clear cell renal cell carcinoma does not depend on survivin expression levels.

The dioxonapthoimidazolium YM155 is a survivin suppressant which has been investigated as an anticancer agent in clinical trials. Here, we investigated its growth inhibitory properties on a panel of immortalized and patient derived renal cell carcinoma (RCC) cell lines which were either deficient in the tumour suppressor von Hippel-Lindau (VHL) protein or possessed a functional copy. Neither the VHL status nor the survivin expression levels of these cell lines influenced their susceptibility to growth inhibition by YM155. Of the various RCC lines, the papillary subtype was more resistant to YM155, suggesting that the therapeutic efficacy of YM155 may be restricted to clear cell subtypes. YM155 was equally potent in cells (RCC786.0) in which survivin expression had been stably silenced or overexpressed, implicating a limited reliance on survivin in the mode of action of YM155. A follow-up in-vitro high throughput RNA microarray identified possible targets of YM155 apart from survivin. Selected genes (ID1, FOXO1, CYLD) that were differentially expressed in YM155-sensitive RCC cells and relevant to RCC pathology were validated with real-time PCR and western immunoblotting analyses. Thus, there is corroboratory evidence that the growth inhibitory activity of YM155 in RCC cell lines is not exclusively mediated by its suppression of survivin. In view of the growing importance of combination therapy in oncology, we showed that a combination of YM155 and sorafenib at ½ x IC50 concentrations was synergistic on RCC786.0 cells. However, when tested intraperitoneally on a murine xenograft model derived from a nephrectomised patient with clear cell RCC, a combination of suboptimal doses of both drugs failed to arrest tumour progression. The absence of synergy in vivo highlighted the need to further optimize the dosing schedules of YM155 and sorafenib, as well as their routes of administration. It also implied that the expression of other oncogenic proteins which YM155 may target is either low or absent in this clear cell RCC.

Antiproliferative, DNA intercalation and redox cycling activities of dioxonaphtho[2,3-d]imidazolium analogs of YM155: A structure-activity relationship study.

The anticancer agent YM155 is widely investigated as a specific survivin suppressant. More recently, YM155 was found to induce DNA damage and this has raised doubts as to whether survivin is its primary target. In an effort to assess the contribution of DNA damage to the anticancer activity of YM155, several analogs were prepared and evaluated for antiproliferative activity on malignant cells, participation in DNA intercalation and free radical generation by redox cycling. The intact positively charged scaffold was found to be essential for antiproliferative activity and intercalation but was less critical for redox cycling where the minimal requirement was a pared down bicyclic quinone. Side chain requirements at the N(1) and N(3) positions of the scaffold were more alike for redox cycling and intercalation than antiproliferative activity, underscoring yet again, the limited structural overlaps for these activities. Furthermore, antiproliferative activities were poorly correlated to DNA intercalation and redox cycling. Potent antiproliferative activity (IC50 9-23 nM), exceeding that of YM155, was found for a minimally substituted methyl analog AB7. Like YM155 and other dioxonaphthoimidazoliums, AB7 was a modest DNA intercalator but with weak redox cycling activity. Thus, the capacity of this scaffold to inflict direct DNA damage leading to cell death may not be significant and YM155 should not be routinely classified as a DNA damaging agent.

CD1d expression in renal cell carcinoma is associated with higher relapse rates, poorer cancer-specific and overall survival.

AIMS: We hypothesised that CD1d expression in renal cell carcinoma (RCC) may play a role in modifying the host immune response. Our aims were to investigate the expression of CD1d and to correlate this with histopathology and clinical outcomes in a cohort study of patients with RCC. METHODS: Gene expression and tissue microarray studies on a panel of RCC tissue were performed. Clinicopathological correlation was analysed using χ(2)/Fisher's exact test. Relapse-free survival, cancer-specific survival and overall survival were calculated for both CD1d high and low expressors. Survival outcomes were estimated with the Kaplan-Meier method and compared using Cox regression analysis. RESULTS: Gene expression microarray showed significant expression of CD1d in RCC versus normal renal tissue. By immunohistochemistry, we found that CD1d expression significantly associated with tumour stage/grade, higher relapse rates, poorer cancer-specific and overall survival. CONCLUSIONS: CD1d expression on RCC correlated with aggressive disease and poorer clinical outcomes.

Combination of the ERK inhibitor AZD6244 and low-dose sorafenib in a xenograft model of human renal cell carcinoma.

Sorafenib, a multikinase inhibitor, is currently used as monotherapy for advanced renal cell carcinoma (RCC). However, adverse effects associated with its use have been experienced by some patients. In this study, we examined the antitumor and antiangiogenic activities of low-dose sorafenib in combination with the MEK inhibitor AZD6244 (sorafenib/AZD6244) in a preclinical model of RCC. Primary RCC 08-0910 and RCC 786-0 cells as well as patient-derived RCC models were used to study the antitumor and antiangiogenic activities of sorafenib/AZD6244. Changes of biomarkers relevant to angiogenesis and cell cycle were determined by western immunoblotting. Microvessel density, apoptosis and cell proliferation were analyzed by immunohistochemistry. Treatment of RCC 786-0 cells with sorafenib/AZD6244 resulted in G1 cell cycle arrest and blockade of serum-induced cell migration. Sorafenib/AZD6244 induced apoptosis in primary RCC 08-0910 cells at low concentrations. In vivo addition of AZD6244 to sorafenib significantly augmented the antitumor activity of sorafenib and allowed dose reduction of sorafenib without compromising its antitumor activity. Sorafenib/AZD6244 potently inhibited angiogenesis and phosphorylation of VEGFR-2, PDGFR-ß and ERK, p90RSK, p70S6K, cdk-2 and retinoblastoma. Sorafenib/AZD6244 also caused upregulation of p27, Bad and Bim but downregulation of survivin and cyclin B1. These resulted in a reduction in cellular proliferation and the induction of tumor cell apoptosis. Our findings showed that AZD6244 and sorafenib complement each other to inhibit tumor growth. This study provides sound evidence for the clinical investigation of low-dose sorafenib in combination with AZD6244 in patients with advanced RCC.