Clinical evaluation of a fully automated and high-throughput molecular testing system for detection of influenza virus

Introduction: We investigated the performance of the cobas® 6800 system and cobas SARS-CoV-2 & Influenza A/B, a fully automated molecular testing system for influenza viruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This enabled an assay in a batch of 96 samples in approximately 3 h. Methods: An assay was performed using the cobas SARS-CoV-2 & Influenza A/B on the cobas 6800 system for samples collected in four facilities between November 2019 and March 2020 in our previous study. The results were compared with those obtained using the reference methods.Results: Of the 127 samples analyzed, the cobas SARS-CoV-2 & Influenza A/B detected influenza A virus in 75 samples, of which 73 were positive using the reference methods. No false negative results were observed. The overall positive and negative percent agreement for influenza A virus detection were 100.0% and 96.3%, respectively. There were no positive results for the influenza B virus or SARS-CoV-2.Conclusion: The cobas 6800 system and cobas SARS-CoV-2 & Influenza A/B showed high accuracy for influenza A virus detection and can be useful for clinical laboratories, especially those that routinely assay many samples

Different Activity Patterns in Retinal Ganglion Cells of TRPM1 and mGluR6 Knockout Mice

TRPM1, the first member of the melanoma-related transient receptor potential (TRPM) subfamily, is the visual transduction channel downstream of metabotropic glutamate receptor 6 (mGluR6) on retinal ON bipolar cells (BCs). Human TRPM1 mutations are associated with congenital stationary night blindness (CSNB). In both TRPM1 and mGluR6 KO mouse retinas, OFF but not ON BCs respond to light stimulation. Here we report an unexpected difference between TRPM1 knockout (KO) and mGluR6 KO mouse retinas. We used a multielectrode array (MEA) to record spiking in retinal ganglion cells (RGCs). We found spontaneous oscillations in TRPM1 KO retinas, but not in mGluR6 KO retinas. We performed a structural analysis on the synaptic terminals of rod ON BCs. Intriguingly, rod ON BC terminals were significantly smaller in TRPM1 KO retinas than in mGluR6 KO retinas. These data suggest that a deficiency of TRPM1, but not of mGluR6, in rod ON bipolar cells may affect synaptic terminal maturation. We speculate that impaired signaling between rod BCs and AII amacrine cells (ACs) leads to spontaneous oscillations. TRPM1 and mGluR6 are both essential components in the signaling pathway from photoreceptors to ON BC dendrites, yet they differ in their effects on the BC terminal and postsynaptic circuitry

Implication of an increased oxidative stress in the formation of advanced glycation end products in patients with end-stage renal failure

Recent studies have demonstrated a marked increase in the level of advanced glycation end products (AGEs) in the plasma, skin and amyloid fibrils of hemodialysis (HD) patients. The presence of AGEs in (beta(2)m) forming amyloid fibrils has been established in a previous immunochemical study relying on a monoclonal anti-AGE antibody. In the present study, Western blot analysis and immunohistochemistry reveal that the epitope recognized by this antibody is N-epsilon-(carboxymethyl)lysine (CML) and that CML is one of the AGE structures present in amyloid fibrils. Thus, two AGE structures, CML and pentosidine, are now recognized in dialysis-related amyloidosis. AGE accumulation in uremia is not accounted for by elevated glucose levels. Since CML and pentosidine formation are closely linked to oxidative processes, we tested the hypothesis that a high oxidative stress enhanced AGE formation in HD patients. We focused on ascorbic acid (AA) because AA is easily oxidized under oxidative stress and its oxidized form (oxiAA) is a source of CML and pentosidine. In vitro incubation of beta(2)m with AA under atmospheric oxygen resulted in: (1) the rapid appearance of characteristic physicochemical properties of AGEs (brown color, fluorescence, polymerization tendency); (2) the transformation of beta(2)m into AGE-modified beta(2)m recognized by a specific monoclonal antibody; and (3) the accelerated formation of CML in beta(2)m and beta(2)m-peptide, recognized by mass spectrometry. A similar in vitro incubation of human serum albumin disclosed a parallel production of pentosidine measured by high-performance liquid chromatographic assay. In HD patients, the degree of AA oxidation, assessed as the ratio of oxiAA to total ascorbate, was more than twice as high as that of normal subjects (0.87 +/- 0.16 vs. 0.35 +/- 0.11, P < 0.0001), suggesting the presence of an increased oxidative stress. interestingly, plasma level of oxiAA was correlated with the plasma levels of protein linked (P < 0.01, r(2) = 0.25) and free (P < 0.05, r(2) = 0.22) pentosidine. Altogether these results demonstrate that AGE, that is, CML and pentosidine, production is accelerated under oxidative stress, even in the absence of glucose. They suggest that, in uremia, CML and pentosidine production is determined both by an increased oxidative stress and the availability of precursors such as oxiAA. Finally, both CML and pentosidine contribute to the AGEs present in dialysis-related amyloid fibrils

Hepatocyte-specific Pten deficiency results in steatohepatitis and hepatocellular carcinomas

PTEN is a tumor suppressor gene mutated in many human cancers, and its expression is reduced or absent in almost half of hepatoma patients. We used the Cre-loxP system to generate a hepatocyte-specific null mutation of Pten in mice (AlbCrePten(flox/flox) mice). AlbCrePten(flox/flox) mice showed massive hepatomegaly and steatohepatitis with triglyceride accumulation, a phenotype similar to human nonalcoholic steatohepatitis. Adipocyte-specific genes were induced in mutant hepatocytes, implying adipogenic-like transformation of these cells. Genes involved in lipogenesis and β-oxidation were also induced, possibly as a result of elevated levels of the transactivating factors PPARγ and SREBP1c. Importantly, the loss of Pten function in the liver led to tumorigenesis, with 47% of AlbCrePten(flox/flox) livers developing liver cell adenomas by 44 weeks of age. By 74–78 weeks of age, 100% of AlbCrePten(flox/flox) livers showed adenomas and 66% had hepatocellular carcinomas. AlbCrePten(flox/flox) mice also showed insulin hypersensitivity. In vitro, AlbCrePten(flox/flox) hepatocytes were hyperproliferative and showed increased hyperoxidation with abnormal activation of protein kinase B and MAPK. Pten is thus an important regulator of lipogenesis, glucose metabolism, hepatocyte homeostasis, and tumorigenesis in the liver