A Tailor-Made Deazaflavin-Mediated Recycling System for Artificial Nicotinamide Cofactor Biomimetics

[Image: see text] Nicotinamide adenine dinucleotide (NAD) and its 2′-phosphorylated form NADP are crucial cofactors for a large array of biocatalytically important redox enzymes. Their high cost and relatively poor stability, however, make them less attractive electron mediators for industrial processes. Nicotinamide cofactor biomimetics (NCBs) are easily synthesized, are inexpensive, and are also generally more stable than their natural counterparts. A bottleneck for the application of these artificial hydride carriers is the lack of efficient cofactor recycling methods. Therefore, we engineered the thermostable F(420):NADPH oxidoreductase from Thermobifida fusca (Tfu-FNO), by structure-inspired site-directed mutagenesis, to accommodate the unnatural N1 substituents of eight NCBs. The extraordinarily low redox potential of the natural cofactor F(420)H(2) was then exploited to reduce these NCBs. Wild-type enzyme had detectable activity toward all selected NCBs, with K(m) values in the millimolar range and k(cat) values ranging from 0.09 to 1.4 min(–1). Saturation mutagenesis at positions Gly-29 and Pro-89 resulted in mutants with up to 139 times higher catalytic efficiencies. Mutant G29W showed a k(cat) value of 4.2 s(–1) toward 1-benzyl-3-acetylpyridine (BAP(+)), which is similar to the k(cat) value for the natural substrate NADP(+). The best Tfu-FNO variants for a specific NCB were then used for the recycling of catalytic amounts of these nicotinamides in conversion experiments with the thermostable ene-reductase from Thermus scotoductus (TsOYE). We were able to fully convert 10 mM ketoisophorone with BAP(+) within 16 h, using F(420) or its artificial biomimetic FOP (FO-2′-phosphate) as an efficient electron mediator and glucose-6-phosphate as an electron donor. The generated toolbox of thermostable and NCB-dependent Tfu-FNO variants offers powerful cofactor regeneration biocatalysts for the reduction of several artificial nicotinamide biomimetics at both ambient and high temperatures. In fact, to our knowledge, this enzymatic method seems to be the best-performing NCB-recycling system for BNAH and BAPH thus far

Field evaluation of tolerance to Tobacco streak virus in sunflower germplasm, and observations of seasonal disease spread

Strong statistical evidence was found for differences in tolerance to natural infections of Tobacco streak virus (TSV) in sunflower hybrids. Data from 470 plots involving 23 different sunflower hybrids tested in multiple trials over 5 years in Australia were analysed. Using a Bayesian Hierarchical Logistic Regression model for analysis provided: (i) a rigorous method for investigating the relative effects of hybrid, seasonal rainfall and proximity to inoculum source on the incidence of severe TSV disease; (ii) a natural method for estimating the probability distributions of disease incidence in different hybrids under historical rainfall conditions; and (iii) a method for undertaking all pairwise comparisons of disease incidence between hybrids whilst controlling the familywise error rate without any drastic reduction in statistical power. The tolerance identified in field trials was effective against the main TSV strain associated with disease outbreaks, TSV-parthenium. Glasshouse tests indicate this tolerance to also be effective against the other TSV strain found in central Queensland, TSV-crownbeard. The use of tolerant germplasm is critical to minimise the risk of TSV epidemics in sunflower in this region. We found strong statistical evidence that rainfall during the early growing months of March and April had a negative effect on the incidence of severe infection with greatly reduced disease incidence in years that had high rainfall during this period

Nummular keratopathy in a patient with Hyper-IgD Syndrome

<p>Abstract</p> <p>Purpose</p> <p>To report a case of recurrent nummular keratitis in a pediatric patient with Hyperimmunoglobulinemia D syndrome.</p> <p>Methods</p> <p>A retrospective chart review.</p> <p>Results</p> <p>A 14-year-old boy with Hyperimmunoglobulinemia D syndrome (HIDS) presented with photophobia and ocular irritation concomitant with disease exacerbation. He was found on exam to have significant nummular keratitis, which responded to a short course of topical steroids. Despite acute response to local immunosuppression, the patient had several recurrent attacks and eventually developed a large corneal scar and decreased vision. After initiation of infliximab therapy his ocular sequelae improved dramatically and his vision returned to 20/20.</p> <p>Conclusion</p> <p>One possible form of end-organ damage associated with HIDS is vision threatening nummular keratopathy.</p

Tamoxifen for the treatment of polycystic liver disease:A case report

RATIONALE: Polycystic liver disease is a rare disease characterized by the growth of numerous cysts in the liver. The liver function remains well preserved, but liver volumes can grow very large, and some patients ultimately need a liver transplantation. Other treatment options are limited and there is an unmet need for new therapeutic options. PATIENT CONCERNS: We describe a 59-year-old patient with pain in the abdomen, especially when bending forward. Five years ago, she was diagnosed with breast cancer and as an incidental finding a couple of large liver cysts were diagnosed, explaining her abdominal pain. DIAGNOSIS: Polycystic liver disease with several large liver cysts. INTERVENTIONS: The patient was treated with tamoxifen, an estrogen receptor modulator, as treatment for her hormone receptor positive breast cancer. One of the liver cysts was aspirated. OUTCOMES: In the 4.6 years after the start of tamoxifen treatment, 20 mg once daily, the volume of her liver cysts decreased remarkably. There was a reduction of combined cyst volume from 311 mL to 22 mL without percutaneous drainage. LESSONS: Epidemiological as well as experimental evidence supports a pivotal role for estrogens as a driver for growth of polycystic livers. Estrogen antagonism has often been proposed as a therapeutic target, but supporting evidence is lacking in the literature. We hypothesize that the decrease in cyst size in this patient was caused by tamoxifen therapy, suggesting an in vivo antagonistic effect on cystic cholangiocytes. This is an important finding because tamoxifen could be a promising new treatment option for polycystic liver disease

Use of the Urine-to-Plasma Urea Ratio to Predict ADPKD Progression

BACKGROUND AND OBJECTIVES: Predicting disease progression in patients with autosomal dominant polycystic kidney disease (ADPKD) poses a challenge, especially in early-stage disease when kidney function is not yet affected. Ongoing growth of cysts causes maximal urine-concentrating capacity to decrease from early on. We therefore hypothesized that the urine-to-plasma urea ratio, as a reflection of the urine-concentrating capacity, can be used as a marker to predict ADPKD progression. DESIGN: The urine-to-plasma urea ratio was calculated by dividing concentrations of early morning fasting spot urine urea by plasma urea. First, this ratio was validated as surrogate marker in 30 patients with ADPKD who underwent a prolonged water deprivation test. Thereafter, association with kidney outcome was evaluated in 583 patients with ADPKD with a broad range of kidney function. Multivariable mixed-model regression was used to assess association with eGFR slope, and logarithmic regression to identify patients with rapidly progressive disease, using a cutoff of -3.0 ml/min per 1.73 m2 per year. The urine-to-plasma urea ratio was compared with established predictors, namely, sex, age, baseline eGFR, Mayo Clinic height-adjusted total kidney volume class, and PKD gene mutation. RESULTS: The maximal urine-concentrating capacity and urine-to-plasma urea ratio correlated strongly (R=0.90; P<0.001). Next, the urine-to-plasma urea ratio was significantly associated with rate of eGFR decline during a median follow-up of 4.0 (interquartile range, 2.6-5.0) years, both crude and after correction for established predictors (β=0.58; P=0.02). The odds ratio of rapidly progressive disease was 1.35 (95% confidence interval, 1.19 to 1.52; P<0.001) for every 10 units decrease in urine-to-plasma urea ratio, with adjustment for predictors. A combined risk score of the urine-to-plasma urea ratio, Mayo Clinic height-adjusted total kidney volume class, and PKD mutation predicted rapidly progressive disease better than each of the predictors separately. CONCLUSIONS: The urine-to-plasma urea ratio, which is calculated from routine laboratory measurements, predicts disease progression in ADPKD in addition to other risk markers. PODCAST: This article contains a podcast at https://www.asn-online.org/media/podcast/CJASN/2021_01_27_CJN10470620_final.mp3

A semi-quantitative reverse transcriptase polymerase chain reaction method for measurement of mRNA for TNF-_ and IL-1ß in whole blood cultures: its application in typhoid fever and exentric exercise

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Optimizing eHealth tools for older patients: Collaborative redesign of a hospital website

Most hospital websites have not been developed in collaboration with patients and, therefore, rarely take into account the preferences and abilities of older patients. This study describes the systematic redesign of an existing hospital website in a co‐design process with patients and professional stakeholders (e.g. researchers, physicians, nurses, department heads, policymakers, website designers), with the aim to make it more user‐friendly for older patients with colorectal cancer (CRC). The redesign process consisted of three phases, where (I) both existing content and design were evaluated among CRC patients; (II) a prototype website was developed based on these insights; which (III) was evaluated again before making final adjustments. Mixed research methods were used for the redesign process. Specifically, insights from existing literature, outcomes from qualitative and quantitative empirical studies conducted by our team, and expert knowledge from relevant stakeholders, were collected and discussed in multidisciplinary consensus meetings, and served as input for the redesigned website. While the existing website was evaluated poorly, the qualitative evaluation of the prototype website in phase 3 showed that the newly redesigned website was usable for older CRC patients. A practical roadmap on how to collaboratively redesign and optimise existing eHealth tools to make them suitable for and operational in clinical settings is provided