A simplified in vitro ligation approach to clone an E1B55k-deleted double-targeted conditionally-replicative adenovirus

<p>Abstract</p> <p>Background</p> <p>Construction of conditionally-replicative Adenovirus (CRAd) is complex and time-consuming. While homologous recombination (HR) using a two-plasmid system in bacteria is commonly used to generate CRAds, alternative methods may be required when HR fails. Previously, <it>in vitro </it>ligation has been suggested to facilitate construction of E1/E3-deleted, replication-incompetent Ad vectors. However, <it>in vitro </it>ligation has only rarely been used to generate CRAds and may be a complex procedure for molecular biologists who are not experts in the field.</p> <p>Methods and Results</p> <p>A modified <it>in vitro </it>ligation approach was developed to construct a double-targeted, E1B55k-deleted CRAd. The method allowed the incorporation of a tumor-specific promoter, e.g. the heat-shock protein 70 (hsp70) promoter, upstream of <it>E1a</it>, deletion of the <it>E1B55k </it>gene, and HR-free cloning of the recombined <it>E1Δ55k </it>gene into the Ad genome. The genetic structure of the CRAd was confirmed using restriction analysis and PCR. The replication rate of the hsp70E1Δ55k CRAd was 1.5–2% of Ad without E1Δ55k deletion.</p> <p>Conclusion</p> <p>A 3-step cloning approach can generate a double-targeted, <it>E1B55k</it>-deleted CRAd using a straight-forward, modified <it>in vitro </it>ligation procedure.</p

Analysis of adenoviral attachment to human platelets

<p>Abstract</p> <p>Background</p> <p>Systemic adenoviral (Ad) vector administration is associated with thrombocytopenia. Recently, Ad interaction with mouse platelets emerged as a key player determining liver uptake and platelet clearance. However, whether Ad can activate platelets is controversial. Thus, <it>in vitro </it>analysis of Ad attachment to platelets is of interest.</p> <p>Methods</p> <p>We developed a direct flow cytometry assay to specifically detect Ad particles adherent to human platelets. The method was pre-validated in nucleated cells. Blocking assays were employed to specifically inhibit Ad attachment to platelets. Platelet activation was analyzed using annexin v flow cytometry.</p> <p>Results</p> <p>We found <it>in vitro </it>that Ad binding to human platelets is synergistically enhanced by the combination of platelet activation by thrombin and MnCl2 supplementation. Of note, Ad binding could activate human platelets. Platelets bound Ad displaying an RGD ligand in the fiber knob more efficiently than unmodified Ad. In contrast to a previous report, CAR expression was not detected on human platelets. Integrins appear to mediate Ad binding to platelets, at least partially. Finally, αIIbβ3-deficient platelets from a patient with Glanzmann thrombasthenia could bind Ad 5-fold more efficiently than normal platelets.</p> <p>Conclusion</p> <p>The flow cytometry methodology developed herein allows the quantitative measurement of Ad attachment to platelets and may provide a useful <it>in vitro </it>approach to investigate Ad interaction with platelets.</p

DNA-Free Recombinant SV40 Capsids Protect Mice from Acute Renal Failure by Inducing Stress Response, Survival Pathway and Apoptotic Arrest

Viruses induce signaling and host defense during infection. Employing these natural trigger mechanisms to combat organ or tissue failure is hampered by harmful effects of most viruses. Here we demonstrate that SV40 empty capsids (Virus Like Particles-VLPs), with no DNA, induce host Hsp/c70 and Akt-1 survival pathways, key players in cellular survival mechanisms. We postulated that this signaling might protect against organ damage in vivo. Acute kidney injury (AKI) was chosen as target. AKI is critical, prevalent disorder in humans, caused by nephrotoxic agents, sepsis or ischemia, via apoptosis/necrosis of renal tubular cells, with high morbidity and mortality. Systemic administration of VLPs activated Akt-1 and upregulated Hsp/c70 in vivo. Experiments in mercury-induced AKI mouse model demonstrated that apoptosis, oxidative stress and toxic renal failure were significantly attenuated by pretreatment with capsids prior to the mercury insult. Survival rate increased from 12% to >60%, with wide dose response. This study demonstrates that SV40 VLPs, devoid of DNA, may potentially be used as prophylactic agent for AKI. We anticipate that these finding may be projected to a wide range of organ failure, using empty capsids of SV40 as well as other viruses

Community-based serum chloride abnormalities predict mortality risk

Introduction This population-based study aimed to investigate the prognostic value of ambulatory serum chloride abnormalities, often ignored by physicians. Methods The study population included all non-hospitalized adult patients, insured by "Clalit" Health Services in Israel’s southern district, who underwent at least 3 serum chloride tests in community-based clinics during 2005–2016. For each patient, each period with low (≤97 mmol/l), high (≥107 mmol/l) or normal chloride levels were recorded. A Cox proportional hazards model was used to estimate the mortality risk of hypochloremia and hyperchloremia periods. Results 664,253 serum chloride tests from 105,655 subjects were analyzed. During a median follow up of 10.8 years, 11,694 patients died. Hypochloremia (≤ 97 mmol/l) was independently associated with elevated all-cause mortality risk after adjusting for age, co-morbidities, hyponatremia and eGFR (HR 2.41, 95%CI 2.16–2.69, pConclusion In the outpatient setting, hypochloremia is independently associated with an increased mortality risk. This risk is dose-dependent where the lower the chloride level, the higher is the risk

The Combined Effect of High Ambient Temperature and Antihypertensive Treatment on Renal Function in Hospitalized Elderly Patients

<div><p>Background</p><p>The aging kidney manifests structural, functional as well as pharmacological changes, rendering elderly patients more susceptible to adverse environmental influences on their health, dehydration in particular.</p><p>Hypothesis</p><p>Higher temperature is associated with renal function impairment in patients 65 years and older who routinely take thiazide and/or ACE-inhibitors/ARBs.</p><p>Methods</p><p>We obtained health data of patients older than 65 who were admitted to a large tertiary center during the years 2006–2011, with a previous diagnosis of hypertension, and treated with thiazide, ACE-inhibitors/ARBs or both. We collected environmental data of daily temperature, available from collaborative public and governmental institutions. In order to estimate the effect of daily temperature on renal function we performed linear mixed models, separately for each treatment group and creatinine change during hospital admission.</p><p>Results</p><p>We identified 26,286 admissions for 14, 268 patients with a mean age of 75.6 (±6.9) years, of whom 53.6% were men. Increment in daily temperature on admission of 5°C had significant effect on creatinine increase in the no treatment (baseline creatinine adjusted 0.824 mg/dL, % change 1.212, % change 95% C.I 0.082–2.354) and dual treatment groups (baseline creatinine adjusted 1.032mg/dL, % change 3.440, % change 95% C.I 1.227–5.700). Sub-analysis stratified by advanced age, chronic kidney disease and primary diagnosis on hospital admission, revealed a significant association within patients admitted due to acute infection and treated with dual therapy.</p><p>Conclusion</p><p>Whereas previous studies analyzed sporadic climate effects during heat waves and/or excluded older population taking anti-hypertensive medications, the present study is novel by showing a durable association of temperature and decreased renal function specifically in elderly patients taking anti-hypertensive medications.</p></div

Factors associated with change in creatinine levels (creatinine levels at the hospitalization compared to the baseline mean creatinine form the preceding year).

<p>Factors associated with change in creatinine levels (creatinine levels at the hospitalization compared to the baseline mean creatinine form the preceding year).</p