Recombinational stability of replicating plasmids in Aspergillus nidulans during transformation,vegetative growth and sexual reproduction

Plasmids containing the AMA1 replicon are capable of autonomous maintenance in Aspergillus nidulans. It has been reported previously that these plasmids can form concatenates by recombination in a transformed mycelium, and up to 10% of molecules are involved in such events. The present study demonstrates that plasmid recombination, although frequent during transformation, rarely occurs during vegetative growth. As a result, the structure and phenotypic stability of AMA1 plasmids generally remains unaltered for many asexual (conidial) generations. It is also evident that plasmid replication does not require specific recombination events in the AMA1 palindrome. However, during sexual reproduction, autonomous plasmids exhibit increased recombination, which results in both plasmid concatenation and integration into the chromosome.     

Integrative and replicative transformation of Penicillium canescens with a heterologous nitrate-reductase gene

A wild isolate of Penicillium canescens was subjected to mutagenesis, and 150 chlorate-resistant mutants were isolated and classified in respect of their ability to utilize various nitrogen sources. Strains supposedly deficient in nitrate reductase have been transformed with the nitrate-reductase gene from Aspergillus niger. Transformation probably occurred by non-homologous integration of the transforming vector into the chromosome. Co-transformation with the AMA1 replicating element from A. nidulans enhanced transformation frequency up to 2000-fold, and was shown to result in autonomous maintenance of replicating concatenates, one of which was re-isolated by transformation of E. coli.     

Safety and tolerability of injectable lipid-lowering drugs: an update of clinical data

Introduction: Cardiovascular (CV) diseases are the leading cause of death and disability in the developed countries. Lipid-lowering therapy is a cornerstone of the CV risk modification strategy. The first line treatment for hyperlipidemia is statins, which decrease low-density lipoprotein cholesterol (LDL-C) by 30–50% and proportionally reduce the CV events. However, they are not always enough to achieve LDL-C goals in many patients, and some patients are statin intolerant. For this reason, new powerful injectable lipid-lowering drugs have been developed.

Areas covered: The aim of this narrative review was to summarize the more recent clinical data on safety and tolerability of injectable lipid-lowering drugs. After an attentive literature search, the authors resumed here information on proprotein convertase subtilisin/kexin 9 inhibitors (evolocumab and alirocumab), small interfering RNA molecule inclisiran, antisense oligonucleotides (mipomersen, volanesorsen, ISIS 681257), and drugs targeting angiopoietin-like protein 3 (evinacumab, IONIS-ANGPTL3_Rx).

Expert opinion: Injectable lipid-lowering therapy for patients at high risk for CV disease complications or with severe inherited hypercholesterolemias can be an important element of the available therapeutic armamentarium. Clinical data prove the favorable risk-benefit profile of evolocumab, alirocumab, and inclisiran. Mipomersen, volanesorsen, ISIS 681257, evinacumab, and IONIS-ANGPTL3_Rx safety is currently less extensively studied, especially in patients with comorbidities and polypharmacotherapy.     

The relationship between angle kappa and astigmatism after phacoemulsification with implanting of spherical and aspheric intraocular lens

Purpose:To determine the significance of any association between either change in angle kappa (K°) or the rectilinear displacement (L, mm) of the first Purkinje image relative to the pupil center and unexpected changes in astigmatism after phacoemulsification.Methods:Orbscan II (Bausch and Lomb) measurements were taken at 1, 2, and 3 months after unremarkable phacoemulsification in patients implanted with spherical (group 1, SA60AT, Alcon) or aspheric (group 2, SN60WF, Alcon) nontoric IOLs. The outputs were used to calculate L. Astigmatism, measured by autorefractometry and subjective refraction, was subjected to vector analysis (polar and cartesian formats) to determine the actual change induced over the periods 1–2 and 2–3 months postop.Results:Chief findings were that the mean (n, ±SD, 95%CI) values for L over each period were as follows: Group 1, 0.407 (38, ±0.340, 0.299–0.521), 0.315 (23, ±0.184, 0.335–0.485); Group 2, 0.442 (45, ±0.423, 0.308–0.577), 0.372 (26, ±0.244, 0.335–0.485). Differences between groups were not significant. There was a significant linear relationship between (A) the change in K (ΔK = value at 1 month-value at 2 months) and K at 1 month (x), where ΔK =0.668-3.794X (r = 0.812, n = 38, P = <0.001) in group 1 and ΔK = 0.263x -1.462 (r = 0.494, n = 45, P = 0.002) in group 2, (B) L and the J₄₅ vector describing the actual change in astigmatism between 1 and 2 months in group 2, where J₄₅ (by autorefractometry) =0.287L-0.160 (r = 0.487, n = 38, P = 0.001) and J₄₅ (by subjective refraction) =0.281L-0.102 (r = 0.490, n = 38, P = 0.002), and (C) J₄₅ and ΔK between 2 and 3 months in group 2, where J₄₅ (by subjective refraction) =0.086ΔK-0.063 (r = 0.378, n = 26, P = 0.020).Conclusion:Changes in the location of the first Purkinje image relative to the pupil center after phacoemulsification contributes to changes in refractive astigmatism. However, the relationship between the induced change in astigmatism resulting from a change in L is not straightforward.     

Evaluation of dyskerin expression and the Cajal body protein WRAP53β as potential prognostic markers for patients with primary vaginal carcinoma

Primary vaginal cancer (PVC) is a rare gynaecological malignancy, which, at present, lacks appropriate biomarkers for prognosis. The proteins dyskerin and WD repeat containing antisense to TP53 (WRAP53β), both of which exert their functions in the telomerase holoenzyme complex, have been shown to be upregulated in different cancer types. These proteins have also been proposed as prognostic markers in some types of cancer. The aim of the present study was to examine the expression patterns of dyskerin and WRAP53β in patients with PVC. Moreover, as part of a search for effective biomarkers to evaluate prognosis in PVC, the expression of these two proteins and their potential association with clinical variables and survival were also evaluated. The expression of dyskerin and WRAP53β was assessed in PVC tumour samples from 68 patients using immunohistochemistry. The majority of tumour samples showed low and moderate expression levels of dyskerin. Upregulation of dyskerin in tumour samples was significantly associated with a shorter survival time and a poorer cancer-specific survival rate. WRAP53β was also expressed in most of the cells but was not significantly associated with clinical variables or survival. This study demonstrates that upregulation of dyskerin is significantly associated with poor prognosis. Thus, dyskerin may serve as a promising prognostic marker and a potential putative therapeutic target in PVC.     

GBT440 increases haemoglobin oxygen affinity,reduces sickling and prolongs RBC half‐life in a murine model of sickle cell disease

A major driver of the pathophysiology of sickle cell disease (SCD) is polymerization of deoxygenated haemoglobin S (HbS), which leads to sickling and destruction of red blood cells (RBCs) and end‐organ damage. Pharmacologically increasing the proportion of oxygenated HbS in RBCs may inhibit polymerization, prevent sickling and provide long term disease modification. We report that GBT440, a small molecule which binds to the N‐terminal α chain of Hb, increases HbS affinity for oxygen, delays in vitro HbS polymerization and prevents sickling of RBCs. Moreover, in a murine model of SCD, GBT440 extends the half‐life of RBCs, reduces reticulocyte counts and prevents ex vivo RBC sickling. Importantly, oral dosing of GBT440 in animals demonstrates suitability for once daily dosing in humans and a highly selective partitioning into RBCs, which is a key therapeutic safety attribute. Thus, GBT440 has the potential for clinical use as a disease‐modifying agent in sickle cell patients.     

Triple fluorescence labelling of neuronal,glial and vascular markers revealing pathological alterations in various animal models

The simultaneous detection of glia, vessels and neurons facilitates insights into the complex chemoarchitecture of the central nervous system. Here, we present a simple, robust and versatile approach for the carbocyanine triple fluorescence labelling of neuronal, vascular and glial markers.The usefulness of this procedure is shown for rat brain tissue under physiological conditions, after traumatic brain injury caused by controlled cortical impact injury, and after stroke following middle cerebral artery occlusion. Moreover, the versatility of the method is verified by its application to sections from old triple transgenic mice with age-dependent β-amyloidosis and tau hyperphosphorylation in the hippocampus, modelling neuropathological alterations in Alzheimer‘s disease. To exemplify the usefulness of the approach for analysis of the enteric nervous system, it was applied to whole mounts from the horse intestine.The biotinylated lectin from potato (Solanum tuberosum) is presented as an excellent tool to detect both vessels and microglia. Furthermore, this lectin revealed macrophages after experimental insults, and senile plaques in aged triple transgenic mice. A large portion of astroglia was demonstrated by immunolabelling of glial fibrillary acidic protein. Neurons were detected by monoclonal antibodies directed against neuronal nuclei and, in horse tissues, mouse-anti-HuC/D recognizing a conserved nuclear protein. Confocal laser-scanning microscopy elucidated spatial relationships of the relevant markers and their pathological alterations after experimental insults and in transgenic mice with Alzheimer-like lesions.