Nuclear heterogeneity is prevalent in high-quality fractionated human sperm cells typically used for assisted conception.

STUDY QUESTION: What is the nuclear heterogeneity of high-density purified human spermatozoa typically used for IVF purposes. SUMMARY ANSWER: The data show that while density gradient separation has improved the overall sperm population, there is still a large degree of nuclear heterogeneity within these cells. WHAT IS KNOWN ALREADY: Chromomycin A3 (CMA3) is an important DNA binding fluorochrome for the assessment of male-factor fertility. It is typically used to predict IVF outcomes on entire sperm ejaculates with very high receiver operating characteristic. Here we used CMA3 to characterise typical populations of human spermatozoa that would be used for IVF purposes after density gradient separation. STUDY DESIGN, SIZE, DURATION: We compared the intensity of CMA3 binding within high-dense sperm populations obtained from men. Binding heterogeneity was confirmed through fluorescence microscopy and FACS analysis independently. We also looked at CMA3 staining directly with head morphology in this sperm population. Finally, we looked at electron micrographs of nuclear heterogeneity (vacuoles, chromatin compaction) of spermatozoa following density gradient sorting of CMA3-stained cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: We used sperm donors who had fathered one or more children. Semen was collected after 2 days abstinence and purified over Percoll gradients. Only the high-quality spermatozoa, the same used for assisted conception, were then used. Cells were stained with CMA3 and sorted using FACS. Following this, electron micrographs were used to assess nuclear heterogeneity of CMA3-dependent sorted spermatozoa. MAIN RESULTS AND THE ROLE OF CHANCE: CMA3 staining occurs within morphologically normal as well as abnormal spermatozoa. High-intensity CMA3-stained sperm possessed large vacuoles that were not seen in the low-CMA3 population. In addition, the high-CMA3 stained cells possess higher amounts of nuclear granulation. LIMITATIONS, REASONS FOR CAUTION: The present study only describes the issues within the chromatin of these cells and does not suggest an alternate selection technique. WIDER IMPLICATIONS OF THE FINDINGS: CMA3 is one of the better reported prognostic assays in predicting pregnancy outcomes, especially in cases where the male is at fault. However, it is clear that even in fractionated populations of human spermatozoa, there are sperm cells that are morphologically normal yet possess high levels of CMA3 staining and chromatin granulation. The implication of this is that the embryologist, whom selects on the basis of sperm morphology, may choose a cell with poor chromatin, which may lead to poor embryo outcomes. STUDY FUNDING/COMPETING INTEREST(S): The project was funded by the National Health and Medical Research council, APP1118943. The authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Correction to: Polymyxin B causes DNA damage in HK-2 cells and mice.

In the original publication of the article, part of Fig. 6 is missing. The missing subpanels, Fig. 6c, d are given below.

Polymyxin B causes DNA damage in HK-2 cells and mice.

Increasing incidence of multidrug-resistant bacteria presents an imminent risk to global health. Polymyxins are 'last-resort' antibiotics against Gram-negative 'superbugs'; however, nephrotoxicity remains a key impediment in their clinical use. Molecular mechanisms underlying this nephrotoxicity remain poorly defined. Here, we examined the pathways which led to polymyxin B induced cell death in vitro and in vivo. Human proximal tubular cells were treated with polymyxin B (12.5-100 µM) for up to 24 h and showed a significant increase in micronuclei frequency, as well as abnormal mitotic events (over 40% in treated cells, p < 0.05). Time-course studies were performed using a mouse nephrotoxicity model (cumulative 72 mg/kg). Kidneys were collected over 48 h and investigated for histopathology and DNA damage. Notable increases in γH2AX foci (indicative of double-stranded breaks) were observed in both cell culture (up to ~ 44% cells with 5+ foci at 24 h, p < 0.05) and mice treated with polymyxin B (up to ~ 25%, p < 0.05). Consistent with these results, in vitro assays showed high binding affinity of polymyxin B to DNA. Together, our results indicate that polymyxin B nephrotoxicity is associated with DNA damage, leading to chromosome missegregation and genome instability. This novel mechanistic information may lead to new strategies to overcome the nephrotoxicity of this important last-line class of antibiotics.

Can Cystic Fibrosis Patients Finally Catch a Breath With Lumacaftor/Ivacaftor?

Cystic fibrosis (CF) is a life-limiting disease caused by defective or deficient cystic fibrosis transmembrane conductance regulator (CFTR) activity. The recent US Food and Drug Administration (FDA) approval of lumacaftor combined with ivacaftor (Orkambi) targets patients with the F508del-CFTR. The question remains: Is this breakthrough combination therapy the "magic-bullet" cure for the vast majority of patients with CF? This review covers the contemporary clinical and scientific knowledge-base for lumacaftor/ivacaftor and highlights the emerging issues from recent conflicting literature reports.

Molecular characterization of the receptor binding structure-activity relationships of influenza B virus hemagglutinin.

Selectivity of α2,6-linked human-like receptors by B hemagglutinin (HA) is yet to be fully understood. This study integrates binding data with structure-recognition models to examine the impact of regional-specific sequence variations within the receptor-binding pocket on selectivity and structure activity relationships (SAR). The receptor-binding selectivity of influenza B HAs corresponding to either B/Victoria/2/1987 or the B/Yamagata/16/88 lineages was examined using surface plasmon resonance, solid-phase ELISA and gel-capture assays. Our SAR data showed that the presence of asialyl sugar units is the main determinant of receptor preference of α2,6 versus α2,3 receptor binding. Changes to the type of sialyl-glycan linkage present on receptors exhibit only a minor effect upon binding affinity. Homology-based structural models revealed that structural properties within the HA pocket, such as a glyco-conjugate at Asn194 on the 190-helix, sterically interfere with binding to avian receptor analogs by blocking the exit path of the asialyl sugars. Similarly, naturally occurring substitutions in the C-terminal region of the 190-helix and near the N-terminal end of the 140-loop narrows the horizontal borders of the binding pocket, which restricts access of the avian receptor analog LSTa. This study helps bridge the gap between ligand structure and receptor recognition for influenza B HA; and provides a consensus SAR model for the binding of human and avian receptor analogs to influenza B HA.

A gel-capture assay for characterizing the sialyl-glycan selectivity of influenza viruses.

Sialic acids (SA) usually linked to galactose (Gal) in an α2,6- or α2,3-configuration are considered the main cell receptors for influenza viruses, in particular for their hemagglutinins (HA). The typing of influenza virus HA receptor selectivity is relevant for understanding the transmissibility of avian and swine viruses to the human population. In this study we developed a simple and inexpensive gel-capture assay (GCA) of the influenza virus HA receptor-binding selectivity. Its principle is the binding of soluble influenza virus to pentasaccharide analogs, representatives of receptors of human and avian influenza viruses, immobilized on a gel resin. The human and avian analogs consisted of a sialyllactose-N-tetraose c (LSTc) [Neu5Ac(α2,6)Gal(ß1-3)GlcNAc(ß1-3)Gal(ß1-4)Glc] and a sialyllactose-N-tetraose a (LSTa) [Neu5Ac(α2,3)Gal(ß1-3)GlcNAc(ß1-3)Gal(ß1-4)Glc], respectively. Following equilibration, the unbound virus is washed away and the bound one is assayed via HA by densitometry as a function of the analog concentration. Using GCA, the receptor selectivity of three influenza viruses of different HA subtype was investigated. The results showed that the egg-adapted A/California/07/2009 (H1N1) virus exhibited an avian α2,3-linked LSTa selectivity, however, it retained the ability to bind to the α2,6-linked LSTc human receptor analog. Influenza B virus B/Florida/4/2006 showed α2,6-linked LSTc selectivity and a poor α2,3-linked LSTa avidity. The H3N2 virus A/Wisconsin/15/2009 displayed almost comparable avidity for both receptor analogs with a marginally greater α2,3-linked LSTa avidity. The described assay protocol provides a simple and rapid method for the characterization of influenza virus HA receptor binding selectivity.