Effect of ionic strength, serum albumin and other macromolecules on the maintenance of motility and the surface of mammalian spermatozoa in a simple medium

The seminal plasma in sperm suspensions from boar, bull, rabbit, ram and stallion was replaced with simple defined media as completely as possible by a combination of centrifugation through Ficoll and dilution. After this process, motility declined and the cells showed a tendency to agglutinate and/or stick to glass. Varying the ionic strength of the medium had little effect upon these parameters but sperm motility was preserved better in the presence of serum albumin. When a number of purified proteins and other macromolecules were tested individually in this way for their motility-preserving ability, bovine or human serum albumin was consistently the most effective. Defatting the albumin or altering its nature by mild reduction, oxidation or alkylation had little detectable effect on its motility-preserving ability; the protein did not appear to be acting as a chelator of metal ions, for it could not be replaced by EDTA. The response of the spermatozoa to replacemrnt of seminal plasma varied between species: bull spermatozoa were particularly sensitive and serum albumin had little effect upon their subsequent motility.     

Physical and genetic analysis of IS110, a transposable element of Streptomyces coelicolor A3(2)

Summary On at least three independent occasions a 1.6 kb segment of Streptomyces coelicolor DNA was detected in apparently the same location in an attP-deleted derivative of the temperature phage C31 that carried a selectable viomycin resistance gene. This sequence (termed IS110) allowed integration of the phage (giving viomycin-resistant transductants) at homologous sequences (detected by Southern hybridisation) at several locations in the S. coelicolor genome. The inserted prophages facilitated genetic mapping of two IS110 copies in the chromosomal linkage map. A third copy did not exhibit simple segregation with chromosomal markers, and there appeared to be a frequent DNA rearrangement close to this copy. Some variation in the number of copies of IS110 and their location has taken place in the pedigree of S. coelicolor derivatives. IS110 did not hybridise to any known S. coelicolor plasmid, nor to any of several other IS-like elements previously described in other Streptomyces plasmids or phages. It hybridised strongly to DNA from only a small minority of other Streptomyces species and was absent from S. lividans, a close relative of S. coelicolor.     

An electro-optic monitor of the behavior of Chlamydomonas reinhardtii cilia

The unicellular green alga Chlamydomonas reinhardtii steers through water with a pair of cilia (eukaryotic flagella). Long-term observation of the beating of its cilia with controlled stimulation is improving our understanding of how a cell responds to sensory inputs. Here we describe how to record ciliary motion continuously for long periods. We also report experiments on the network of intracellular signaling that connects the environment inputs with response outputs. Local spatial changes in ciliary response on the time scale of the underlying biochemical dynamics are observed. Near-infrared light monitors the cells held by a micropipette. This condition is tolerated well for hours, not interfering with ciliary beating or sensory transduction. A computer integrates the light stimulation of the eye of Chlamydomonas with the ciliary motion making possible long-term correlations. Measures of ciliary responses include the beating frequency, stroke velocity, and stroke duration of each cilium, and the relative phase of the cis and trans cilia. The stationarity and dependence of the system on light intensity was investigated. About 150,000,000 total beat cycles and up to 8 h on one cell have been recorded. Each beat cycle is resolved so that each asynchronous beat is detected. Responses extend only a few hundred milliseconds, but there is a persistence of momentary changes that last much longer. Interestingly, we see a response that is linear with absolute light intensity as well as different kinds of response that are clearly nonlinear, implying two signaling pathways from the cell body to the cilia.     

Trypanosoma cruzi Survival following Cold Storage: Possible Implications for Tissue Banking

While Trypanosoma cruzi, the etiologic agent of Chagas disease, is typically vector-borne, infection can also occur through solid organ transplantation or transfusion of contaminated blood products. The ability of infected human cells, tissues, and cellular and tissue-based products (HCT/Ps) to transmit T. cruzi is dependent upon T. cruzi surviving the processing and storage conditions to which HCT/Ps are subjected. In the studies reported here, T. cruzi trypomastigotes remained infective 24 hours after being spiked into blood and stored at room temperature (N = 20); in 2 of 13 parasite-infected cultures stored 28 days at 4°C; and in samples stored 365 days at −80°C without cryoprotectant (N = 28), despite decreased viability compared to cryopreserved parasites. Detection of viable parasites after multiple freeze/thaws depended upon the duration of frozen storage. The ability of T. cruzi to survive long periods of storage at +4 and −80°C suggests that T. cruzi-infected tissues stored under these conditions are potentially infectious.     

Selection of low-variance expressed Malus x domestica (apple) genes for use as quantitative PCR reference genes (housekeepers)

To accurately measure gene expression using PCR-based approaches, there is the need for reference genes that have low variance in expression (housekeeping genes) to normalise the data for RNA quantity and quality. For non-model species such as Malus x domestica (apples), previously, the selection of reference genes relied on using homology to reference genes in model species. In this study, a genomics approach was used to identify apple genes with low variance in expression in 217 messenger RNA (mRNA)-seq data sets covering different tissues, during fruit development, and treated with a range of different stress conditions. Ten potential reference genes were chosen for validation by quantitative PCR (qPCR) over 29 different tissue types and treatments. From the combined mRNA-seq and qPCR results, three potential reference genes are proposed that can be used as good controls for PCR based expression studies. The three genes show homology to lipid transfer proteins, phytochrome protein phosphatase and the ubiquitination pathway. With the progression of research away from non-model species, this approach provides a robust method for selecting candidate genes for use as reference genes in qPCR.     

Predicting substrate exchange in marine diatom-heterocystous cyanobacteria symbioses

In the open ocean, some phytoplankton establish symbiosis with cyanobacteria. Some partnerships involve diatoms as hosts and heterocystous cyanobacteria as symbionts. Heterocysts are specialized cells for nitrogen fixation, and a function of the symbiotic cyanobacteria is to provide the host with nitrogen. However, both partners are photosynthetic and capable of carbon fixation, and the possible metabolites exchanged and mechanisms of transfer are poorly understood. The symbiont cellular location varies from internal to partial to fully external, and this is reflected in the symbiont genome size and content. In order to identify the membrane transporters potentially involved in metabolite exchange, we compare the draft genomes of three differently located symbionts with known transporters mainly from model free-living heterocystous cyanobacteria. The types and numbers of transporters are directly related to the symbiont cellular location: restricted in the endosymbionts and wider in the external symbiont. Three proposed models of metabolite exchange are suggested which take into account the type of transporters in the symbionts and the influence of their cellular location on the available nutrient pools. These models provide a basis for several hypotheses that given the importance of these symbioses in global N and C budgets, warrant future testing.     

Incidence and causes of end-stage renal disease among Aboriginal children and young adults

Background:

Although Aboriginal adults have a higher risk of end-stage renal disease than non-Aboriginal adults, the incidence and causes of end-stage renal disease among Aboriginal children and young adults are not well described.

Methods:

We calculated age- and sex-specific incidences of end-stage renal disease among Aboriginal people less than 22 years of age using data from a national organ failure registry. Incidence rate ratios were used to compare rates between Aboriginal and white Canadians. To contrast causes of end-stage renal disease by ethnicity and age, we calculated the odds of congenital diseases, glomerulonephritis and diabetes for Aboriginal people and compared them with those for white people in the following age strata: 0 to less than 22 years, 22 to less than 40 years, 40 to less than 60 years and older than 60 years.

Results:

Incidence rate ratios of end-stage renal disease for Aboriginal children and young adults (age < 22 yr, v. white people) were 1.82 (95% confidence interval [CI] 1.40–2.38) for boys and 3.24 (95% CI 2.60–4.05) for girls. Compared with white people, congenital diseases were less common among Aboriginal people aged less than 22 years (odds ratio [OR] 0.56, 95% CI 0.36–0.86), and glomerulonephritis was more common (OR 2.18, 95% CI 1.55–3.07). An excess of glomerulonephritis, but not diabetes, was seen among Aboriginal people aged 22 to less than 40 years. The converse was true (higher risk of diabetes, lower risk of glomerulonephritis) among Aboriginal people aged 40 years and older.

Interpretation:

The incidence of end-stage renal disease is higher among Aboriginal children and young adults than among white children and young adults. This higher incidence may be driven by an increased risk of glomerulonephritis in this population.Compared with white Canadians, Aboriginal Canadians have a higher prevalence of end-stage renal disease,^1,2 which is generally attributed to their increased risk for diabetes. However, there has been limited investigation of the incidence and causes of end-stage renal disease among Aboriginal children and young adults. Because most incident cases of diabetes are identified in middle-aged adults, an excess risk of end-stage renal disease in young people would not be expected if the high risk of diabetes is responsible for higher overall rates of end-stage renal disease among Aboriginal people. About 12.3% of children with end-stage renal disease in Canada are Aboriginal,³ but only 6.1% of Canadian children (age < 19 yr) are Aboriginal.^4,5A few reports suggest that nondiabetic renal disease is common among Aboriginal populations in North America.^2,6–8 Aboriginal adults in Saskatchewan are twice as likely as white adults to have end-stage renal disease caused by glomerulonephritis,^7,8 and an increased rate of mesangial proliferative glomerulonephritis has been reported among Aboriginal people in the United States.^6,9 These studies suggest that diabetes may be a comorbid condition rather than the sole cause of kidney failure among some Aboriginal people in whom diabetic nephropathy is diagnosed using clinical features alone.We estimated incidence rates of end-stage renal disease among Aboriginal children and young adults in Canada and compared them with the rates seen among white children and young adults. In addition, we compared relative odds of congenital renal disease, glomerulonephritis and diabetic nephropathy in Aboriginal people with the relative odds of these conditions in white people.     

Single cell RNA‐seq in the sea urchin embryo show marked cell‐type specificity in the Delta/Notch pathway

Sea urchin embryos are excellent for in vivo functional studies because of their transparency and tractability in manipulation. They are also favorites for pharmacological approaches since they develop in an aquatic environment and addition of test substances is straightforward. A concern in many pharmacological tests though is the potential for pleiotropic effects that confound the conclusions drawn from the results. Precise cellular interpretations are often not feasible because the impact of the perturbant is not known. Here we use single‐cell mRNA (messenger RNA) sequencing as a metric of cell types in the embryo and to determine the selectivity of two commonly used inhibitors, one each for the Wnt and the Delta‐Notch pathways, on these nascent cell types. We identified 11 distinct cell types based on mRNA profiling, and that the cell lineages affected by Wnt and Delta/Notch inhibition were distinct from each other. These data support specificity and distinct effects of these signaling pathways in the embryo and illuminate how these conserved pathways selectively regulate cell lineages at a single cell level. Overall, we conclude that single cell RNA‐seq analysis in this embryo is revealing of the cell types present during development, of the changes in the gene regulatory network resulting from inhibition of various signaling pathways, and of the selectivity of these pathways in influencing developmental trajectories.     

Planarian MBD2/3 is required for adult stem cell pluripotency independently of DNA methylation

Planarian adult stem cells (pASCs) or neoblasts represent an ideal system to study the evolution of stem cells and pluripotency as they underpin an unrivaled capacity for regeneration. We wish to understand the control of differentiation and pluripotency in pASCs and to understand how conserved, convergent or divergent these mechanisms are across the Bilateria. Here we show the planarian methyl-CpG Binding Domain 2/3 (mbd2/3) gene is required for pASC differentiation during regeneration and tissue homeostasis. The genome does not have detectable levels of 5-methylcytosine (5^mC) and we find no role for a potential DNA methylase. We conclude that MBD proteins may have had an ancient role in broadly controlling animal stem cell pluripotency, but that DNA methylation is not involved in planarian stem cell differentiation.     

Analysis of peptidoglycan structure from vegetative cells of Bacillus subtilis 168 and role of PBP 5 in peptidoglycan maturation.

The composition and fine structure of the vegetative cell wall peptidoglycan from Bacillus subtilis were determined by analysis of its constituent muropeptides. The structures of 39 muropeptides, representing 97% of the total peptidoglycan, were elucidated. About 99% analyzed muropeptides in B. subtilis vegetative cell peptidoglycan have the free carboxylic group of diaminopimelic acid amidated. Anhydromuropeptides and products missing a glucosamine at the nonreducing terminus account for 0.4 and 1.5%, respectively, of the total muropeptides. These two types of muropeptides are suggested to end glycan strands. An unexpected feature of B. subtilis muropeptides was the occurrence of a glycine residue in position 5 of the peptide side chain on monomers or oligomers, which account for 2.7% of the total muropeptides. This amount is, however, dependent on the composition of the growth media. Potential attachment sites for anionic polymers to peptidoglycan occur on dominant muropeptides and account for 2.1% of the total. B. subtilis peptidoglycan is incompletely digested by lysozyme due to de-N-acetylation of glucosamine, which occurs on 17.3% of muropeptides. The cross-linking index of the polymer changes with the growth phase. It is highest in late stationary phase, with a value of 33.2 or 44% per muramic acid residue, as determined by reverse-phase high-pressure liquid chromatography or gel filtration, respectively. Analysis of the muropeptide composition of a dacA (PBP 5) mutant shows a dramatic decrease of muropeptides with tripeptide side chains and an increase or appearance of muropeptides with pentapeptide side chains in monomers or oligomers. The total muropeptides with pentapeptide side chains accounts for almost 82% in the dacA mutant. This major low-molecular-weight PBP (DD-carboxypeptidase) is suggested to play a role in peptidoglycan maturation.