Segregation products of male mice doubly heterozygous for the RB(6.16) and RB(16.17) translocations: Influence of sperm karyotype on fertilizing competence under varying mating frequencies

The meiotic segregants of male mice heterozygous for Rb(6.16)24Lub and Rb(16.17)7Bnr were viewed, for the first time, at first cleavage metaphase. Chromosomes were analyzed after G-banding, C-banding, and karyotyping. To study sperm aging effects, chromosomes of 202 one-cell zygotes derived from males mating at intervals of approximately 3,14, and 21 days were examined. At least 89.6% of sperm-derived complements were products of 2:2 segregation; at most, a possible 6.4% were 3:1 segregants. The six expected types of 2:2 segregants, both balanced and unbalanced, were equifrequent in the total zygote population derived from sperm of all ages. When the data were analyzed according to mating frequency, the 3-day sperm population considered most likely to be fresh showed a deficiency of the segregant nullisomic for chromosome 6 and disomic for chromosome 17, when compared with the reciprocal segregant (P < 0.025) as well as to all other 2:2 segregants (P < 0.05). However, these sperm fertilized in greater numbers (P < 0.01) than their reciprocal segregant (disomic for 6 and nullisomic for 17) in the 14-day sperm population. While sperm with chromosomal abnormalities are capable of fertilization, the competence of segregants nullisomic for 6 and disomic for 17 apparently depends on the prior storage period in the male. Further, the results suggest that the effect of aneuploidy on sperm function is dependent on the specific chromosome(s) involved.     

The prognostic value of CD3+ tumor-infiltrating lymphocytes for stage II colon cancer according to use of adjuvant chemotherapy: A large single-institution cohort study

BackgroundHigh tumor infiltrating lymphocytes (TILs) density was previously shown to be associated with favorable prognosis for patients with colon cancer (CC). However, the impact of TILs on overall survival (OS) of stage II CC patients who received adjuvant chemotherapy (ADJ) or not (no-ADJ) is unknown. We assessed the prognostic value of CD3+ TILs in stage II CC patients according to whether they had ADJ or not.MethodsPatients treated with curative surgery for stage II CC (2002–2013) were selected from the Santa Maria alle Scotte Hospital registry. TILs at the invasive front, center of tumor, and stroma were determined by immunohistochemistry and manually quantified as the rate of TILs/total tissue areas. High TILs (H-TILs) was defined as >20%. Patients were categorized as high or low TILs (L-TILs) and ADJ or no-ADJ.ResultsOf the 678 patients included, 137 (20%) received ADJ and 541 (80%) did not. The distribution of the 4 groups were: 16% (L-TIL/ADJ), 64% (L-TIL/no-ADJ), 5% (H-TIL/ADJ), 15% (H-TIL/no-ADJ). Compared to H-TILs/no-ADJ, ADJ patients showed a significantly increased OS (P<.01) regardless of the TILs rate whereas L-TILs/no-ADJ had significantly decreased OS and higher risk of death (HR=1.41; 95% CI, 1.06–1.88; P<.0001). On multivariable analysis, the unfavorable prognostic value of L-TILs (vs. H-TILs) for no-ADJ patients was confirmed (HR=1.36; 95% CI 1.02, 1.82; P=.0373).ConclusionLow CD3+ TILs rate was associated with shorter OS in those with stage II colon cancer who did not receive adjuvant therapy. Low CD3+ TILs could be considered an additional risk factor for still ADJ-untreated stage II CC patients, which could facilitate clinical decision making.     

A PKS/NRPS/FAS Hybrid Gene Cluster from Serratia plymuthica RVH1 Encoding the Biosynthesis of Three Broad Spectrum,Zeamine-Related Antibiotics

Serratia plymuthica strain RVH1, initially isolated from an industrial food processing environment, displays potent antimicrobial activity towards a broad spectrum of Gram-positive and Gram-negative bacterial pathogens. Isolation and subsequent structure determination of bioactive molecules led to the identification of two polyamino antibiotics with the same molecular structure as zeamine and zeamine II as well as a third, closely related analogue, designated zeamine I. The gene cluster encoding the biosynthesis of the zeamine antibiotics was cloned and sequenced and shown to encode FAS, PKS as well as NRPS related enzymes in addition to putative tailoring and export enzymes. Interestingly, several genes show strong homology to the pfa cluster of genes involved in the biosynthesis of long chain polyunsaturated fatty acids in marine bacteria. We postulate that a mixed FAS/PKS and a hybrid NRPS/PKS assembly line each synthesize parts of the backbone that are linked together post-assembly in the case of zeamine and zeamine I. This interaction reflects a unique interplay between secondary lipid and secondary metabolite biosynthesis. Most likely, the zeamine antibiotics are produced as prodrugs that undergo activation in which a nonribosomal peptide sequence is cleaved off.     

A global review and meta-analysis of applications of the freshwater Fish Invasiveness Screening Kit

Reviews in Fish Biology and Fisheries - The freshwater Fish Invasiveness Screening Kit (FISK) has been applied in 35 risk assessment areas in 45 countries across the six inhabited continents (11...     

Beta-HPV 5 and 8 E6 promote p300 degradation by blocking AKT/p300 association

The E6 oncoprotein from high-risk genus alpha human papillomaviruses (α-HPVs), such as HPV 16, has been well characterized with respect to the host-cell proteins it interacts with and corresponding signaling pathways that are disrupted due to these interactions. Less is known regarding the interacting partners of E6 from the genus beta papillomaviruses (β-HPVs); however, it is generally thought that β-HPV E6 proteins do not interact with many of the proteins known to bind to α-HPV E6. Here we identify p300 as a protein that interacts directly with E6 from both α- and β-HPV types. Importantly, this association appears much stronger with β-HPV types 5 and 8-E6 than with α-HPV type 16-E6 or β-HPV type 38-E6. We demonstrate that the enhanced association between 5/8-E6 and p300 leads to p300 degradation in a proteasomal-dependent but E6AP-independent manner. Rather, 5/8-E6 inhibit the association of AKT with p300, an event necessary to ensure p300 stability within the cell. Finally, we demonstrate that the decreased p300 protein levels concomitantly affect downstream signaling events, such as the expression of differentiation markers K1, K10 and Involucrin. Together, these results demonstrate a unique way in which β-HPV E6 proteins are able to affect host-cell signaling in a manner distinct from that of the α-HPVs.     

Optimal stimulation settings for CMAP scan registrations

Background

The CMAP (Compound Muscle Action Potential) scan is a non-invasive electrodiagnostic tool, which provides a quick and visual assessment of motor unit potentials as electrophysiological components that together constitute the CMAP. The CMAP scan records the electrical activity of the muscle (CMAP) in response to transcutaneous stimulation of the motor nerve with gradual changes in stimulus intensity. Large MUs, including those that result from collateral reinnervation, appear in the CMAP scan as so-called steps, i.e., clearly visible jumps in CMAP amplitude. The CMAP scan also provides information on nerve excitability. This study aims to evaluate the influence of the stimulation protocol used on the CMAP scan and its quantification.

Methods

The stimulus frequency (1, 2 and 3 Hz), duration (0.05, 0.1 and 0.3 ms), or number (300, 500 and 1000 stimuli) in CMAP scans of 23 subjects was systematically varied while the other two parameters were kept constant. Pain was measured by means of a visual analogue scale (VAS). Non-parametric paired tests were used to assess significant differences in excitability and step variables and VAS scores between the different stimulus parameter settings.

Results

We found no effect of stimulus frequency on CMAP scan variables or VAS scores. Stimulus duration affected excitability variables significantly, with higher stimulus intensity values for shorter stimulus durations. Step variables showed a clear trend towards increasing values with decreasing stimulus number.

Conclusions

A protocol delivering 500 stimuli at a frequency of 2 Hz with a 0.1 ms pulse duration optimized CMAP scan quantification with a minimum of subject discomfort, artefact and duration of the recording. CMAP scan variables were influenced by stimulus duration and number; hence, these need to be standardized in future studies.     

Spatial proximity of proteins surrounding zyxin under force-bearing conditions

Sensing physical forces is a critical first step in mechano-transduction of cells. Zyxin, a LIM domain-containing protein, is recruited to force-bearing actin filaments and is thought to repair and strengthen them. Yet, the precise force-induced protein interactions surrounding zyxin remain unclear. Using BioID analysis, we identified proximal proteins surrounding zyxin under normal and force-bearing conditions by label-free mass spectrometry analysis. Under force-bearing conditions, increased biotinylation of α-actinin 1, α-actinin 4, and AFAP1 were detected, and these proteins accumulated along force-bearing actin fibers independently from zyxin, albeit at a lower intensity than zyxin. VASP also accumulated along force-bearing actin fibers in a zyxin-dependent manner, but the biotinylation of VASP remained constant regardless of force, supporting the model of a free zyxin–VASP complex in the cytoplasm being corecruited to tensed actin fibers. In addition, ARHGAP42, a RhoA GAP, was also identified as a proximal protein of zyxin and colocalized with zyxin along contractile actin bundles. The overexpression of ARHGAP42 reduced the rate of small wound closure, a zyxin-dependent process. These results demonstrate that the application of proximal biotinylation can resolve the proximity and composition of protein complexes as a function of force, which had not been possible with traditional biochemical analysis.     

Heme oxygenase activity increases after exercise in healthy volunteers

Heme oxygenase (HO) is an essential, rate-limiting protein which catalyses the breakdown of heme to iron, carbon monoxide (CO), and biliverdin. The alpha methene bridge of the heme is eliminated as CO which can be measured as blood carboxyhaemoglobin (COHb). Using blood concentrations of COHb as a measure reflecting HO activity, we tested the postulate that the activity of HO changes with exercise. Ten healthy, nonsmoking volunteers (5 females and 5 males with a mean age?±?standard deviation of 25.7?±?3.2 years), lifetime nonsmokers with no history of respiratory diseases and not taking any medication, were included in the study. Subjects were exposed to filtered air for 2?hrs while alternating exercise for 15?minutes on a cycle ergometer with rest for 15?minutes. Workload was adjusted so that subjects breathed at a ventilatory rate, normalised for body surface area, of 25?L/m²/minute. Immediately before, immediately after, and the day following exercise, blood was drawn by standard venipuncture technique. COHb was determined using the interleukin (IL) 682 Co-Oximeter (Instrumentation Laboratory, Bedford, MA). COHb increased in each participant during the exercise session with the mean value (± standard deviation) almost doubling (1.1?±?1.6 to 2.1?±?1.6%) and returned to baseline by the following day (1.3?±?1.3%). We conclude that exercise increases HO activity.     

Zinc transport by respiratory epithelial cells and interaction with iron homeostasis

Despite recurrent exposure to zinc through inhalation of ambient air pollution particles, relatively little information is known about the homeostasis of this metal in respiratory epithelial cells. We describe zinc uptake and release by respiratory epithelial cells and test the postulate that Zn²⁺ transport interacts with iron homeostasis in these same cells. Zn²⁺ uptake after 4 and 8 h of exposure to zinc sulfate was concentration- and time-dependent. A majority of Zn²⁺ release occurred in the 4 h immediately following cell exposure to ZnSO₄. Regarding metal importers, mRNA for Zip1 and Zip2 showed no change after respiratory epithelial cell exposure to zinc while mRNA for divalent metal transporter (DMT)1 increased. Western blot assay for DMT1 protein supported an elevated expression of this transport protein following zinc exposure. RT-PCR confirmed mRNA for the metal exporters ZnT1 and ZnT4 with the former increasing after ZnSO₄. Cell concentrations of ferritin increased with zinc exposure while oxidative stress, measured as lipid peroxides, was decreased supporting an anti-oxidant function for Zn²⁺. Increased DMT1 expression, following pre-incubations of respiratory epithelial cells with TNF-α, IFN-γ, and endotoxin, was associated with significantly decreased intracellular zinc transport. Finally, incubations of respiratory epithelial cells with both zinc sulfate and ferric ammonium citrate resulted in elevated intracellular concentrations of both metals. We conclude that exposure to zinc increases iron uptake by respiratory epithelial cells. Elevations in cell iron can possibly affect an increased expression of DMT1 and ferritin which function to diminish oxidative stress. Comparable to other metal exposures, changes in iron homeostasis may contribute to the biological effects of zinc in specific cells and tissues.