The influenza A virus genome consists of eight single-stranded negative-sense RNA (vRNA) segments. Although genome segmentation provides advantages such as genetic reassortment, which contributes to the emergence of novel strains with pandemic potential, it complicates the genome packaging of progeny virions. Here we elucidate, using electron tomography, the three-dimensional structure of ribonucleoprotein complexes (RNPs) within progeny virions. Each virion is packed with eight well-organized RNPs that possess rod-like structures of different lengths. Multiple interactions are found among the RNPs. The position of the eight RNPs is not consistent among virions, but a pattern suggests the existence of a specific mechanism for assembly of these RNPs. Analyses of budding progeny virions suggest two independent roles for the viral spike proteins: RNP association on the plasma membrane and the subsequent formation of the virion shell. Our data provide further insights into the mechanisms responsible for segmented-genome packaging into virions.

No abstract

Filopodia are finger-like protrusions at the leading edge of migrating cells that play a crucial antennal function during cell motility. It is known that actin filaments are bundled hexagonally and provide rigidity to filopodia by virtue of fascin, which plays a central role in actin filament bundling. However, the molecular mechanisms underlying their formation remain unclear. Here, we observed the filopodia of intact whole cells fixed by rapid freezing and revealed their three-dimensional structure by cryo-electron tomography and image processing; the actin filament bundling structure by fascin was clarified at high resolution under physiological conditions. It was found that actin filaments in vivo were more numerous than in bundles reconstructed in vitro, and each filopodial actin filament had limited variability in helical twisting. In addition, statistical analysis of actin filament bundles unveiled their detailed architecture. In filopodia, actin filaments had highly ordered structures, and the shift between cross-links of each adjacent actin filament was approximately 2.7 nm, similar to the monomer repeat of actin filaments. We then proposed a plausible actin-fascin cross-link model at the amino acid level and identified three fascin binding sites on two adjacent actin filaments: one filament bound fascin at two discrete, widely separated regions and the other bound fascin in a single small region. We propose that these two different binding modalities should confer rigid bundles that retain flexibility and dynamic performance. © 2016 Wiley Periodicals, Inc.

Radiation therapy can result in severe side-effects, including the development of radiation resistance. The aim of this study was to validate the use of oxygen nanobubble water to overcome resistance to radiation in cancer cell lines via the suppression of the hypoxia-inducible factor 1-α (HIF-1α) subunit. Oxygen nanobubble water was created using a newly developed method to produce nanobubbles in the single-nanometer range with the ΣPM-5 device. The size and concentration of the oxygen nanobubbles in the water was examined using a cryo-transmission electron microscope. The nanobubble size was ranged from 2 to 3 nm, and the concentration of the nanobubbles was calculated at 2×1018 particles/ml. Cell viability and HIF-1α levels were evaluated in EBC-1 lung cancer and MDA-MB-231 breast cancer cells treated with or without the nanobubble water and radiation under normoxic and hypoxic conditions in vitro. The cancer cells grown in oxygen nanobubble-containing media exhibited a clear suppression of hypoxia-induced HIF-1α expression compared to the cells grown in media made with distilled water. Under hypoxic conditions, the EBC-1 and MDA-MB231 cells displayed resistance to radiation compared to the cells cultured under normoxic cells. The use of oxygen nanobubble medium significantly suppressed the hypoxia-induced resistance to radiation compared to the use of normal medium at 2, 6, 10 and 14 Gy doses. Importantly, the use of nanobubble media did not affect the viability and radiation sensitivity of the cancer cell lines, or the non-cancerous cell line, BEAS-2B, under normoxic conditions. This newly created single-nanometer range oxygen nanobubble water, without any additives, may thus prove to be a promising agent which may be used to overcome the hypoxia-induced resistance of cancer cells to radiation via the suppression of HIF-1α.

Teachers’ diagnostic practice on students-difficulties is one of the important steps in designing and managing classroom lessons. The purpose of this study was to explore teachers’ perception and practices regarding diagnosing students’ learning difficulties. The participants of the study were 28 Indonesian mathematics teachers of Junior High School. The data was collected through a Focus Group Discussion and a teacher questionnaire. The data were analyzed qualitatively to describe how the teachers perceive learning difficulties and how the teachers diagnosed students’ learning difficulties. The results of the analysis reveal that the teachers do not yet perform an in-depth diagnosis of students’ difficulties in learning mathematics. The teachers only focus on the mathematics topics and non-mathematical issues, instead of on students’ thinking process. The teachers also do not differentiate the diagnosis, evaluation, and prediction test. With regard to the strategies used by the teachers to diagnose students’ difficulties, analyzing students’ responses to tests was the majority. In this respect, observing students’ learning process during classroom activities is rarely done by the teachers in the purpose of diagnosing students’ learning difficulties. The results of the diagnosis are mainly used as the basis for remedial and drill and practices. The results imply that more support is needed for teachers to improve their competences particularly in diagnosing students’ thinking process difficulties when learning mathematics.

Outer dense fibre 2 (Odf2 or ODF2) is a cytoskeletal protein required for flagella (tail)-beating and stability to transport sperm cells from testes to the eggs. There are infertile males, including human patients, who have a high percentage of decapitated and decaudated spermatozoa (DDS), whose semen contains abnormal spermatozoa with tailless heads and headless tails due to head-neck separation. DDS is untreatable in reproductive medicine. We report for the first time a new type of Odf2-DDS in heterozygous mutant Odf2+/− mice. Odf2+/− males were infertile due to haploinsufficiency caused by heterozygous deletion of the Odf2 gene, encoding the Odf2 proteins. Odf2 haploinsufficiency induced sperm neck-midpiece separation, a new type of head-tail separation, leading to the generation of headneck sperm cells or headnecks composed of heads with necks and neckless tails composed of only the main parts of tails. The headnecks were immotile but alive and capable of producing offspring by intracytoplasmic headneck sperm injection (ICSI). The neckless tails were motile and could induce capacitation but had no significant forward motility. Further studies are necessary to show that ICSI in humans, using headneck sperm cells, is viable and could be an alternative for infertile patients suffering from Odf2-DDS.

We examine the cost in router complexity of adaptivity and virtual lanes in wormhole routers, using f-flat adaptive routers (based on a generalization of planar-adaptive routing) which include routers with a range of routing freedom. Our studies show that adaptivity is expensive because it requires additional virtual channels and much larger crossbar switches for both adaptivity and deadlock prevention. Increases of 50 to 100% in channel utilization are required to justify additional degrees of routing freedom.Three internal router architectures for virtual lanes are examined and the fully expanded crossbar is found to be most effective because it gives simplest control and minimal internal blocking. Examining router designs with from 1–16 virtual lanes indicates that 30% improvements in channel utilization are required to justify each additional virtual lane. These studies combined with published simulation results indicate that only modest numbers of virtual lanes are likely to be cost effective.

No abstract