We developed a methodology using 3D bio-printing technology to create a functional in vitro vascular channel with perfused open lumen using only cells and biological matrices. The fabricated vasculature has a tight, confluent endothelium lining, presenting barrier function for both plasma protein and high-molecular weight dextran molecule. The fluidic vascular channel is capable of supporting the viability of tissue up to 5mm in distance at 5 million cells/mL density under the physiological flow condition. In static-cultured vascular channels, active angiogenic sprouting from the vessel surface was observed whereas physiological flow strongly suppressed this process. Gene expression analysis were reported in this study to show the potential of this vessel model in vascular biology research. The methods have great potential in vascularized tissue fabrication using 3D bio-printing technology as the vascular channel is simultaneously created while cells and matrix are printed around the channel in desired 3D patterns. It can also serve as a unique experimental tool for investigating fundamental mechanisms of vascular remodeling with extracellular matrix and maturation process under 3D flow condition.

Kinesin-like proteins are integral to formation and function of a conserved mitotic spindle apparatus that directs chromosome segregation and precedes cell division. Ubiquitous to the mechanism of spindle assembly and stability are balanced Kinesin-5 promoting and Kinesin-14 opposing forces. Distinct Kinesin-14 roles in bipolarity in eukaryotes have not been shown, but are suggested by gamma-tubulin-based pole interactions that affect establishment and by microtubule cross-linking and sliding that maintain bipolarity and spindle length. Distinct roles also imply specialized functional domains. By cross-species analysis of compatible mechanisms in establishing mitotic bipolarity we demonstrate that Kinesin-14 human HSET (HsHSET) functionally replaces Schizosaccharomyces pombe Pkl1 and its action is similarly blocked by mutation in a Kinesin-14 binding site on gamma-tubulin. Drosophila DmNcd localizes preferentially to bundled interpolar microtubules in fission yeast and does not replace SpPkl1. Analysis of twenty-six Kinesin-14 derivatives, including Tail, Stalk or Neck-Motor chimeras, for spindle localization, spindle assembly and mitotic progression defined critical domains. The Tail of SpPkl1 contains functional elements enabling its role in spindle assembly that are distinct from but transferable to DmNcd, whereas HsHSET function utilizes both Tail and Stalk features. Our analysis is the first to demonstrate distinct mechanisms between SpPkl1 and DmNcd, and reveal that HsHSET shares functional overlap in spindle pole mechanisms.

SummaryMicrotubule-organizing centers such as the c-tubulin ring complex (c-TuRC) act as a template for polarized growth and regulation of microtubules that are essential for diverse cellular structures and processes in eukaryotes. New structural models of the budding yeast ctubulin small complex (c-TuSC) of the c-TuRC combined with functional studies done in multiple eukaryotes are revealing the first mechanistic clues into control of microtubule nucleation and organization. Cross-species studies of human and budding yeast c-TuSC proteins in fission yeast revealed conserved and divergent structural and functional features of the c-TuSC. We show genetically that GCP3/Spc98 function is fully conserved with Alp6 across species but that functional differences exist between GCP2/Spc97 and Alp4. By further analysis of human c-TuSC proteins, we found that GCP3 assembles normally into the .2000 kDa fission yeast c-TuRC and that the GCP3 gene replaces fission yeast alp6. Interestingly, human GCP2 replaces the essential alp4 gene but is unable to rescue a normally recessive G1 defect of the alp4-1891 allele that results in loss of c-TuRC from poles in subsequent cell cycles. Biochemically, GCP2 incorporation into fission yeast c-TuRC is limited in the presence of Alp4; instead, the bulk of GCP2 fractionates as smaller complexes. By generating a functional Alp4-GCP2 chimeric protein we determined that the GCP2 N-terminal domain limits its ability to fully displace or compete with Alp4 during c-TuRC assembly. Our findings have broad importance for understanding the essential domains of c-TuSC proteins in the c-TuRC mechanism.

BackgroundThe Maasai are the most widely known African ethnic group located in Kenya and northern Tanzania. Most spend their days either barefoot or in their traditional shoes made of car tires. Although they walk long distances of up to sixty kilometers a day, they do not suffer from any foot ailments. Little is known about their foot structure and gait. The goal of this investigation was to characterize various aspects of Maasai foot in standing and walking.MethodsFoot length, calf circumference, hindfoot alignment, step length, cadence, and walking velocity were obtained from 1,096 adult Maasai people (545 males and 551 females; mean age, 40.28 ± 14.69 years; age range, 16 to 65 years). All included subjects were from rural areas, where the primary terrain was sandy soil, who spend most of their lifetime barefoot, walking. They all denied any medical history or previous symptoms related to foot problems. A trained clinician scanned all feet for deformities. Static (standing) and dynamic (walking) Harris mat footprints were taken to determine the distribution of forefoot pressure patterns during walking.ResultsThe average foot length was 250.14 ± 18.12 mm (range, 210 to 295 mm) and calf circumference was 32.50 ± 3.22 cm (range, 25 to 41 cm). The mean hindfoot alignment was 6.21° ± 1.55° of valgus. Sixty-four subjects (5.84%) had bilateral flat-shaped feet with a low medial longitudinal arch that exactly matched the broad pattern of their static footprints. Step length, cadence, and walking velocity were 426.45 ± 88.73 cm (range, 200 to 690 cm), 94.35 steps/min (range, 72 to 111 steps/min), and 40.16 ± 8.36 m/min (range, 18.20 to 63.36 m/min), respectively. A total of 83.39% subjects showed unilateral or bilateral deformities of multiple toes regardless of age. The most frequent deformity was clawing (98.79%) of which the highest incidence occurred with the fifth toe (93.23%). Dynamic footprints showed even pressure patterns throughout the forefoot (64.87%), followed by lateral forefoot pressure concentration patterns (21.81%).ConclusionsOur study shows the distinct parameters that provide more insight into the Maasai foot.

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We have here investigated the effect of varying neck-linker length on the motile properties of Eg5Kin. As truncated versions of Eg5 contain the native 18 amino acids of the neck linker, we generated six Eg5Kin constructs comprising of 13 to up to the 18 amino acids of the native Eg5 neck linker, possibly providing a physiological context. Using single-molecule fluorescence, we found that all six constructs are active motor molecules capable of processive motility. In a first set of experiments, we found that the neck-linker length influences the run length, but not the velocity of the motor. We thus confirm the findings of Shastry and Hancock (2010, Curr. Biol. 20:939) with a different motor. In addition we used optical-trap assays to investigate the change in the average force the motor constructs generated and found only a small variation. Our data thus suggest that the neck-linker length of Eg5 is at least not the sole determinant for speed and force generation.