Effects of Coaxial Nozzle's Inner Nozzle Diameter on Filament Strength and Gelation in Extrusion-Based 3D Printing with In Situ Ionic Crosslinking.

This study systematically investigates the effects of the coaxial nozzle's inner nozzle diameter on the strength and gelation of filaments produced via extrusion-based 3D printing with in situ ionic crosslinking. In this system, bioink (sodium alginate solution) was extruded through the outer nozzle, and the ionic crosslinking solution (calcium chloride solution) was extruded through the inner nozzle. The outer nozzle diameter was fixed at 2.16 mm, and the inner nozzle diameter was varied among 1.19, 0.84, and 0.584 mm. The results indicate that, as the inner nozzle diameter decreased, filament strength decreased, and filament gelation became poorer. These findings highlight the importance of optimizing inner nozzle diameter for improved filament strength and gelation in extrusion-based 3D printing with in situ ionic crosslinking.

Experimental Study on Compatibility of Human Bronchial Epithelial Cells in Collagen-Alginate Bioink for 3D Printing.

This paper reports an experimental study on the compatibility of human bronchial epithelial (HBE) cells in a collagen-alginate bioink. The compatibility was assessed using the culture well method with three bioink compositions prepared from a 10% alginate solution and neutralized TeloCol-10 mg/mL collagen stock solution. Cell viability, quantified by (live cell count-dead cell count)/live cell count within the HBE cell-laden hydrogel, was evaluated using the live/dead assay method from Day 0 to Day 6. Experimental results demonstrated that the collagen-alginate 4:1 bioink composition exhibited the highest cell viability on Day 6 (85%), outperforming the collagen-alginate 1:4 bioink composition and the alginate bioink composition, which showed cell viability of 75% and 45%, respectively. Additionally, the live cell count was highest for the collagen-alginate 4:1 bioink composition on Day 0, a trend that persisted through Days 1 to 6, underscoring its superior performance in maintaining cell viability and promoting cell proliferation. These findings show that the compatibility of HBE cells with the collagen-alginate 4:1 bioink composition was higher compared with the other two bioink compositions.

Life table construction for crapemyrtle bark scale (Acanthococcus lagerstroemiae): the effect of different plant nutrient conditions on insect performance.

Crapemyrtle Bark Scale (Acanthococcus lagerstroemiae; CMBS) is an invasive pest species that primarily infest crapemyrtles (Lagerstroemia spp.) in the United States. Recent reports have revealed the dire threat of CMBS to attack not only crapemrytles but also the U.S. native species with expanded host plants such as American beautyberry (Callicarpa spp.) and Hypericum kalmianum L. (St. Johnswort). A better understanding of plant-insect interaction will provide better and environmental-friendly pest management strategies. In this study, we constructed the first comprehensive life table for CMBS to characterize its biological parameters, including developmental stages, reproductive behavior, and fecundity. The indirect effects of three plant nutrient conditions (water, 0.01MS, and 0.1MS) on CMBS populations were examined using the age-stage, two-sex life table. The demographic analyses revealed that the plant nutrient conditions had significantly altered CMBS development in terms of the intrinsic rate of increase (r), the finite rate of increase (λ), the net reproductive rate (R0), and mean generation time (T). Higher r, λ, and R0 were recorded under nutrient-deficient conditions (water), while CMBS reared on plants with healthier growing conditions (0.1MS) had the most prolonged T. Overall, CMBS shows better insect performance when reared on plants under nutrient-deficient conditions.

Real-Time Feeding Behavior Monitoring by Electrical Penetration Graph Rapidly Reveals Host Plant Susceptibility to Crapemyrtle Bark Scale (Hemiptera: Eriococcidae).

Host range confirmation of invasive hemipterans relies on the evaluation of plant susceptibility though greenhouse or field trials, which are inefficient and time-consuming. When the green industry faces the fast-spreading threat of invasive pests such as crapemyrtle bark scale (Acanthococcus lagerstroemiae), it is imperative to timely identify potential host plants and evaluate plant resistance/susceptibility to pest infestation. In this study, we developed an alternative technology to complement the conventional host confirmation methods. We used electrical penetration graph (EPG) based technology to monitor the A. lagerstroemiae stylet-tip position when it was probing in different plant tissues in real-time. The frequency and relative amplitude of insect EPG waveforms were extracted by an R programming-based software written to generate eleven EPG parameters for comparative analysis between plant species. The results demonstrated that the occurrences of phloem phase and xylem phase offered conclusive evidence for host plant evaluation. Furthermore, parameters including the percentage of insects capable of accessing phloem tissue, time duration spent on initiating phloem phase and ingesting phloem sap, provided insight into why host plant susceptibility differs among similar plant species. In summary, this study developed a novel real-time diagnostic tool for quick A. lagerstroemiae host confirmation, which laid the essential foundation for effective pest management.

Biosynthesis of Linear Protein Nanoarrays Using the Flagellar Axoneme.

Applications in biotechnology and synthetic biology often make use of soluble proteins, but there are many potential advantages of anchoring enzymes to a stable substrate, including stability and the possibility for substrate channeling. To avoid the necessity of protein purification and chemical immobilization, there has been growing interest in bio-assembly of protein-containing nanoparticles, exploiting the self-assembly of viral capsid proteins or other proteins that form polyhedral structures. However, these nanoparticles are limited in size, which constrains the packaging and the accessibility of the proteins. An axoneme, the insoluble protein core of the eukaryotic flagellum or cilium, is a highly ordered protein structure that can be several microns in length, orders of magnitude larger than other types of nanoparticles. We show that when proteins of interest are fused to specific axonemal proteins and expressed in living Chlamydomonas reinhardtii cells, they become incorporated into linear arrays, which have the advantages of high protein loading capacity and single-step purification with retention of biomass. The arrays can be isolated as membrane-enclosed vesicles or as exposed protein arrays. The approach is demonstrated for both a fluorescent protein and an enzyme (beta-lactamase), showing that incorporation into axonemes retains protein function in a stable, easily isolated array form.

Host Suitability for Crapemyrtle Bark Scale (Acanthococcus lagerstroemiae) Differed Significantly among Crapemyrtle Species.

Crapemyrtle bark scale (CMBS, Acanthococcus lagerstroemiae), an invasive polyphagous sap-sucking hemipteran, has spread across 14 states of the United States since 2004. The infestation of CMBS has negatively impacted the flowering of ornamental plants and even the fruiting of some crops. Host identification is critical for determining potential risks in ecosystems and industries and helps develop strategic management. A host confirmation test was performed over 25 weeks using six Lagerstroemia species (L. caudata, L. fauriei 'Kiowa', L. indica 'Dynamite', L. limii, L. speciosa, and L. subcostata) and California loosestrife (Lythrum californicum). The 25-week observations confirmed all tested plants as the hosts. The repeated measures of analysis of variance (ANOVA; Tukey's HSD, α = 0.05) indicated that the average number of CMBS females differed significantly between L. limii and L. speciosa. The highest number of the females observed on L. limii was 576 ± 25 (mean ± SE) at 17 weeks after inoculation (WAI), while the highest number was 57 ± 15 on L. speciosa at 19 WAI. In addition, L. subcostata and L. speciosa had significantly high and low numbers of males, respectively, among the Lagerstroemia species. Our results suggest that L. speciosa could be incorporated in developing new cultivars with low CMBS suitability.

Genotoxic properties of materials used for endoprostheses: Experimental and human data.

Approximately 2 million endoprostheses are implanted annually and metal ions as well as particles are released into the body from the materials which are used. This review describes the results of studies concerning genotoxic damage caused by artificial joints. DNA damage leads to various adverse long-term health effects in humans including cancer. Experiments with mammalian cells showed that metal ions and particles from orthopedic materials cause DNA damage. Induction of chromosomal aberrations (CA) was found in several in vitro experiments and in studies with rodents with metals from orthopedic materials. Human studies focused mainly on induction of CA (7 studies). Only few investigations (4) concerned sister chromatid exchanges, oxidative DNA damage (2) and micronucleus formation (1). CA are a reliable biomarker for increased cancer risks in humans) and were increased in all studies in patients with artificial joints. No firm conclusion can be drawn at present if the effects in humans are due to oxidative stress and if dissolved metal ions or release particles play a role. Our findings indicate that patients with artificial joints may have increased cancer risks due to damage of the genetic material. Future studies should be performed to identify safe materials and to study the molecular mechanisms in detail.

Feeding Preference of Crapemyrtle Bark Scale (Acanthococcus lagerstroemiae) on Different Species.

Crapemyrtle bark scale (CMBS; Acanthococcus lagerstroemiae) is an exotic pest species that causes aesthetic and economic damage to crapemyrtles and poses potential threats to other horticultural crops in the United States. Although previous studies reported the infestation of CMBS on several alternative hosts across multiple families in Asia, its potential threats to other documented alternative hosts remain elusive and yet to be confirmed. In this study, feeding preference studies of CMBS were conducted on forty-nine plant species and cultivars in 2016 and 2019, in order to gain insight into the expansion of CMBS distribution in the United States, as well as other regions of the world. The infestations of CMBS were confirmed on apple (Malus domestica), Chaenomeles speciosa, Disopyros rhombifolia, Heimia salicifolia, Lagerstroemia 'Spiced Plum', M. angustifolia, and twelve out of thirty-five pomegranate cultivars. However, the levels of CMBS infestation on these test plant hosts in this study is very low compared to Lagerstroemia, and may not cause significant damage. No sign of CMBS infestation was observed on Rubus 'Arapaho', R. 'Navaho', R. idaeus 'Dorman Red', R. fruticosus, B. microphylla var. koreana × B. sempervirens, B. harlandii, or D. virginiana.

Speed and Diffusion of Kinesin-2 Are Competing Limiting Factors in Flagellar Length-Control Model.

Flagellar length control in Chlamydomonas is a tractable model system for studying the general question of organelle size regulation. We have previously proposed that the diffusive return of the kinesin motor that powers intraflagellar transport can play a key role in length regulation. Here, we explore how the motor speed and diffusion coefficient for the return of kinesin-2 affect flagellar growth kinetics. We find that the system can exist in two distinct regimes, one dominated by motor speed and one by diffusion coefficient. Depending on length, a flagellum can switch between these regimes. Our results indicate that mutations can affect the length in distinct ways. We discuss our theory's implication for flagellar growth influenced by beating and provide possible explanations for the experimental observation that a beating flagellum is usually longer than its immotile mutant. These results demonstrate how our simple model can suggest explanations for mutant phenotypes.

O-GlcNAcylation Regulates Primary Ciliary Length by Promoting Microtubule Disassembly.

The sensory organelle cilium is involved in sensing and transducing important signaling cascades in almost all cells of our body. These ciliary-mediated pathways affect cellular homeostasis and metabolisms profoundly. However, it is almost completely unknown whether the cellular metabolic state affects the assembly of cilia. This study is to investigate how O-linked ß-N-acetylglucosamine (O-GlcNAc), a sensor of cellular nutrients, regulates the cilia length. Pharmacologic or genetic inhibition of O-GlcNAcylation led to longer cilia, and vice versa. Further biochemical assays revealed that both α-tubulin and HDAC6 (histone deacetylase 6) were O-GlcNAcylated in vivo. In vitro enzymatic assays showed that O-GlcNAcylation of either tubulin or HDAC6 promoted microtubule disassembly, which likely in turn caused ciliary shortening. Taken together, these results uncovered a negative regulatory role of O-GlcNAc in modulating the ciliary microtubule assembly. The cross talk between O-GlcNAc and cilium is likely critical for fine-tuning the cellular response to nutrients.

H+- and Na+- elicited rapid changes of the microtubule cytoskeleton in the biflagellated green alga Chlamydomonas.

Although microtubules are known for dynamic instability, the dynamicity is considered to be tightly controlled to support a variety of cellular processes. Yet diverse evidence suggests that this is not applicable to Chlamydomonas, a biflagellate fresh water green alga, but intense autofluorescence from photosynthesis pigments has hindered the investigation. By expressing a bright fluorescent reporter protein at the endogenous level, we demonstrate in real time discreet sweeping changes in algal microtubules elicited by rises of intracellular H+ and Na+. These results from this model organism with characteristics of animal and plant cells provide novel explanations regarding how pH may drive cellular processes; how plants may respond to, and perhaps sense stresses; and how organisms with a similar sensitive cytoskeleton may be susceptible to environmental changes.

IFT57 stabilizes the assembled intraflagellar transport complex and mediates transport of motility-related flagellar cargo.

Intraflagellar transport (IFT) is essential for the assembly and maintenance of flagella and cilia. Recent biochemical studies have shown that IFT complex B (IFT-B) is comprised of two subcomplexes, IFT-B1 and IFT-B2. The IFT-B2 subunit IFT57 lies at the interface between IFT-B1 and IFT-B2. Here, using a Chlamydomonasreinhardtii mutant for IFT57, we tested whether IFT57 is required for IFT-B complex assembly by bridging IFT-B1 and IFT-B2 together. In the ift57-1 mutant, levels of IFT57 and other IFT-B proteins were greatly reduced at the whole-cell level. However, strikingly, in the protease-free flagellar compartment, while the level of IFT57 was reduced, the levels of other IFT particle proteins were not concomitantly reduced but were present at the wild-type level. The IFT movement of the IFT57-deficient IFT particles was also unchanged. Moreover, IFT57 depletion disrupted the flagellar waveform, leading to cell swimming defects. Analysis of the mutant flagellar protein composition showed that certain axonemal proteins were altered. Taken together, these findings suggest that IFT57 does not play an essential structural role in the IFT particle complex but rather functions to prevent it from degradation. Additionally, IFT57 is involved in transporting specific motility-related proteins.

[Biomechanical research on upper femur with vertical and bending stress].

OBJECTIVE: To study the bone state with ultimate stress by examining biomechanical distribution of upper femur in Chinese, in order to accumulate more experiences for clinical work. METHODS: Totally 60 Chinese femurs from fresh cadavers were randomly divided into two groups. All the femurs were cleaned, and the body age ranged from 36 to 72 years old, averaged 56.4 years, including 41 from males, and 19 from females. These two groups underwent mechanical stress and bending stress tests. Special mechanical laboratory and machines were used to get the information. Results about the loading value at each testing point under stress were collected. RESULTS: The four faces of the upper femur suffered different stress under external forces. The bone on upper femur can tolerate more mechanical stress than bending stress. Medial and lateral region of the femur neck and the rear side of the small tuberosity section were themain position enduring the vertical stress. The rear position of the base femur neck and the small tuberosity section were the main regions enduring the bending stress. Those main positions had strong cancellous bones. The intertrochanteric fracture fixation and artificial femoral stems were designed depending on this biomechanical basis. CONCLUSION: According to our experiment result, doctors need to chose more effective fixations for upper femur fracture, and femoral stems for the patients. More information should be collected by further researches.

[Analysis on morphology and stability of femoral hip prosthesis based on the revision operation].

OBJECTIVE: According to the information obtained from the revision of total hip arthroplasty, the situations of the artificial femoral stem under physiological stress were analyzed preliminarily. In order to accumulate clinical experience, the discussions about how to enlongate the life of artifical joints were performed. METHODS: Fifty-three patients required revision operations were selected, including 28 males and 25 females,with an average age of 74.66 years old (61 to 84 years old). The average life of artificial joints was 18.24 years (3 to 27 years). The initial weightbearing was started 11 days (5 to 16 days) after the first operation. The main reasons for the revision were the spontaneous loosening and trauma. Based on imaging and surgical findings, as well as the histological pathology, the situations of the stems in human bodies were determined. RESULTS: Femoral prosthesis would sink under physiological stress. The design of femoral stems, motion of patients', and bone growth determined the secondary stability. Straight stems were helpful for physiological subsidence of prosthesis, achieving the stability with close bone-implant contact by bone growth,which allowed the patient to do early landing exercise. The collared prosthesis and prosthesis combined with internal fixation limited the subsidence of femoral stems. Their stability depends on the time of initial placement and the bone growth for supporting. Delaying the inital weightbearing was suggested for patients with these protheses. CONCLUSION: According to the actual situation of the patients, the appropriate chosen of femoral stems and time to perform the weightbearing can extend the life of the femoral prosthesis.

TTC26/DYF13 is an intraflagellar transport protein required for transport of motility-related proteins into flagella.

Cilia/flagella are assembled and maintained by the process of intraflagellar transport (IFT), a highly conserved mechanism involving more than 20 IFT proteins. However, the functions of individual IFT proteins are mostly unclear. To help address this issue, we focused on a putative IFT protein TTC26/DYF13. Using live imaging and biochemical approaches we show that TTC26/DYF13 is an IFT complex B protein in mammalian cells and Chlamydomonas reinhardtii. Knockdown of TTC26/DYF13 in zebrafish embryos or mutation of TTC26/DYF13 in C. reinhardtii, produced short cilia with abnormal motility. Surprisingly, IFT particle assembly and speed were normal in dyf13 mutant flagella, unlike in other IFT complex B mutants. Proteomic and biochemical analyses indicated a particular set of proteins involved in motility was specifically depleted in the dyf13 mutant. These results support the concept that different IFT proteins are responsible for different cargo subsets, providing a possible explanation for the complexity of the IFT machinery. DOI: http://dx.doi.org/10.7554/eLife.01566.001.

FAP20 is an inner junction protein of doublet microtubules essential for both the planar asymmetrical waveform and stability of flagella in Chlamydomonas.

The axoneme-the conserved core of eukaryotic cilia and flagella-contains highly specialized doublet microtubules (DMTs). A long-standing question is what protein(s) compose the junctions between two tubules in DMT. Here we identify a highly conserved flagellar-associated protein (FAP), FAP20, as an inner junction (IJ) component. The flagella of Chlamydomonas FAP20 mutants have normal length but beat with an abnormal symmetrical three-dimensional pattern. In addition, the mutant axonemes are liable to disintegrate during beating, implying that interdoublet connections may be weakened. Conventional electron microscopy shows that the mutant axonemes lack the IJ, and cryo-electron tomography combined with a structural labeling method reveals that the labeled FAP20 localizes at the IJ. The mutant axonemes also lack doublet-specific beak structures, which are localized in the proximal portion of the axoneme and may be involved in planar asymmetric flagellar bending. FAP20 itself, however, may not be a beak component, because uniform localization of FAP20 along the entire length of all nine DMTs is inconsistent with the beak's localization. FAP20 is the first confirmed component of the IJ. Our data also suggest that the IJ is important for both stabilizing the axoneme and scaffolding intra-B-tubular substructures required for a planar asymmetrical waveform.

Isolation of intraflagellar transport particle proteins from Chlamydomonas reinhardtii.

Cilia, the hair-like protrusions found on most eukaryotic cells, were once considered vestigial organelles. The recent renaissance of research in cilia arose from the discoveries of intraflagellar transport (IFT) and the involvement of IFT particle proteins in human diseases. Many IFT particle proteins have since been identified, and research on IFT particle complexes and their protein components continues to provide insight into the mechanism of IFT and the etiology of ciliopathies. In this chapter, we describe the methods of isolating IFT particles from the flagella of Chlamydomonas reinhardtii. Two methods, sucrose density gradient fractionation and immunoprecipitation, are explained in detail. Troubleshooting information is presented to illustrate the critical steps of the procedure to ensure successful implementation of these methods in individual labs.

Dissecting the sequential assembly and localization of intraflagellar transport particle complex B in Chlamydomonas.

Intraflagellar transport (IFT), the key mechanism for ciliogenesis, involves large protein particles moving bi-directionally along the entire ciliary length. IFT particles contain two large protein complexes, A and B, which are constructed with proteins in a core and several peripheral proteins. Prior studies have shown that in Chlamydomonas reinhardtii, IFT46, IFT52, and IFT88 directly interact with each other and are in a subcomplex of the IFT B core. However, ift46, bld1, and ift88 mutants differ in phenotype as ift46 mutants are able to form short flagella, while the other two lack flagella completely. In this study, we investigated the functional differences of these individual IFT proteins contributing to complex B assembly, stability, and basal body localization. We found that complex B is completely disrupted in bld1 mutant, indicating an essential role of IFT52 for complex B core assembly. Ift46 mutant cells are capable of assembling a relatively intact complex B, but such complex is highly unstable and prone to degradation. In contrast, in ift88 mutant cells the complex B core still assembles and remains stable, but the peripheral proteins no longer attach to the B core. Moreover, in ift88 mutant cells, while complex A and the anterograde IFT motor FLA10 are localized normally to the transition fibers, complex B proteins instead are accumulated at the proximal ends of the basal bodies. In addition, in bld2 mutant, the IFT complex B proteins still localize to the proximal ends of defective centrioles which completely lack transition fibers. Taken together, these results revealed a step-wise assembly process for complex B, and showed that the complex first localizes to the proximal end of the centrioles and then translocates onto the transition fibers via an IFT88-dependent mechanism.