Finite element modelling of primary hip stem stability: the effect of interference fit

The most commonly reported complications related to cementless hip stems are loosening and thigh pain; both of these have been attributed to high levels of relative micromotion at the bone-implant interface due to insufficient primary fixation. Primary fixation is believed by many to rely on achieving a sufficient interference fit between the implant and the bone. However, attempting to achieve a high interference fit not infrequently leads to femoral canal fracture either intra-operatively or soon after. The appropriate range of diametrical interference fit that ensures primary stability without risking femoral fracture is not well understood. In this study, a finite element model was constructed to predict micromotion and, therefore, instability of femoral stems. The model was correlated with an in vitro micromotion experiment carried out on four cadaver femurs. It was confirmed that interference fit has a very significant effect on micromotion and ignoring this parameter in an analysis of primary stability is likely to underestimate the stability of the stem. Furthermore, it was predicted that the optimal level of interference fit is around 50 microm as this is sufficient to achieve good primary fixation while having a safety factor of 2 against femoral canal fracture. This result is of clinical relevance as it indicates a recommendation for the surgeon to err on the side of a low interference fit rather than risking femoral fracture.     

Demonstrating the Manufacture of Human CAR‐T Cells in an Automated Stirred‐Tank Bioreactor

Chimeric antigen receptor T‐cell (CAR‐T) therapies have proven clinical efficacy for the treatment of hematological malignancies. However, CAR‐T cell therapies are prohibitively expensive to manufacture. The authors demonstrate the manufacture of human CAR‐T cells from multiple donors in an automated stirred‐tank bioreactor. The authors successfully produced functional human CAR‐T cells from multiple donors under dynamic conditions in a stirred‐tank bioreactor, resulting in overall cell yields which were significantly better than in static T‐flask culture. At agitation speeds of 200 rpm and greater (up to 500 rpm), the CAR‐T cells are able to proliferate effectively, reaching viable cell densities of >5 × 10⁶ cells ml‐1 over 7 days. This is comparable with current expansion systems and significantly better than static expansion platforms (T‐flasks and gas‐permeable culture bags). Importantly, engineered T‐cells post‐expansion retained expression of the CAR gene and retained their cytolytic function even when grown at the highest agitation intensity. This proves that power inputs used in this study do not affect cell efficacy to target and kill the leukemia cells. This is the first demonstration of human CAR‐T cell manufacture in stirred‐tank bioreactors and the findings present significant implications and opportunities for larger‐scale allogeneic CAR‐T production.     

Morphology of Salmonella enterica serovar Heidelberg typing phages

Eleven tailed phages are described. They belong to the Myoviridae, Siphoviridae, or Podoviridae families and represent the ViI, T1, T5, Jersey, N4, and P22 species of enterobacterial phages. Morphology is correlated with host range.     

Geochemistry and Bacterial Recovery from Hindu Kush Range Glacier and Their Potential for Metal Resistance and Antibiotic Production

In this study, we isolated and characterized bacteria and analyzed the geochemistry of unexplored non-polar Tirich Mir glacier, Chitral, Pakistan. The glacier is located in the Hindu Kush mountains range with the highest peak of 7708 m. Three types of samples, i.e. glacial ice, sediment, and melt water were collected. The geochemical analysis demonstrated a higher concentration of heavy metals Fe⁺², Mn⁺², and Cr⁺², cations Na⁺², Ca⁺², and Mg⁺², anions SO₄^?2 and Cl^?1. While among the dissolved free amino acids, tyrosine was detected in high concentration. A total of 43 strains were isolated based on colony morphology. Molecular identification revealed these isolates belong to four phyla, i.e. Proteobacteria 23(54%), Bacteroidetes 4(9%), Actinobacteria 7(16%), and Firmicutes 9(21). Gram-negative bacteria were enrich relative to Gram-positive bacteria. The bacterial communities were more abundant in sediment (21) than in water (12) and ice (10) samples. The isolates also exhibited potent activity against both ATCC and multidrug-resistant clinical isolates. The increased level of tolerance against heavy metals (Cd⁺², Cr⁺³, Hg⁺², Fe⁺³, Ar⁺³, and Ni⁺²) and NaCl was observed. Tirich Mir glacier was found to have a diverse bacterial community structure, with potential candidates having ability to produce bioactive metabolites with possible industrial applications.     

Functional requirement for symmetric assembly of archaeal box C/D small ribonucleoprotein particles

Box C/D small ribonucleoprotein particles (sRNPs) are archaeal homologs of small nucleolar ribonucleoprotein particles (snoRNPs) in eukaryotes that are responsible for site specific 2'-O-methylation of ribosomal and transfer RNAs. The function of box C/D sRNPs is characterized by step-wise assembly of three core proteins around a box C/D RNA that include fibrillarin, Nop5p, and L7Ae. The most distinct structural feature in all box C/D RNAs is the presence of two conserved box C/D motifs accompanied by often a single, and sometimes two, antisense elements located immediately upstream of either the D or D' box. Despite this asymmetric distribution of antisense elements, the bipartite feature of the box C/D motifs appears to be in pleasing agreement with a recently reported three-dimensional structure of the core protein complex between fibrillarin and Nop5p. This investigates functional implications of the symmetric features both in box C/D RNAs and in the fibrillarin-Nop5p complex. Site-directed mutagenesis was employed to generate box C/D RNAs lacking one of the two box C/D motifs and a mutant fibrillarin-Nop5p complex deficient in self-association. The ability of the mutated components to assemble and to direct methyl transfer reactions was assessed by gel mobility-shift, analytical ultracentrifugation, and in vitro catalysis studies. The results presented here suggest that, while a box C/D sRNP is capable of asymmetrical assembly, the symmetries in both the box C/D RNA and in the fibrillarin-Nop5p complex are required for efficient catalysis. These findings underscore the importance of functional assembly in methyl transfer reactions.     

Synergistic plant-microbes interactions in the rhizosphere: a potential headway for the remediation of hydrocarbon polluted soils

Soil pollution is an unavoidable evil; many crude-oil exploring communities have been identified to be the most ecologically impacted regions around the world due to hydrocarbon pollution and their concurrent health risks. Several clean-up technologies have been reported on the removal of hydrocarbons in polluted soils but most of them are either very expensive, require the integration of advanced mechanization and/or cannot be implemented in small scale. However, “Bioremediation” has been reported as an efficient, cost-effective and environment-friendly technology for clean-up of hydrocarbon”s contaminated soils. Here, we suggest the implementation of synergistic mechanism of bioremediation such as the use of rhizosphere mechanism which involves the actions of plant and microorganisms, which involves the exploitation of plant and microorganisms for effective and speedy remediation of hydrocarbon”s contaminated soils. In this mechanism, plant”s action is synergized with the soil microorganisms through the root rhizosphere to promote soil remediation. The microorganisms benefit from the root metabolites (exudates) and the plant in turn benefits from the microbial recycling/solubilizing of mineral nutrients. Harnessing the abilities of plants and microorganisms is a potential headway for cost-effective clean-up of hydrocarbon”s polluted sites; such technology could be very important in countries with great oil producing activities/records over many years but still developing.     

In vivo responses to vaccination with Mage-b,GM-CSF and thioglycollate in a highly metastatic mouse breast tumor model, 4T1

Metastatic breast cancer is an important contributor to morbidity and mortality. Hence, new therapies are needed that target breast cancer metastases. Here, we focus on Mage-b as a possible vaccine target to prevent the development of breast cancer metastases, through activation of Mage-b-specific cytotoxic T lymphocytes (CTL). The syngeneic cell line 4T1, highly expressing Mage-b, was used as a pre-clinical metastatic mouse breast tumor model. BALB/c mice received three preventive intraperitoneal immunizations with Mage-b DNA vaccine mixed with plasmid DNA, secreting granulocyte–macrophage colony stimulating factor (GM-CSF). In addition, antigen-presenting cells were more efficiently recruited to the peritoneal cavity by the injection of thioglycollate broth (TGB), prior to each immunization. Immunization with Mage-b/GM-CSF/TGB significantly reduced the number of metastases by 67% compared to the saline/GM-CSF/TGB and by 69% compared to the vector control/GM-CSF/TGB. Also, tumor growth was significantly reduced by 45% in mice vaccinated with Mage-b/GM-CSF/TGB compared to the saline/GM-CSF/TGB and by 47% compared to the control vector/GM-CSF/TGB group. In vivo, the number of CD8 T cells significantly increased in the primary tumors and metastases of mice vaccinated with Mage-b/GM-CSF/TGB compared to the saline/GM-CSF/TGB and the control vector/GM-CSF/TGB group, while the number of CD4 T cells significantly decreased. The combination of Mage-b, GM-CSF and TGB did not only induce significantly higher levels of IFNγ in the lymph nodes of vaccinated compared to control mice, but also induced significantly higher expression levels of Fas-ligand (FasL) in the primary tumors (expressing Fas protein constitutively), compared to the control mice. Whether the interaction between Fas and FasL may have contributed to the smaller tumors needs to be further analyzed.     

Overlapping roles of JIP3 and JIP4 in promoting axonal transport of lysosomes in human iPSC-derived neurons

The dependence of neurons on microtubule-based motors for the movement of lysosomes over long distances raises questions about adaptations that allow neurons to meet these demands. Recently, JIP3/MAPK8IP3, a neuronally enriched putative adaptor between lysosomes and motors, was identified as a critical regulator of axonal lysosome abundance. In this study, we establish a human induced pluripotent stem cell (iPSC)-derived neuron model for the investigation of axonal lysosome transport and maturation and show that loss of JIP3 results in the accumulation of axonal lysosomes and the Alzheimer’s disease–related amyloid precursor protein (APP)-derived Aβ42 peptide. We furthermore reveal an overlapping role of the homologous JIP4 gene in lysosome axonal transport. These results establish a cellular model for investigating the relationship between lysosome axonal transport and amyloidogenic APP processing and more broadly demonstrate the utility of human iPSC–derived neurons for the investigation of neuronal cell biology and pathology.     

Process development of human multipotent stromal cell microcarrier culture using an automated high‐throughput microbioreactor

Microbioreactors play a critical role in process development as they reduce reagent requirements and can facilitate high‐throughput screening of process parameters and culture conditions. Here, we have demonstrated and explained in detail, for the first time, the amenability of the automated ambr15 cell culture microbioreactor system for the development of scalable adherent human mesenchymal multipotent stromal/stem cell (hMSC) microcarrier culture processes. This was achieved by first improving suspension and mixing of the microcarriers and then improving cell attachment thereby reducing the initial growth lag phase. The latter was achieved by using only 50% of the final working volume of medium for the first 24 h and using an intermittent agitation strategy. These changes resulted in >150% increase in viable cell density after 24 h compared to the original process (no agitation for 24 h and 100% working volume). Using the same methodology as in the ambr15, similar improvements were obtained with larger scale spinner flask studies. Finally, this improved bioprocess methodology based on a serum‐based medium was applied to a serum‐free process in the ambr15, resulting in >250% increase in yield compared to the serum‐based process. At both scales, the agitation used during culture was the minimum required for microcarrier suspension, N_JS. The use of the ambr15, with its improved control compared to the spinner flask, reduced the coefficient of variation on viable cell density in the serum containing medium from 7.65% to 4.08%, and the switch to serum free further reduced these to 1.06–0.54%, respectively. The combination of both serum‐free and automated processing improved the reproducibility more than 10‐fold compared to the serum‐based, manual spinner flask process. The findings of this study demonstrate that the ambr15 microbioreactor is an effective tool for bioprocess development of hMSC microcarrier cultures and that a combination of serum‐free medium, control, and automation improves both process yield and consistency. Biotechnol. Bioeng. 2017;114: 2253–2266. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.