A Ratio of Spore to Viable Organisms: A Case Study of the JPL-SAF Cleanroom

Spacecraft surfaces that are destined to land on potential life-harboring celestial bodies are required to be rigorously cleaned and continuously monitored for spore bioburden as a proxy for spacecraft cleanliness. The NASA standard assay (NSA), used for spacecraft bioburden estimates, specifically measures spores that are cultivable, aerobic, resistant to heat shock, and grow at 30 C in a nutrient-rich medium. Since the vast majority of microorganisms cannot be cultivated using the NSA, it is necessary to utilize state-of-the art molecular techniques to better understand the presence of all viable microorganisms, not just those measured with the NSA. In this study, the nutrient-deprived low biomass cleanrooms, where spacecraft are assembled, were used as a surrogate for spacecraft surfaces to measure the ratio of NSA spores in relation to the total viable microorganism population in order to make comparisons with the 2006 Space Studies Board (SSB) estimate of 1 spore per approximately 50,000 viable organisms. Ninety-eight surface wipe samples were collected from the Spacecraft Assembly Facility (SAF) cleanroom at the Jet Propulsion Laboratory (JPL) over a 6-month period. The samples were processed and analyzed using classical microbiology along with molecular methodology. Traditional microbiology plating methods were used to determine the cultivable bacterial, fungal, and spore populations. Molecular assays were used to determine the total organisms (TO, dead and live) and the viable organisms (VO, live). The TO was measured using adenosine triphosphate (ATP) and quantitative polymerase chain reaction (qPCR) assays. The VO was measured using internal ATP, propidium monoazide (PMA)-qPCR, and flow cytometry (after staining for viable microorganisms) assays. Based on the results, it was possible to establish a ratio between spore counts and VO for each viability assay. The ATP-based spore to VO ratio ranged from 149-746, and the bacterial PMA-qPCR assay-based ratio ranged from 314-1,491 VO, per spore. The most conservative estimate came from fluorescent-assisted cell sorting (FACS), which estimated the ratio to be 12,091 VO per 1 NSA spore. Since archaeal (less than 1%) and fungal (approximately 2%) populations were negligible, the spore to VO ratios were based on bacterial population estimates. The most conservative ratio from this study can be used as a replacement for the SSB estimate on nutrient-deprived (oligotrophic) desiccated spacecraft surfaces, to estimate the VO from NSA measurements without utilizing state-of-the art molecular methods that are costly and require more biomass than is typically found on spacecraft surfaces

Authorship Trends Over the Past 30-Years in the Annals of Biomedical Engineering

In academia, manuscripts serve as an important component of career development. The past several years have seen heightened evaluation of the role of the gender gap in career advancement, as well as other bibliometric changes in publications. We therefore analyzed authorship and publication trends in the Annals of Biomedical Engineering over the past three decades (one complete year of manuscripts for each decade; 1986, 1996, 2006, and 2016). The variables analyzed were number of authors per manuscript, numerical position of the corresponding author, number of collaborating institutions and countries, number of references, and number of citations per manuscript. The gender of both the first and corresponding authors was identified and analyzed over time and by region. Globally, the percentage of female first and corresponding authors significantly increased from 0% in 1986 to 28.6% (p = 0.003) and 20.4% (p = 0.0009), respectively, in 2016. Although there were significant differences regarding female first and corresponding author over time, they did not vary by region of origin (p = 0.5 and 0.2, respectively). Overall, these findings highlight the improvements made and the challenges that still exist related to publishing within the bioengineering field

Quantitative Proteomic Analysis of Bacillus pumilus from Spores that Survived in Outer Space Conditions

The hardy spores of Bacillus species are well known for their resistance to unfavorable conditions such as UV and gamma radiation, hydrogen peroxide desiccation, chemical disinfection, or starvation. In particular, Bacillus pumilus strain SAFR-032 that was originally recovered from the Jet Propulsion Lab Spacecraft Assembly Facility has shown to exhibit unusually high resistance to UV radiation and peroxide treatment compared to other Bacillus species. To further understand the resistance of bacterial endospores to relevant outer space environments, spores of B. pumilus SAFR-032 were exposed for 1.5 years to selected parameters of space on board of the International Space Station (ISS). Here we are using a quantitative proteomics approach to gain insights into the resistance mechanism of B. pumilus

Skeletal adaptations in young male mice after 4 weeks aboard the International Space Station

Gravity has an important role in both the development and maintenance of bone mass. This is most evident in the rapid and intense bone loss observed in both humans and animals exposed to extended periods of microgravity in spaceflight. Here, cohabitating 9-week-old male C57BL/6 mice resided in spaceflight for ~4 weeks. A skeletal survey of these mice was compared to both habitat matched ground controls to determine the effects of microgravity and baseline samples in order to determine the effects of skeletal maturation on the resulting phenotype. We hypothesized that weight-bearing bones would experience an accelerated loss of bone mass compared to non-weight-bearing bones, and that spaceflight would also inhibit skeletal maturation in male mice. As expected, spaceflight had major negative effects on trabecular bone mass of the following weight-bearing bones: femur, tibia, and vertebrae. Interestingly, as opposed to the bone loss traditionally characterized for most weight-bearing skeletal compartments, the effects of spaceflight on the ribs and sternum resembled a failure to accumulate bone mass. Our study further adds to the insight that gravity has site-specific influences on the skeleton

Build your own closed loop: Graph-based proof of concept in closed loop for autonomous networks

Next Generation Networks (NGNs) are expected to handle heterogeneous technologies, services, verticals and devices of increasing complexity. It is essential to fathom an innovative approach to automatically and efficiently manage NGNs to deliver an adequate end-to-end Quality of Experience (QoE) while reducing operational expenses. An Autonomous Network (AN) using a closed loop can self-monitor, self-evaluate and self-heal, making it a potential solution for managing the NGN dynamically. This study describes the major results of building a closed-loop Proof of Concept (PoC) for various AN use cases organized by the International Telecommunication Union Focus Group on Autonomous Networks (ITU FG-AN). The scope of this PoC includes the representation of closed-loop use cases in a graph format, the development of evolution/exploration mechanisms to create new closed loops based on the graph representations, and the implementation of a reference orchestrator to demonstrate the parsing and validation of the closed loops. The main conclusions and future directions are summarized here, including observations and limitations of the PoC

Morphological changes associated with the genesis and development of an excitatory glutemergic synapse: An integrated framework model

The genesis of an excitatory synapse has its inception when a dendritic filopodium makes a tactile contact with a presynaptic specialisation (bouton). The subsequent maturation of the synapse takes place via a series of interrelated biochemical and biophysical signalling pathways which controls the actin polymerisation in the presynaptic and the postsynaptic sites. Although individual models of many of these signalling transductions have been proposed, a holistic model integrating the various signalling pathways to the morphological plasticity associated with the genesis and development of synapses has not. In this poster an attempt has been made towards establishing a framework for an integrated model such as the one aforementioned, encompassing several signalling pathways which control the morphology and the efficacy of the synapse. Predominant pathways include those triggered by NMDA and AMPA receptors, Trkb-BDNF, Integrin and Epherin. Also, steps towards a model that elucidates the change in shape of the synapse carried out by zonal actin polymerisation (ZAP) governed by the "wastage" of neurotransmitters during exo cum endocytosis processes and the assimilation of the postsynaptic density (PSD) and cell adhesion molecules with emphasis on Neurexin-Neuriligin, have been explored. The cannabinoid receptors in the PAZ have extracellular lipophilic domains. Endocannabinoid receptors are triggered by the retrograde signalling cues which negatively affect the cAMP dependent mechanisms. Apart from this, autoreceptors also pilot a feedback mechanism via secondary messengers with Ca 2+ ion concentration and neurotransmitter concentration in the synaptic cleft as its stakeholders. Feedback signals of autoreceptors which functions in accordance to “Lock and Key Mechanism” plays a vital role in fine-tuning the plasticity of the synapse and in controlling the presynaptic release probability by invoking PKA dependent pathways. In a future continuation of this framework, we would be focusing on working towards an integrated model of the pre-genesis and genesis and development of the presynaptic and the postsynaptic processes that we have explored. The first aspect that we would further be exploring into, would be a segue from the pre-genesis to the genesis part. In the postsynapse, we intend to link the stochastic model of receptor trafficking with our hypothesis on the Nlg-1 based shape change control of the postsynaptic active zone in a three dimensional space such that the various actin related cascading signalling pathways are zonally localised. In the presynapse, we wish to develop a model which holistically integrates the neurotransmitter exocytosis, endocytosis, vesicle reuptake and repacking processes, the autoreceptor control mechanism and the retrograde signalling mechanism. The symbiotic and fictile relationship between the presyanpse and the postsynapse is one that requires further study although lack of experimental data in this area poses a hurdle

Characterization of magnetically impelled arc butt welded T11 tubes for high pressure applications

Magnetically impelled arc butt (MIAB) welding is a pressure welding process used for joining of pipes and tubes with an external magnetic field affecting arc rotation along the tube circumference. In this work, MIAB welding of low alloy steel (T11) tubes were carried out to study the microstructural changes occurring in thermo-mechanically affected zone (TMAZ). To qualify the process for the welding applications where pressure could be up to 300 bar, the MIAB welds are studied with variations of arc current and arc rotation time. It is found that TMAZ shows higher hardness than that in base metal and displays higher weld tensile strength and ductility due to bainitic transformation. The effect of arc current on the weld interface is also detailed and is found to be defect free at higher values of arc currents. The results reveal that MIAB welded samples exhibits good structural property correlation for high pressure applications with an added benefit of enhanced productivity at lower cost. The study will enable the use of MIAB welding for high pressure applications in power and defence sectors

Optimal isolation of functional Foxp3+ induced regulatory T cells using DEREG mice.

Foxp3 reporter mice including DEREG (DEpletion of REGulatory T cells) mice have greatly helped in exploring the biology of Foxp3(+) Tregs. DEREG mice express a DTR-eGFP fusion protein under the control of a bacterial artificial chromosome (BAC)-encoded Foxp3 promoter, allowing the viable isolation and inducible depletion of Foxp3(+) Tregs. Adaptive Tregs differentiated in vitro to express Foxp3 (iTregs) are gaining high interest as potential therapeutics for inflammatory conditions such as autoimmunity, allergy and transplant rejection. However, selective isolation of Foxp3(+) iTregs with a stable phenotype still remains to be a problem, especially in the human setting. While screening for culture conditions to generate stable CD4(+)Foxp3(+) iTregs from DEREG mice, with maximum suppressive activity, we observed an unexpected dichotomy of eGFP and Foxp3 expression which is not seen in ex vivo isolated cells from DEREG mice. Further characterization of eGFP(+)Foxp3(-) cells revealed relatively lower CD25 expression and a lack of suppressive activity in vitro. Similarly, eGFP(-) cells isolated from the same cultures were not suppressive despite of a broad CD25 expression reflecting mere T cell activation. In contrast, eGFP(+)Foxp3(+) iTregs exhibited potent suppressive activity comparable to that of natural eGFP(+)Foxp3(+) Tregs, emphasizing the importance of isolating Foxp3 expressing iTregs. Interestingly, the use of plate-bound anti-CD3 and anti-CD28 or Flt3L-driven BMDC resulted in considerable resolution of the observed dichotomy. In summary, we defined culture conditions for efficient generation of eGFP(+)Foxp3(+) iTregs by use of DEREG mice. Isolation of functional Foxp3(+) iTregs using DEREG mice can also be achieved under sub-optimal conditions based on the magnitude of surface CD25 expression, in synergy with transgene encoded eGFP. Besides, the reported phenomenon may be of general interest for exploring Foxp3 gene regulation, given that Foxp3 and eGFP expression are driven from distinct Foxp3 loci and because this dichotomy preferentially occurs only under defined in vitro conditions