bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluating turnover intention as a proxy for behavior in the federal public service: Evidence from two surveys of senior civil servants
Authors: Limbocker Scott , Richardson Mark D.
Published: October 2023

Studies of turnover in the federal public service often study intention rather than behavior and studies that evaluate the reliability of intention as a proxy for that behavior are rare. This article uses a novel data set that measures exit intention and behavior for the same senior civil servants to evaluate two aspects of the reliability of turnover intention as a proxy for behavior: (1) prediction error and (2) similarity of marginal effects. Overall, exit intention is a problematic proxy for behavior because it consistently overestimates the probability of exit. While coefficients from models of intent tend to have the correct sign, the magnitudes of marginal effects can be misleading, tending to overestimate corresponding marginal effects in models of exit behavior. Moreover, coefficients do sometimes change signs between models. The article discusses the implications of these findings and suggests the next steps to better use exit intention to understand exit behavior.
Cluster analysis of multiple impairment measures in evidence‐based classification for para‐alpine sit skiers
Authors: Liu Kaiqi , Ji Linhong , Ma Han , Lu Yijia
Published: October 2023

The International Paralympic Committee has been promoting the development of evidence‐based classification to reduce the subjectivity in current decision‐making systems. The current study aimed to evaluate the validity of the impairment and performance tests for para‐alpine sit skiing classification, and whether cluster analysis of the measures would produce a valid classification structure. Thirty‐eight para‐alpine sit skiers with different disabilities completed seven tests. During these tests, isometric trunk strength, trunk muscle excitation, trunk range of movement (ROM), and simulated skiing performance (board tilt angle) were assessed. Correlations between the measures and the board tilt angle were calculated. To group athletes, K‐means cluster analysis was performed according to how much the impairment measures affected the board tilting. There were significant correlations between all measures and the maximal board tilt angle (r = 0.35–0.81, p < 0.05). The cluster analysis revealed that the introduction of ROM and muscle excitation was an effective supplement to strength measures in improving the classification accuracy (53%–79%). It produced four clusters with strong structures (mean silhouette coefficient = 0.81) and large and significant inter‐cluster differences in most measures and performance between clusters (p < 0.05). The cluster analysis produced classes comprising athletes with similar degrees of activity limitation. All tests reported can help establish a more transparent classification system for para‐alpine sit skiers. This study also provides a reference for evidence‐based classification systems in other Para sports.
The Formation and Consequences of Political Generations in Social Movements: Cases of Feminist Activism in Ecuador and Peru1,2
Authors: Coe Anna‐Britt
Published: October 2023

Anchored in Mannheim's theory, the concept of political generations captures how new movement recruits respond to shifting political contexts and become agents of change within a social movement. A key challenge when using this concept in generational analyses is to link context with agency. In this article, I make this link by focusing on the interactions between political contexts and movement agency. My study among two generations of feminist activism in Ecuador and Peru found that both cohorts interacted with two sociopolitical conditions—prevailing gender relations and notions of political action—when they were initially mobilized. These interactions took different forms for each cohort, thereby shaping their distinct understandings and practices of feminist activism, and continuing to have consequences for movement goals, strategies, and relationships overtime. For the earlier generation, which became active between the late 1970s and early 1990s, consequences meant practicing militancy to achieve goals, deploying vanguardism to execute a comprehensive strategy, and exerting autonomy to manage the actions of the powerful. I theorize the interactions between movement agency and political contexts as a mesostructure, where process and structure meet, thereby providing a more comprehensive account of the mechanism of change bringing about political generations.
Evaluation of eyebrow position and upper eyelid laxity after endolift laser treatment
Authors: Lotfi Elaheh , DellAvanzato Roberto , Ahramiyanpour Najmeh , Shadmanyazdi Fatemeh , Khosravi Somayeh
Published: October 2023

The periorbital area is one of the most sensitive areas in the face, and many techniques are used to change the eyelid laxity and position of the eyebrows. Recently the use of noninvasive or minimally invasive techniques is developed so, the amount of surgical procedures has decreased. In the present study. We evaluate the effect of Endolift laser as a non‐invasive method on upper eyelid laxity and eyebrow position. Nine patients underwent Endolift laser and evaluated for eyelid laxity and change in eyebrow position. Two blinded physicians assessed the cosmetic outcome of improvement in upper eyelid tightening via photography 6 months post‐treatment. The physicians evaluated the patient's improvement degree using the photographs by two clinical factors: skin laxity and total cosmetic result. Also, eyebrow height from the center of the pupil (CPEBH), Central eyebrow height (CEBH), Lateral eyebrow height (LEBH), Medial eyebrow height (MEBH) were measured by ImagJ before and 6 months post Endolift laser treatment. Our results showed eyelid laxity and eyebrow height were significantly changed after treatment.
Characterization of the novel HLA‐C*07:402:02 allele detected in a potential hematopoietic stem cell donor
Authors: Ananeva Anastasiia , Nizamov Shamil , Andryushkina Anna V. , Shagimardanova Elena I.
Published: October 2023

The novel HLA‐C*07:402:02 allele was characterized using next‐generation sequencing technology.
Identification of the novel HLA‐A*03:02:07 allele in a Russian individual
Authors: Ananeva Anastasiia , Sergeeva Iuliia , Andryushkina Anna V. , Shagimardanova Elena I.
Published: October 2023

One nucleotide substitution in codon 279 of HLA‐A*03:02:01:01 results in the novel HLA‐A*03:02:07 allele.
A role for fermentation in aerobic conditions as revealed by computational analysis of maize root metabolism during growth by cell elongation
Authors: Hunt Hilary , Leape Stefan , Sidhu Jagdeep Singh , Ajmera Ishan , Lynch Jonathan P. , Ratcliffe R. George , Sweetlove Lee J.
Published: October 2023

The root is a well‐studied example of cell specialisation, yet little is known about the metabolism that supports the transport functions and growth of different root cell types. To address this, we used computational modelling to study metabolism in the elongation zone of a maize lateral root. A functional‐structural model captured the cell‐anatomical features of the root and modelled how they changed as the root elongated. From these data, we derived constraints for a flux balance analysis model that predicted metabolic fluxes of the 11 concentric rings of cells in the root. We discovered a distinct metabolic flux pattern in the cortical cell rings, endodermis and pericycle (but absent in the epidermis) that involved a high rate of glycolysis and production of the fermentation end‐products lactate and ethanol. This aerobic fermentation was confirmed experimentally by metabolite analysis. The use of fermentation in the model was not obligatory but was the most efficient way to meet the specific demands for energy, reducing power and carbon skeletons of expanding cells. Cytosolic acidification was avoided in the fermentative mode due to the substantial consumption of protons by lipid synthesis. These results expand our understanding of fermentative metabolism beyond that of hypoxic niches and suggest that fermentation could play an important role in the metabolism of aerobic tissues.
Techno-Economic Analysis of the Solid Oxide Semi-Closed CO2 Cycle and Comparison With Other Power Generation Cycles With CO2 Capture
Authors: Martinelli Matteo , Scaccabarozzi Roberto , Gatti Manuele , Campanari Stefano , Martelli Emanuele
Published: October 2023

This work presents the techno-economic analysis of the solid oxide semi-closed CO2 cycle (SOS-CO2), a hybrid semi-closed cycle with solid oxide fuel cells (SOFC) recently developed by Politecnico di Milano for power generation from natural gas with near-zero CO2 emissions. According to previous studies, the cycle is able to achieve an outstanding net electric efficiency of 75.7%. while capturing more than 99% of the generated CO2. All the cycles components, including the fuel cell and the turbine, have been designed and sized with the aim of assessing the performance and the capital cost. The energy and economic key performance indicators are compared with those of two benchmark cycles with CO2 capture: the Allam cycle and a combined cycle with amines. Moreover, a sensitivity analysis is performed on the forecasted cost of the natural gas and the SOFC stacks. The results indicate that the specific total capital requirement of the SOS-CO2 cycle (2504 €/kWel) is considerably higher than the Allam cycle (1926 €/kWel) and combined cycle with amines (1981 €/kWel). On the other hand, the SOS-CO2 cycle benefits from its far higher efficiency (74.0% versus 53.9% of the Allam cycle and 52.8% of the combined cycle with amines) which makes the cycle less sensitive to the fuel cost and CO2 tax. In terms of cost of electricity, the SOS-CO2 cycle turns out to be the best technology for natural gas prices above 8 €/GJ, while the Allam cycle appears to be the preferred option at lower fuel prices. For a fuel cost of 12 €/GJ, the SOS-CO2 cycle features a cost of electricity of 106.2 €/MWh, lower than the Allam cycle (119.1 €/MWh) and the combined cycle with amines (126.9 €/MWh).
Numerical Analysis of Flame Shape Impact on the Performance of Fuel Staging in a Lean-Burn Aeronautical Burner
Authors: Mesquita Léo C. C. , Vié Aymeric , Ducruix Sébastien
Published: October 2023

The BIMER combustor is a lab-scale burner investigating fuel staging techniques as a stabilization strategy for lean premixed prevaporized combustion for aeronautical applications. Two stages compose its injection system: the pilot and the multipoint stages. The staging factor is defined as the ratio of fuel mass flowrate injected through the pilot stage over the total one. As three flame shapes were found experimentally, Large-Eddy Simulations are performed in this study to assess the impact of the flame shape on the combustion regime and stability of the burner. Two operating conditions were explored experimentally (pilot-only and multipoint-dominated) to validate the simulations and compare the three flames. An additional multipoint-only condition is also investigated for the V flame. The combustion regimes (premixed and non-premixed) and noise signatures (as a function of fuel staging) were compared to check whether these flames could benefit from the staging strategy. The M and Tulip flame combustion regimes are little affected by fuel staging, remaining mostly premixed and non-premixed, respectively, regardless of fuel staging. In opposition, the V flame changes from being mostly non-premixed to completely premixed when the injection is changed from pilot-only to multipoint-only. For the same staging factor values, the V flame also emits less noise compared to the other two flame shapes. These results show that the V flame shape is the only one that allows this burner to benefit from an efficient fuel staging strategy.
Evaluation of Water-Cooling Effect in Hydrogen-Fed SOFC for High-Efficiency Combined System Design
Authors: Kim Hye Rim , Kim Tong Seop
Published: October 2023

If pure hydrogen is used as a fuel in high-temperature fuel cells, waste heat must be removed by air cooling, which requires increased power consumption for supplying excess air. This study presents a hydrogen-fed solid oxide fuel cell (SOFC) that uses water instead of air for stack cooling and improved system performance. A novel SOFC system with energy cascade utilization is also proposed using cooling water as the working fluid for a steam turbine. Water cooling for the SOFC stack cooling reduced the stack power and efficiency but significantly reduced the power consumption for supplying excess air by more than 60%. Under ambient SOFC operating pressure, the net power and efficiency of the proposed system were increased by 25.6% and 12.2%p compared to the air-cooled system, respectively. At an SOFC operating pressure of 1000 kPa, the proposed hybrid system with energy cascade utilization achieved improvements of 10.2% in net power and 7.5%p in net efficiency, leading to efficiency higher than 73%. This study is significant in that it proposes a novel high-efficiency SOFC system with energy cascade utilization by using two-phase water as a cooling medium and working fluid of steam turbine.
A Biphasic Fluid–Structure Interaction Model of Backflow During Infusion Into Agarose Gel
Authors: Ayers Arthur D. , Smith Joshua H.
Published: October 2023

The efficacy of convection-enhanced delivery as a technique to treat disorders of the central nervous system is limited by backflow, in which the infused fluid flows backward along surface of the catheter rather than toward the targeted area. In order to improve treatment protocols, finite element models of backflow have been developed to understand the underlying physics. García et al. (2013, “Description and Validation of a Finite Element Model of Backflow During Infusion Into a Brain Tissue Phantom,” ASME J. Comput. Nonlinear Dyn., 8(1), p. 011017) presented a finite element model that accounted for the flow in the annular gap that develops between the tissue and the outer surface of the catheter by using a layer of biphasic elements with a formula for the axial hydraulic conductivity to represent annular Poiseuille flow. In this study, we present a generalization of that model using fluid-FSI and biphasic-FSI elements that are recently available in febio. We demonstrate that our model of a 0.98 mm radius catheter is able to reproduce experimental backflow lengths and maximum fluid pressures for infusions into a brain tissue surrogate and that it agrees well with the previous model by García et al. (2013, “Description and Validation of a Finite Element Model of Backflow During Infusion Into a Brain Tissue Phantom,” ASME J. Comput. Nonlinear Dyn., 8(1), p. 011017). The model predicts that the backflow length and the total amount of flow into the hemispherical region forward of the catheter tip is comparable for two different catheter sizes, albeit at a higher fluid pressure for the smaller catheter. This biphasic-FSI model has the potential to be extended to a stepped catheter geometry, which has been shown in experiments to be successful in controlling backflow.
Identification of dust particles on a periodic nanostructured substrate using scanning electron microscope imaging
Authors: Tunell Andrew , Micklow Lauren , Scott Nichole , Furst Stephen , Chang Chih-Hao
Published: October 2023

Dust-mitigating surfaces typically consist of high-aspect-ratio structures that separate particles from resting on the bulk material, thereby limiting adhesion due to short-range van der Waals forces. These surfaces can find uses in solar-panel coatings and a variety of dust-resistant optics. The current method for quantifying surface contamination is optical microscopy, but this method is inadequate for observing particles at the submicrometer scale due to the diffraction limit. Furthermore, regardless of the microscopy technique, particle identification becomes problematic as the particle contaminates approach the same length scale of the surface structures. In this work, we demonstrate a method to identify micro-/nanoparticle contaminates on nanostructured surfaces using electron microscopy and image processing. This approach allows the characterization of particles that approach the length scale of the surface structures. Image processing, including spectrum filters and edge detection, is used to remove the periodic features of the surface nanostructure to omit them from the particle counting. The detection of these small particles using electron microscopy leads to an average of 5.62 particles/100 μm2 detected compared to 0.63 particles/100 μm2 detected for the traditional confocal optical detection method. Beyond dust-mitigation nanostructures, the demonstrated particle detection technique can find applications in nanobiology, the detection of ice nucleation on a structured surface, and semiconductor mask inspections.
Coinhibition of topoisomerase 1 and BRD4-mediated pause release selectively kills pancreatic cancer via readthrough transcription
Authors: Cameron Donald P. , Grosser Jan , Ladigan Swetlana , Kuzin Vladislav , Iliopoulou Evanthia , Wiegard Anika , Benredjem Hajar , Jackson Kathryn , Liffers Sven T. , Lueong Smiths , Cheung Phyllis F. , Vangala Deepak , Pohl Michael , Viebahn Richard , Teschendorf Christian , Wolters Heiner , Usta Selami , Geng Keyi , Kutter Claudia , Arsenian-Henriksson Marie , Siveke Jens T. , Tannapfel Andrea , Schmiegel Wolff , Hahn Stephan A. , Baranello Laura
Published: October 2023

Pancreatic carcinoma lacks effective therapeutic strategies resulting in poor prognosis. Transcriptional dysregulation due to alterations in KRAS and MYC affects initiation, development, and survival of this tumor type. Using patient-derived xenografts of KRAS- and MYC-driven pancreatic carcinoma, we show that coinhibition of topoisomerase 1 (TOP1) and bromodomain-containing protein 4 (BRD4) synergistically induces tumor regression by targeting promoter pause release. Comparing the nascent transcriptome with the recruitment of elongation and termination factors, we found that coinhibition of TOP1 and BRD4 disrupts recruitment of transcription termination factors. Thus, RNA polymerases transcribe downstream of genes for hundreds of kilobases leading to readthrough transcription. This occurs during replication, perturbing replisome progression and inducing DNA damage. The synergistic effect of TOP1 + BRD4 inhibition is specific to cancer cells leaving normal cells unaffected, highlighting the tumor’s vulnerability to transcriptional defects. This preclinical study provides a mechanistic understanding of the benefit of combining TOP1 and BRD4 inhibitors to treat pancreatic carcinomas addicted to oncogenic drivers of transcription and replication.
The persistence of smoke VOCs indoors: Partitioning, surface cleaning, and air cleaning in a smoke-contaminated house
Authors: Li Jienan , Link Michael F. , Pandit Shubhrangshu , Webb Marc H. , Mayer Kathryn J. , Garofalo Lauren A. , Rediger Katelyn L. , Poppendieck Dustin G. , Zimmerman Stephen M. , Vance Marina E. , Grassian Vicki H. , Morrison Glenn C. , Turpin Barbara J. , Farmer Delphine K.
Published: October 2023

Wildfires are increasing in frequency, raising concerns that smoke can permeate indoor environments and expose people to chemical air contaminants. To study smoke transformations in indoor environments and evaluate mitigation strategies, we added smoke to a test house. Many volatile organic compounds (VOCs) persisted days following the smoke injection, providing a longer-term exposure pathway for humans. Two time scales control smoke VOC partitioning: a faster one (1.0 to 5.2 hours) that describes the time to reach equilibrium between adsorption and desorption processes and a slower one (4.8 to 21.2 hours) that describes the time for indoor ventilation to overtake adsorption-desorption equilibria in controlling the air concentration. These rates imply that vapor pressure controls partitioning behavior and that house ventilation plays a minor role in removing smoke VOCs. However, surface cleaning activities (vacuuming, mopping, and dusting) physically removed surface reservoirs and thus reduced indoor smoke VOC concentrations more effectively than portable air cleaners and more persistently than window opening.
The cryo-EM structure of the human ERAD retrotranslocation complex
Authors: Rao Bing , Wang Qian , Yao Deqiang , Xia Ying , Li Wenguo , Xie Yuming , Li Shaobai , Cao Mi , Shen Yafeng , Qin An , Zhao Jie , Cao Yu
Published: October 2023

Endoplasmic reticulum–associated degradation (ERAD) maintains protein homeostasis by retrieving misfolded proteins from the endoplasmic reticulum (ER) lumen into the cytosol for degradation. The retrotranslocation of misfolded proteins across the ER membrane is an energy-consuming process, with the detailed transportation mechanism still needing clarification. We determined the cryo-EM structures of the hetero-decameric complex formed by the Derlin-1 tetramer and the p97 hexamer. It showed an intriguing asymmetric complex and a putative coordinated squeezing movement in Derlin-1 and p97 parts. With the conformational changes of p97 induced by its ATP hydrolysis activities, the Derlin-1 channel could be torn into a “U” shape with a large opening to the lipidic environment, thereby forming an entry for the substrates in the ER membrane. The EM analysis showed that p97 formed a functional protein complex with Derlin-1, revealing the coupling mechanism between the ERAD retrotranslocation and the ATP hydrolysis activities.
Thrombospondin-1 proteomimetic polymers exhibit anti-angiogenic activity in a neovascular age-related macular degeneration mouse model
Authors: Choi Wonmin , Nensel Ashley K. , Droho Steven , Fattah Mara A. , Mokashi-Punekar Soumitra , Swygart David I. , Burton Spencer T. , Schwartz Greg W. , Lavine Jeremy A. , Gianneschi Nathan C.
Published: October 2023

Neovascular age-related macular degeneration (nAMD) is the leading cause of blindness in the developed world. Current therapy includes monthly intraocular injections of anti-VEGF antibodies, which are ineffective in up to one third of patients. Thrombospondin-1 (TSP1) inhibits angiogenesis via CD36 binding, and its down-regulated expression is negatively associated with the onset of nAMD. Here, we describe TSP1 mimetic protein-like polymers (TSP1 PLPs). TSP1 PLPs bind CD36 with high affinity, resist degradation, show prolonged intraocular half-lives (13.1 hours), have no toxicity at relevant concentrations in vivo (40 μM), and are more efficacious in ex vivo choroidal sprouting assays compared to the peptide sequence and Eylea (aflibercept), the current standard of care anti-VEGF treatment. Furthermore, PLPs exhibit superior in vivo efficacy in a mouse model for nAMD compared to control PLPs consisting of scrambled peptide sequences, using fluorescein angiography and immunofluorescence. Since TSP-1 inhibits angiogenesis by VEGF-dependent and independent mechanisms, TSP1 PLPs are a potential therapeutic for patients with anti-VEGF treatment–resistant nAMD.
Methanogens acquire and bioaccumulate nickel during reductive dissolution of nickelian pyrite
Authors: Spietz Rachel L. , Payne Devon , Boyd Eric S.
Published: October 2023

Nickel (Ni) is a key component of the active site metallocofactors of numerous enzymes required for methanogenesis, including [NiFe]-hydrogenase, carbon monoxide dehydrogenase, and methyl CoM reductase, leading to a high demand for Ni among methanogens. However, methanogens often inhabit euxinic environments that favor the sequestration of nickel as metal-sulfide minerals, such as nickelian pyrite [(Ni,Fe)S
2
], that have low solubilities and that are not considered bioavailable. Recently, however, several different model methanogens (
Methanosarcina barkeri
,
Methanococcus voltae
,
Methanococcus maripaludis
) were shown to reductively dissolve pyrite (FeS
2
) and to utilize dissolution products to meet iron and sulfur biosynthetic demands. Here, using
M. barkeri
Fusaro, and laboratory-synthesized (Ni,Fe)S
2
that was physically isolated from cells using dialysis membranes, we show that trace nickel (<20 nM) abiotically solubilized from the mineral can support methanogenesis and limited growth, roughly fivefold less than the minimum concentration known to support methanogenesis. Furthermore, when provided direct contact with (Ni,Fe)S
2
,
M. barkeri
promoted the reductive dissolution of (Ni,Fe)S
2
and assimilated solubilized nickel, iron, and sulfur as its sole source of these elements. Cells that reductively dissolved (Ni,Fe)S
2
bioaccumulated approximately fourfold more nickel than those grown with soluble nickel and sulfide but had similar metabolic coupling efficiencies. While the mechanism for Ni uptake in archaeal methanogens is not known, homologs of the bacterial Nik uptake system were shown to be ubiquitous across methanogen genomes. Collectively, these observations indicate that (Ni,Fe)S
2
is bioavailable in anoxic environments and that methanogens can convert this mineral into nickel-, iron-, and sulfur-containing metalloenzymes to support methanogenesis and growth.

IMPORTANCE

Nickel is an essential metal, and its availability has changed dramatically over Earth history due to shifts in the predominant type of volcanism in the late Archean that limited its availability and an increase in euxinic conditions in the early Proterozoic that favored its precipitation as nickel sulfide minerals. Observations presented herein indicate that the methanogen,
Methanosarcina barkeri
, can acquire nickel at low concentration (<20 nM) from soluble and mineral sources. Furthermore,
M. barkeri
was shown to actively reduce nickelian pyrite; use dissolution products to meet their iron, sulfur, and nickel demands; and bioaccumulate nickel. These data help to explain how
M. barkeri
(and possibly other methanogens and anaerobes) can acquire nickel in contemporary and past anoxic or euxinic environments.
Hyperimmune bovine colostrum containing lipopolysaccharide antibodies (IMM124-E) has a nondetrimental effect on gut microbial communities in unchallenged mice
Authors: Gore Rachele , Mohsenipour Mitra , Wood Jennifer L. , Balasuriya Gayathri K. , Hill-Yardin Elisa L. , Franks Ashley E.
Published: October 2023

Enterotoxigenic
Escherichia coli
(ETEC) is a leading cause of bacterial diarrhea with the potential to cause long-term gastrointestinal (GI) dysfunction. Preventative treatments for ETEC-induced diarrhea exist, yet the effects of these treatments on GI commensals in healthy individuals are unclear. Whether administration of a prophylactic preventative treatment for ETEC-induced diarrhea causes specific shifts in gut microbial populations in controlled environments is also unknown. Here, we studied the effects of a hyperimmune bovine colostrum (IMM-124E) used in the manufacture of Travelan (AUST L 106709) on GI bacteria in healthy C57BL/6 mice. Using next-generation sequencing, we aimed to test the onset and magnitude of potential changes to the mouse gut microbiome in response to the antidiarrheagenic hyperimmune bovine colostrum product, rich in immunoglobulins against select ETEC strains (Travelan, Immuron Ltd). We show that in mice administered colostrum containing lipopolysaccharide (LPS) antibodies, there was an increased abundance of potentially gut-beneficial bacteria, such as
Akkermansia
and
Desulfovibrio,
without disrupting the underlying ecology of the GI tract. Compared to controls, there was no difference in overall weight gain, body or cecal weights, or small intestine length following LPS antibody colostrum supplementation. Overall, dietary supplementation with colostrum containing LPS antibodies produced subtle alterations in the gut bacterial composition of mice. Primarily, Travelan LPS antibody treatment decreased the ratio of Firmicutes/Bacteroidetes in gut microbial populations in unchallenged healthy mice. Further studies are required to examine the effect of Travelan LPS antibody treatment to engineer the microbiome in a diseased state and during recovery.
Responsible and ethical conduct of research (RECR) diagnostic survey using case scenarios from biology course-based undergraduate research experiences (CUREs)
Authors: Monthofer Elizabeth G. , Ito Jewel M. , Battaglia Samantha , Diaz-Martinez Laura A.
Published: October 2023

Course-based undergraduate research experiences (CUREs) are increasingly becoming the first, and perhaps only, research experience for many biology students. Responsible and ethical conduct of research (RECR) is crucial for the integrity of scientific research and essential for students to have an understanding of the scientific process at any academic level. However, there is a current lack of RECR education in biology CUREs. To understand the level of RECR knowledge and skills in undergraduate students, we created a diagnostic survey that uses case scenarios designed to illustrate RECR issues in the CURE classroom. Analysis of students’ responses indicated that the overall percentage of students who are able to effectively use RECR terminology and identify the impact of RECR violations on science integrity and ultimately on society is low. Furthermore, some students equated RECR violations to academic dishonesty, indicating difficulties separating the research and academic aspects of CUREs. This diagnostic tool can aid instructors in identifying gaps in student RECR knowledge for the subsequent development of RECR educational interventions, particularly to ensure the integrity of the research performed in CURE settings.
Adenoviral-vectored universal influenza vaccines administered intranasally reduce lung inflammatory responses upon viral challenge 15 months post-vaccination
Authors: Misplon Julia A. , Lo Chia-Yun , Crabbs Torrie A. , Price Graeme E. , Epstein Suzanne L.
Published: October 2023

New approaches to vaccination are needed for protection against rapidly evolving viruses because emerging variant strains often evade neutralizing antibodies. Alternative strategies include targeting highly conserved antigens that change more slowly over time, which could provide the basis for universal vaccines. Recombinant adenovirus (rAd)-based vaccines expressing conserved influenza virus antigens nucleoprotein (NP) and matrix 2 (M2) provide powerful, long-lasting protection against challenge with diverse influenza strains, especially if administered intranasally. After intranasal administration of these rAd-based vaccines, animals are protected against morbidity and mortality in part by strong local T cell responses. However, concerns have been raised that these local responses could result in immune-mediated lung damage. In this study, we vaccinated mice intranasally with rAd vectors expressing influenza A/NP and/or M2, or influenza B/NP as a control, and then challenged them with an influenza A virus 15 months later. Protection persisted, as shown by accelerated viral clearance. Vaccination reduced the incidence and/or severity of histopathologic findings associated with influenza infection. Vaccination with A/NP+M2-rAd or A/NP-rAd reduced cytokine responses in the lungs following challenge. Granzyme B levels peaked and resolved earlier in animals vaccinated with A/NP+M2-rAd or A/NP-rAd than in other groups, consistent with a rapid CD8 T cell response that resolved more quickly. An influx of neutrophils, shown by a rise in neutrophil elastase, occurred in all groups following the challenge but returned to baseline more quickly in the two A/NP-vaccinated groups. Thus, vaccines inducing potent local immunity in the respiratory tract provided protection, rather than damage, to the lungs.

IMPORTANCE

Vaccines targeting highly conserved proteins can protect broadly against diverse viral strains. When a vaccine is administered to the respiratory tract, protection against disease is especially powerful. However, it is important to establish that this approach is safe. When vaccinated animals later encounter viruses, does reactivation of powerful local immunity, including T cell responses, damage the lungs? This study investigates the safety of mucosal vaccination of the respiratory tract. Non-replicating adenoviral vaccine vectors expressing conserved influenza virus proteins were given intranasally. This vaccine-induced protection persists for at least 15 months. Vaccination did not exacerbate inflammatory responses or tissue damage upon influenza virus infection. Instead, vaccination with nucleoprotein reduced cytokine responses and histopathology, while neutrophil and T cell responses resolved earlier. The results are promising for safe vaccination at the site of infection and thus have implications for the control of influenza and other respiratory viruses.
Xylella fastidiosa
modulates exopolysaccharide polymer length and the dynamics of biofilm development with a β-1,4-endoglucanase
Authors: Castro Claudia , Ndukwe Ikenna , Heiss Christian , Black Ian , Ingel Brian M. , Guevara Matthew , Sun Yuling , Azadi Parastoo , Sun Qiang , Roper M. Caroline
Published: October 2023

Xylella fastidiosa
is a Gram-negative bacterium that causes disease in many economically important crops. It colonizes the plant host xylem and the mouthparts of its insect vectors where it produces exopolysaccharide (EPS) and forms robust biofilms. Typically, the ability to form a biofilm enhances virulence, but
X. fastidiosa
does not fit neatly into that paradigm. Instead,
X. fastidiosa
enters into biofilms to attenuate its movement in the xylem, which, in turn, slows disease progression. In most of its over 600 known plant hosts,
X. fastidiosa
behaves as a benign commensal, but in some hosts like
Vitis vinifera
grapevines, it acts as a pathogen. Its ability to attenuate its own virulence in susceptible hosts may be a remnant of its commensal lifestyle in other hosts. Here, we demonstrate that
X. fastidiosa
utilizes a β-1,4 endoglucanase to cleave its self-produced β-1,4-glucan exopolysaccharide polymer to process it from a higher molecular weight to a lower molecular weight polymer. This processing mediates surface adherence of the cells and ultimately governs overall biofilm architecture, indicating enzymatic pruning of the EPS plays a key role in biofilm-mediated attenuation of
X. fastidiosa in planta
and, thus, is a key vestige that links its commensal behaviors to its parasitic behaviors in specific hosts.

IMPORTANCE

It is well established that exopolysaccharide (EPS) is an integral structural component of bacterial biofilms necessary for assembly and maintenance of the three-dimensional architecture of the biofilm. However, the process and role of EPS turnover within a developing biofilm is not fully understood. Here, we demonstrated that
Xylella fastidiosa
uses a self-produced endoglucanase to enzymatically process its own EPS to modulate EPS polymer length. This enzymatic processing of EPS dictates the early stages of
X. fastidiosa
’s biofilm development, which, in turn, affects its behavior
in planta
. A deletion mutant that cannot produce the endoglucanase was hypervirulent, thereby linking enzymatic processing of EPS to attenuation of virulence in symptomatic hosts, which may be a vestige of
X. fastidiosa
’s commensal behavior in many of its other non-symptomatic hosts.
The cystic fibrosis treatment Trikafta affects the growth, viability, and cell wall of
Aspergillus fumigatus
biofilms
Authors: Jones Jane T. , Morelli Kaesi A. , Vesely Elisa M. , Puerner Charles T. S. , Pavuluri Chetan K. , Ross Brandon S. , van Rhijn Norman , Bromley Michael J. , Cramer Robert A.
Published: October 2023

People with cystic fibrosis (pwCF) commonly test positive for the pathogenic fungus
Aspergillus fumigatus
, which is associated with a decline in lung function. Trikafta is a recently approved therapy for pwCF that improves quantity and function of the CFTR protein; however, it is not known how Trikafta affects microbial communities in the lung. Therefore, the aim of this study was to determine whether Trikafta directly affects
A. fumigatus
growth and biology. While Trikafta did not impact the viability of
A. fumigatus
conidia, treatment of
A. fumigatus
biofilms with Trikafta reduced overall biofilm biomass. This finding was associated with increased membrane permeability, decreased viability, and reduced metabolic activity following long-term treatment of biofilms. Trikafta-induced membrane permeability, and biomass reduction was partially blocked with the calcium channel inhibitor verapamil and fully blocked by the mammalian CFTR inhibitor GlyH-101. Trikafta-induced biomass reduction and metabolic activity was shown to be regulated by the high-osmolarity glycerol pathway gene
sakA
. Trikafta treatment also induced resistance to the cell wall stressor calcofluor white, susceptibility to the anti-fungal drug caspofungin, and decreased inflammatory responses from murine bone marrow cells. Collectively, these results reveal that Trikafta affects infection-relevant
A. fumigatus
biology and host-microbial interactions.

IMPORTANCE

PwCF commonly test positive for pathogenic fungi, and more than 90% of the cystic fibrosis patient population is approved for the modulator treatment, Trikafta. Therefore, it is critical to understand how fungal communities, specifically
A. fumigatus
, respond to Trikafta exposure. Therefore, we sought to determine whether Trikafta impacted the biology of
A. fumigatus
biofilms. Our data demonstrate that Trikafta reduces biomass in several laboratory strains as well as clinical strains isolated from the expectorated sputum of pwCF. Furthermore, Trikafta reduces fungal viability and the capacity of biofilms to recover following treatment. Of particular importance, Trikafta affects how
A. fumigatus
biofilms respond to cell wall stressors, suggesting that Trikafta modulates components of the cell wall. Since the cell wall directly affects how a host immune system will respond to and effectively neutralize pathogens, our work, demonstrating that Trikafta impacts the
A. fumigatus
cell wall, is potentially highly relevant to fungal-induced disease pathogenesis.
Epstein-Barr virus infectious particles initiate B cell transformation and modulate cytokine response
Authors: Baccianti Francesco , Masson Charlène , Delecluse Susanne , Li Zhe , Poirey Remy , Delecluse Henri-Jacques
Published: October 2023

The Epstein-Barr virus (EBV) efficiently transforms primary B cells. Here, we show that this process starts immediately after cellular exposure to infectious viral particles. Virus binding to B cells led to the activation of intracytoplasmic tyrosine kinases and STAT3. Tegument proteins within the virion in turn activated the p38-MK2 pathway upon cell entry, independently of the viral DNA. Engagement of STAT3 and p38/MK2, two pro-inflammatory pathways, was essential for expression of the key EBV transforming gene EBNA2 but also facilitated IL-6 and TNFα release. However, these pathways simultaneously activated ZFP36L1, a stress response protein that targets transcripts with an AU-rich 3′UTR, to reduce IL-6 and TNFα transcription in infected cells. Expression of viral latent proteins after infection amplified the viral effects on p38 and MK2, but also on ZFP36L1, altogether resulting in a transitory and limited increase in IL-6 and TNFα transcription and release. Thus, EBV virions are not merely vehicles that allow injection of the viral DNA into the nucleus but manipulate cellular pathways to initiate transformation while limiting cytokine release.

IMPORTANCE

The Epstein-Barr virus efficiently infects and transforms B lymphocytes. During this process, infectious viral particles transport the viral genome to the nucleus of target cells. We show here that these complex viral structures serve additional crucial roles by activating transcription of the transforming genes encoded by the virus. We show that components of the infectious particle sequentially activate proinflammatory B lymphocyte signaling pathways that, in turn, activate viral gene expression but also cause cytokine release. However, virus infection activates expression of ZFP36L1, an RNA-binding stress protein that limits the length and the intensity of the cytokine response. Thus, the infectious particles can activate viral gene expression and initiate cellular transformation at the price of a limited immune response.
Thioflavin-T does not report on electrochemical potential and memory of dormant or germinating bacterial spores
Authors: Li Yong-qing , He Lin , Aryal Makunda , Wicander James , Korza George , Setlow Peter
Published: October 2023

Bacterial spores are metabolically dormant, resistant to microbicides, and vectors of food spoilage and diseases, while germinated spores are easy to kill. Consequently, understanding germination mechanisms may facilitate the development of “germinate-to-eradicate” strategies. Spores germinate in response to many compounds (called germinants). They can also retain the memory of a germinant exposure, such that a second exposure triggers more efficient germination, but how is not clear. A recent high-profile paper [Science (2022) 378:43] suggested that increasing spore electrochemical potential is how memory is “stored” based on measurements of
Bacillus subtilis
spores’ accumulation of the dye thioflavin-T after germinant exposure. Indeed, we found that wild-type spores of three
Bacillus
and one
Clostridioides
species all exhibited this early thioflavin-T accumulation during nutrient pulses. However, our data indicate that inferring spores’ electrochemical potential from thioflavin-T accumulation is problematic. We found that
B. subtilis
spores lacking their proteinaceous coat exhibited memory but did not accumulate thioflavin-T prior to germinant addition or during nutrient pulses. Furthermore, wild-type
Bacillus
spores germinating with dodecylamine, which also elicits memory, showed no thioflavin-T accumulation. Finally, we found that thioflavin-T accumulation by a germinating spore is outside the spore core at early stages but inside the spore core as germination proceeds. These findings suggest that thioflavin-T accumulation during the early stages of germination is due to its binding to one or more protein in the spore coat rather than to changes in spores’ electrochemical potential; thus, thioflavin-T is not a potentiometric dye for the study of spore memory of germinant pulses.

IMPORTANCE

Bacillus
and
Clostridium
spores cause food spoilage and disease because of spores’ dormancy and resistance to microbicides. However, when spores “come back to life” in germination, their resistance properties are lost. Thus, understanding the mechanisms of spore germination could facilitate the development of “germinate to eradicate” strategies. One germination feature is the memory of a pulsed germinant stimulus leading to greater germination following a second pulse. Recent observations of increases in spore binding of the potentiometric dye thioflavin-T early in their germination of spores led to the suggestion that increasing electrochemical potential is how spores “remember” germinant pulses. However, new work finds no increased thioflavin-T binding in the physiological germination of Coatless spores or of intact spores germinating with dodecylamine, even though spore memory is seen in both cases. Thus, using thioflavin-T uptake by germinating spores to assess the involvement of electrochemical potential in memory of germinant exposure, as suggested recently, is questionable.
Genomic, transcriptomic, and phenotypic differences among archetype
Shigella flexneri
strains of serotypes 2a, 3a, and 6
Authors: Gabor Caitlin E. , Hazen Tracy H. , Delaine-Elias BreOnna C. , Rasko David A. , Barry Eileen M.
Published: October 2023

Shigella
is one of the leading causes of diarrheal disease in low-to-middle income countries, where it accounts for significant disease burden in children under the age of five.
Shigella flexneri
, one of four
Shigella
species, is composed of 15 serotypes and caused 65.9% of all shigellosis cases within the Global Enteric Multicenter Study.
S. flexneri
serotypes 2a, 3a, and 6 are the three leading serotypes implicated in clinical disease, yet there are limited studies examining the variation of pathogenesis and virulence between serotypes. Our data revealed through
in silico
and
in vitro
analysis that several serotype-pecific differences exist between archetype strains 2457T (
S. flexneri
serotype 2a [
Sf
2a]), J17B (
S. flexneri
serotype 3a [
Sf
3a), and CCH060 (
S. flexneri
serotype 6 [
Sf6
]). Comparative genomics of these archetype strains demonstrated that CCH060 contains the greatest amount of unique genomic content compared to the
Sf
3a and
Sf
2a archetype strains, as well as lacks several previously identified
Shigella
virulence genes. The pINV virulence plasmid contains a highly conserved core genome irrespective of the archetype reference strain with the greatest strain-specific genomic features. The transcriptional responses of each archetype strain to bile salt stimulus were unique in a strain-dependent manner. Phenotypic analysis revealed that while archetype strains adhere at similar levels to cultured HT-29 intestinal cells, there are differences in the production and secretion of IpaB, IpaC, and IpaD, as well as reduced invasion by CCH060 compared to 2457T and J17B. Our results identify strain-specific features that support further large-scale analysis to identify unique serotype-specific host-pathogen interactions.

IMPORTANCE

Given the genomic diversity between
S. flexneri
serotypes and the paucity of data to support serotype-specific phenotypic differences, we applied
in silico
and
in vitro
functional analyses of archetype strains of 2457T (
Sf
2a), J17B (
Sf
3a), and CH060 (
Sf
6). These archetype strains represent the three leading
S. flexneri
serotypes recommended for inclusion in multivalent vaccines. Characterizing the genomic and phenotypic variation among these clinically prevalent serotypes is an important step toward understanding serotype-specific host-pathogen interactions to optimize the efficacy of multivalent vaccines and therapeutics. This study underpins the importance for further large-scale serotype-targeted analyses.