bluuebunny/crossref_metadata_2025 huggingface dataset:
Examining the effects of response monitoring instructions on endorsements of suicidal ideation in an online study
Authors: Brydon Cassidy , Waitz‐Kudla Sydney N. , Fox Hailey , Smith April , Witte Tracy K.
Published: February 2024

Introduction

It is common practice for researchers to monitor responses to items assessing suicidal ideation and follow‐up with high‐risk participants, when their identities are known. However, it is becoming increasingly common for researchers to administer fully anonymous online surveys that do not allow for follow‐ups with participants at higher risk. The aim of the current study was to investigate whether these two different approaches—monitoring and follow‐up versus no monitoring or follow‐up—affect the willingness of participants to endorse suicidal ideation.

Methods

The sample included N = 555 undergraduate students, who were randomly assigned to the monitoring (n = 275) or anonymous (n = 280) instruction conditions, with the monitoring condition shifting to anonymous, non‐monitored responses at Time 2.

Results

There were no significant differences in self‐reported suicidal ideation between those in the monitoring and anonymous condition at Time 1. At Time 2, no significant interaction was identified between condition and time, suggesting that the change in instructions across timepoints for the monitoring condition had no impact on endorsement of suicidal ideation.

Conclusion

Findings indicate that both monitoring and anonymous instruction methods should elicit the same pattern of endorsements of suicidal ideation.
The novel HLA‐B*51:389 allele, identified by Sanger dideoxy nucleotide sequencing in a Chinese individual
Authors: Lou Xiao , Zhao Chuanhua , An Lin , Yu Changlin , Guo Mei
Published: February 2024

HLA‐B*51:389 differs from HLA‐B*51:02:01:01 by one nucleotide in exon 2.
Molecular relapse monitoring reveals the domination of impaired NK cell education over impaired inhibition in missing KIR‐ligand recognition in patients after unrelated hematopoietic stem cell transplantation for malignant diseases
Authors: Nowak Jacek , Witkowska Agnieszka , Rogatko‐Koroś Marta , Malinowska Agnieszka , Graczyk‐Pol Elżbieta , Nestorowicz‐Kałużna Klaudia , Flaga Anna , Szlendak Urszula , Wnorowska Anna , Gawron Agnieszka
Published: February 2024

Transplantation of HLA and/or KIR mismatched allogeneic hematopoietic stem cells can lead NK cells to different states of activation/inhibition or education/resetting and change anti‐tumor immunosurveillance. In this study, we used molecular relapse monitoring to investigate a correlation between either missing ligand recognition or variation of the cognate iKIR‐HLA pairs with clinical outcomes in patients with hematological malignancies requiring allogeneic hematopoietic stem cell transplantation (allo‐HSCT). Patients (N = 418) with acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), myelodysplastic syndrome (MDS), or lymphoma receiving T‐cell repleted graft from HLA‐matched or partly mismatched unrelated donors between 2012 and 2020 in our center were included in this study. Missing‐ligand recognition was assessed through the presence or absence of recipients' HLA ligand for a particular inhibitory KIR (iKIR) exhibited by the donor. Inhibitory KIR‐HLA pair number variation was defined by loss or gain of a new cognate pair of HLA‐KIR within the new HLA environment of the recipient, compared with the donor's one. Considering the results of our research, we drew the following conclusions: (i) loss of iKIR‐HLA cognate pair for C1, C2, and/or Bw4 groups led to significant deterioration of disease‐free survival (DFS), molecular relapse, overall survival (OS) and non‐relapse mortality (NRM) for patients undergoing allo‐HSCT in the standard phase of the disease. This phenomenon was not observed in patients who underwent transplantation in advanced hematological cancer. (ii) The missing ligand recognition had no impact if the proportion of HLA mismatches was not considered; however, adjustments of HLA mismatch level in the compared groups highlighted the adverse effect of the missing ligand constellation. (iii) The adverse effect of adjusted missing ligand suggests a predominance of lost NK cell education over lost NK cell inhibition in posttransplant recipients' new HLA environment. Our results suggested that donors with the loss of an iKIR‐HLA cognate pair after transplantation should be avoided, and donors who provided an additional iKIR‐HLA cognate pair should be preferred in the allo‐HSCT donor selection process.
Next generation sequencing identifies two novel HLA alleles, HLA‐B*51:395 and HLA‐DQB1*06:478
Authors: Loginova Maria , Druzhinina Svetlana , Paramonov Igor
Published: February 2024

We identified two novel HLA alleles by NGS, HLA‐B*51:395 and HLA‐DQB1*06:478.
The novel HLA‐DRB1*08:127 allele, identified in a deceased organ donor of Scandinavian descent
Authors: Sharma A. , Beaune D. , Fielder D. , Al Agbar S. , Sidahmed A. M. E.
Published: February 2024

HLA‐DRB1*08:127 differs from HLA‐DRB1*08:01:01:01 by a single substitution in exon 4.
Student challenges during third‐year perioperative clerkships through the lens of faculty and residents: A qualitative study
Authors: Wile Rachel K. , Barnes Katherine E. , Charondo Leslie B. , Breyer Kristine , Lager Jeannette , Campbell Andre , O'Sullivan Patricia S.
Published: February 2024

Purpose

Studies of medical students suggest they often find the transition from the pre‐clinical curriculum to clinical rotations particularly challenging during perioperative clerkships. Educators could add a new perspective into students' clerkship experiences and potential interventions to improve them. The purpose of this study was to examine the educator perspective on students' experiences in perioperative clerkships. The findings could inform potential curricular interventions to facilitate student transition from a didactic environment into perioperative clerkships.

Methods

Semi‐structured qualitative interviews were conducted with 16 faculty and residents in the departments of anaesthesia, obstetrics and gynaecology (OBGYN), and general surgery across multiple clinical teaching sites at one institution. Interview questions explored their perceptions of the challenges students face during their transition into perioperative clerkships and probed thoughts on curriculum interventions they believed would be the most beneficial. Interviews were recorded, transcribed and analysed thematically.

Findings

Three themes were identified. Faculty and residents perceive that student experiences on perioperative clerkships are shaped by (1) students' ability to adapt to the specialty and operating room norms on these clerkships, (2) students' understanding of how they can meaningfully contribute to the clinical team, and (3) dedicated teaching time constraints. Interventions were suggested to address educator expectations and student gaps, such as implementing a pre‐clerkship orientation across anaesthesia, general surgery and OBGYN.

Conclusions

To facilitate the medical student transition to perioperative clerkships, interventions should aid students in adapting to clerkship norms for these specialties and clarifying their role and expectations within the care team.
Coupled U–Pb and 40Ar/39Ar chronology of late‐stage intrusions at Elba Island (Italy) supports late Miocene long‐lived magma reservoirs in the Tyrrhenian upper crust
Authors: Mazzarini F. , Bracciali L. , Musumeci G. , Wijbrans J. R. , Kuiper K. , Horstwood M. S. A.
Published: February 2024

The late Miocene Monte Capanne and Porto Azzurro plutons are investigated by means of coupled U–Pb zircon and 40Ar/39Ar white mica dating to test the occurrence of long‐lived magmatic systems in the upper crust. Zircon crystallized for >1 Ma in both plutonic systems, with supersolidus conditions overlapping for ~220 ka indicating previously unrecognized co‐existence of the two reservoirs. The development of the Porto Azzurro high T‐aureole is post‐dated by continuous igneous zircon crystallization until ~6.0 Ma. By linking crystallization to post‐emplacement cooling of late‐stage pulses in both western and eastern Elba we constrain long‐lived sizeable reservoirs (possibly the same reservoir) in the Tyrrhenian upper crust between ~8 and 6 Ma.
WRKY48 negatively regulates plant acclimation to a combination of high light and heat stress
Authors: Balfagón Damián , Pascual Lidia S. , Sengupta Soham , Halliday Karen J. , Gómez‐Cadenas Aurelio , Peláez‐Vico María Ángeles , Sinha Ranjita , Mittler Ron , Zandalinas Sara I.
Published: February 2024

Plants growing under natural conditions experience high light (HL) intensities that are often accompanied by elevated temperatures. These conditions could affect photosynthesis, reduce yield, and negatively impact agricultural productivity. The combination of different abiotic challenges creates a new type of stress for plants by generating complex environmental conditions that often exceed the impact of their individual parts. Transcription factors (TFs) play a key role in integrating the different molecular signals generated by multiple stress conditions, orchestrating the acclimation response of plants to stress. In this study, we show that the TF WRKY48 negatively controls the acclimation of Arabidopsis thaliana plants to a combination of HL and heat stress (HL + HS), and its expression is attenuated by jasmonic acid under HL + HS conditions. Using comparative physiological and transcriptomic analyses between wild‐type and wrky48 mutants, we further demonstrate that under control conditions, WRKY48 represses the expression of a set of transcripts that are specifically required for the acclimation of plants to HL + HS, hence its suppression during the HL + HS stress combination contributes to plant survival under these conditions. Accordingly, mutants that lack WRKY48 are more resistant to HL + HS, and transgenic plants that overexpress WRKY48 are more sensitive to it. Taken together, our findings reveal that WRKY48 is a negative regulator of the transcriptomic response of Arabidopsis to HL + HS and provide new insights into the complex regulatory networks of plant acclimation to stress combination.
Marginal resection and infracyanine green‐mediated photodynamic therapy in the management of feline eyelid squamous cell carcinoma: Two cases
Authors: Sebbag Lionel , Pe'er Oren
Published: February 2024

Objective

Describe the presenting features, surgical procedure, and clinical outcomes of two cats managed with marginal resection and photodynamic therapy (PDT) for eyelid squamous cell carcinoma (SCC).

Animals Studied

A 12‐year‐old female spayed domestic shorthair cat (case 1) and a 10‐year‐old female spayed domestic shorthair cat (case 2).

Procedures

Following marginal resection of the eyelid neoplasm, hemostasis was achieved using a handheld cautery unit then 1 mL of infracyanine green was injected into the surgical wound bed. Photodynamic therapy was performed using an 810 nm diode laser in two consecutive steps: (i) six cycles at 500 mW for 30 s per cycle, using a rapid movement; then (ii) one (case 1) or two cycles (case 2) of 30 s at 2000 mW, using a slow deliberate movement to effect (charred surface).

Results

Histopathology was consistent with SCC resected with incomplete margins in both cats. Follow‐up duration was 416 and 161 days in case 1 and case 2, respectively. Consecutive exams and photo‐documentation (in clinic or by owners) showed appropriate healing of the lower eyelid, with a smooth lid margin, and no evidence of tumor regrowth or ocular irritation. Subtle trichiasis was noted in case 1 on day 185, but not at the last follow‐up.

Conclusions and Clinical Relevance

Marginal resection followed by PDT may be a valid alternative to complete surgical resection of periocular SCC in cats. The procedure was easy to perform, post‐operative recovery was uncomplicated, and neither cat developed recurrent disease during the follow‐up period.
Enlargement of the hepatic artery is present in dogs with a congenital extrahepatic portosystemic shunt and is independent of shunt insertion into the systemic circulation
Authors: Humphreys William J. E. , Sumping Jessica C. , Maddox Thomas W. , Marwood Rachel
Published: February 2024

The accurate diagnosis of portovascular anomalies has been facilitated by improvements in diagnostic imaging technology. In humans, hepatic arterial blood flow changes in response to the reduction in portal blood flow. The hepatic arterial buffer response characterizes an intrinsic regulatory mechanism in response to reduced portal venous blood flow, which results in hepatic arterial enlargement. At the authors’ institution, enlargement of the hepatic artery has been anecdotally observed in a population of dogs with extrahepatic portosystemic shunting, consistent with previous literature that documents variability in hepatic arterial size. In this retrospective, blinded, analytical study, a hepatic artery:aorta (Ha:Ao) ratio was assessed on CT studies from 112 dogs, with (n = 43) and without (n = 69) an extrahepatic congenital portosystemic shunt in order to compare the hepatic artery size independent of body weight between the two populations. A significant increase in the Ha:Ao ratio was documented in dogs with an extrahepatic portosystemic shunt (EHPSS) compared with those dogs with no EHPSS independent of the location of shunt insertion into the systemic circulation (P < .001). Three cases had repeat CT after surgery, and all had Ha:Ao ratio reductions following treatment. The authors propose that this may be an additional imaging feature observed in dogs with an EHPSS.
The rainstorm influence on ecological restoration: A novel response ecological model for an urban shallow lake
Authors: Yan Zhiqiang , Wu Luming , Lu Fenghui , Huang Jingwen , Long Qingwu
Published: February 2024

Environmental processes in cities and suburbs are significantly impacted by climate change. The development of reliable ecological models may successfully direct agricultural activities. Numerous models have been put forth as of late; however, because to the complexity of environmental microorganisms, their use in complex systems is still restricted. For a better understanding of the ecological restoration of an urban lake system that had been disturbed by rainfall, an improved ecological dynamic model that took into account inundation plants, phytoplankton and microorganisms was proposed based on the field survey. Observed data from a shallow urban lake with a surface area of approximately 66 600 m2 in the heart of Shunde district, Foshan, in South China, was used to validate the model. In this model, five hypotheses—phytoplankton, microorganisms, NH3‐N, COD and TP in water—were selected as experimental variables. To assess the model's correctness and dependability, the correlation coefficients (R) and root mean square error‐observations standard deviation ratio (RSR) were computed. The results from the establish model (0.446 < R < 0.985, RSR < 0.7) are very similar to those of actual observations. In addition, four microbe species (Aquabacium, Bradyrhizobium japonicum, Curvibacter and Cyanobacteria) multiplied when pollutant concentration dropped. Our model provides a useful tool for managing urban shallow water lakes by properly simulating the dynamic changes of aquatic species and microbes in urban shallow water lakes.
Species delimitation and phylogenetic relationships of the Prionospio complex (Annelida, Spionidae) in the Northeast Atlantic
Authors: Hektoen Martin M. , Bakken Torkild , Ekrem Torbjørn , Radashevsky Vasily I. , Dunshea Glenn
Published: February 2024

The Prionospio complex comprises the most diverse and complex group within the polychaete family Spionidae. The phylogenetic relationships within the group are still poorly understood, and the generic breakdown is unstable. In this study, we assessed the diversity, relationships, and distribution of species of the Prionospio complex occurring in Norwegian waters. We analysed mitochondrial genomes and nuclear ribosomal DNA assembled via whole‐genome shotgun sequencing, and Sanger sequenced fragments of COI and 16S rDNA. Sanger sequencing proved challenging in the group, where COI was only amplified successfully in 14% of specimens. By molecular species delimitation algorithms, our study revealed the presence of four well‐supported but currently undescribed species of Prionospio in Norwegian waters. We observed a novel distribution pattern of polychaetes in coastal waters, where certain species demonstrated distribution ranges spanning over 7000 km. Such wide distribution parallels patterns of deep‐sea Prionospio species, suggesting that factors beyond recent anthropogenic translocations are involved. Our analysis of 38 mitochondrial genomes and ribosomal nuclear DNA enabled us to hypothesise on the phylogenetic relationships of 14 species of the Prionospio complex. The analysis suggested that two characters previously used to designate genera: the beginning of the branchiae from chaetiger 3 and the presence of pinnules on the branchiae, might have evolved more than one time within the complex. We return Aurospio banyulensis to the genus Prionospio according to the diagnosis of Aurospio resulting tree where this species was nested among Prionospio species. Our findings provide new insights into the diversity and distribution patterns of Prionospio species and contribute to a better understanding of marine benthic biodiversity and the importance of taxonomic accuracy in conservation and management practices.
On the geometry of rod packings in the 3‐torus
Authors: Hui Connie On Yu , Purcell Jessica S.
Published: February 2024

Rod packings in the 3‐torus encode information of some crystal structures in crystallography. They can be viewed as links in the 3‐torus, and tools from 3‐manifold geometry and topology can be used to study their complements. In this paper, we initiate the use of geometrisation to study such packings. We analyse the geometric structures of the complements of simple rod packings, and find families that are hyperbolic and Seifert fibred.
A Comparative Study of Machine Learning and Algorithmic Approaches to Automatically Identify the Yield Point in Normal and Aneurysmal Human Aortic Tissues
Authors: Chung Timothy K. , Kim Joseph , Gueldner Pete H. , Vorp David A. , Raghavan M. L.
Published: February 2024

The stress–strain curve of biological soft tissues helps characterize their mechanical behavior. The yield point on this curve is when a specimen breaches its elastic range due to irreversible microstructural damage. The yield point is easily found using the offset yield method in traditional engineering materials. However, correctly identifying the yield point in soft tissues can be subjective due to its nonlinear material behavior. The typical method for yield point identification is visual inspection, which is investigator-dependent and does not lend itself to automation of the analysis pipeline. An automated algorithm to identify the yield point objectively assesses soft tissues' biomechanical properties. This study aimed to analyze data from uniaxial extension testing on biological soft tissue specimens and create a machine learning (ML) model to determine a tissue sample's yield point. We present a trained machine learning model from 279 uniaxial extension curves from testing aneurysmal/nonaneurysmal and longitudinal/circumferential oriented tissue specimens that multiple experts labeled through an adjudication process. The ML model showed a median error of 5% in its estimated yield stress compared to the expert picks. The study found that an ML model could accurately identify the yield point (as defined) in various aortic tissues. Future studies will be performed to validate this approach by visually inspecting when damage occurs and adjusting the model using the ML-based approach.
A Case Study of a Senior High-Performance Leader Managing Psychological Effects During the COVID-19 Pandemic
Authors: Friesen Andrew P. , Patrick Tom
Published: February 2024

The potential scope for qualified sport psychology service providers is broadening. Recent empirical research and associated commentaries have highlighted the opportunity for applied sport psychology professionals to apply concepts and experiences toward having a positive effect on athletes’ training environment. This case study details a high-performance leader within an Australian national training institute who is also a trained sport psychology practitioner, attempting to optimize psychological effects in the time leading up to the Tokyo 2020 (i.e., 2021) Olympic Games. The objective was to meet or exceed medal targets though the provision of a strong, team-led approach where activity to achieve high-performance outcomes was prioritized but in a manner that leveraged strong professional relationships and through the prioritization of everyone’s mental and emotional health whenever practicably achievable.
AMPK phosphorylation of FNIP1 (S220) controls mitochondrial function and muscle fuel utilization during exercise
Authors: Xiao Liwei , Yin Yujing , Sun Zongchao , Liu Jing , Jia Yuhuan , Yang Likun , Mao Yan , Peng Shujun , Xie Zhifu , Fang Lei , Li Jingya , Xie Xiaoduo , Gan Zhenji
Published: February 2024

Exercise-induced activation of adenosine monophosphate–activated protein kinase (AMPK) and substrate phosphorylation modulate the metabolic capacity of mitochondria in skeletal muscle. However, the key effector(s) of AMPK and the regulatory mechanisms remain unclear. Here, we showed that AMPK phosphorylation of the folliculin interacting protein 1 (FNIP1) serine-220 (S220) controls mitochondrial function and muscle fuel utilization during exercise. Loss of FNIP1 in skeletal muscle resulted in increased mitochondrial content and augmented metabolic capacity, leading to enhanced exercise endurance in mice. Using skeletal muscle–specific nonphosphorylatable FNIP1 (S220A) and phosphomimic (S220D) transgenic mouse models as well as biochemical analysis in primary skeletal muscle cells, we demonstrated that exercise-induced FNIP1 (S220) phosphorylation by AMPK in muscle regulates mitochondrial electron transfer chain complex assembly, fuel utilization, and exercise performance without affecting mechanistic target of rapamycin complex 1–transcription factor EB signaling. Therefore, FNIP1 is a multifunctional AMPK effector for mitochondrial adaptation to exercise, implicating a mechanism for exercise tolerance in health and disease.
Broadband and large-aperture metasurface edge encoders for incoherent infrared radiation
Authors: Swartz Brandon T. , Zheng Hanyu , Forcherio Gregory T. , Valentine Jason
Published: February 2024

The prevalence of computer vision systems necessitates hardware-based approaches to relieve the high computational demand of deep neural networks in resource-limited applications. One solution would be to off-load low-level image feature extraction, such as edge detection, from the digital network to the analog imaging system. To that end, this work demonstrates incoherent, broadband, low-noise optical edge detection of real-world scenes by combining the wavefront shaping of a 24-mm aperture metasurface with a refractive lens. An inverse design approach is used to optimize the metasurface for Laplacian-based edge detection across the 7.5- to 13.5-μm LWIR imaging band, allowing for facile integration with uncooled microbolometer-based LWIR imagers to encode edge information. A polarization multiplexed approach leveraging a birefringent metasurface is also demonstrated as a single-aperture implementation. This work could be applied to improve computer vision capabilities of resource-constrained systems by leveraging optical preprocessing to alleviate the computational requirements for high-accuracy image segmentation and classification.
MutSβ-MutLβ-FANCJ axis mediates the restart of DNA replication after fork stalling at cotranscriptional G4/R-loops
Authors: Isik Esin , Shukla Kaustubh , Pospisilova Michaela , König Christiane , Andrs Martin , Rao Satyajeet , Rosano Vinicio , Dobrovolna Jana , Krejci Lumir , Janscak Pavel
Published: February 2024

Transcription-replication conflicts (TRCs) induce formation of cotranscriptional RNA:DNA hybrids (R-loops) stabilized by G-quadruplexes (G4s) on the displaced DNA strand, which can cause fork stalling. Although it is known that these stalled forks can resume DNA synthesis in a process initiated by MUS81 endonuclease, how TRC-associated G4/R-loops are removed to allow fork passage remains unclear. Here, we identify the mismatch repair protein MutSβ, an MLH1-PMS1 heterodimer termed MutLβ, and the G4-resolving helicase FANCJ as factors that are required for MUS81-initiated restart of DNA replication at TRC sites in human cells. This DNA repair process depends on the G4-binding activity of MutSβ, the helicase activity of FANCJ, and the binding of FANCJ to MLH1. Furthermore, we show that MutSβ, MutLβ, and MLH1-FANCJ interaction mediate FANCJ recruitment to G4s. These data suggest that MutSβ, MutLβ, and FANCJ act in conjunction to eliminate G4/R-loops at TRC sites, allowing replication restart.
ForceGen: End-to-end de novo protein generation based on nonlinear mechanical unfolding responses using a language diffusion model
Authors: Ni Bo , Kaplan David L. , Buehler Markus J.
Published: February 2024

Through evolution, nature has presented a set of remarkable protein materials, including elastins, silks, keratins and collagens with superior mechanical performances that play crucial roles in mechanobiology. However, going beyond natural designs to discover proteins that meet specified mechanical properties remains challenging. Here, we report a generative model that predicts protein designs to meet complex nonlinear mechanical property-design objectives. Our model leverages deep knowledge on protein sequences from a pretrained protein language model and maps mechanical unfolding responses to create proteins. Via full-atom molecular simulations for direct validation, we demonstrate that the designed proteins are de novo, and fulfill the targeted mechanical properties, including unfolding energy and mechanical strength, as well as the detailed unfolding force-separation curves. Our model offers rapid pathways to explore the enormous mechanobiological protein sequence space unconstrained by biological synthesis, using mechanical features as the target to enable the discovery of protein materials with superior mechanical properties.
DRAK2 suppresses autophagy by phosphorylating ULK1 at Ser
56
to diminish pancreatic β cell function upon overnutrition
Authors: Lu Yuting , Xu Junyu , Li Yufeng , Wang Ruoran , Dai Chengqiu , Zhang Bingqian , Zhang Xinwen , Xu Lei , Tao Yunhua , Han Ming , Guo Ren , Wu Qingqian , Wu Linshi , Meng Zhuoxian , Tan Minjia , Li Jingya
Published: February 2024

Impeded autophagy can impair pancreatic β cell function by causing apoptosis, of which DAP-related apoptosis-inducing kinase-2 (DRAK2) is a critical regulator. Here, we identified a marked up-regulation of DRAK2 in pancreatic tissue across humans, macaques, and mice with type 2 diabetes (T2D). Further studies in mice showed that conditional knockout (cKO) of DRAK2 in pancreatic β cells protected β cell function against high-fat diet feeding along with sustained autophagy and mitochondrial function. Phosphoproteome analysis in isolated mouse primary islets revealed that DRAK2 directly phosphorylated unc-51–like autophagy activating kinase 1 (ULK1) at Ser
56
, which was subsequently found to induce ULK1 ubiquitylation and suppress autophagy. ULK1-S56A mutation or pharmacological inhibition of DRAK2 preserved mitochondrial function and insulin secretion against lipotoxicity in mouse primary islets, Min6 cells, or INS-1E cells. In conclusion, these findings together indicate an indispensable role of the DRAK2-ULK1 axis in pancreatic β cells upon metabolic challenge, which offers a potential target to protect β cell function in T2D.
BECC438b TLR4 agonist supports unique immune response profiles from nasal and muscular DTaP pertussis vaccines in murine challenge models
Authors: DeJong Megan A. , Wolf M. Allison , Bitzer Graham J. , Hall Jesse M. , Fitzgerald Nicholas A. , Pyles Gage M. , Huckaby Annalisa B. , Petty Jonathan E. , Lee Katherine , Barbier Mariette , Bevere Justin R. , Ernst Robert K. , Damron F. Heath
Published: February 2024

The protection afforded by acellular pertussis vaccines wanes over time, and there is a need to develop improved vaccine formulations. Options to improve the vaccines involve the utilization of different adjuvants and administration via different routes. While intramuscular (IM) vaccination provides a robust systemic immune response, intranasal (IN) vaccination theoretically induces a localized immune response within the nasal cavity. In the case of a
Bordetella pertussis
infection, IN vaccination results in an immune response that is similar to natural infection, which provides the longest duration of protection. Current acellular formulations utilize an alum adjuvant, and antibody levels wane over time. To overcome the current limitations with the acellular vaccine, we incorporated a novel TLR4 agonist, BECC438b, into both IM and IN acellular formulations to determine its ability to protect against infection in a murine airway challenge model. Following immunization and challenge, we observed that DTaP + BECC438b reduced bacterial burden within the lung and trachea for both administration routes when compared with mock-vaccinated and challenged (MVC) mice. Interestingly, IN administration of DTaP + BECC438b induced a Th1-polarized immune response, while IM vaccination polarized toward a Th2 immune response. RNA sequencing analysis of the lung demonstrated that DTaP + BECC438b activates biological pathways similar to natural infection. Additionally, IN administration of DTaP + BECC438b activated the expression of genes involved in a multitude of pathways associated with the immune system. Overall, these data suggest that BECC438b adjuvant and the IN vaccination route can impact efficacy and responses of pertussis vaccines in pre-clinical mouse models.
The
Bacillus subtilis
cell envelope stress-inducible
ytpAB
operon modulates membrane properties and contributes to bacitracin resistance
Authors: Willdigg Jessica R. , Patel Yesha , Arquilevich Briana E. , Subramanian Chitra , Frank Matthew W. , Rock Charles O. , Helmann John D.
Published: February 2024

Antibiotics that inhibit peptidoglycan synthesis trigger the activation of both specific and general protective responses. σ
M
responds to diverse antibiotics that inhibit cell wall synthesis. Here, we demonstrate that cell wall-inhibiting drugs, such as bacitracin and cefuroxime, induce the σ
M
-dependent
ytpAB
operon. YtpA is a predicted hydrolase previously proposed to generate the putative lysophospholipid antibiotic bacilysocin (lysophosphatidylglycerol), and YtpB is the branchpoint enzyme for the synthesis of membrane-localized C
35
terpenoids. Using targeted lipidomics, we reveal that YtpA is not required for the production of lysophosphatidylglycerol. Nevertheless,
ytpA
was critical for growth in a mutant strain defective for homeoviscous adaptation due to a lack of genes for the synthesis of branched chain fatty acids and the Des phospholipid desaturase. Consistently, overexpression of
ytpA
increased membrane fluidity as monitored by fluorescence anisotropy. The
ytpA
gene contributes to bacitracin resistance in mutants additionally lacking the
bceAB
or
bcrC
genes, which directly mediate bacitracin resistance. These epistatic interactions support a model in which σ
M
-dependent induction of the
ytpAB
operon helps cells tolerate bacitracin stress, either by facilitating the flipping of the undecaprenyl phosphate carrier lipid or by impacting the assembly or function of membrane-associated complexes involved in cell wall homeostasis.

IMPORTANCE

Peptidoglycan synthesis inhibitors include some of our most important antibiotics. In
Bacillus subtilis
, peptidoglycan synthesis inhibitors induce the σ
M
regulon, which is critical for intrinsic antibiotic resistance. The σ
M
-dependent
ytpAB
operon encodes a predicted hydrolase (YtpA) and the enzyme that initiates the synthesis of C
35
terpenoids (YtpB). Our results suggest that YtpA is critical in cells defective in homeoviscous adaptation. Furthermore, we find that YtpA functions cooperatively with the BceAB and BcrC proteins in conferring intrinsic resistance to bacitracin, a peptide antibiotic that binds tightly to the undecaprenyl-pyrophosphate lipid carrier that sustains peptidoglycan synthesis.
Oxidative stress changes the effectiveness of artemisinin in
Plasmodium falciparum
Authors: Pires Camilla Valente , Cassandra Debora , Xu Shulin , Laleu Benoit , Burrows Jeremy N. , Adams John H.
Published: February 2024

Malaria parasites have adaptive mechanisms to modulate their intracellular redox status to tolerate the enhanced oxidizing effects created by malaria fever, hemoglobinopathies and other stress conditions, including antimalaria drugs. Emerging artemisinin (ART) resistance in
Plasmodium falciparum
is a complex phenotype linked to the parasite’s tolerance of the activated drug’s oxidative damage along with changes in vesicular transport, lipid metabolism, DNA repair, and exported proteins. In an earlier study, we discovered that many of these metabolic processes are induced in
P. falciparum
to respond to the oxidative damage caused by artemisinin, which exhibited a highly significant overlap with the parasite’s adaptive response mechanisms to survive febrile temperatures. In addition, there was a significant overlap with the parasite’s survival responses to oxidative stress. In this study, we investigated these relationships further using an
in vitro
model to evaluate if oxidative stress and heat-shock conditions could alter the parasite’s response to artemisinin. The results revealed that compared to ideal culture conditions, the antimalarial efficacy of artemisinin was significantly reduced in parasites growing in intraerythrocytic oxidative stress but not in heat-shock condition. In contrast, heat shock significantly reduced the efficacy of lumefantrine that is an important ART combination therapy partner drug. We propose that prolonged exposure to intraerythrocytic microenvironmental oxidative stress, as would occur in endemic regions with high prevalence for sickle trait and other hemoglobinopathies, can predispose malaria parasites to develop tolerance to the oxidative damage caused by antimalarial drugs like artemisinin.

IMPORTANCE

Emerging resistance to the frontline antimalarial drug artemisinin represents a significant threat to worldwide malaria control and elimination. The patterns of parasite changes associated with emerging resistance represent a complex array of metabolic processes evident in various genetic mutations and altered transcription profiles. Genetic factors identified in regulating
P. falciparum
sensitivity to artemisinin overlap with the parasite’s responses to malarial fever, sickle trait, and other types of oxidative stresses, suggesting conserved inducible survival responses. In this study we show that intraerythrocytic stress conditions, oxidative stress and heat shock, can significantly decrease the sensitivity of the parasite to artemisinin and lumefantrine, respectively. These results indicate that an intraerythrocytic oxidative stress microenvironment and heat-shock condition can alter antimalarial drug efficacy. Evaluating efficacy of antimalarial drugs under ideal
in vitro
culture conditions may not accurately predict drug efficacy in all malaria patients.
Structural basis of DNA recognition of the
Campylobacter jejuni
CosR regulator
Authors: Zhang Zhemin , Yan Yuqi , Pang Jinji , Dai Lei , Zhang Qijing , Yu Edward W.
Published: February 2024

Campylobacter jejuni
is a foodborne pathogen commonly found in the intestinal tracts of animals. This pathogen is a leading cause of gastroenteritis in humans. Besides its highly infectious nature,
C. jejuni
is increasingly resistant to a number of clinically administrated antibiotics. As a consequence, the Centers for Disease Control and Prevention has designated antibiotic-resistant
Campylobacter
as a serious antibiotic resistance threat in the United States. The
C. jejuni
CosR regulator is essential to the viability of this bacterium and is responsible for regulating the expression of a number of oxidative stress defense enzymes. Importantly, it also modulates the expression of the CmeABC multidrug efflux system, the most predominant and clinically important system in
C. jejuni
that mediates resistance to multiple antimicrobials. Here, we report structures of apo-CosR and CosR bound with a 21 bp DNA sequence located at the
cmeABC
promotor region using both single-particle cryo-electron microscopy and X-ray crystallography. These structures allow us to propose a novel mechanism for CosR regulation that involves a long-distance conformational coupling and rearrangement of the secondary structural elements of the regulator to bind target DNA.

IMPORTANCE

Campylobacter jejuni
has emerged as an antibiotic-resistant threat worldwide. CosR is an essential regulator for this bacterium and is important for
Campylobacter
adaptation to various stresses. Here, we describe the structural basis of CosR binding to target DNA as determined by cryo-electron microscopy and X-ray crystallography. Since CosR is a potential target for intervention, our studies may facilitate the development of novel therapeutics to combat
C. jejuni
infection.
Machine learning analysis of RB-TnSeq fitness data predicts functional gene modules in
Pseudomonas putida
KT2440
Authors: Borchert Andrew J. , Bleem Alissa C. , Lim Hyun Gyu , Rychel Kevin , Dooley Keven D. , Kellermyer Zoe A. , Hodges Tracy L. , Palsson Bernhard O. , Beckham Gregg T.
Published: February 2024

There is growing interest in engineering
Pseudomonas putida
KT2440 as a microbial chassis for the conversion of renewable and waste-based feedstocks, and metabolic engineering of
P. putida
relies on the understanding of the functional relationships between genes. In this work, independent component analysis (ICA) was applied to a compendium of existing fitness data from randomly barcoded transposon insertion sequencing (RB-TnSeq) of
P. putida
KT2440 grown in 179 unique experimental conditions. ICA identified 84 independent groups of genes, which we call fModules (“functional modules”), where gene members displayed shared functional influence in a specific cellular process. This machine learning-based approach both successfully recapitulated previously characterized functional relationships and established hitherto unknown associations between genes. Selected gene members from fModules for hydroxycinnamate metabolism and stress resistance, acetyl coenzyme A assimilation, and nitrogen metabolism were validated with engineered mutants of
P. putida
. Additionally, functional gene clusters from ICA of RB-TnSeq data sets were compared with regulatory gene clusters from prior ICA of RNAseq data sets to draw connections between gene regulation and function. Because ICA profiles the functional role of several distinct gene networks simultaneously, it can reduce the time required to annotate gene function relative to manual curation of RB-TnSeq data sets.

IMPORTANCE

This study demonstrates a rapid, automated approach for elucidating functional modules within complex genetic networks. While
Pseudomonas putida
randomly barcoded transposon insertion sequencing data were used as a proof of concept, this approach is applicable to any organism with existing functional genomics data sets and may serve as a useful tool for many valuable applications, such as guiding metabolic engineering efforts in other microbes or understanding functional relationships between virulence-associated genes in pathogenic microbes. Furthermore, this work demonstrates that comparison of data obtained from independent component analysis of transcriptomics and gene fitness datasets can elucidate regulatory-functional relationships between genes, which may have utility in a variety of applications, such as metabolic modeling, strain engineering, or identification of antimicrobial drug targets.
A novel mechanism for dissimilatory nitrate reduction to ammonium in
Acididesulfobacillus acetoxydans
Authors: Egas Reinier A. , Kurth Julia M. , Boeren Sjef , Sousa Diana Z. , Welte Cornelia U. , Sánchez-Andrea Irene
Published: February 2024

The biological route of nitrate reduction has important implications for the bioavailability of nitrogen within ecosystems. Nitrate reduction via nitrite, either to ammonium (ammonification) or to nitrous oxide or dinitrogen (denitrification), determines whether nitrogen is retained within the system or lost as a gas. The acidophilic sulfate-reducing bacterium (aSRB)
Acididesulfobacillus acetoxydans
can perform dissimilatory nitrate reduction to ammonium (DNRA). While encoding a Nar-type nitrate reductase,
A. acetoxydans
lacks recognized nitrite reductase genes. In this study,
A. acetoxydans
was cultivated under conditions conducive to DNRA. During cultivations, we monitored the production of potential nitrogen intermediates (nitrate, nitrite, nitric oxide, hydroxylamine, and ammonium). Resting cell experiments were performed with nitrate, nitrite, and hydroxylamine to confirm their reduction to ammonium, and formed intermediates were tracked. To identify the enzymes involved in DNRA, comparative transcriptomics and proteomics were performed with
A. acetoxydans
growing under nitrate- and sulfate-reducing conditions. Nitrite is likely reduced to ammonia by the previously undescribed nitrite reductase activity of the NADH-linked sulfite reductase AsrABC, or by a putatively ferredoxin-dependent homolog of the nitrite reductase NirA (DEACI_1836), or both. We identified enzymes and intermediates not previously associated with DNRA and nitrosative stress in aSRB. This increases our knowledge about the metabolism of this type of bacteria and helps the interpretation of (meta)genome data from various ecosystems on their DNRA potential and the nitrogen cycle.

IMPORTANCE

Nitrogen is crucial to any ecosystem, and its bioavailability depends on microbial nitrogen-transforming reactions. Over the recent years, various new nitrogen-transforming reactions and pathways have been identified, expanding our view on the nitrogen cycle and metabolic versatility. In this study, we elucidate a novel mechanism employed by
Acididesulfobacillus acetoxydans
, an acidophilic sulfate-reducing bacterium, to reduce nitrate to ammonium. This finding underscores the diverse physiological nature of dissimilatory reduction to ammonium (DNRA).
A. acetoxydans
was isolated from acid mine drainage, an extremely acidic environment where nitrogen metabolism is poorly studied. Our findings will contribute to understanding DNRA potential and variations in extremely acidic environments.
Multi-omics analysis reveals the associations between altered gut microbiota, metabolites, and cytokines during pregnancy
Authors: Huang Ting , Liang Xinyuan , Bao Han , Ma Guangyu , Tang Xiaomei , Luo Huijuan , Xiao Xiaomin
Published: February 2024

For embryo implantation and fetal development, the maternal immune system undergoes dramatic changes. The mechanisms involved in inducing alterations of maternal immunity have not been fully clarified. Gut microbiome and metabolites were thought to influence the host immune response. During normal pregnancy, notable changes occur in the gut microbiota and metabolites. However, the relationship of these alterations to immune function during pregnancy remains unclear. In this study, we examined gut microbiota, fecal metabolites, plasma metabolites, and cytokines in pregnant women and non-pregnant women. Our findings revealed that, in comparison to non-pregnant women, pregnant women exhibit a significant increase in the relative abundance of
Actinobacteriota
and notable differences in metabolic pathways related to bile acid secretion. Furthermore, there was a marked reduction in pro-inflammatory cytokines levels in pregnant women. Correlation analyses indicated that these alterations in cytokines may be linked to specific gut bacteria and metabolites. Bacteria within the same microbial modules exhibited consistent effects on cytokines, suggesting that gut bacteria may function as functional groups. Mediation analysis further identified that certain bacteria might influence cytokines through metabolites, such as bile acids and arachidonic acid. Our findings propose potential biological connections between bacteria, metabolites, and immunity, which require further validation in future studies.

IMPORTANCE

A great number of studies have focused on diseases induced by intestinal microecological disorders and immune imbalances. However, the understanding of how intestinal microbiota interacts with immunity during normal pregnancy, which is fundamental to studying pathological pregnancies related to intestinal microbiota disturbances, has not been well elucidated. Our study employed multi-omics analysis to discover that changes in gut microbiota and metabolites during pregnancy can impact immune function. In addition, we identified several metabolites that may mediate the effect of gut microbes on plasma cytokines. Our study offered new insights into our understanding of the connections between the gut microbiome, metabolome, and the immune system during pregnancy.
Inter-laboratory ring trial to compare four quantitative polymerase chain reaction assays employed for detection of
Mycobacterium avium
subspecies
paratuberculosis
Authors: Worsley L. , Davies P. L.
Published: February 2024

Johne’s disease is an infectious enteric disease caused by
Mycobacterium avium
subspecies
paratuberculosi
s (MAP) affecting ruminant species worldwide. In Project 1, an independent performance comparison ring trail was conducted between three different commercial MAP quantitative polymerase chain reaction (qPCR) assay services (B, C, and D) currently marketed in Great Britain by three separate laboratories against each other and against a fourth assay (A) not available commercially in Great Britain. A total of 205 individual ovine and bovine samples from five farms were analyzed to give 41 sets of pooled results (pool size five) from each laboratory according to their specific protocols. The numbers of positive pools for assays A–D were 18, 12, 11, and 1 (43.9%, 29.2%, 26.8%, and 2.4%), respectively. Assessment of interrater reliability produced a Fleiss’ kappa coefficient of 0.15, indicating very poor overall agreement between the four laboratories. Laboratories A–D diagnosed 4, 3, 2, and 1 flocks at the farm level, respectively, as MAP positive. In Project 2, 38 pooled ovine samples from 10 flocks were analyzed to compare the performance of laboratories A and B. The numbers of positive results for laboratories A and B were 24 (63.1%) and 17 (44.7%), respectively (Cohen’s kappa 0.54), indicating that laboratory A was more sensitive than B in line with results from Project 1. Variation between laboratories offering MAP qPCR assays is a significant concern, and further work is warranted to validate and standardize the performance of assays between laboratories for both ovine and bovine samples.

IMPORTANCE

Our study reports the findings of an inter-laboratory ring trial comparing the performance of four different quantitative polymerase chain reaction (qPCR) assay services for detecting
Mycobacterium avium
subspecies
paratuberculosis
(MAP) infection in cattle and sheep. MAP is the causative agent of Johne’s disease (also known as paratuberculosis), a significant production-limiting disease in livestock populations with a worldwide distribution. The content of this paper is significant and novel as it is the first to highlight the marked variation between the diagnostic sensitivity and reproducibility of the three principal commercial laboratories offering MAP qPCR diagnostic and screening services in Great Britain. The low sensitivity and high variability between the laboratories are of great concern and relevance to veterinary practitioners and livestock producers.