bluuebunny/crossref_metadata_2025 huggingface dataset:
Perceived Control and Inflammation: Mediating and Moderating Effects in the Relationship Between Cumulative Trauma and Depression
Authors: Magin Zachary E. , Park Crystal L. , Burke Jeffrey D. , Infurna Frank J.
Published: January 2024

Objective

The effects of trauma exposure on depression risk and severity are well established, but psychosocial and biological factors that impact or explain those relationships remain poorly understood. This study examined the moderating and mediating effects of perceived control and inflammation in the relationship between trauma and depression.

Methods

Moderation analyses and longitudinal mediation analyses were conducted on data from 945 adults who completed all three waves (spanning around 19 years) of the Midlife Development in the United States (MIDUS) study and the MIDUS Biomarker Study. Data were collected during a phone interview, self-report surveys distributed in the mail, and an in-person blood draw. Two dimensions of perceived control—mastery and constraints—were examined separately in all analyses.

Results

Perceived control did not significantly moderate the relationship between trauma and depression severity at MIDUS 2 (b = 0.03, SE = .02, p = .091). Constraints significantly mediated the relationship between trauma and MIDUS 3 depression (indirect effect = 0.03, SE = 0.01, p = .016) but not after accounting for MIDUS 2 depression. Perceived control did not have a significant moderating effect in the relationships between trauma and inflammation or inflammation and depression.

Conclusions

Findings from this study revealed that perceived control may be better characterized as an explanatory factor rather than a buffer in trauma-associated depression. Perceived constraints in particular may be a useful treatment target for trauma-associated depression. Further research is needed to examine whether these results generalize to populations other than among mostly non-Hispanic White adults in the United States.
How the COVID-19 pandemic and the health system's response reduced HIV testing and increased late diagnoses in Mexico
Authors: Macías-González Fernando , Vermandere Heleen , Piñeirúa-Menendez Alicia , Bautista-Arredondo Sergio
Published: January 2024

Objectives:

This study aims to evaluate the disruption in HIV screening and diagnoses due to the coronavirus disease 2019 (COVID-19) pandemic and to investigate the pandemic's subsequent influence on the HIV epidemic.

Design:

A retrospective examination of testing and confirmed diagnoses time series was undertaken from 2011 to 2022. The analysis encompassed testing, positive tests, positivity rates, and diagnosis outcomes, including new HIV diagnoses, asymptomatic HIV diagnoses, and symptomatic HIV diagnoses.

Methods:

We used Autoregressive Integrated Moving Average (ARIMA) models to estimate the COVID-19 epidemic's impact on screening and diagnosis outcomes. We gauged the pandemic's effect between January 2020 and December 2022 by comparing modeled predicted results with actual outcomes.

Results:

The advent of COVID-19 prompted a reduction of 50.7% in HIV testing, followed by a monthly escalation in testing afterward, estimated at 30.2 and 65.1% for 2021 and 2022, respectively. Although new diagnoses reported between 2020 and 2022 gradually increased to prepandemic levels, we estimate a gap of 13 207 new diagnoses, with symptomatic detections increasing more than proportionally in 2021 and 2022.

Conclusion:

Our results suggest that the COVID-19 pandemic resulted in missed HIV diagnoses and a rise in late HIV diagnoses. Implementing tailored post-COVID-19 strategies to accelerate timely HIV testing and prevention is needed to avert additional burdens and remain on track toward achieving the 2030 HIV management goals.
Muscle Pressure Correlates With Pain Levels in Fibromyalgia Patients
Authors: Katz Robert S. , Adler Seth , Small Ben J. , Cherny Katya , Small Alexandra Katz
Published: January 2024

Background/Objective

We evaluated patients with fibromyalgia syndrome (FMS) to determine whether there is a correlation between pain scores based on a 0- to 10-point visual analog scale (VAS) and muscle pressure.

Methods

One hundred forty-two patients who satisfied the American College of Rheumatology classification criteria for FMS and 38 non-FMS controls comprised the study groups. Muscle pressure was measured in mm Hg using a pressure gauge attached to a no. 22 needle inserted into the midportion of the trapezius muscle. The muscle pressure was then correlated with the VAS pain score of 0 to 10, some with an increment of 0.5. A second muscle pressure was obtained from 19 patients at a subsequent visit, which was compared with their pain scores.

Results

The mean (SD) pain score for 142 patients with FMS was 6.6 (SD, 1.84) on a 0- to 10-point VAS. The mean pain score in the non-FMS subjects was 0.7 (SD, 1.26). The mean muscle pressure in the FMS group was 32.9 (SD, 6.57) mm Hg. The mean muscle pressure in the non-FMS subjects was 10.6 (SD, 3.85) mm Hg. The calculated Pearson correlation coefficient for muscle pressure versus pain score was 0.8312 (p < 0.0001). This indicates a highly significant association between subjects' muscle pressure and pain scores. For the repeat muscle pressures, the change in muscle pressure was correlated with the change in pain score, and the resulting Pearson correlation coefficient was 0.9255 (p < 0.0001). These results again indicate a highly significant association between subjects' muscle pressure and pain scores.

Conclusion

The results show that increased muscle pressure may be a significant cause of pain in FMS, and the etiology of the pain may have a large peripheral component in addition to a centralized origin of the pain.
A New Iterative Metal Artifact Reduction Algorithm for Both Energy-Integrating and Photon-Counting CT Systems
Authors: Anhaus Julian A. , Heider Maximilian , Killermann Philipp , Hofmann Christian , Mahnken Andreas H.
Published: January 2024

Objectives

The aim of this study was to introduce and evaluate a new metal artifact reduction framework (iMARv2) that addresses the drawbacks (residual artifacts after correction and user preferences for image quality) associated with the current clinically applied iMAR.

Materials and Methods

A new iMARv2 has been introduced, combining the current iMAR with new modular components to remove residual metal artifacts after image correction. The postcorrection image impression is adjustable with user-selectable strength settings. Phantom scans from an energy-integrating and a photon-counting detector CT were used to assess image quality, including a Gammex phantom and anthropomorphic phantoms. In addition, 36 clinical cases (with metallic implants such as dental fillings, hip replacements, and spinal screws) were reconstructed and evaluated in a blinded and randomized reader study.

Results

The Gammex phantom showed lower HU errors compared with the uncorrected image at almost all iMAR and iMARv2 settings evaluated, with only minor differences between iMAR and the different iMARv2 settings. In addition, the anthropomorphic phantoms showed a trend toward lower errors with higher iMARv2 strength settings. On average, the iMARv2 strength 3 performed best of all the clinical reconstructions evaluated, with a significant increase in diagnostic confidence and decrease in artifacts. All hip and dental cases showed a significant increase in diagnostic confidence and decrease in artifact strength, and the improvements from iMARv2 in the dental cases were significant compared with iMAR. There were no significant improvements in the spine.

Conclusions

This work has introduced and evaluated a new method for metal artifact reduction and demonstrated its utility in routine clinical datasets. The greatest improvements were seen in dental fillings, where iMARv2 significantly improved image quality compared with conventional iMAR.
Identifying Stigmatizing Language Used by Israelis and Germans With a Mild Neurocognitive Disorder, Their Relatives, and Caregivers of People With Alzheimer’s Disease
Authors: Werner Perla , Ulitsa Natalie , Alpinar-Sencan Zümrüt , Shefet Daphna , Schicktanz Silke
Published: January 2024

Objectives:

The purpose of the study was to examine and compare the dementia-related discourse and language used by people with mild neurocognitive disorder (MND), their family members, and family caregivers of people with Alzheimer’s disease in Israel and Germany.

Design:

This secondary qualitative analysis included focus groups and semistructured interviews. Thematic analysis was used to reveal the main discourses and stigmatic language used regarding dementia and people with dementia.

Setting:

Israeli and German stakeholders.

Participants:

Forty-four Israeli and 44 German participants: 28 people with MND, 20 family members of people with MND, and 40 family caregivers of people with Alzheimer’s disease.

Results:

Two main discourses were identified: the tragedy and the biomedical discourse. The tragedy discourse included several subthemes: “Dementia as the worst-case scenario,” “Nothing can be done about dementia,” and “People with dementia are not human.” The biomedical discourse stressed pathologic aspects of the condition. Similarities and differences were found between Israeli and German participants.

Conclusions:

The study indicates that, despite culture and language differences, the use of stigmatic discourse and stigmatizing language is common among people with close exposure to dementia in Israel and Germany. Efforts should be increased to develop a person-centered and dementia-friendly language.
Implementing evidence-based pre-operative education in hospitalized patients undergoing total knee replacement: a best practice implementation project
Authors: Yang Miao-Miao , Wang Lin , Yu Zheng-Hong , Ying-Zhang
Published: January 2024

Introduction:

Pre-operative education is a key factor influencing post-operative recovery, patient expectations of surgery, and patient satisfaction. Although there are guidelines for the pre-operative education of patients undergoing knee replacement, pre-operative education of patients remains suboptimal.

Objectives:

This study aimed to improve the knowledge and skills of health care providers and patients undergoing elective knee replacement. The project used pre-operative education to increase compliance with best practices.

Methods:

This project was conducted from January to October 2022 in the orthopedic department of a tertiary hospital in China. The project was conceptually guided by the JBI Model of Evidence-Based Healthcare, while the JBI Evidence Implementation Framework was used to guide the implementation. Two audit criteria were developed for the baseline and follow-up audits, which assessed 20 patients undergoing elective knee replacement.

Results:

A comparison of the audit outcomes revealed that for Criterion 1, the percentage of patients receiving pre-operative education in both audits was 100%. For Criterion 2, the compliance rate increased significantly from 0% in the baseline audit to 90% in the follow-up audit.

Conclusions:

A clinical audit is an effective approach to improving compliance with recommended best practices. Leadership, team resource management, education, and monitoring were helpful in implementing the best practices. Further audits will need to be conducted to maintain practice changes and ensure that the project is sustainable.
Examination of SARS-CoV-2 serological test results from multiple commercial and laboratory platforms with an in-house serum panel
Authors: Lester Sandra N. , Stumpf Megan , Freeman Brandi D. , Mills Lisa , Schiffer Jarad , Semenova Vera , Jia Tao , Desai Rita , Browning Peter , Alston Bailey , Ategbole Muyiwa , Bolcen Shanna , Chen Alexander , David Ebenezer , Manitis Panagiotis , Tatum Heather , Qin Yunlong , Zellner Briana , Drobeniuc Jan , Tejada-Strop Alexandra , Chatterjee Payel , Shrivastava-Ranjan Punya , Jenks M. Harley , McMullan Laura K. , Flint Mike , Spiropoulou Christina F. , Niemeyer Glenn P. , Werner Bonnie J. , Bean Christopher J. , Johnson Jeffrey A. , Hoffmaster Alex R. , Satheshkumar Panayampalli S. , Schuh Amy J. , Owen S. Michele , Thornburg Natalie J.
Published: January 2024

Severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) is a novel human coronavirus that was identified in 2019. SARS-CoV-2 infection results in an acute, severe respiratory disease called coronavirus disease 2019 (COVID-19).  The emergence and rapid spread of SARS-CoV-2 has led to a global public health crisis, which continues to affect populations across the globe. Real time reverse transcription polymerase chain reaction (rRT-PCR) is the reference standard test for COVID-19 diagnosis. Serological tests are valuable tools for serosurveillance programs and establishing correlates of protection from disease. This study evaluated the performance of one in-house enzyme linked immunosorbent assay (ELISA) utilizing the pre-fusion stabilized ectodomain of SARS-CoV-2 spike (S), two commercially available chemiluminescence assays Ortho VITROS Immunodiagnostic Products Anti-SARS-CoV-2 Total Reagent Pack and Abbott SARS-CoV-2 IgG assay and one commercially available Surrogate Virus Neutralization Test (sVNT), GenScript USA Inc., cPass SARS-CoV-2 Neutralization Antibody Detection Kit for the detection of SARS-CoV-2 specific antibodies.  Using a panel of rRT-PCR confirmed COVID-19 patients’ sera and a negative control group as a reference standard, all three immunoassays demonstrated high comparable positivity rates and low discordant rates.  All three immunoassays were highly sensitive with estimated sensitivities ranging from 95.4%-96.6%. ROC curve analysis indicated that all three immunoassays had high diagnostic accuracies with area under the curve (AUC) values ranging from 0.9698-0.9807. High positive correlation was demonstrated among the conventional microneutralization test (MNT) titers and the sVNT inhibition percent values.  Our study indicates that independent evaluations are necessary to optimize the overall utility and the interpretation of the results of serological tests.  Overall, we demonstrate that all serological tests evaluated in this study are suitable for the detection of SARS-CoV-2 antibodies.
A toolbox for manipulating the genome of the major goat pathogen, Mycoplasma capricolum subsp. capripneumoniae
Authors: Gourgues Géraldine , Manso-Silván Lucía , Chamberland Catherine , Sirand-Pugnet Pascal , Thiaucourt François , Blanchard Alain , Baby Vincent , Lartigue Carole
Published: January 2024

Mycoplasma capricolum

subspecies

capripneumoniae

(Mccp) is the causative agent of contagious caprine pleuropneumonia (CCPP), a devastating disease listed by the World Organisation for Animal Health (WOAH) as a notifiable disease and threatening goat production in Africa and Asia. Although a few commercial inactivated vaccines are available, they do not comply with WOAH standards and there are serious doubts regarding their efficacy. One of the limiting factors to comprehend the molecular pathogenesis of CCPP and develop improved vaccines has been the lack of tools for Mccp genome engineering. In this work, key synthetic biology techniques recently developed for closely related mycoplasmas were adapted to Mccp. CReasPy-Cloning was used to simultaneously clone and engineer the Mccp genome in yeast, prior to whole-genome transplantation into

M. capricolum

subsp.

capricolum

recipient cells. This approach was used to knock out an S41 serine protease gene recently identified as a potential virulence factor, leading to the generation of the first site-specific Mccp mutants. The Cre–lox recombination system was then applied to remove all DNA sequences added during genome engineering. Finally, the resulting unmarked S41 serine protease mutants were validated by whole-genome sequencing and their non-caseinolytic phenotype was confirmed by casein digestion assay on milk agar. The synthetic biology tools that have been successfully implemented in Mccp allow the addition and removal of genes and other genetic features for the construction of seamless targeted mutants at ease, which will pave the way for both the identification of key pathogenicity determinants of Mccp and the rational design of novel, improved vaccines for the control of CCPP.
Retention of an endosymbiont for the production of a single molecule
Authors: Garber Arkadiy I. , de la Filia Molina Andrés Garcia , Vea Isabelle , Mongue Andrew J. , Ross Laura , McCutcheon John P.
Published: January 2024

Sap-feeding insects often maintain multiple nutritional endosymbionts, which act in concert to produce compounds essential for insect survival. Many mealybugs have endosymbionts in a nested configuration: one or two bacterial species reside within the cytoplasm of another bacterium, and to-gether these bacteria have genomes which encode interdependent but complete sets of genes needed to produce key nutritional molecules. Here we show that the mealybugPseudococcus viburnihas three endosymbionts, one of which contributes only two genes that produce a single host nutrition-related molecule. All three bacterial endosymbionts have tiny genomes, suggesting that they have been co-evolving inside their insect host for millions of years.

Significance

Nutritional endosymbionts synthesize (or contribute to the synthesis of) key metabolites such as essential amino acids and vitamins for their host organism. These nutrients are required by hosts because of their restricted diets, which in the case of mealybugs consists solely of plant phloem sap. Genome sequencing of insect endosymbionts has shown that their genomes can be very small, encoding few genes outside of core bacterial processes and nutrient provisioning. Here we highlight an example that has taken this reductive process to the extreme: a mealybug endosymbiont contributes only a single essential compound, chorismate, to the symbiosis.
The recombination landscape of introgression in yeast
Authors: Schwarzkopf Enrique J. , Brandt Nathan , Heil Caiti Smukowski
Published: January 2024

Meiotic recombination is an evolutionary force that acts by breaking up genomic linkage, increasing the efficacy of selection. Recombination is initiated with a double-strand break which is resolved via a crossover, which involves the reciprocal exchange of genetic material between homologous chromosomes, or a non-crossover, which results in small tracts of non-reciprocal exchange of genetic material. Crossover and non-crossover rates vary between species, populations, individuals, and across the genome. In recent years, recombination rate has been associated with the distribution of ancestry derived from past interspecific hybridization (introgression) in a variety of species. We explore this interaction of recombination and introgression by sequencing spores and detecting crossovers and non-crossovers from two crosses of the yeastSaccharomyces uvarum. One cross is between strains which each contain introgression from their sister species,S. eubayanus, while the other cross has no introgression present. We find that the recombination landscape is significantly different betweenS. uvarumcrosses, and that some of these differences can be explained by the presence of introgression in one cross. Crossovers are reduced and non-crossovers are increased in heterozygous introgression compared to syntenic regions in the cross without introgression. This translates to reduced allele shuffling within introgressed regions, and an overall reduction of shuffling on most chromosomes with introgression compared to the syntenic regions and chromosomes without introgression. Our results suggest that hybridization can significantly influence the recombination landscape, and that the reduction in allele shuffling contributes to the initial purging of introgression in the generations following a hybridization event.
Epithelial cytokeratin 6a restricts secretory autophagy of proinflammatory cytokines by interacting with Sec16A
Authors: Bhushan Anand , Chan Jonathan K. , Sun Yan , Ghiamati Fariba , Crabb Jack S , Jang Geeng-Fu , Willard Belinda , Crabb John W , Tam Connie
Published: January 2024

Epithelial cells form a crucial barrier against harmful microbes and inflammatory stimuli. Restraining inflammatory responses at the corneal barrier is necessary for avoiding sight-threatening tissue damage. Yet, epithelial cell-intrinsic mechanisms that dampen inflammation are largely unexplored. Keratin 6a (K6a) is a common type II cytokeratin highly expressed in corneal and other stratified epithelial cells. In a mouse model of sterile corneal inflammation, K6a knockout mice exhibit disease exacerbation. Here, we investigated cell-intrinsic mechanisms by which cytoplasmic K6a curbs corneal inflammation. We stimulated wild-type (WT) and K6a siRNA-knockdown (K6a-KD) human corneal epithelial (hTCEpi) cells with inflammatoryP. aeruginosaculture supernatant. Our results showed that, under both basal and inflammatory conditions, K6a-KD cells secreted higher levels of cytokines and chemokines (IL-1α, IL-6, IL-8, CXCL1, CCL20) as compared to WT cells. K6a-KD cells also had increased level of LC3-II, a marker for autophagosomes, while autophagic degradation of SQSTM1/p62 remained unchanged. In K6a-KD cells, the majority of LC3-II puncta were associated with non-acidified autophagosomes rather than acidified autolysosomes. Upon stimulation, IL-8 was found to co-localize with LC3-II by confocal microscopy. Mechanistically, mass spectrometric analysis of K6a immunoprecipitates identified Sec16A, a protein involved in secretory autophagy, as an interacting partner of K6a. Further experiments showed that knocking down key proteins involved in autophagosome formation (ATG5) and the secretory autophagy process (Sec16A, GRASP55, Rab8) abolished the augmentative effect of K6a-KD on cytokine and chemokine secretion. These findings reveal a novel repressive role of K6a in secretory autophagy-mediated proinflammatory cytokine secretion and provide new insights into cell-intrinsic mechanisms of inflammation control at epithelial barriers.
Movement-integrated habitat selection reveals wolves balance ease of travel with human avoidance in a risk-reward trade-off
Authors: Kingdon Katrien A. , Prokopenko Christina M. , Dupont Daniel L. J. , Turner Julie , Wiens Jonathan , Harriman Vanessa B. , Wal Eric Vander
Published: January 2024

Anthropogenic linear features are often linked to alterations in wildlife behaviour and movement. Landscape features such as habitat can have important mediating effects on wildlife response to disturbance and yet is rarely explicitly considered in how it interacts with other features. This study tests the effects of habitat variation on the space-use responses of GPS-collared wolves to linear features in eastern Manitoba. We simultaneously model wolf movement and selection within a conditional logistic regression framework (integrated Step Selection Analysis) with explicit consideration for how habitat alters these responses through either added cover or friction to movement. Classifying linear features based on the selection and movement response of wolves revealed that pairing transmission lines and primary roads increased the avoidance response to be greater than either feature on their own and provided evidence of a semi-permeable barrier to movement. In contrast, features with reduced human activity, including secondary and tertiary roads were highly selected for and provided access to movement corridors. Explicit parameterization of habitat provides evidence that where a linear feature is routed and which habitats it interacts with will have the greatest implications for wolf behavioural responses. Reduction of avoidance behavioural in highly risky environments signifies the importance of habitat for maintaining landscape connectivity, particularly when routing multiple features together. Natural regrowth along these features reduced movement advantages by increasing friction, indicating that actively decommissioning other features such as secondary roads would have important implications for reducing wolf encounters with prey. Knowing the influence of adjacent habitats on the likelihood of wolves selecting for a given linear feature creates context to minimize the impact of new anthropogenic features on behaviour.
The CD4 T cell epigenetic JUNB+ state is associated with proliferation and exhaustion
Authors: Mihai Ionut Sebastian , Selinger Martin , Boucheron Nicole , Forsell Mattias , Magalhaes Isabelle , Trygg Johan , Henriksson Johan
Published: January 2024

Adoptive cell therapy (ACT) requires the in vitro expansion of T cells, a process where currently several variables are poorly controlled. As the state and quality of the cells affects the treatment outcome, the lack of insight is problematic. To get a better understanding of the production process and its degrees of freedom, we have generated a multiome CD4 T cell single-cell atlas. We find in particular a JUNB+ epigenetic state, orthogonal to traditional CD4 T cell subtype categorization. This new state is present but overlooked in previous transcriptomic CD4 T cell atlases. We characterize it to be highly proliferative, having condensed and actively remodeled chromatin, and correlating with exhaustion. JUNB+ subsets are also linked to memory formation, as well as circadian rhythm, connecting several important processes into one state. To dissect JUNB regulation, we also derived a gene regulatory network (GRN) and developed a new explainable machine learning package, Nando. We propose potential upstream drivers of JUNB, verified by other atlases and orthogonal data. We expect our results to be relevant for optimizing in vitro ACT conditions as well as modulation of gene expression through novel gene editing.
Temporal information of subsecond sensory stimuli in primary visual cortex is encoded via high dimensional population vectors
Authors: Post Sam , Mol William , Rahmatullah Noorhan , Goel Anubhuti
Published: January 2024

Whether in music, language, baking, or memory, our experience of the world is fundamentally linked to time. However, it is unclear how temporal information is encoded, particularly in the range of milliseconds to seconds. Temporal processing at this scale is critical to prediction and survival, such as in a prey anticipating not only where a charging predator will go but alsowhenthe predator will arrive at that location. Several models of timing have been proposed that suggest that either time is encoded intrinsically in the dynamics of a network or that time is encoded by mechanisms that are explicitly dedicated to temporal processing. To determine how temporal information is encoded, we recorded neural activity in primary visual cortex (V1) as mice (male and female) performed a goal directed sensory discrimination task, in which patterns of subsecond stimuli differed only in their temporal profiles. We found that temporal information was encoded in the changing population vector of the network and that the space between these vectors was maximized in learned sessions. Our results suggest that temporal information in the subsecond range is encoded intrinsically and does not rely upon specialized timing mechanisms.

SIGNIFICANCE STATEMENT

Our experience of the world is fundamentally linked to time, but it is unclear how temporal information is encoded, particularly in the range of milliseconds to seconds. Using a sensory discrimination task in which patterns of subsecond stimuli differed in their temporal profiles, we found that primary visual cortex encodes temporal information via the changing population vector of the network. As temporal processing via population encoding has been shown to rely on inhibitory activity in computational models, our results may provide insight into temporal processing deficits in disorders such as autism spectrum disorder in which there is inhibitory-excitatory imbalance. Furthermore, our results may underlie processing of higher-order sensory stimuli, such as language, that are characterized by complex temporal sequences.
A Combinatorial Strategy for HRV 3C Protease Engineering to Achieve the N-terminal Free Cleavage
Authors: Mei Meng , Fan Xian , Zhou Yu , Zhang Faying , Zhang Guimin , Yi Li
Published: January 2024

Human rhinovirus 3C protease (HRV 3C-P) has a high specificity against the substrate of LEVLFQ↓G at P1’ site, which plays an important role in biotechnology and academia as a fusion tag removal tool. However, a non-ignorable limitation is that an extra residue of Gly would remain at the N terminus of the recombinant target protein after cleavage with HRV 3C-P, thus potentially causing protein mis-functionality or immunogenicity. Here, we developed a combinatorial strategy by integrating structure-guided library design and high-throughput screening of eYESS approach for HRV 3C-P engineering to expand its P1’ specificity. Finally, a C3 variant was obtained, exhibiting a broad substrate P1’ specificity to recognize 20 different amino acids with the highest activity against LEVLFQ↓M (kcat/KM= 3.72 ± 0.04 mM-1·s-1). Further biochemical and NGS-mediated substrate profiling analysis showed that C3 variant still kept its substrate stringency at P1 site and good residue tolerance at P2’ site, but with an expanded P1’ specificity. Structural simulation of C3 indicated a reconstructed S1’ binding pocket as well as new interactions with the substrates. Overall, our studies here prompt not only the practical applications and understanding of substrate recognition mechanisms of HRV 3C-P, also provide new tools for other enzyme engineering.

Abstract graphic
In-silicoevaluation of Mirror repeats in some selected genes ofCandida albicans
Authors: Sehrawat Barkha , Yadav Priya , Sarjeet Mustak , Yadav Sakshi , Yadav Vidhi , Goyal Nupur , Alam Parvej , Ahlawat Meghali , Yadav Sandeep
Published: January 2024

All cellular processes in a living cell are controlled by its genetic material. DNA in majority of the domains acts as a regulatory molecule by controlling various vital functions. The genetic makeup of DNA or RNA (in viruses) is unique in all the domains. Their uniqueness is determined by the presence of various types of repetitive patterns of bases. These includes inverted repeats, tandem repeats, VNTR’s, palindromes etc. Among many repetitive pattern types, Mirror repeats (MR) found to be dispersed throughout the genes or genomes. These sequences are associated with various functional features like their involvement in H-DNA formation, in replication & transcription, nervous system related diseases development etc. The major focus of this investigation is to identify MR sequences from some selected genes ofCandida albicansusing a bioinformatics based pipeline. The approach refers to as FPCB which utilized some manual steps to extract out MR sequence from any targeted gene or genome. The current study find out that the identified Mirror repeats found to be dispersed throughout the selected genes along with variable length. Among them the maximum & minimum MR sequences were reported in the gene FAS2 (108) & HIS1 (15) respectively. The present study will be helpful to provide a new insight for molecular as well as computational based studies ofCandida albicansas well as other related fungal groups.
Characterization of a Unique Spontaneous Calcifying Cell Line (CJ): a Novel Tool for the Study of Ectopic Calcification
Authors: Ida Nobutaka , Yamane Yoshihisa
Published: January 2024

Purpose

Due to the lack of an appropriate in vitro evaluation system, there is no effective prevention or treatment for ectopic calcification diseases (ECD). We obtained canine renal adenocarcinoma cells (CJ cells) that spontaneously form large amounts of calcified precipitates (CaP-ppt) and examined whether they could serve as a model for the early stages of ectopic calcification (EC).

Methods

Dispersed cells obtained by collagenase-DNase digestion were cultured in 10% FBS, antibiotic-containing DMEM-high glucose medium (standard medium). CaP-ppt was stained with Alizarin Red (AR) and colorimetrically quantified after acid extraction. Cell volume was determined by Crystal violet (CV) staining followed by extraction and colorimetric quantification. Calcium (Ca) and phosphate (PO4) were determined with a commercial kit to obtain the Ca/P ratio. Screening of anti-CaP-ppt compounds was performed in the same 96-well plate in the order of cell culture ⇒ CaP-ppt quantification ⇒ cell quantification and evaluated by IC50value.

Results

CJ cells produced large amounts of CaP-ppt on standard medium alone without external phosphate addition; CaP-ppt formation was not accompanied by cell death, but on the contrary, CaP-ppt increased at physiological pH values around pH 7.4 due to active cell metabolism. The Ca2+and PO43-partitioning kinetics to CaP-ppt were found, and the Ca/P ratio of CaP-ppt was stable at 1.35. The anti-Cap-ppt effects of bisphosphonates and all-trans retinoic acid (ATRA) were also confirmed in this CJ cell CaP-ppt system.

Discussion

There was doubt about the vascular calcifying cell model with the addition of high concentrations of phosphoric acid, but there were no suitable alternative cells. Spontaneously calcifying CJ cells provide a fundamental solution to this problem. Anti-CaP-ppt screening also eliminates the need for medium exchange, thus saving labor and cost. The Ca/P ratio of CaP-ppt in CJ cells is 1.35, the same as that of amorphous Ca phosphate (ACP), corresponding to the early (reversible) stage of EC. Therefore, it has favorable conditions as an evaluation system for drug discovery.

Conclusion

CJ cells, which calcify at physiological phosphate concentrations (0.9 mM) in standard media, are useful and novel research material for basic and preventive studies of ECD and for drug development studies.
A weighted and cumulative point system for accurate scoring of intestinal pathology in a piglet model of necrotizing enterocolitis
Authors: Offersen Simone Margaard , Henriksen Nicole Lind , Brunse Anders
Published: January 2024

Background

Necrotizing enterocolitis (NEC) is a serious condition, primarily affecting premature infants, in which a portion of the gut undergoes inflammation and necrosis. Symptoms of NEC are unspecific, and together with a rapid progression, the disease remains a significant concern. The preterm pig develops NEC spontaneously, making it a suitable model for exploring novel treatments. During piglet necropsy, NEC-lesions closely resemble the pathologies found during surgery or autopsy of preterm infants. As such, the systematic gross inspection enables direct evaluation of gut lesions, which is not possible in the medical preterm patient. Here, we introduce a revised intestinal scoring system with an expanded score range and more detailed descriptive features to accurately describe the diversity of NEC-lesions in the preterm piglet model.

Methods

We included 333 preterm piglets from four separate experiments, each delivered via cesarian section at 90% gestation. The pigs were fed either a gently processed (GP) or harshly processed (HP) milk formula for 96 hours and were subsequently euthanized. At necropsy, the gastrointestinal tract was assessed with 1) an established 6-grade scoring system and 2) a systematic and descriptive approach focusing on the distribution and severity of hyperemia, hemorrhage, pneumatosis intestinalis (intramural gas), and necrosis. Lesion biopsies were sampled for cytokine measurement and a subset (n= 62) was sampled for histopathological assessment.

Results

The systematic and descriptive registrations were evaluated and converted into a weighted and cumulative point (WCP) score. Compared to the 6-grade score, the WCP score enabled a higher discrepancy in severity levels, especially among organs with more prominent NEC lesions. IL-1β in small intestinal lesions and both IL-8 and IL-1β in colon lesions correlated positively with the WCP scale. A histopathological grade system (0-8) was established and revealed mucosal lesions not recognized macroscopically. Finally, the WCP score showed a higher NEC-promoting effect of the HP formula compared to the GP formula.

Conclusion

The validation of the weighted and cumulative scoring system demonstrated an expanded score range, enhancing the accuracy in describing NEC-lesions in gastrointestinal segments of preterm pigs. This approach may increase the efficiency of preclinical NEC experiments.
CRISPR-repressed toxin-antitoxin provides population-level immunity against diverse anti-CRISPR elements
Authors: Shu Xian , Wang Rui , Li Zhihua , Xue Qiong , Wang Jiajun , Liu Jingfang , Cheng Feiyue , Liu Chao , Zhao Huiwei , Hu Chunyi , Li Jie , Ouyang Songying , Li Ming
Published: January 2024

Prokaryotic CRISPR-Cas systems are highly vulnerable to phage-encoded anti-CRISPR (Acr) factors. How CRISPR-Cas systems protect themselves remains unclear. Here, we uncovered a broad-spectrum anti-anti-CRISPR strategy involving a phage-derived toxic protein. Transcription of this toxin is normally reppressed by the CRISPR-Cas effector, but is activated to halt cell division when the effector is inhibited by any anti-CRISPR proteins or RNAs. We showed that this abortive infection-like effect efficiently expels Acr elements from bacterial population. Furthermore, we exploited this anti-anti-CRISPR mechanism to develop a screening method for specific Acr candidates for a CRISPR-Cas system, and successfully identified two distinct Acr proteins that enhance the binding of CRISPR effector to non-target DNA. Our data highlight the broad-spectrum role of CRISPR-repressed toxins in counteracting various types of Acr factors, which illuminates that the regulatory function of CRISPR-Cas confers host cells herd immunity against Acr-encoding genetic invaders, no matter they are CRISPR-targeted or not.
Investigating thecis-Regulatory Basis of C3and C4Photosynthesis in Grasses at Single-Cell Resolution
Authors: Mendieta John Pablo , Tu Xiaoyu , Jiang Daiquan , Yan Haidong , Zhang Xuan , Marand Alexandre P. , Zhong Silin , Schmitz Robert J.
Published: January 2024

While considerable knowledge exists about the enzymes pivotal for C4photosynthesis, much less is known about thecis-regulation important for specifying their expression in distinct cell types. Here, we use single-cell-indexed ATAC-seq to identify cell-type-specific accessible chromatin regions (ACRs) associated with C4enzymes for five different grass species. This study spans four C4species, covering three distinct photosynthetic subtypes:Zea maysandSorghum bicolor(NADP-ME),Panicum miliaceum(NAD-ME),Urochloa fusca(PEPCK), along with the C3outgroupOryza sativa. We studied thecis-regulatory landscape of enzymes essential across all C4species and those unique to C4subtypes, measuring cell-type-specific biases for C4enzymes using chromatin accessibility data. Integrating these data with phylogenetics revealed diverse co-option of gene family members between species, showcasing the various paths of C4evolution. Besides promoter proximal ACRs, we found that, on average, C4genes have two to three distal cell-type-specific ACRs, highlighting the complexity and divergent nature of C4evolution. Examining the evolutionary history of these cell-type-specific ACRs revealed a spectrum of conserved and novel ACRs, even among closely related species, indicating ongoing evolution ofcis-regulation at these C4loci. This study illuminates the dynamic and complex nature of CRE evolution in C4photosynthesis, particularly highlighting the intricatecis-regulatory evolution of key loci. Our findings offer a valuable resource for future investigations, potentially aiding in the optimization of C3crop performance under changing climatic conditions.
Joint estimation of paternity, sibships and pollen dispersal in a snapdragon hybrid zone
Authors: Ellis Thomas James , Field David Luke , Barton Nicholas H.
Published: January 2024

1

The distribution of pollen dispersal distances sets the scale for plant population dynamics. A useful approach for inferring the distribution dispersal distances is to infer the distances between mates by paternity or parentage reconstruction. This is most powerful when information about multiple properties or data types are inferred in a joint analysis. We describe an approach to jointly infer paternity, sibling relationships and population parameters, with the example of the pollen dispersal kernel in a natural population of the yellow-floweredAntirrhinum majus striatumand the magenta-floweredA. m. pseudomajus. Pollen dispersal is lep-tokurtic, with half of mating events occurring within 30m, but with a long tail of mating events up to 747m. We also find tentative evidence that fathers tend to be to the East of mothers, indicating that there is a bias in pollen dispersal fromA. m. pseudomajusintoJoint estimation of paternity, sibships and pollen dispersal in a snapdragon hybrid zone A. m. striatum. The scale of pollen dispersal is large enough that pollinators should encounter the full range of hybrid phenotypes in the hybrid zone, and would be sufficient for any pollinator-mediated selection to influence male or female fitness.
A limited concentration range of diaphorin, a polyketide produced by a bacterial symbiont of the Asian citrus psyllid, promotes thein vitrogene expression with bacterial ribosomes
Authors: Takasu Rena , Izu Takashi , Nakabachi Atsushi
Published: January 2024

Diaphorin is a polyketide produced by “CandidatusProfftella armatura” (Gammaproteobacteria:Burkholderiales), an obligate symbiont of a devastating agricultural pest, the Asian citrus psyllidDiaphorina citri(Hemiptera: Psyllidae). Physiological concentrations of diaphorin, whichD. citricontains at levels as high as 2–20 mM, are inhibitory to various eukaryotes andBacillus subtilis(Firmicutes:Bacilli) but promote the growth and metabolic activity ofEscherichia coli(Gammaproteobacteria:Enterobacterales). Our previous study demonstrated that five-millimolar diaphorin, which exhibits significant inhibitory and promoting effects on culturedB. subtilisandE. coli, respectively, inhibitsin vitrogene expression utilizing purifiedB. subtilisandE. coliribosomes. This suggested that the adverse effects of diaphorin onB. subtilisare partly due to its influence on gene expression. However, the result appeared inconsistent with the positive effects onE. coli. Moreover, the diaphorin concentration in bacterial cells, where genes are expressedin vivo, may be lower than in culture media. Therefore, the present study analyzed the effects of 50 and 500 μM of diaphorin on bacterial gene expression using the same analytical method. The result revealed that this concentration range of diaphorin, in contrast to five-millimolar diaphorin, promotes thein vitrotranslation with theB. subtilisandE. coliribosomes, suggesting that the positive effects of diaphorin onE. coliare due to its direct effects on translation. This study demonstrated for the first time that a pederin-type compound promotes gene expression, establishing a basis for utilizing its potential in pest management and industrial applications.

Importance

This study revealed that a limited concentration range of diaphorin, a secondary metabolite produced by a bacterial symbiont of an agricultural pest, promotes cell-free gene expression utilizing substrates and proteins purified from bacteria. The unique property of diaphorin, which is inhibitory to various eukaryotes andBacillus subtilisbut promotes the growth and metabolic activity ofEscherichia coli, may affect the microbial flora of the pest insect, potentially influencing the transmission of devastating plant pathogens. Moreover, the activity may be exploited to improve the efficacy of industrial production byE. coli, which is often used to produce various important materials, including pharmaceuticals, enzymes, amino acids, and biofuels. This study elucidated a part of the mechanism by which the unique activity of diaphorin is expressed, constructing a foundation for applying the unique property to pest management and industrial use.
Human cell surface-AAV interactomes identify LRP6 as blood-brain-barrier transcytosis receptor and immune cytokine IL3 as AAV9 binder
Authors: Shay Timothy F. , Jang Seongmin , Chen Xinhong , Walker Beth , Tebbutt Claire , Wolfe Damien A. , Brittain Tyler J. , Arokiaraj Cynthia M. , Sullivan Erin E. , Ding Xiaozhe , Wang Ting-Yu , Lei Yaping , Chuapoco Miguel R. , Chou Tsui-Fen , Gradinaru Viviana
Published: January 2024

Adeno-associated viruses (AAVs) are foundational gene delivery tools for basic science and clinical therapeutics. However, lack of mechanistic insight, especially for engineered vectors created by directed evolution, can hamper their application. Here, we adapted an unbiased human cell microarray platform to determine the extracellular and cell surface interactomes of natural and engineered AAVs. We identified a naturally-evolved and serotype-specific interaction between the AAV9 capsid and human interleukin 3 (IL3), with possible roles in host immune modulation, as well as lab-evolved low-density-lipoprotein-receptor-related-protein 6 (LRP6) interactions specific to engineered capsids that cross the blood-brain barrier in non-human primates after intravenous administration. The unbiased cell microarray screening approach also allowed us to identify off-target tissue binding interactions of engineered brain-enriched AAV capsids that may inform vectors’ peripheral organ tropism and side effects. These results allow confident application of engineered AAVs in diverse organisms and unlock future target-informed engineering of improved viral and non-viral vectors for non-invasive therapeutic delivery to the brain.
GENOMIC PROFILING OF SARS-COV-2 STRAINS CIRCULATING IN SOUTH EASTERN REGION OF INDIA DURING THREE WAVES OF PANDEMIC
Authors: Kandhasamy Vanathy , Balakrishna Pillai Agieshkumar , Mariappan Vignesh , Ramalingam Malarvizhi , Raganadin Pajanivel , Ramadoss Ramya , Moovarkumudalvan Balasubramanian , Easow Joshy M , Vasudevan Madavan , S.R. Rao
Published: January 2024

Continuous bio-surveillance of SARS-CoV-2 is an ongoing task at local, national and global levels since the pandemic onset for understanding genetic evolution and vaccine efficacy. Present study was designed to track the emergence of new variants along the duration of three peaks of infection in the city of Puducherry, India. A total of 128 samples were subjected to Illumina deep RNA sequencing. The results showed predominance of uncommon, delta and omicron variants in first, second and third waves respectively. The most common pangolin lineage was B.1.560 and B.1.617.2. The study observed a total of 3133 common and 11 new mutations. The most common is in the Spike_D614G. A new set of mutations was observed in key viral factors such as NS16 that are implicated to be involved in immune evasion. This may have impact on enhanced disease virulence, vaccine efficiency and possible tolerance to current antivirals. This warrants further in vitro studies to understand the significance of the mutations. While the results presented would also augment the ongoing research on evolutionary and the genetic epidemiology of SARS-CoV-2, it also emphasizes the need for continuous genetic monitoring to predict the forthcoming threats due to the emergence of new or existing variants.