bluuebunny/crossref_metadata_2025 huggingface dataset:
Hypertonic Dextrose Prolotherapy, an Alternative to Intra-Articular Injections With Hyaluronic Acid in the Treatment of Knee Osteoarthritis
Authors: Arias-Vázquez Pedro Iván , Tovilla-Zárate Carlos Alfonso , Castillo-Avila Rosa Giannina , Legorreta-Ramírez Blanca Gabriela , López-Narváez María Lilia , Arcila-Novelo Russell , González-Castro Thelma Beatriz
Published: November 2021

Intra-articular injections with hyaluronic acid are recommended in the treatment of knee osteoarthritis; however, hypertonic dextrose prolotherapy has been reported as effective and safe. The aim was to evaluate the effectiveness of hypertonic dextrose prolotherapy for pain reduction and improvement of function in individuals with knee osteoarthritis in comparison with hyaluronic acid by meta-analysis. The search was performed in electronic databases. Six studies were included (395 participants). No statistically significant differences were found between prolotherapy and hyaluronic acid in pain control in the short-term; however, in the subanalysis that included only the studies that used intra-articular injection within the prolotherapy scheme, an effect was found in favor of the prolotherapy groups (d = −1.33; 95% confidence interval, −2.50 to −0.16; P (z) = 0.03). Also, an effect was found in favor of the prolotherapy group in the improvement in function (d = −1.05; 95% confidence interval, −2.03 to −0.08; P (z) = 0.03). No major adverse reactions or side effects were reported in any of the studies. Hypertonic dextrose prolotherapy seems to be an effective intervention to decrease pain and improve function in knee osteoarthritis, with efficacy similar to intra-articular injections with hyaluronic acid in the short-term follow-up. Nonetheless, better-quality clinical trials are necessary.
δPKC-Mediated DRP1 Phosphorylation Impacts Macrophage Mitochondrial Function and Inflammatory Response to Endotoxin
Authors: Lin Amanda J. , Joshi Amit U. , Mukherjee Riddhita , Tompkins Carly A. , Vijayan Vijith , Mochly-Rosen Daria , Haileselassie Bereketeab
Published: November 2021

Background:

Recent studies have demonstrated that alterations in mitochondrial dynamics can impact innate immune function. However, the upstream mechanisms that link mitochondrial dynamics to innate immune phenotypes have not been completely elucidated. This study asks if Protein Kinase C, subunit delta (δPKC)-mediated phosphorylation of dynamin-related protein 1 (Drp1), a key driver of mitochondrial fission, impacts macrophage pro-inflammatory response following bacterial-derived lipopolysaccharide (LPS) stimulation.

Methods:

Using RAW 264.7 cells, bone marrow-derived macrophages from C57BL/6J mice, as well as human monocyte-derived macrophages, we first characterized changes in δPKC-mediated phosphorylation of Drp1 following LPS stimulation. Next, using rationally designed peptides that inhibit δPKC activation (δV1-1) and δPKC-Drp1 interaction (ψDrp1), we determined whether δPKC-mediated phosphorylation of Drp1 impacts LPS-induced changes in mitochondrial morphology, mitochondrial function, and inflammatory response.

Results:

Our results demonstrated that δPKC-dependent Drp1 activation is associated with increased mitochondrial fission, impaired cellular respiration, and increased mitochondrial reactive oxygen species in LPS-treated macrophages. This is reversed using a rationally designed peptide that selectively inhibits δPKC phosphorylation of Drp1 (ψDrp1). Interestingly, limiting excessive mitochondrial fission using ψDrp1 reduced LPS-triggered pro-inflammatory response, including a decrease in NF-κB nuclear localization, decreased iNOS induction, and a reduction in pro-inflammatory cytokines (IL-1β, TNFα, IL-6).

Conclusion:

These data suggest that inhibiting Drp1 phosphorylation by δPKC abates the excessive mitochondrial fragmentation and mitochondrial dysfunction that is seen following LPS treatment. Furthermore, these data suggest that limiting δPKC-dependent Drp1 activation decreases the pro-inflammatory response following LPS treatment. Altogether, δPKC-dependent Drp1 phosphorylation might be an upstream mechanistic link between alterations in mitochondrial dynamics and innate immune phenotypes, and may have therapeutic potential.
The Impact of Indocyanine Green Fluorescence Angiography on Intraoperative Decision Making in Right Hemicolectomy: A Case-Control Study
Authors: Aawsaj Yousif , Mustafa Abdalla , Winstanley Joseph , O’loughlin Paul
Published: November 2021

Introduction:

Anastomotic leak (AL) after right hemicolectomy remains a significant clinical challenge with an incidence of 4.2% to 8.2% in European series. Near infrared imaging with indocyanine green (NIR-ICG) allows real-time assessment of bowel perfusion. However, there is a lack of published data assessing the clinical utility of this new technology in right sided colonic resection.

Materials and Methods:

Data from electronic records were retrospectively analyzed for consecutive patients undergoing right hemicolectomy in a single center between March 1, 2016 and October 31, 2019. Primary outcomes were the incidence of AL and the frequency with which ICG-NIR imaging altered the intraoperative course.

Results:

Our study included 127 patients, with 65 in the NIR-ICG group and 62 in the control group. Median length of follow-up was 24 months. There was no significant difference in demographic or pathologic characteristics between the 2 cohorts. There was no significant difference in operation length between the NIR-ICG and control groups (164.7 vs. 162.9 min, P=0.88). The use of NIR-ICG altered the intraoperative course in 4/65 (6.2%) patients. The rate of AL was lower in the NIR-ICG group (1.5% vs. 4.8%), although this did not reach statistical significance.

Conclusion:

The use of NIR-ICG altered the intraoperative course for notable subset of patients undergoing right hemicolectomy without prolonging operative time. Larger prospective studies are required to evaluate the potential for the routine use of this technology to reduce AL rate in right hemicolectomy.
A new trauma frontier: Exploratory pilot study of platelet transcriptomics in trauma patients
Authors: Fields Alexander T. , Lee Man-Cheung , Mayer Fahima , Santos Yale A. , Bainton Cedric M.V. , Matthay Zachary A. , Callcut Rachael A. , Mayer Nasima , Cuschieri Joseph , Kober Kord M. , Bainton Roland J. , Kornblith Lucy Zumwinkle
Published: November 2021

BACKGROUND

The earliest measurable changes to postinjury platelet biology may be in the platelet transcriptome, as platelets are known to carry messenger ribonucleic acids (RNAs), and there is evidence in other inflammatory and infectious disease states of differential and alternative platelet RNA splicing in response to changing physiology. Thus, the aim of this exploratory pilot study was to examine the platelet transcriptome and platelet RNA splicing signatures in trauma patients compared with healthy donors.

METHODS

Preresuscitation platelets purified from trauma patients (n = 9) and healthy donors (n = 5) were assayed using deep RNA sequencing. Differential gene expression analysis, weighted gene coexpression network analysis, and differential alternative splicing analyses were performed. In parallel samples, platelet function was measured with platelet aggregometry, and clot formation was measured with thromboelastography.

RESULTS

Differential gene expression analysis identified 49 platelet RNAs to have differing abundance between trauma patients and healthy donors. Weighted gene coexpression network analysis identified coexpressed platelet RNAs that correlated with platelet aggregation. Differential alternative splicing analyses revealed 1,188 splicing events across 462 platelet RNAs that were highly statistically significant (false discovery rate <0.001) in trauma patients compared with healthy donors. Unsupervised principal component analysis of these platelet RNA splicing signatures segregated trauma patients in two main clusters separate from healthy controls.

CONCLUSION

Our findings provide evidence of finetuning of the platelet transcriptome through differential alternative splicing of platelet RNA in trauma patients and that this finetuning may have relevance to downstream platelet signaling. Additional investigations of the trauma platelet transcriptome should be pursued to improve our understanding of the platelet functional responses to trauma on a molecular level.
Patient-reported outcomes 6 to 12 months after isolated rib fractures: A nontrivial injury pattern
Authors: Heindel Patrick , Ordoobadi Alexander , El Moheb Mohamad , Serventi-Gleeson Jessica , Garvey Shannon , Heyman Annie , Patel Nikita , Sanchez Sabrina , Kaafarani Haytham M.A. , Herrera-Escobar Juan , Salim Ali , Nehra Deepika
Published: November 2021

BACKGROUND

Despite the ubiquity of rib fractures in patients with blunt chest trauma, long-term outcomes for patients with this injury pattern are not well described.

METHODS

The Functional Outcomes and Recovery after Trauma Emergencies (FORTE) project has established a multicenter prospective registry with 6- to 12-month follow-up for trauma patients treated at participating centers. We combined the FORTE registry with a detailed retrospective chart review investigating admission variables and injury characteristics. All trauma survivors with complete FORTE data and isolated chest trauma (Abbreviated Injury Scale score of ≤1 in all other regions) with rib fractures were included. Outcomes included chronic pain, limitation in activities of daily living, physical limitations, exercise limitations, return to work, and both inpatient and discharge pain control modalities. Multivariable logistic regression models were built for each outcome using clinically relevant demographic and injury characteristic univariate predictors.

RESULTS

We identified 279 patients with isolated rib fractures. The median age of the cohort was 68 years (interquartile range, 56–78 years), 59% were male, and 84% were White. Functional and quality of life limitations were common among survivors of isolated rib fractures even 6 to 12 months after injury. Forty-three percent of patients without a preexisting pain disorder reported new daily pain, and new chronic pain was associated with low resilience. Limitations in physical functioning and exercise capacity were reported in 56% and 51% of patients, respectively. Of those working preinjury, 28% had not returned to work. New limitations in activities of daily living were reported in 29% of patients older than 65 years. Older age, higher number of rib fractures, and intensive care unit admission were independently associated with higher odds of receiving regional anesthesia. Receiving a regional nerve block did not have a statistically significant association with any patient-reported outcome measures.

CONCLUSION

Isolated rib fractures are a nontrivial trauma burden associated with functional impairment and chronic pain even 6 to 12 months after injury.

LEVEL OF EVIDENCE

Prognostic/epidemiologic, level III.
Social determinants of health and patient-level mortality prediction after trauma
Authors: Phelos Heather M. , Kass Nicolas M. , Deeb Andrew-Paul , Brown Joshua B.
Published: November 2021

BACKGROUND

Social determinants of health (SDOH) impact patient outcomes in trauma. Census data are often used to account for SDOH; however, there is no consensus on which variables are most important. Social vulnerability indices offer the advantage of combining multiple constructs into a single variable. Our objective was to determine if incorporation of SDOH in patient-level risk-adjusted outcome modeling improved predictive performance.

METHODS

We evaluated two social vulnerability indices at the zip code level: Distressed Community Index (DCI) and National Risk Index (NRI). Individual variable combinations from Agency for Healthcare Research and Quality's SDOH data set were used for comparison. Patients were obtained from the Pennsylvania Trauma Outcomes Study 2000 to 2020. These measures were added to a validated base mortality prediction model with comparison of area under the curve and Bayesian information criterion. We performed center benchmarking using risk-standardized mortality ratios to evaluate change in rank and outlier status based on SDOH. Geospatial analysis identified geographic variation and autocorrelation.

RESULTS

There were 449,541 patients included. The DCI and NRI were associated with an increase in mortality (adjusted odds ratio, 1.02; 95% confidence interval, 1.01–1.03 per 10% percentile rank increase; p < 0.01, respectively). The DCI, NRI, and seven Agency for Healthcare Research and Quality variables also improved base model fit but discrimination was similar. Two thirds of centers changed mortality ranking when accounting for SDOH compared with the base model alone. Outlier status changed in 7% of centers, most representing an improvement from worse-than-expected to nonoutlier or nonoutlier to better-than-expected. There was significant geographic variation and autocorrelation of the DCI and NRI (DCI; Moran's I 0.62, p = 0.01; NRI; Moran's I 0.34, p = 0.01).

CONCLUSION

Social determinants of health are associated with an individual patient's risk of mortality after injury. Accounting for SDOH may be important in risk adjustment for trauma center benchmarking.

LEVEL OF EVIDENCE

Prognostic/Epidemiologic, level IV.
Health-related quality of life and functional outcome measures for pediatric multiple injury: A systematic review and narrative synthesis
Authors: Collins Kathryn C. , Burdall Oliver , Kassam Jamila , Firth Gregory , Perry Daniel , Ramachandran Manoj
Published: November 2021

BACKGROUND

Pediatric multiple injury is a major cause of mortality and morbidity worldwide, with varied long-term sequelae. To improve care, a better understanding of the outcome tools used following multiple injury is needed.

OBJECTIVES

This study aimed to (1) identify the outcome tools used to assess functional and health-related quality of life outcomes in pediatric multiple injury and to (2) describe the tool domains and validity.

METHODS

Eligible studies were those that included pediatric participants aged 0 to 17 years who experienced multiple injury or severe trauma based on Injury Severity Score/Abbreviated Injury Scale score and a functional outcome tool was used to assess outcomes (e.g., physical, psychological, quality of life). Excluded study designs were editorials, narrative, and systematic reviews.

RESULTS

Twenty-two papers were included encompassing 16,905 participants and 34 different outcome tools. Ten tools were validated in children of which 4 were multiple injury specific; 18 were validated in adults of which 8 were trauma specific, and 6 were previously unvalidated. The tools were a mixture of patient reported (7 of 10 validated in children and 13 of 18 validated in adults) and clinician reported (3 of 10 validated in children and 2 of 13 validated in adults). Pediatric tool domains assessed were function, mobility, activities of daily living, pain, school, cognition, emotional domains, mental health, behavior, and high-risk behaviors. Using adult-validated tools to assess children may not capture the children's true function and health-related quality of life.

CONCLUSION

There was a lack of consistency in the outcome tools used following multiple injury in children. Adult-validated measures may not accurately capture pediatric outcomes after multiple injury. To fully understand the impact of pediatric multiple injury and make comparisons between studies, development of a core outcome set is required.

LEVEL OF EVIDENCE

Quantitative observational studies, level III.
Desulfomarina profundi gen. nov., sp. nov., a novel mesophilic, hydrogen-oxidizing, sulphate-reducing chemolithoautotroph isolated from a deep-sea hydrothermal vent chimney
Authors: Hashimoto Yurina , Tame Akihiro , Sawayama Shigeki , Miyazaki Junichi , Takai Ken , Nakagawa Satoshi
Published: November 2021

A novel mesophilic, strictly anaerobic, chemolithoautotrophic sulphate-reducing bacterium, designated strain KT2T, was isolated from a deep-sea hydrothermal vent chimney at the Suiyo Seamount in the Izu-Bonin Arc. Strain KT2Tgrew at 25–40 °C (optimum 35 °C) and pH 5.5–7.0 (optimum 6.6) in the presence of 25–45 g l−1NaCl (optimum 30 g l−1). Growth occurred with molecular hydrogen as the electron donor and sulphate, thiosulphate, and sulphite as the electron acceptors. The isolate utilized CO2as the sole carbon source for chemolithoautotrophic growth on H2. Glycerol, succinate, fumarate, malate, glutamate, or casamino acids could serve as an alternative electron donor in the presence of CO2. Malate, citrate, glutamate, and casamino acids were used as fermentative substrates for weak growth. The G+C content of genomic DNA was 46.1 %. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain KT2Tis a member of the familyDesulfobulbaceae, showing a sequence similarity of 94.3 % withDesulforhopalus singaporensis. Phylogenomic analysis based on concatenated 156 single-copy marker genes confirmed the same topology as the 16S rRNA gene phylogeny. The ANI and AAI values between strain KT2Tand related genera of the familyDesulfobulbaceaewere 65.6–68.6 % and 53.1–62.9 %. Based on the genomic, molecular, and physiological characteristics, strain KT2Trepresents a novel genus and species within the familyDesulfobulbaceae, for which the nameDesulfomarina profundigen. nov., sp. nov. is proposed, with KT2T(=JCM 34118T= DSM 111364T) as the type strain.
Clinical characteristics and prognostic risk factors of mortality in patients with interstitial lung diseases and viral infection: a retrospective cohort study
Authors: Li Lijuan , Wang Chulei , Sun Lingxiao , Zhang Xiaoqi , Yang Guoru
Published: November 2021

Introduction. Patients with interstitial lung disease (ILD) who subsequently develop a viral infection have high rates of morbidity and mortality.

Hypothesis/Gap Statement. Few large-scale epidemiological studies have investigated potential prognostic factors for morbidity and mortality in this patient group.

Aim. To evaluate the risk factors for morbidity and mortality in hospitalized patients with ILD and viral infection, as well as the clinical characteristics.

Methodology. This retrospective cohort study included patients with ILD who were hospitalized for a viral infection in two tertiary academic hospitals in China, between 1 January 2013 and 31 December 2019. We analysed the prevalence of comorbidities, clinical characteristics, 30 day mortality rates, and prognostic risk factors.

Results. A total of 282 patients were included; 195 and 87 were immunocompromised and immunocompetent, respectively. The most common underlying interstitial diseases were idiopathic pulmonary fibrosis (42.9 %) and connective tissue disease (36.9 %). The 30 day mortality rate was 20.6 %. During the influenza season, an increase in influenza virus (IFV) (25.7 %), respiratory syncytial virus (14.9 %) and cytomegalovirus (CMV) (11.3 %) cases was observed in the immunocompromised group. The most frequently detected virus in the immunocompetent group was IFV (44.8 %), followed by respiratory syncytial virus (11.5 %), and human rhinovirus (9.2 %). During the non-influenza season, CMV (34.4 %) was the main virus detected in the immunocompromised group. The 30 day mortality rates of non-IFV patients were higher than those of IFV patients. Older age (>60 years), respiratory failure, persistent lymphocytopenia, invasive mechanical ventilation and non-IFV virus infection were significantly associated with increased 30 day mortality.

Conclusion. Patients with ILD who develop viral infection have high rates of morbidity and mortality, which are associated with increased age (>60 years), respiratory failure, mechanical ventilation, persistent lymphocytopenia and non-IFV virus infection. These risk factors should be carefully considered when determining treatment strategies for this patient population.
The enigma of Pacini’s Vibrio cholerae discovery
Authors: Carboni Gian Piero
Published: November 2021

During the 1854 cholera outbreak in Florence, Italy, Filippo Pacini documented that the cause of the infection was a bacterium. This conclusion was also independently reached by John Snow during the 1854 cholera outbreak in London. By using an epidemiological method, Snow found that the infection spread through a polluted water network. Snow identified a water pump as the source of the disease. After removing the infected handle of this pump, the cases of cholera rapidly began to decrease. A microscopic examination of the water showed organic impurities but no bacteria. This discovery was ignored during Snow’s lifetime. In contrast, through microscopy during the autopsies of cholera victims, Pacini observed that the disruption of their intestinal mucosa was closely associated with millions of the bacteria that he calledVibrio cholerae. Via histological techniques, Pacini detected that intestinal mucosa reabsorption dysfunction was the cause of debilitating diarrhoea, vomiting, severe dehydration and death. Nevertheless, his discovery ofVibrio choleraewas ignored during Pacini’s lifetime. A survey of Pacini’s autographic manuscripts suggests that Pacini and Snow may have shared mutual knowledge within their respective seminal papers. This survey also facilitates, for the first time, the creation of maps that illustrate the worldwide distribution of Pacini’s cholera papers from 1854 to 1881. The consistent neglect of Pacini’s discovery remains a true enigma.
Bakta: rapid and standardized annotation of bacterial genomes via alignment-free sequence identification
Authors: Schwengers Oliver , Jelonek Lukas , Dieckmann Marius Alfred , Beyvers Sebastian , Blom Jochen , Goesmann Alexander
Published: November 2021

Command-line annotation software tools have continuously gained popularity compared to centralized online services due to the worldwide increase of sequenced bacterial genomes. However, results of existing command-line software pipelines heavily depend on taxon-specific databases or sufficiently well annotated reference genomes. Here, we introduce Bakta, a new command-line software tool for the robust, taxon-independent, thorough and, nonetheless, fast annotation of bacterial genomes. Bakta conducts a comprehensive annotation workflow including the detection of small proteins taking into account replicon metadata. The annotation of coding sequences is accelerated via an alignment-free sequence identification approach that in addition facilitates the precise assignment of public database cross-references. Annotation results are exported in GFF3 and International Nucleotide Sequence Database Collaboration (INSDC)-compliant flat files, as well as comprehensive JSON files, facilitating automated downstream analysis. We compared Bakta to other rapid contemporary command-line annotation software tools in both targeted and taxonomically broad benchmarks including isolates and metagenomic-assembled genomes. We demonstrated that Bakta outperforms other tools in terms of functional annotations, the assignment of functional categories and database cross-references, whilst providing comparable wall-clock runtimes. Bakta is implemented in Python 3 and runs on MacOS and Linux systems. It is freely available under a GPLv3 license at https://github.com/oschwengers/bakta. An accompanying web version is available at https://bakta.computational.bio.
Proteomics of spatially identified tissues in whole organs
Authors: Bhatia Harsharan Singh , Brunner Andreas-David , Rong Zhouyi , Mai Hongcheng , Thielert Marvin , Al-Maskari Rami , Paetzold Johannes Christian , Kofler Florian , Todorov Mihail Ivilinov , Ali Mayar , Molbay Muge , Kolabas Zeynep Ilgin , Kaltenecker Doris , Müller Stephan , Lichtenthaler Stefan F. , Menze Bjoern H. , Theis Fabian J. , Mann Matthias , Ertürk Ali
Published: November 2021

Spatial molecular profiling of complex tissues is essential to investigate cellular function in physiological and pathological states. However, methods for molecular analysis of biological specimens imaged in 3D as a whole are lacking. Here, we present DISCO-MS, a technology combining whole-organ imaging, deep learning-based image analysis, and ultra-high sensitivity mass spectrometry. DISCO-MS yielded qualitative and quantitative proteomics data indistinguishable from uncleared samples in both rodent and human tissues. Using DISCO-MS, we investigated microglia activation locally along axonal tracts after brain injury and revealed known and novel biomarkers. Furthermore, we identified initial individual amyloid-beta plaques in the brains of a young familial Alzheimer’s disease mouse model, characterized the core proteome of these aggregates, and highlighted their compositional heterogeneity. Thus, DISCO-MS enables quantitative, unbiased proteome analysis of target tissues following unbiased imaging of entire organs, providing new diagnostic and therapeutic opportunities for complex diseases, including neurodegeneration.

Graphical Abstract

Highlights

DISCO-MS combines tissue clearing, whole-organ imaging, deep learning-based image analysis, and ultra-high sensitivity mass spectrometry

DISCO-MS yielded qualitative and quantitative proteomics data indistinguishable from fresh tissues

DISCO-MS enables identification of rare pathological regions & their subsequent molecular analysis

DISCO-MS revealed core proteome of plaques in 6 weeks old Alzheimer‘s disease mouse model Supplementary Video can be seen at: http://discotechnologies.org/DISCO-MS/
Fast and robust imputation for miRNA expression data using constrained least squares
Authors: Webber James W. , Elias Kevin M.
Published: November 2021

High dimensional transcriptome profiling, whether through next generation sequencing techniques or high-throughput arrays, may result in scattered variables with missing data. Data imputation is a common strategy to maximize the inclusion of samples by using statistical techniques to fill in missing values. However, many data imputation methods are cumbersome and risk introduction of systematic bias. Here we present a new data imputation method using constrained least squares and algorithms from the inverse problems literature and present applications for this technique in miRNA expression analysis. The proposed technique is shown to offer an imputation orders of magnitude faster, with greater than or equal accuracy when compared to similar methods from the literature.
Expression of coffee florigenCaFT1reveals a sustained floral induction window associated with asynchronous flowering in tropical perennials
Authors: Cardon Carlos Henrique , de Oliveira Raphael Ricon , Lesy Victoria , Cherubino Ribeiro Thales Henrique , Fust Catherine , Pereira Luísa Peloso , Colasanti Joseph , Chalfun-Junior Antonio
Published: November 2021

The behavior of florigen(s) and environment-influenced regulatory pathways that control flowering in tropical perennials with complex phenological cycles is poorly understood. Understanding the mechanisms underlying this process is important for food production in the face of climate change. To explore this, homologs ofArabidopsisflorigenFLOWERING LOCUS T(CaFT1) and environment-related regulatorsCONSTANS(CO), PHYTOCHROME INTERACTING FACTOR 4(PIF4) andFLOWERING LOCUS C(FLC) were isolated fromCoffeasp. L. (Rubiaceae). Overexpression ofCaFT1inArabidopsisshowed typical early-flowering and yeast two hybrid studies indicated CaFT1 binding to bZIP floral regulator, FD, demonstrates that CaFT1 is a coffee orthologue of florigen. Expression ofCaFT1and floral regulators were evaluated over one year using three contrasting genotypes: twoC. arabicaand oneC. canephora. All genotypes showed activeCaFT1transcription from February until October, indicating a potential window for floral induction.CaCOexpression, as expected, varied over the day period and monthly with day length, whereas expression of temperature-responsive homologs,CaFLCandCaPIF4, did not correlate with temperature changes. Using coffee as a model, we suggest a continuum of floral induction that allows different starting points for floral activation, which explains developmental asynchronicity and prolonged anthesis events in tropical perennial species.

Highlight

Coffee florigen CaFT1 and related regulators revealed an extended floral induction window clarifying the asynchronicity and influence of environment for flowering in tropical perennial crops, providing perspectives to its control.
Coronavirus RNA synthesis takes place within membrane-bound sites
Authors: Doyle Nicole , Simpson Jennifer , Hawes Philippa C , Maier Helena J
Published: November 2021

Infectious bronchitis virus (IBV), a gammacoronavirus, is an economically important virus to the poultry industry as well as a significant welfare issue for chickens. As for all positive strand RNA viruses, IBV infection causes rearrangements of the host cell intracellular membranes to form replication organelles. Replication organelle formation is a highly conserved and vital step in the viral life cycle. Here, we investigate the localization of viral RNA synthesis and the link with replication organelles in host cells. We have shown that sites of viral RNA synthesis and virus-related dsRNA are associated with one another and, significantly, that they are located within a membrane-bound compartment within the cell. We have also shown that some viral RNA produced early in infection remains within these membranes throughout infection. Importantly, we demonstrate conservation across all four coronavirus genera, including SARS-CoV-2. Under-standing more about the replication of these viruses is imperative in order to effectively find ways to control them.
Conservation of the direct and indirect pathways dichotomy in mouse caudal striatum with uneven distribution of dopamine receptor D1- and D2-expressing neurons
Authors: Ogata Kumiko , Kadono Fuko , Hirai Yasuharu , Inoue Ken-ichi , Takada Masahiko , Karube Fuyuki , Fujiyama Fumino
Published: November 2021

The striatum is one of the key nuclei for adequate control of voluntary behaviors and reinforcement learning. Two striatal projection neuron types, expressing either dopamine receptor D1 (D1R) or dopamine receptor D2 (D2R) constitute two independent output routes: the direct or indirect pathways, respectively. These pathways co-work in balance to achieve coordinated behavior. Two projection neuron types are equivalently intermingled in most striatal space. However, recent studies revealed two atypical zones in the caudal striatum: the zone in which D1R-neurons are the minor population (D1R-poor zone) and that in which D2R-neurons are the minority (D2R-poor zone). It remains obscure as to whether these imbalanced zones have similar properties on axonal projections and electrophysiology compared to other striatal regions. Based on morphological experiments in mice using immunofluorescence, in situ hybridization, and neural tracing, here, we revealed that the poor zones densely projected to the globus pallidus and substantia nigra pars lateralis, with a few collaterals in substantia nigra pars reticulata and compacta. Similar to that in other striatal regions, D1R-neurons were the direct pathway neurons. We also showed membrane properties of projection neurons in the poor zones were largely similar to those in the conventional striatum using in vitro electrophysiological recording. In addition, the poor zones existed irrespective of the age or sex of mice. We also identified the poor zones in the common marmoset as well as other rodents. These results suggest that the poor zones in the caudal striatum follow the conventional projection patterns irrespective of imbalanced distribution of projection neurons. The poor zones could be an innate structure and common in mammals. The unique striatal zones possessing highly restricted projections could relate to functions different from those of motor-related striatum.
Augmenting Recombinant Antibody Production in HEK293E Cells: Optimising Transfection and Culture Parameters
Authors: Heng Zealyn Shi-Lin , Yeo Joshua Yi , Koh Darius Wen-Shuo , Gan Samuel Ken-En , Ling Wei-Li
Published: November 2021

Background

Optimising recombinant antibody production is important for cost-effective therapeutics and diagnostics. With impact on commercialisation, higher productivity beyond laboratory scales is highly sought, where efficient production can also accelerate antibody characterisations and investigations.

Methods

Investigating HEK293E cells for mammalian antibody production, various transfection and culture parameters were systematically analysed for antibody light chain production before evaluating them for whole antibody production. Transfection parameters investigated include seeding cell density, the concentration of the transfection reagent and DNA, complexation time, temperature, and volume, as well as culture parameters such as medium replacement, serum deprivation, use of cell maintenance antibiotic, incubation temperature, medium volume, post-transfection harvest day and common nutrient supplements.

Results

Using 2 mL adherent HEK293E cell culture transfections with 25 kDa linear Polyethylenimine in the most optimised parameters, we demonstrated a ~2-fold production increase for light chain alone and for whole antibody production reaching 536 and 49 μg respectively in a cost-effective manner. With the addition of peptone, κ light chain increased by ~4-fold to 1032 μg while whole antibody increased to a lesser extent by ~2.5-fold to 51 μg, with benefits potentially for antibodies limited by their light chains in production.

Conclusions

Our optimised findings show promise for a more efficient and convenient antibody production method through transfection and culture optimisations that can be incorporated to scale up processes and with potential transferability to other mammalian-based recombinant protein production using HEK293E cells.

Statement of Significance

Recombinant antibody production is crucial for antibody research and development. Systematically investigating transfection and culture parameters such as PEI/DNA concentrations, complexation time, volume, and temperature, supplements, etc., we demonstrated a ~4-fold light chain alone production increase to 1032 μg and a 2.5-fold whole antibody production increase to 51 μg from 2 mL transfections.
Comparisons of ribonuclease HI homologs and mutants uncover a multi-state model for substrate recognition
Authors: Martin James A. , Palmer Arthur G.
Published: November 2021

Ribonuclease HI (RNHI) non-specifically cleaves the RNA strand in RNA:DNA hybrid duplexes in a myriad of biological processes, including retroviral reverse transcription. Several RNHI homologs contain an extended domain, termed the handle region, that is critical to substrate binding. Nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations have suggested a kinetic model in which the handle region can exist in open (substrate-binding competent) or closed (substrate-binding incompetent) states in homologs containing arginine or lysine at position 88 (using sequence numbering of E. coli RNHI), while the handle region populates a state intermediate between the open and closed conformers in homologs with asparagine at residue 88 [Stafford, K. A., et al., PLoS Comput. Biol.2013, 9, 1-10]. NMR parameters characterizing handle region dynamics are highly correlated with enzymatic activity for RNHI homologs with two-state (open/closed) handle regions [Martin, J. A., et al., Biochemistry2020, 59, 3201-3205]. The work presented herein shows that homologs with one-state (intermediate) handle regions display distinct structural features compared with their two-state counterparts. Comparisons of RNHI homologs and site-directed mutants with arginine at position 88 support a kinetic model for handle region dynamics that includes 12 unique transitions between eight conformations. Overall, these findings present an example of the structure-function relationships of enzymes and spotlight the use of NMR spectroscopy and MD simulations in uncovering fine details of conformational preferences.
Rapid quantification of tissue perfusion properties with a two-stage look-up table: a simulation study
Authors: Yang Bin
Published: November 2021

Tissue perfusion properties reveal crucial information pertinent to clinical diagnosis and treatment. Multispectral spatial frequency domain imaging (SFDI) is an emerging imaging technique that has been widely used to quantify tissue perfusion properties. However, slow processing speed limits its usefulness in real-time imaging applications. In this study, we present a two-stage look-up table (LUT) approach that accurately and rapidly quantifies optical (absorption and reduced scattering maps) and perfusion (total hemoglobin and oxygen saturation maps) properties using stage-1 and stage-2 LUTs, respectively, based on reflectance images at 660nm and 850nm. The two-stage LUT can be implemented on both CPU and GPU computing platforms. Quantifying tissue perfusion properties using the simulated diffuse reflectance images, we achieved a quantification speed of 266, 174, and 74 frames per second for three image sizes 512×512, 1024×1024, and 2048×2048 pixels, respectively. Quantification of tissue perfusion properties was highly accurate with only 3.5% and 2.5% error for total hemoglobin and oxygen saturation quantification, respectively. The two-stage LUT has the potential to be adopted in existing SFDI applications to enable real-time imaging capability of tissue hemodynamics.
CRISPR/Cas13 effectors have differing extents of off-target effects that limit their utility in eukaryotic cells
Authors: Ai Yuxi , Liang Dongming , Wilusz Jeremy E.
Published: November 2021

CRISPR/Cas13 effectors have garnered increasing attention as easily customizable tools for detecting and depleting RNAs of interest. Near perfect complementarity between a target RNA and the Cas13-associated guide RNA is required for activation of Cas13 ribonuclease activity. Nonetheless, the specificity of Cas13 effectors in eukaryotic cells has been debated as the Cas13 nuclease domains can be exposed on the enzyme surface, providing the potential for promiscuous cleavage of nearby RNAs (so-called collateral damage). Here, using co-transfection assays in Drosophila and human cells, we found that the off-target effects of RxCas13d, a commonly used Cas13 effector, can be as strong as the level of on-target RNA knockdown. The extent of off-target effects is positively correlated with target RNA expression levels, and collateral damage can be observed even after reducing RxCas13d/guide RNA levels. The PspCas13b effector showed improved specificity and, unlike RxCas13d, can be used to deplete a Drosophila circular RNA without affecting the expression of the associated linear RNA. PspCas13b nonetheless still can have off-target effects and we notably found that the extent of off-target effects for Cas13 effectors differs depending on the cell type and target RNA examined. In total, these results highlight the need for caution when designing and interpreting Cas13-based knockdown experiments.
Mutant-SETBP1 activates transcription of Myc programs to accelerate CSF3R-driven myeloproliferative neoplasms
Authors: Carratt Sarah A. , Kong Garth L. , Smith Brittany M. , Schonrock Zachary , Maloney Lauren , Baris Adrian , Druker Brian J. , Braun Theodore P. , Maxson Julia E.
Published: November 2021

Colony stimulating factor 3 receptor (CSF3R) mutations lead to JAK pathway activation and are the molecular hallmark of chronic neutrophilic leukemia (CNL). Approximately half of CNL patients also have mutations in SET binding protein 1 (SETBP1). In this study, we developed models of SETBP1-mutant leukemia to understand the role that SETBP1 plays in CNL. SETBP1 mutations promote self-renewal of CSF3R-mutant hematopoietic progenitors in vitro and prevent cells from undergoing terminal differentiation. In vivo, SETBP1 mutations accelerate leukemia progression, leading to the rapid development of hepatosplenomegaly and granulocytosis. Through transcriptomic and epigenomic profiling, we found that SETBP1 enhances progenitor-associated programs—most strongly upregulating Myc and Myc target genes. This upregulation of Myc can be reversed by epigenetic modulatory drugs. In summary, we find that SETBP1 mutations promote aggressive hematopoietic cell expansion when expressed with mutant CSF3R through the upregulation of Myc-associated gene expression programs.

Statement of Significance

SETBP1 is frequently mutated in chronic neutrophilic leukemia, but its role in the biology of this disease is unclear. We find that mutant SETBP1 enhances transcription of Myc and Myc target genes to promote aggressive disease biology, and that these oncogenic transcriptional programs can be reversed by epigenetic modulatory drugs.
Raman microspectroscopy imaging analysis of extracellular vesicles (EVs) biogenesis by filamentous fungusPenicilium chrysogenum
Authors: Samuel Ashok Zachariah , Horii Shumpei , Nakashima Takuji , Shibata Naoko , Ando Masahiro , Takeyama Haruko
Published: November 2021

Mechanism of production of extracellular vesicles (EVs) and their molecular contents are of great interest owing to their diverse roles in biological systems and are far from being completely understood. Even though, cellular cargo release mediated by EVs have been demonstrated in several cases, their role in secondary metabolite production and release remains elusive. In this study we investigate this aspect in detail using Raman micro-spectroscopic imaging. We provide considerable evidence to suggest that the release of antibiotic penicillin by filamentous fungusPenicillium chrysogenuminvolves EVs. Morphological modifications of the fungal body during biogenesis, changes in cell composition at the locus of biogenesis, and major molecular contents of the released EVs are also revealed in this study.

Importance

Extracellular vesicles (EVs) play a key role in cellular communications. EVs role in functioning of fungi are relatively less explored. Here we show selective enrichment of chemical contents at certain locations of mycelium ofP. chrysogenumforming protruding regions. Secondary metabolite penicillin is excessively localized in them. We provide evidence to show that EVs are released from these protrusions. Raman imaging has been applied for molecular profiling of the mycelium and for characterizing chemical contents of the EVs. Our study suggests a possible general role of EVs in the release of antibiotics from the producing organisms.
Shank promotes action potential repolarization by recruiting BK channels to calcium nanodomains
Authors: Gao Luna , Zhao Jian , Ardiel Evan , Hall Qi , Nurrish Stephen , Kaplan Joshua M.
Published: November 2021

Mutations altering the scaffolding protein Shank are linked to several psychiatric disorders, and to synaptic and behavioral defects in mice. Among its many binding partners, Shank directly binds CaV1 voltage activated calcium channels. Here we show that the C. elegans SHN-1/Shank promotes CaV1 coupling to calcium activated potassium channels. Mutations inactivating SHN-1, and those preventing SHN-1 binding to EGL-19/CaV1 all increase action potential durations in body muscles. Action potential repolarization is mediated by two classes of potassium channels: SHK-1/KCNA and SLO-1 and SLO-2 BK channels. BK channels are calcium-dependent, and their activation requires tight coupling to EGL-19/CaV1 channels. SHN-1’s effects on AP duration are mediated by changes in BK channels. In shn-1 mutants, SLO-2 currents and channel clustering are significantly decreased in both body muscles and neurons. Finally, increased and decreased shn-1 gene copy number produce similar changes in AP width and SLO-2 current. Collectively, these results suggest that an important function of Shank is to promote nanodomain coupling of BK with CaV1.
Crystal structure and self-assembly on graphite of a pyrazolo[1,5-c]pyrimidine derivative
Authors: Trivedi Laxmikant , Gupta Kratika , Mishra Vipin , Gopakumar Thiruvancheril G. , Gupta Atul , Vasudev Prema G.
Published: November 2021

The crystal structure of the heterocyclic compound 2-(4-methoxyphenyl)-7-phenylpyrazolo[1,5-c]pyrimidine, C19H15N3O, has been determined and its self-assembly on the surface of graphite has been examined using atomic force microscopy (AFM). The title compound crystallized in the monoclinic space group P21/c, with two independent molecules in the asymmetric unit. The packing of the L-shaped molecules in the crystal is governed by arene interactions, in the absence of any conventional hydrogen-bonding interactions. The packing arrangement reveals four types of dimeric motifs stabilized by π–π and C—H...π interactions. At low coverage, molecules assemble into long needle-like islands on the graphite surface. High-resolution AFM images reveal that the molecules interact through weak noncovalent interactions between the aromatic H atoms and the methoxy O atoms.
A homochiral coordination polymer of cobalt(II) and L-serine
Authors: Dzesse T. Christelle N. , Majoumo-Mbe Felicite , Nfor Emmanuel N. , Bourne Susan A.
Published: November 2021

A one-dimensional chiral cobalt(II) coordination polymer, namely, catena-poly[[[(S)-2-amino-3-hydroxypropanoato-κ2
N,O
1]cobalt(II)]-μ-(S)-2-amino-3-hydroxypropanoato-κ4
O
1,O
3:N,O
1′], [Co(C3H6NO3)2]
n
or Δ-[Co(L-Ser-κ2
N,O)2]
n
(L-Ser = L-serine) (1), has been synthesized and characterized using elemental and thermal analyses, IR spectroscopy and single-crystal and powder X-ray diffraction techniques. The asymmetric unit of 1 consists of two serine anions which are coordinated to a Co2+ ion to give three chelate rings. These extend the structure into a helical chain with pendant chelate rings which participate in interchain hydrogen bonding. The ability of 1 to undergo transmetallation was evaluated. Among a range of divalent metal ions, only copper(II) partially replaced cobalt(II).
Crystal structure of (+)-(1S,5S,6S,7S,10S,11S,16S)-16-hydroxy-7-(methoxymethoxy)-11,15,18,18-tetramethyl-3,13-dioxo-2,4-dioxatetracyclo[12.3.1.01,5.06,11]octadec-14-en-10-yl benzoate
Authors: Oishi Takeshi , Fukaya Keisuke , Sato Takaaki , Chida Noritaka
Published: November 2021

In the fused tetracyclic system of the title compound, C29H36O9, the five-membered dioxolane ring adopts a twist conformation; the two adjacent C atoms deviate alternately from the mean plane of the other three atoms by −0.252 (6) and 0.340 (6) Å. The cyclohexane, cyclohexene and central cyclooctane rings show chair, half-chair and boat-chair forms, respectively. There are three intramolecular C—H...O interactions supporting the molecular conformation, with one S(6) and two S(7) graph-set motifs. In the crystal, intermolecular O—H...O hydrogen bonds connect the molecules into a helical chain running along the c-axis direction, generating a C(7) graph-set motif. The chains are further linked by intermolecular C—H...O interactions to construct a three-dimensional network. There is no valid C—H...π interaction.