bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isolation, purification and PEG-mediated transient expression of mesophyll protoplasts in Camellia oleifera
Authors: Li Sufang , Zhao Rui , Ye Tianwen , Guan Rui , Xu Linjie , Ma Xiaoling , Zhang Jiaxi , Xiao Shixin , Yuan Deyi 
Published: December 2022

Abstract
Background
Camellia oleifera (C. oleifera) is a woody edible oil crop of great economic importance. Because of the lack of modern biotechnology research, C. oleifera faces huge challenges in both breeding and basic research. The protoplast and transient transformation system plays an important role in biological breeding, plant regeneration and somatic cell fusion. The objective of this present study was to develop a highly efficient protocol for isolating and purifying mesophyll protoplasts and transient transformation of C. oleifera. Several critical factors for mesophyll protoplast isolation from C. oleifera, including starting material (leaf age), pretreatment, enzymatic treatment (type of enzyme, concentration and digestion time), osmotic pressure and purification were optimized. Then the factors affecting the transient transformation rate of mesophyll protoplasts such as PEG molecular weights, PEG4000 concentration, plasmid concentration and incubation time were explored.

Results
The in vitro grown seedlings of C. oleifera ‘Huashuo’ were treated in the dark for 24 h, then the 1st to 2nd true leaves were picked and vacuumed at − 0.07 MPa for 20 min. The maximum yield (3.5 × 107/g·FW) and viability (90.9%) of protoplast were reached when the 1st to 2nd true leaves were digested in the enzymatic solution containing1.5% (w/v) Cellulase R-10, 0.5% (w/v) Macerozyme R-10 and 0.25% (w/v) Snailase and 0.4 M mannitol for 10 h. Moreover, the protoplast isolation method was also applicable to the other two cultivars, the protoplast yield for ‘TXP14’ and ‘DP47’ was 1.1 × 107/g·FW and 2.6 × 107/g·FW, the protoplast viability for ‘TXP14’ and ‘DP47’ was 90.0% and 88.2%. The purification effect was the best when using W buffer as a cleaning agent by centrifugal precipitation. The maximum transfection efficiency (70.6%) was obtained with the incubation of the protoplasts with 15 µg plasmid and 40% PEG4000 for 20 min.

Conclusion
In summary, a simple and efficient system for isolation and transient transformation of C. oleifera mesophyll protoplast is proposed, which is of great significance in various aspects of C. oleifera research, including the study of somatic cell fusion, genome editing, protein function, signal transduction, transcriptional regulation and multi-omics analyses.
Effects of cushion box and closed let-down ladder usage on damage to corn during handling: physiological deterioration
Authors: Shahbazi Reza , Shahbazi Feizollah 
Published: December 2022

Abstract
Background
Corn seeds have a high susceptibility to mechanical damage due to their large size and mass. The main purpose of this study was to evaluate the effects of the cushion box and closed let-down ladder usage in minimizing the negative influence of the free fall on the storage potential of corn seeds. Corn seeds were evaluated for the extent of physiological damage by measuring the seed deterioration by the accelerated aging test (percentage loss in germination in the accelerated aging test), using three drop methods (free fall, with cushion box, and with closed let-down ladder) at three drop heights (5, 10, and 15 m) and five different moisture contents (10, 15, 20 and 25%).

Results
The drop methods had a significant effect on the storage potential of corn seeds. Sample seeds dropped without a ladder (free fall) had a significantly higher average physiological quality loss of 13.87% (loss in accelerated aging germination). In the use of the cushion box, the average percentage loss in germination was calculated to be 11.38%, which was decreased by about 18% more than the free fall. Sample seeds dropped with the closed let-down ladder had a lower average percentage loss in the germination of 8.78%, which showed that the closed let-down ladder significantly helped to reduce mechanical damage to corn seeds by about 37% comparing free fall and by about 23% to the use of the cushion box. The amounts of loss in physiological quality of corn seeds increased significantly with increasing drop height and moisture content, but the use of the cushion box and closed let-down ladder systems somewhat reduced the adverse effect of the above factors. Empirical models were developed for the dependency of physiological damage to corn seeds due to the impact caused by free fall, on the drop height and moisture content at different drop methods.

Conclusions
To minimize mechanical damage to seeds as they fall into the bin, a let-down ladder should be installed in the bin so that it can receive seeds from the filling spout with minimum damage.
Identification of an emerging cucumber virus in Taiwan using Oxford nanopore sequencing technology
Authors: Dong Zi-Xuan , Lin Chian-Chi , Chen Yuh-Kun , Chou Chia-Cheng , Chen Tsung-Chi 
Published: December 2022

Abstract
Background
In June 2020, severe symptoms of leaf mosaic and fruit malformation were observed on greenhouse-grown cucumber plants in Xizhou Township of Changhua County, Taiwan. An unknown virus, designated CX-2, was isolated from a diseased cucumber sample by single lesion isolation on Chenopodium quinoa leaves. Identification of CX-2 was performed. Moreover, the incidence of cucumber viruses in Taiwan was also investigated.

Methods
Transmission electron microscopy was performed to examine virion morphology. The portable MinION sequencer released by Oxford Nanopore Technologies was used to detect viral sequences in dsRNA of CX-2-infected leaf tissue. The whole genome sequence of CX-2 was completed by Sanger sequencing and analyzed. Reverse transcription-polymerase chain reaction (RT-PCR) with species-specific primers and indirect enzyme-linked immunosorbent assay (ELISA) with anti-coat protein antisera were developed for virus detection in the field [see Additional file 1].

Results
Icosahedral particles about 30 nm in diameter were observed in the crud leaf sap of CX-2-infected C. quinoa plant. The complete genome sequence of CX-2 was determined as 4577 nt long and shared 97.0–97.2% of nucleotide identity with that of two cucumber Bulgarian latent virus (CBLV) isolates in Iran and Bulgaria. Therefore, CX-2 was renamed CBLV-TW. In 2020–2022 field surveys, melon yellow spot virus (MYSV) had the highest detection rate of 74.7%, followed by cucurbit chlorotic yellows virus (CCYV) (32.0%), papaya ringspot virus virus watermelon type (PRSV-W) (10.7%), squash leaf curl Philippines virus (SLCuPV) (9.3%), CBLV (8.0%) and watermelon silver mottle virus (WSMoV) (4.0%). Co-infection of CBLV and MYSV could be detected in field cucumbers.

Conclusion
The emerging CBLV-TW was identified by nanopore sequencing. Whole genome sequence analysis revealed that CBLV-TW is closely related, but phylogenetically distinct, to two known CBLV isolates in Bulgaria and Iran. Detection methods including RT-PCR and indirect ELISA have been developed to detect CBLV and to investigate cucumber viruses in central Taiwan. The 2020–2022 field survey results showed that MYSV and CCYV were the main threats to cucumbers, with CBLV, SLCuPV and WSMoV were occasionally occurring.
Impact of the COVID-19 pandemic on alcohol or drug use symptoms and service need among youth: a cross-sectional sample from British Columbia, Canada
Authors: Marchand Kirsten , Liu Guiping , Mallia Emilie , Ow Nikki , Glowacki Krista , Hastings Katherine G. , Mathias Steve , Sutherland Jason M. , Barbic Skye 
Published: December 2022

Abstract
Background
Concerns about youth alcohol and drug use have risen since the declaration of the global COVID-19 pandemic due to the pandemic’s impact on known risk and protective factors for substance use. However, the pandemic’s immediate and long-term impact on youths’ substance use patterns has been less clear. Thus, this study sought to determine how the COVID-19 pandemic impacted alcohol or drug use and its risk and protective factors among youth accessing integrated youth services.

Methods
We conducted a repeated cross-sectional study of patient-reported outcomes data collected between May 2018 and February 2022 among youth (n = 6022) ages 10–24 accessing a provincial network of integrated youth services in Canada. The main exposure of interest was the COVID-19 pandemic (March 2020 – February 2022) compared with a pre-pandemic period (May 2018 – February 2020). As measured by the Global Appraisal of Individual Needs – Short Screener, outcomes included the average number of past month alcohol or drug use symptoms and past month likelihood of service need for alcohol/drug use (moderate/high vs. low need). Interrupted time series (ITS) examined change in average monthly alcohol/drug use symptoms between the pre- and pandemic periods. Stratified multivariable logistic regression investigated how the pandemic modified the effects of established risk/protective factors on likelihood of alcohol/drug use service need.

Results
Fifty-percent of youth met the criteria for moderate/high likelihood of alcohol/drug use service need, with the odds being 2.39 times (95% confidence interval = 2.04, 2.80) greater during the pandemic compared to the pre-pandemic period. Results from the ITS indicated significant immediate effects of the pandemic on monthly substance use symptoms (p = 0.01). Significant risk/protective factors for service need included exposure to violence, engagement in meaningful activities, and self-rated physical and mental health; and the direction of their effects remained consistent across pandemic and pre-pandemic periods.

Conclusions
This study demonstrated that the COVID-19 pandemic corresponded with increased alcohol or drug use among youth accessing integrated services. This signals an urgent need for increased clinical capacity in existing youth services and policies that can respond to risk/protective factors for substance use earlier.
Saturation-scale functional evidence supports clinical variant interpretation in Lynch syndrome
Authors: Scott Anthony , Hernandez Felicia , Chamberlin Adam , Smith Cathy , Karam Rachid , Kitzman Jacob O. 
Published: December 2022

Abstract
Background
Lynch syndrome (LS) is a cancer predisposition syndrome affecting more than 1 in every 300 individuals worldwide. Clinical genetic testing for LS can be life-saving but is complicated by the heavy burden of variants of uncertain significance (VUS), especially missense changes.

Result
To address this challenge, we leverage a multiplexed analysis of variant effect (MAVE) map covering >94% of the 17,746 possible missense variants in the key LS gene MSH2. To establish this map’s utility in large-scale variant reclassification, we overlay it on clinical databases of >15,000 individuals with LS gene variants uncovered during clinical genetic testing. We validate these functional measurements in a cohort of individuals with paired tumor-normal test results and find that MAVE-based function scores agree with the clinical interpretation for every one of the MSH2 missense variants with an available classification. We use these scores to attempt reclassification for 682 unique missense VUS, among which 34 scored as deleterious by our function map, in line with previously published rates for other cancer predisposition genes. Combining functional data and other evidence, ten missense VUS are reclassified as pathogenic/likely pathogenic, and another 497 could be moved to benign/likely benign. Finally, we apply these functional scores to paired tumor-normal genetic tests and identify a subset of patients with biallelic somatic loss of function, reflecting a sporadic Lynch-like Syndrome with distinct implications for treatment and relatives’ risk.

Conclusion
This study demonstrates how high-throughput functional assays can empower scalable VUS resolution and prospectively generate strong evidence for variant classification.
Advances and opportunities in integrating economic and environmental performance of renewable products
Authors: Meramo Samir , Fantke Peter , Sukumara Sumesh 
Published: December 2022

AbstractThere is a growing global need to transition from a fossil-based to a bio-based economy to produce fuels, chemicals, food, and materials. In the specific context of industrial biotechnology, a successful transition toward a sustainable development requires not only steering investment toward a bioeconomy, but also responsibly introducing bio-based products with lower footprints and competitive market prices. A comprehensive sustainability assessment framework applied along various research stages to guide bio-based product development is urgently needed but currently missing. To support holistic approaches to strengthen the global bioeconomy, the present study discusses methodologies and provides perspectives on the successful integration of economic and environmental performance aspects to guide product innovation in biotechnology. Efforts on quantifying the economic and environmental performance of bio-based products are analyzed to highlight recent trends, challenges, and opportunities. We critically analyze methods to integrate Techno-Economic Assessment (TEA) and Life Cycle Assessment (LCA) as example tools that can be used to broaden the scope of assessing biotechnology systems performance. We highlight the lack of social assessment aspects in existing frameworks. Data need for jointly applying TEA and LCA of succinic acid as example commodity chemical are assessed at various Technology readiness levels (TRLs) to illustrate the relevance of the level of integration and show the benefits of the use of combined assessments. The analysis confirms that the implementation of integrated TEA and LCA at lower TRLs will provide more freedom to improve bio-based product’s sustainability performance. Consequently, optimizing the system across TRLs will guide sustainability-driven innovation in new biotechnologies transforming renewable feedstock into valuable bio-based products.
Evolutionary trade-offs associated with copy number variations in resistance alleles in Culex pipiens mosquitoes
Authors: Milesi Pascal , Claret Jean-Loup , Unal Sandra , Weill Mylène , Labbé Pierrick 
Published: December 2022

AbstractOrganophosphate and carbamate insecticides have largely been used worldwide to control mosquito populations. As a response, the same amino acid substitution in theace-1gene (G119S), conferring resistance to both insecticides, has been selected independently in many mosquito species. InAnopheles gambiae, it has recently been shown that the G119S mutation is actually part of homogeneous duplications that associate multiple resistance copies of theace-1gene. In this study, we showed that duplications of resistance copies of theace-1gene also exist in theCulex pipiensspecies complex. The number of copies is variable, and different numbers of copies are associated with different phenotypic trade-offs: we used a combination of bioassays and competition in population cages to show that having more resistance copies conferred higher resistance levels, but was also associated with higher selective disadvantage (or cost) in the absence of insecticide. These results further show the versatility of the genetic architecture of resistance to organophosphate and carbamate insecticides around theace-1locus and its role in fine-tuned adaptation to insecticide treatment variations.Graphical Abstract
The presence of SARS-CoV-2 in multiple clinical specimens of a fatal case of COVID-19: a case report
Authors: Sokolovska Liba , Terentjeva-Decuka Anna , Cistjakovs Maksims , Nora-Krukle Zaiga , Gravelsina Sabine , Vilmane Anda , Vecvagare Katrine , Murovska Modra 
Published: December 2022

Abstract
Background
The risk of developing severe and even fatal coronavirus disease 2019 (COVID-19) increases with various factors such as advanced age and chronic diseases, especially those treated with immunosuppressive drugs. Viral ribonucleic acid (RNA) and viral load detection in extra-pulmonary specimens have been proposed to indicate disease severity.

Case presentation
Here we describe a fatal COVID-19 case of an 83-year-old Caucasian male patient with various underlying comorbidities, including cardiovascular and autoimmune disorders, as well as immunosuppression due to lymphoma treatment. Upon admission, the patient was radiologically diagnosed with severe COVID-19. The patient was febrile and presented with diarrhea, continued dyspnea, tachypnea, and low blood oxygen saturation, treated with high-concentration oxygen supplementation and antibacterial therapy. Overall the patient was treated for COVID-19 for 19 days. Blood tests were performed upon admission, on the fifth, 10th, 13th, and 19th day. In addition, nasopharyngeal swab, blood, urine, and fecal samples were collected from the patient on the 14th day for virological and immunological investigations. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was detectable in all samples collected from this patient, including blood plasma and peripheral blood mononuclear cells (PBMC), with very high viral loads. However, neither virus-specific IgA, IgM, nor IgG antibodies were detectable.

Conclusions
The various cardiovascular, autoimmune, and oncological disorders, advanced age, and the high levels of inflammatory markers predisposed the patient to severe COVID-19 and determined the fatal outcome of the disease. We believe that the multiple specimen SARS-CoV-2 positivity and extremely high viral loads in nasopharyngeal swab and fecal samples to be the result of COVID-19 severity, the inability of viral clearance and weakened immune response due to advanced age, comorbidities, and the presence of non-Hodgkin's lymphoma and the immunosuppressive treatment for it, highlighting the risks of COVID-19 in such patients.
Leaves litterfall and nutrient inputs from four multipurpose tree/shrub species of homegarden agroforestry systems
Authors: Kassa Gezahegn , Bekele Tamrat , Demissew Sebsebe , Abebe Tesfaye 
Published: December 2022

AbstractBackgroundAs compared to natural forest ecosystems, soils under agroecosystems are more subjected to changes in physical and biochemical attributes. But, tree species that are retained and/or planted on these agroecosystems replenish soil fertility through the influence of leaves litterfall quantity and quality. Tree species-related differences in the quantity and seasonality of leaves litterfall and the associated amounts of annual returns of nutrient elements were assessed from four multipurpose tree/shrub species (Erythrina bruceiSchweinf,Cordia africanaLam.,Grevillea robustaR. Br. andMalus sylvestrisMill.) in homegarden agroforestry systems. Five replicates of two leaf litterfall traps per species were randomly positioned beneath the subcanopy area for 12 months, totaling 40 litterfall traps distributed among the four species.ResultsThe results of this study suggested that total annual leaves litterfall varied significantly among species (ranging from 171.18 to 962 kg ha− 1), with the combined per tree mean monthly (28.13 kg tree− 1), per tree annual (331.10 kg tree− 1 year− 1) and per area basis total annual (9439.33 kg ha− 1) leaves litterfall production. A clear seasonal leaves litterfall pattern with varying peaks was observed for the multipurpose tree/shrub species in the present study, with a bimodal distribution pattern forE. bruceiandC. africanaand a unimodal distribution pattern forG. robustaandM. sylvestris. Similarly, both the concentrations and inputs of nutrients from the leaves᾽ litterfall showed considerable variations among species (p < 0.05). Among the investigated species,C. africanaandE. bruceiwere found to be the best in terms of carbon (C) and nitrogen (N) return,G. robustathe best in terms of phosphorous (P) return, andC. africanaas well best for potassium (K) return. The combined mean annual nutrient inputs recorded for all the species were 3125.32 kg C ha− 1 year− 1, 149.70 kg N ha− 1 year− 1, 54.86 kg K ha− 1 year− 1, and 3.56 kg P ha− 1 year− 1.ConclusionsLeaves litterfall amount differed among tree species as influenced by tree species characteristics (canopy area, diameter at breast height (DBH), height), but no cause-effect relationship with age and climate (monthly precipitation and temperature).
Lithium induced hypercalcemia: an expert opinion and management algorithm
Authors: Kovacs Zoltan , Vestergaard Peter , W. Licht Rasmus , P. V. Straszek Sune , Hansen Anne Sofie , H. Young Allan , Duffy Anne , Müller-Oerlinghausen Bruno , Seemueller Florian , Sani Gabriele , Rubakowski Janusz , Priller Josef , Vedel Kessing Lars , Tondo Leonardo , Alda Martin , Manchia Mirko , Grof Paul , Ritter Phillip , Hajek Tomas , Lewitzka Ute , Bergink Veerle , Bauer Michael , Nielsen René Ernst 
Published: December 2022

Abstract
Background
Lithium is the gold standard prophylactic treatment for bipolar disorder. Most clinical practice guidelines recommend regular calcium assessments as part of monitoring lithium treatment, but easy-to-implement specific management strategies in the event of abnormal calcium levels are lacking.

Methods
Based on a narrative review of the effects of lithium on calcium and parathyroid hormone (PTH) homeostasis and its clinical implications, experts developed a step-by-step algorithm to guide the initial management of emergent hypercalcemia during lithium treatment.

Results
In the event of albumin-corrected plasma calcium levels above the upper limit, PTH and calcium levels should be measured after two weeks. Measurement of PTH and calcium levels should preferably be repeated after one month in case of normal or high PTH level, and after one week in case of low PTH level, independently of calcium levels. Calcium levels above 2.8 mmol/l may require a more acute approach. If PTH and calcium levels are normalized, repeated measurements are suggested after six months. In case of persistent PTH and calcium abnormalities, referral to an endocrinologist is suggested since further examination may be needed.

Conclusions
Standardized consensus driven management may diminish the potential risk of clinicians avoiding the use of lithium because of uncertainties about managing side-effects and consequently hindering some patients from receiving an optimal treatment.