bluuebunny/crossref_metadata_2025_split huggingface dataset:
Heme biosensor-guided in vivo pathway optimization and directed evolution for efficient biosynthesis of heme
Authors: Zhang Jian , Li Qingbin , Wang Qi , Zhao Jingyu , Zhu Yuan , Su Tianyuan , Qi Qingsheng , Wang Qian 
Published: March 2023

Abstract
Background
Heme has attracted much attention because of its wide applications in medicine and food. The products of genes hemBCDEFY convert 5-aminolevulinic acid to protoporphyrin IX (PPIX; the immediate precursor of heme); protoporphyrin ferrochelatase (FECH) inserts Fe2+ into PPIX to generate heme. Biosynthesis of heme is limited by the need for optimized expression levels of multiple genes, complex regulatory mechanisms, and low enzymatic activity; these problems need to be overcome in metabolic engineering to improve heme synthesis.

Results
We report a heme biosensor-guided screening strategy using the heme-responsive protein HrtR to regulate tcR expression in Escherichia coli, providing a quantifiable link between the intracellular heme concentration and cell survival in selective conditions (i.e., the presence of tetracycline). This system was used for rapid enrichment screening of heme-producing strains from a library with random ribosome binding site (RBS) variants and from a FECH mutant library. Through up to four rounds of iterative evolution, strains with optimal RBS intensities for the combination of hemBCDEFY were screened; we obtained a PPIX titer of 160.8 mg/L, the highest yield yet reported in shaken-flask fermentation. A high-activity FECH variant was obtained from the saturation mutagenesis library. Fed-batch fermentation of strain SH20C, harboring the optimized hemBCDEFY and the FECH mutant, produced 127.6 mg/L of heme.

Conclusion
We sequentially improved the multigene biosynthesis pathway of PPIX and performed in vivo directed evolution of FECH, based on a heme biosensor, which demonstrated the effectiveness of the heme biosensor-based pathway optimization strategy and broadens our understanding of the mechanism of heme synthesis.
EBS-seq: enrichment-based method for accurate analysis of 5-hydroxymethylcytosine at single-base resolution
Authors: Lee Jaywon , Lee Dongin , Kim Hwang-Phill , Kim Tae-You , Bang Duhee 
Published: March 2023

Abstract
Background
A growing body of research has emphasized 5-hydroxymethylcytosine (5hmC) as an important epigenetic mark. High-resolution methods to detect 5hmC require high sequencing depth and are therefore expensive. Many studies have used enrichment-based methods to detect 5hmC; however, conventional enrichment-based methods have limited resolution. To overcome these limitations, we developed EBS-seq, a cost-efficient method for 5hmC detection with single-base resolution that combines the advantages of high-resolution methods and enrichment-based methods.

Results
EBS-seq uses selective labeling of 5hmC, deamination of cytosine and 5-methylcytosine, pull-down of labeled 5hmC, and C-to-T conversion during DNA amplification. Using this method, we profiled 5hmC in HEK293T cells and two colorectal cancer samples. Compared with conventional enrichment-based 5hmC detection, EBS-seq improved 5hmC signals by localizing them at single-base resolution. Furthermore, EBS-seq was able to determine 5hmC levels in CpG-dense regions where distortion of signals can occur, such as CpG islands and CpG shores. Comparing EBS-seq and conventional high-resolution 5hmC detection by ACE-seq, we showed that EBS-seq is more effective at finding 5hmC sites. Using EBS-seq, we found strong associations between gene expression and gene-body 5hmC content in both HEK293T cells and colorectal cancer samples.

Conclusions
EBS-seq is a reliable and cost-efficient method for 5hmC detection because it simultaneously enriches 5hmC-containing DNA fragments and localizes 5hmC signals at single-base resolution. This method is a promising choice for 5hmC detection in challenging clinical samples with low 5hmC levels, such as cancer tissues.
Long-term follow-up of Mycoplasma hyopneumoniae-specific immunity in vaccinated pigs
Authors: Biebaut Evelien , Beuckelaere Lisa , Boyen Filip , Haesebrouck Freddy , Gomez-Duran Charles-Oliver , Devriendt Bert , Maes Dominiek 
Published: March 2023

AbstractMycoplasma hyopneumoniaeis the primary agent of enzootic pneumonia in pigs. To minimize the economic losses caused by this disease,M. hyopneumoniaevaccination is commonly practiced. However, the persistence ofM. hyopneumoniaevaccine-induced immunity, especially the cell-mediated immunity, till the moment of slaughter has not been investigated yet. Therefore, on two commercial farms, 25 pigs (n = 50) received a commercial bacterin intramuscularly at 16 days of age. Each month, the presence ofM. hyopneumoniae-specific serum antibodies was analyzed and the proliferation of and TNF-α, IFN-γ and IL-17A production by different T cell subsets in blood was assessed using recall assays. Natural infection withM. hyopneumoniaewas assumed in both farms. However, the studied pigs remainedM. hyopneumoniaenegative for almost the entire trial. Seroconversion was not observed after vaccination and all pigs became seronegative at two months of age. The kinetics of the T cell subset frequencies was similar on both farms.Mycoplasma hyopneumoniae-specific cytokine-producing CD4+CD8+T cells were found in blood of pigs from both farms at one month of age but decreased significantly with increasing age. On the other hand, T cell proliferation after in vitroM. hyopneumoniaestimulation was observed until the end of the fattening period. Furthermore, differences in humoral and cell-mediated immune responses afterM. hyopneumoniaevaccination were not seen between pigs with and without maternally derived antibodies. This study documents the long-termM. hyopneumoniaevaccine-induced immune responses in fattening pigs under field conditions. Further research is warranted to investigate the influence of a natural infection on these responses.
RETRACTED ARTICLE: Loan repayment performance and its determinants: evidence from micro and small enterprises operating in Dire-Dawa, Ethiopia
Authors: Kiros Yitbarek 
Published: March 2023

AbstractMicrofinance Institutions (MFIs) reach a large number of poor people who are not served by formal financial institutions and have been a prime element in the economic growth of countries like Ethiopia. To operate successfully MFIs have to make sure that the loan disbursed has to be repaid to have a sustainable and viable financial operation and contribute their own its share in reducing unemployment and poverty reduction. In light of this, this research study was conducted to investigate the factors affecting loan repayment performance and factors affecting it in Micro and Small Enterprises (MSEs) financed by Microfinance Institutions by taking lender characteristics. Primary data was collected using questionnaires and interviews. A total of 175 Micro and Small Enterprises were selected using purposive sampling. Secondary data was acquired from annual reports and financial statements of Microfinance institutions and other institutions. Descriptive analysis as well as econometric analysis was used to analyze the effect of the literature-driven variables on the loan repayment performance of borrowers. The binary logistic regression result revealed that loan repayment period, grace period, and timeliness of loan release have a statistically significant effect on the loan repayment performances of borrowers. Loan size has a statistically insignificant effect on the loan repayment performance of borrowers.
Standardization of Rehmannia glutinosa (Gaertn.) DC. steam processing and evaluation of its chemical, anti-oxidant, and anti-inflammatory properties
Authors: Chung Yuseong , Rahmat Endang , Nam Hyeon Hwa , Lee Ayeong , Park Jun Hong , Moon Byeong Cheol , Kang Youngmin 
Published: March 2023

AbstractRehmannia glutinosa (Gaertn.) DC., belonging to the family Scrophulariaceae, is an important medicinal herb cultivated in East Asia. Traditionally, R. glutinosa is steam processed to increase its efficacy in treating various ailments such as diabetes, hematinic deficiencies and adrenal disorder. However, standardization of processed R. glutinosa is highly needed to increase its quality to fulfill global market demand that is safe and possess high level of efficacy. Therefore, this study aimed to optimize the R. glutinosa steam processing methods by evaluating some key parameters such as steaming temperature, number of steaming times, steaming duration, and additive supplementation. R. glutinosa samples were steam processed at different temperatures (100 °C, 110 °C, and 120 °C), various steaming times (1 to 5 times), several steaming duration (1 to 4 h), and additives supplementation (rice wine, 5% EtOH, 10% EtOH, 20% EtOH, 30% EtOH, and 40% EtOH). As the result, 2 h, 3 replications, and supplementation with 20% EtOH at 120 °C were identified as the optimal conditions for R. glutinosa steam processing. Optimized processed R. glutinosa (SPRR 20%EtOH) resulted in significantly higher content of 5-HMF (7648.60 ± 150.08 µg/g) and iso-verbacoside (203.80 ± 10.72 µg/g) compared with unprocessed R. glutinosa (UPR). Compared to those of other samples, SPRR 20% EtOH samples had higher total flavonoid (55.36 ± 1.68 mg/g) and phenolic (69.24 ± 4.56 mg/g) contents and stronger DPPH antioxidant activity (56%). Furthermore, SPRR 20% EtOH had excellent anti-inflammatory activity, as evidenced by the suppression of inducible nitric oxide synthase (iNOS) caused by activation of nuclear factor-κB (NF-κB) through p-p65 pathway in LPS-stimulated RAW 264.7 cells. These findings will provide a basis towards industrialization of R. glutinosa processing technology that will be very helpful for oriental medication field.
When complex movement yields simple dispersal: behavioural heterogeneity, spatial spread and parasitism in groups of micro-wasps
Authors: Burte Victor , Cointe Melina , Perez Guy , Mailleret Ludovic , Calcagno Vincent 
Published: March 2023

Abstract
Background
Understanding how behavioural dynamics, inter-individual variability and individual interactions scale-up to shape the spatial spread and dispersal of animal populations is a major challenge in ecology. For biocontrol agents, such as the microscopic Trichogramma parasitic wasps, an understanding of movement strategies is also critical to predict pest-suppression performance in the field.

Methods
We experimentally studied the spatial propagation of groups of parasitoids and their patterns of parasitism. We investigated whether population spread is density-dependent, how it is affected by the presence of hosts, and whether the spatial distribution of parasitism (dispersal kernel) can be predicted from the observed spread of individuals. Using a novel experimental device and high-throughput imaging techniques, we continuously tracked the spatial spread of groups of parasitoids over large temporal and spatial scales (8 h; and 6 m, ca. 12,000 body lengths). We could thus study how population density, the presence of hosts and their spatial distribution impacted the rate of population spread, the spatial distribution of individuals during population expansion, the overall rate of parasitism and the dispersal kernel (position of parasitism events).

Results
Higher population density accelerated population spread, but only transiently: the rate of spread reverted to low values after 4 h, in a “tortoise-hare” effect. Interestingly, the presence of hosts suppressed this transiency and permitted a sustained high rate of population spread. Importantly, we found that population spread did not obey classical diffusion, but involved dynamical switches between resident and explorer movement modes. Population distribution was therefore not Gaussian, though surprisingly the distribution of parasitism (dispersal kernel) was.

Conclusions
Even homogenous asexual groups of insects develop behavioural heterogeneities over a few hours, and the latter control patterns of population spread. Behavioural switching between resident and explorer states determined population distribution, density-dependence and dispersal. A simple Gaussian dispersal kernel did not reflect classical diffusion, but rather the interplay of several non-linearities at individual level. These results highlight the need to take into account behaviour and inter-individual heterogeneity to understand population spread in animals.
Highly fluorescent hydroxyl groups functionalized graphitic carbon nitride for ultrasensitive and selective determination of mercury ions in water and fish samples
Authors: El-Reash Y. G. Abou , Ghaith Eslam A. , El-Awady Osama , Algethami Faisal K. , Lin Haiqing , Abdelrahman Ehab A. , Awad Fathi S. 
Published: March 2023

AbstractHeavy metal ion pollution is always a serious problem worldwide. Therefore, monitoring heavy metal ions in environmental water is a crucial and difficult step to ensure the safety of people and the environment. A mercury ion (Hg2+) fluorescence probe with excellent sensitivity and selectivity is described here. The functionalized graphitic carbon nitride nanosheets (T/G-C3N4) fluorescence probe was fabricated using melamine as a precursor by the pyrolysis technique, followed by a rapid KOH heat treatment method for 2 min. The chemical structure and morphology of the T/G-C3N4 probe were characterized using multiple analytical techniques including UV–Vis, SEM, XPS, XRD, and fluorometer spectroscopy. Geometry optimization of T/G-C3N4 as a modified probe was performed to assess its stability and interaction ability with Hg(II) via using the density function approach. The T/G-C3N4 probe showed a linear response based on quenching over the range 0–1.25 × 103 nM Hg(II); the detection limit was 27 nM. The remarkable sensitivity of T/G-C3N4 towards the Hg2+ ions was explained by the intense coordination and fast chelation kinetics of Hg2+ with the NH2, CN, C=N, and OH groups of T/G-C3N4 nanoprobe. The T/G-C3N4 probe demonstrates exceptional selectivity for Hg2+ ions among other metal ions including (Na+, Ag+, Mg2+, Fe2+, Fe3+, Co2+, Ni2+, Cd2+, K+, Ca2+, Cu2+, Pb2+, Mn2+ and Hg2+) and over a broad pH range (6–10), together with remarkable long-term fluorescence stability in water (> 30 days) and minimal toxicity. T/G-C3N4 was used to detect and quantify Hg2+ ions in tuna and mackerel fish and the results compared to ICP-AES. The results obtained offer a new simple and green technique for the design of multifunctional fluorescent probe appropriate for environmental applications.
Graphical Abstract
Engineering a norcoclaurine synthase for one-step synthesis of (S)-1-aryl-tetrahydroisoquinolines
Authors: Zhang Man , Huang Zheng-Yu , Su Ying , Chen Fei-Fei , Chen Qi , Xu Jian-He , Zheng Gao-Wei 
Published: March 2023

AbstractTetrahydroisoquinoline alkaloids (THIQAs) are ubiquitous compounds with important pharmaceutical and biological activity. Their key N-heterocyclic structural motifs are synthesised via Pictet–Spengler (P–S) reaction by norcoclaurine synthases (NCS) in plants. The synthesis of 1-aryl-tetrahydroisoquinoline alkaloids has attracted increasing attention due to their antitumor and antivirus activities. Herein, the L68T/M97V mutant of NCS from Thalictrum flavum with improved activity was developed by semi-rational design. This mutant not only showed higher catalytic performance (> 96% conversion) toward benzaldehyde and dopamine over the wild-type enzyme, but also catalysed the P–S reaction of the bulky substrate 4-biphenylaldehyde and dopamine with high conversion (> 99%) for the effective synthesis of 1-aryl-THIQA. In terms of stereoselectivity, all products synthesised by the L68T/M97V mutant showed high optical purity (92–99% enantiomeric excess).
Graphical Abstract
Kinematics of platform stabilization using a 3-PRS parallel manipulator
Authors: Udomsap Tossaporn , Chinchouryrang Sakda , Liampipat Siwat , Chanthasopeephan Teeranoot 
Published: March 2023

AbstractIn this paper, a 3-PRS (prismatic, revolute, and spherical) parallel manipulator for platform stabilization is designed. The main purpose of this device is to stabilize visual equipment, which is placed on top of a car to inspect electrical transmission cables, as part of routine maintenance. Due to the bulky and heavy infrared cameras used during inspections, a stabilizer platform has been designed to handle the weight of camera equipment up to 10 kg. This device consists of two major mechanisms. The first mechanism is able to adjust the angle of the camera. Thus, the user can focus the camera along the electric transmission lines. The second mechanism is stabilization. The mechanism serves to stabilize the orientation and position of the camera in the roll, pitch, and heave directions. To test the performance of the stabilization mechanism, the device is fed with the known value of the angle with regard to the input. As such, the device is trying to compensate for the change in angle. The results show that the errors between the input angles and compensated angles are in the range of 0.4–3%. Errors are seen to be within an acceptable range. It is significant that the resultant errors do not affect the orientation of the camera.
Cerebral infarction by paradoxical gas embolism detected after laparoscopic partial hepatectomy with an insufflation management system: a case report
Authors: Shimizu Kenji , Usuda Masahiro , Kakizaki Yuta , Narita Tomohiro , Suzuki On , Fukuoka Kengo 
Published: March 2023

Abstract
Background
Laparoscopic surgery has reduced surgical morbidity and postoperative duration of hospital stay. Gas embolism is commonly known as a risk factor for all laparoscopic procedures. We report a case of severe cerebral infarction presumably caused by paradoxical CO2 embolism in laparoscopic partial hepatectomy with an insufflation management system.

Case presentation
A male in his 60 s was diagnosed with recurrence of liver metastasis in the right hepatic lobe after laparoscopic lower anterior resection for rectal cancer. We performed laparoscopic partial hepatectomy with an AirSeal® under 10 mmHg of intra-abdominal pressure. During the surgery, the patient’s end-tidal CO2 and percutaneous oxygen saturation dropped from approximately 40–20 mmHg and 100–90%, respectively, while the heart rate increased from 60 to 120 beats/min; his blood pressure remained stable. Postoperatively, the patient developed right hemiplegia and aphasia. Brain magnetic resonance imaging showed cerebral infarction in the broad area of the left cerebral cortex. Thereafter, transesophageal echocardiography revealed a patent foramen ovale, suggesting cerebral infarction due to paradoxical gas embolism.

Conclusions
A patent foramen ovale is found in approximately 15–20% of healthy individuals. While gas embolism is a rare complication of laparoscopic surgery, cerebral infarction must be considered a possible complication even if the intra-abdominal pressure is constant under 10 mmHg with an insufflation management system.
Composition and variability of core phyllosphere fungal mycobiota on field-grown broccoli
Authors: Kim Min-Soo , Park Eun-Jin 
Published: March 2023

AbstractBackgroundFresh vegetables harbor an assemblage of different microorganisms on their surfaces. The phyllosphere microbiota is important for maintaining plant health and managing crop quality before and after harvest. However, the diversity and ecology of fungal communities are largely unexplored in fresh vegetables. This study investigated the phyllosphere mycobiota of field-grown broccoli florets (n = 66) collected from 22 farms across four regions in Korea, using culturing, amplicon sequencing of the internal transcribed spacer region, and microbial network analysis.ResultsMicrobial network analysis identified core genera (Purpureocillium,Filobasidium,Cystofilobasidium,Papiliotrema,Aureobasidium, and unclassified genera of Capnodiales) specific to the broccoli phyllosphere. The composition and network complexity of core and unique populations varied among farming regions, and was associated with local agro-meteorological conditions. The complexity of microbial associations was higher in mature communities than in immature communities, but complexity was lost upon development of plant pathogenic disease. Broccoli mycobiota were classified according to the dominance ofPurpurecillium. WhilePurpurecillium-type microbiota were prevalent in normal samples,Filobasidium-type microbiota were frequently observed in immature, damaged, or postharvest samples.ConclusionsTogether, fungal communities were important components of phyllosphere microbiota on fresh vegetables, and have substantial potential for exploitation to enhance and stabilize plant health and growth.
Development of human-machine language interfaces for the visual analysis of complex biologics and RNA modalities and associated experimental data
Authors: Kunz Roxanne K. , Rojnuckarin Atipat , Schmidt Christian Marc , Miranda Les P. 
Published: March 2023

AbstractThe advent of recombinant protein-based therapeutic agents in the 1980s and subsequent waves of innovation in molecular biology and engineering of biologics has permitted the production of an increasingly broad array of complex, high molecular weight constructs. While this has opened a powerful new toolbox of molecular scaffolds with which to probe and interdict biological processes, it also makes deciphering the architectural nuances between individual constructs intuitively difficult. Key to downstream data processes for the detection of data trends is the ability to unambiguously identify, compare, and communicate the nature of molecular compositions. Existing small molecule orientated software tools are not intended for structures such as peptides, proteins, antibodies, and RNA, and do not contain adequate atomistic or domain-level detail to appropriately convey their higher structural complexity. Similarly, there is a paucity of large molecule-focused data analysis and visualization tools. This article will describe four new approaches we developed for the graphical representation and analysis of complex large molecules and experimental data. These tools help fulfill key needs in scientific communication and structure-property analysis of complex biologics and modified oligonucleotide-based drug candidates.
Anti-Trypanosoma cruzi activity of Coptis rhizome extract and its constituents
Authors: Tayama Yuki , Mizukami Shusaku , Toume Kazufumi , Komatsu Katsuko , Yanagi Tetsuo , Nara Takeshi , Tieu Paul , Huy Nguyen Tien , Hamano Shinjiro , Hirayama Kenji 
Published: March 2023

Abstract
Background
Current therapeutic agents, including nifurtimox and benznidazole, are not sufficiently effective in the chronic phase of Trypanosoma cruzi infection and are accompanied by various side effects. In this study, 120 kinds of extracts from medicinal herbs used for Kampo formulations and 94 kinds of compounds isolated from medicinal herbs for Kampo formulations were screened for anti-T. cruzi activity in vitro and in vivo.

Methods
As an experimental method, a recombinant protozoan cloned strain expressing luciferase, namely Luc2-Tulahuen, was used in the experiments. The in vitro anti-T. cruzi activity on epimastigote, trypomastigote, and amastigote forms was assessed by measuring luminescence intensity after treatment with the Kampo extracts or compounds. In addition, the cytotoxicity of compounds was tested using mouse and human feeder cell lines. The in vivo anti-T. cruzi activity was measured by a murine acute infection model using intraperitoneal injection of trypomastigotes followed by live bioluminescence imaging.

Results
As a result, three protoberberine-type alkaloids, namely coptisine chloride, dehydrocorydaline nitrate, and palmatine chloride, showed strong anti-T. cruzi activities with low cytotoxicity. The IC50 values of these compounds differed depending on the side chain, and the most effective compound, coptisine chloride, showed a significant effect in the acute infection model.

Conclusions
For these reasons, coptisine chloride is a hit compound that can be a potential candidate for anti-Chagas disease drugs. In addition, it was expected that there would be room for further improvement by modifying the side chains of the basic skeleton.