Whole-cell bioconversion for producing thymoquinone by engineered Saccharomyces cerevisiae.

Thymoquinone, extracted from the black seeds of Nigella sativa, is a natural substance with highly beneficial effects against various human diseases. In this study, we aimed to construct a Saccharomyces cerevisiae strain that, produce thymoquinone from thymol, a relatively inexpensive substrate. To achieve this, cytochrome P450 from Origanum vulgare was expressed in S. cerevisiae for the bioconversion of thymol to thymoquinone, with the co-expression of cytochrome P450 reductase (CPR) from Arabidopsis thaliana, ATR1. Additionally, flexible linkers were used to connect these two enzymes. Furthermore, modifications were performed to expand the endoplasmic reticulum (ER) space, leading to increased thymoquinone production. After integrating the genes into the chromosome and optimizing the media components, a significant improvement in the thymol-to-thymoquinone conversion rate and yield were achieved. This study represents a possibility of the production of thymoquinone, a bioactive ingredient of a plant, using an engineered microbial cell.

Effect of smartphone use on cervical spine stability.

Using a smartphone often involves a sustained head-forward tilt posture, which may deteriorate the mechanism of muscle reaction efficiency or reduce the stiffness of connective tissues of the cervical spine. These changes in muscular and connective tissues can impair cervical spine stability and contribute to developing neck pain symptoms. In this experiment, change in the cervical spine stability associated with a sustained smartphone use posture was evaluated by quantifying the effective stiffness and the reflexive responses of the head to sudden perturbations. Seventeen young smartphone users maintained their heads tilted forward approximately 30° for 30 min while watching videos on their smartphones in sitting. Data show that the measures of cervical spine stability did not change significantly after the smartphone use task despite developing mild to moderate neck and upper body discomfort symptoms. Study findings imply that keeping the head tilt posture for 30 min for smartphone use did not significantly alter spinal stability, rejecting its association with neck discomfort.

Fast field echo resembling CT using restricted echo-spacing (FRACTURE) MR sequence can provide craniocervical region images comparable to a CT in dogs.

Magnetic resonance imaging (MRI) is essential for evaluating cerebellar compression in patients with craniocervical junction abnormalities (CJA). However, it is limited in depicting cortical bone because of its short T2 relaxation times, low proton density, and organized structure. Fast field echo resembling a computed tomography (CT) scan using restricted echo-spacing (FRACTURE) MRI, is a new technique that offers CT-like bone contrast without radiation. This study aimed to assess the feasibility of using FRACTURE MRI for craniocervical junction (CCJ) assessment compared with CT and conventional MRI, potentially reducing the need for multiple scans and radiation exposure, and simplifying procedures in veterinary medicine. CT and MRI of the CCJ were obtained from five healthy beagles. MRI was performed using three-dimensional (3D) T1-weighted, T2-weighted, proton density-weighted (PDW), single echo-FRACTURE (sFRACTURE), and multiple echo-FRACTURE (mFRACTURE) sequences. For qualitative assessment, cortical delineation, trabecular bone visibility, joint space visibility, vertebral canal definition, overall quality, and artifacts were evaluated for each sequence. The geometrical accuracy, signal-to-noise ratio (SNR), and contrast-to-noise ratio (CNR) were quantified. Both sFRACTURE and CT images provided significantly higher scores for cortical delineation and trabecular bone visibility than conventional MRI. Joint space visibility and vertebral canal definition were similar to those observed on CT images, regardless of the MR sequence. In the quantitative assessment, the distances measured on T2-weighted images differed significantly from those measured on CT. There were no significant differences between the distances taken using T1-weighted, PD-weighted, sFRACTURE, mFRACTURE and those taken using CT. T1-weighted and sFRACTURE had a higher SNR for trabecular bone than CT. The CNR between the cortical bone and muscle was high on CT and FRACTURE images. However, the CNR between the cortical and trabecular bones was low in mFRACTURE. Similar to CT, FRACTURE sequences showed higher cortical delineation and trabecular bone visibility than T2-weighted, T1-weighted, and PDW CCJ sequences. In particular, sFRACTURE provided a high signal-to-noise ratio (SNR) of the trabecular bone and a high CNR between the cortical bone and muscle and between the cortical and trabecular bones. FRACTURE sequences can complement conventional MR sequences for bone assessment of the CCJ in dogs.

Fast field echo resembling a CT using restricted echo-spacing (FRACTURE) sequence for shoulder joint in normal dogs.

Shoulder disease is a common cause of forelimb lameness in dogs. Determining the precise underlying cause of shoulder lameness can be challenging, especially in veterinary practice. Computerized tomography (CT) is often the preferred imaging modality for bone evaluation; however, it uses ionizing radiation and provides limited soft tissue contrast. Conversely, magnetic resonance imaging (MRI) offers excellent soft tissue contrast but has limitations in bone imaging. This study aimed to introduce a new technical innovation that enhances cortical and trabecular bone contrast on MRI, which we refer to as Fast Field Echo Resembling a CT Using Restricted Echo-Spacing (FRACTURE). In this prospective pilot study, we aimed to evaluate the use of FRACTURE, CT, and conventional MRI sequences in assessing the normal canine shoulder using a 3.0 Tesla MRI scanner. Five research beagle dogs were included, and the following pulse sequences were acquired for each dog (1): three-dimensional (3D) FRACTURE, (2) T2-weighted (T2W) images using 3D turbo spin echo (TSE), (3) T1-weighted (T1W) images using 3D TSE, (4) PD-weighted (PDW) images using 3D TSE, and (5) CT. Various parameters, including the delineation of cortical bone (intertubercular groove, greater tubercle, and lesser tubercle), conspicuity of the trabecular bone, shoulder joint visualization, and image quality, were measured for each dog and sequence. In all sequences, the shoulder joint was successfully visualized in all planes with mild motion artifacts. The intertubercular groove was best visualized on CT and FRACTURE. Both the greater and lesser tubercles were easily identified on the CT, FRACTURE, and PDW images. The trabecular pattern scored significantly higher in the CT and FRACTURE images compared to the T1W, T2W, and PDW images. Overall, the visualization of the shoulder joint was excellent in all sequences except for T1W. The use of FRACTURE in combination with conventional MRI sequences holds promise for facilitating not only soft tissue evaluation but also cortical and trabecular bone assessment. The findings from this study in normal dogs can serve as a foundation for further FRACTURE studies in dogs with shoulder diseases.

Utility of Cell Saver in Trauma Compared to Cardiac Surgery.

While reperfusion of autologous blood using the Cellsaver (CS) device is routine in cardiothoracic surgery, there is a paucity of evidence-based literature regarding its use in trauma. Utility of CS was compared in these two distinct populations at a Level 1 trauma center from 2017 to 2022. CS was successfully used in 97% and 74% of cardiac and trauma cases, respectively. The proportion of blood requirements provided by CS, compared to allogenic transfusion, was also significantly higher in cardiac surgery. However, there was still net benefit for CS in trauma surgery, with median salvaged transfusion volume of one unit, in both general & orthopedic trauma. Therefore, in centers where the cost of setting up CS, both in terms of equipment and personnel, is less than the cost of one unit of blood from blood bank, use of CS in trauma operations should be considered.

The range of visual detection of ground-level cues during distracted walking: Effect of cue contrast and walking speed.

Walking while distracted by a smartphone has been a major safety concern for pedestrians. Visual and cognitive attention paid to the smartphone while walking with the head tilted downward would affect the ability to perceive walkway hazards and elevate risks for pedestrian accidents associated with physical contact with obstacles. A laboratory experiment was conducted to evaluate the performance of detecting ground-level visual cues during texting while walking. Forty young smartphone users performed walking trials at faster, preferred, and slower speeds for the dual-task walking on a treadmill and detected approaching cues of three contrast levels. Detection distance was quantified from the location of cue detection to the participants to assess the effects of walking speed and cue contrast on detection performance. Results show that detection distance varied from 1.7 m to 2.9 m for Low to High contrast cues and from 2.3 m to 2.5 m for Slower to Faster walking speeds, and the effects of contrast and speed were statistically significant (p < 0.05). Study findings suggest that higher contrast fixtures or in-ground signals and slower walking would help smartphone users perceive walkway hazards and in-ground safety signals earlier during their distracted walking.

Effect of hand loads on upper extremity muscle activity during pushing and pulling motions.

Manual pushing or pulling with a hand tool is a coordinated action by various upper extremity muscles. The objective of this experimental study was to examine the effects of horizontal and vertical hand loads on upper extremity muscle activity during concentric pushing and pulling exertions. Twenty young female participants conducted repetitive pushing and pulling trials with three horizontal loads (1 kg, 2 kg, 3 kg) and two vertical loads (0.6 kg, 1.3 kg) in a seated posture, while the myoelectric activity of seven upper extremity and shoulder muscles were quantified. Study results indicate that the shoulder flexor and extensor muscles were more strongly associated with horizontal load, and elbow flexors were more sensitive to vertical load. The empirical data from this systematic evaluation can offer initial insights for ergonomic design and evaluation of hand tools or occupational tasks that involve repetitive pushing or pulling.

User discomfort while using a virtual reality headset as a personal viewing system for text-intensive office tasks.

Ergonomics issues while using virtual reality (VR) headsets for text-intensive applications have not been studied. Measures of neck and shoulder discomfort and simulator sickness symptoms were quantified while participants were performing a document creation task for 60 min using a VR headset and a desktop monitor. During the task with the headset, participants rotated the head 2.7 times more frequently and used the neck extensor muscles 25.9% more, in average. They also rated the neck and shoulder discomfort 60% and 17.5% higher after the task. The simulator sickness symptoms were also rated significantly higher (p < .05) for the headset condition, with more pronounced differences in the symptoms related to visual discomfort. Results indicate that the physical discomforts due to the frequent head rotations and the headset weight, and visual discomforts due to difficulty in reading texts were the main issues of the VR headset for common office tasks. Practitioner summary: Ergonomics issues associated with the use of a VR headset for conducting office productivity work tasks have been evaluated in an experiment. Study results indicate that the development in the neck physical discomfort and visual discomfort may be the main barriers to the use of current VR headsets for office works. Abbreviations: VR: virtual reality; VDT: video display terminal; EMG: electromyography; MVC: maximum voluntary contraction; SSQ: simulator sickness questionnaire; ECG: electrocardiogram; NEMG: normalised electromyography.

Performance of ground-level signal detection when using a phone while walking.

Ground-level traffic lights or safety signs have been introduced recently as a prevention measure for smartphone-related pedestrian accidents. However, quantitative evaluation of smartphone users' detection performance during distracted walking has been scarce. A laboratory experiment was conducted to evaluate the performance of detecting ground-level visual cues during the concurrent use of a smartphone while walking. Thirty-eight young smartphone users performed ground-level visual cue detection trials, 1) while walking upright on a treadmill without using a smartphone; 2) when conducting one-handed browsing while walking; and, 3) when conducting two-handed texting while walking. Visual cues were presented on the ground at 24 locations by a ceiling-mounted projector, and participants were asked to respond verbally when they perceived the appearance of each cue. Study results show that the concurrent use of a smartphone decreased the detection rate significantly (p < 0.05) from 93.5 % to 76.3∼74.1 %, and increased the reaction time from 0.90 s to 1.04∼1.15 s. Among the 24 cue locations, cues that were presented closer to participants were detected significantly (p < 0.05) more often and faster than cues that were shown at further locations. The results of this laboratory-based study imply that the ground-level signals might not be detected well by smartphone users, specifically when they were conducting more demanding tasks such as texting while walking. However, the laboratory conditions were confined to a specific usage environment; therefore, future research should be focused on the situation awareness of smartphone users under various usage scenarios and more realistic environments.

Changes in Low Back Muscle Activity and Spine Kinematics in Response to Smartphone Use During Walking.

STUDY DESIGN: Within-subject design of an experimental study. OBJECTIVE: The aim of this study was to determine the effects of smartphone use on the activity level of the lumbar erector spinae muscles and spine kinematics during walking. SUMMARY OF BACKGROUND DATA: Using a smartphone while walking makes the user hold the phone steady and look downward to interact with the phone. Walking with this non-natural posture of the head and the arms may alter the spine kinematics and increase the muscular load on the low back extensor muscles. METHODS: Twenty healthy young individuals participated in the laboratory experiment. Each participant walked on a treadmill in five different conditions: normal walking without using a phone, conducting one-handed browsing while walking, two-handed texting while walking, walking with one arm bound, and walking with both arms bound. Spine kinematics variables and the myoelectric activity levels of the lumbar erector spinae muscles were quantified and compared between the five walking conditions. RESULTS: Participants walked with significantly (P < 0.05) more thoracic kyphosis and lumbar lordosis when using a phone compared to when walking without using the phone. The median level of muscle activity was also 16.5% (browsing) to 31.8% (texting) greater for the two smartphone use conditions than for the normal walking condition, and the differences were significant (P < .05). Between the normal walking and the two bound arm walking conditions, no significant difference in the muscle activity was found. CONCLUSION: Study results show that the concurrent use of a smartphone while walking could pose a larger muscular load to the lumbar erector spinae muscles than that of normal walking. Habitual use of a smartphone while walking could be a risk factor for low back musculoskeletal problems.Level of Evidence: N/A.

Creation of bladder assembloids mimicking tissue regeneration and cancer.

Current organoid models are limited by their inability to mimic mature organ architecture and associated tissue microenvironments1,2. Here we create multilayer bladder 'assembloids' by reconstituting tissue stem cells with stromal components to represent an organized architecture with an epithelium surrounding stroma and an outer muscle layer. These assembloids exhibit characteristics of mature adult bladders in cell composition and gene expression at the single-cell transcriptome level, and recapitulate in vivo tissue dynamics of regenerative responses to injury. We also develop malignant counterpart tumour assembloids to recapitulate the in vivo pathophysiological features of urothelial carcinoma. Using the genetically manipulated tumour-assembloid platform, we identify tumoural FOXA1, induced by stromal bone morphogenetic protein (BMP), as a master pioneer factor that drives enhancer reprogramming for the determination of tumour phenotype, suggesting the importance of the FOXA1-BMP-hedgehog signalling feedback axis between tumour and stroma in the control of tumour plasticity.

Coexistence of Growth Hormone Deficiency and Pituitary Microadenoma in a Child with Unique Mosaic Turner Syndrome: A Case Report and Literature Review.

Turner syndrome (TS) is a genetic disorder with phenotypic heterogeneity caused by the monosomy or structural abnormalities of the X chromosome, and it has a prevalence of about 1/2500 females live birth. The variable clinical features of TS include short stature, gonadal failure, and skeletal dysplasia. The association with growth hormone (GH) deficiency or other hypopituitarism in TS is extremely rare, with only a few case reports published in the literature. Here, we report the first case of a patient with mosaic TS with complete GH deficiency and pituitary microadenoma, and we include the literature review. During the work-up of the patient for severe short stature, three GH provocation tests revealed peak GH levels of less than 5 ng/mL, which was compatible with complete GH deficiency. Sella magnetic resonance imaging showed an 8 mm non-enhancing pituitary adenoma with mild superior displacement of the optic chiasm. Karyotyping revealed the presence of ring chromosome X and monosomy X (46,X,r(X)/45,X/46,X,psu dic r(X;X)), which indicated a mosaic TS. It is important to consider not only chromosome analyses in females with short stature, but also the possibility of the coexistence of complete GH deficiency accompanying pituitary lesions in TS. In conclusion, the present study reports the first case of GH deficiency and pituitary adenoma in a patient with rare mosaic TS, which extends the genotype-phenotype spectrum for TS.

Use of inkjet-printed single cells to quantify intratumoral heterogeneity.

Quantification of intratumoral heterogeneity is essential for designing effective therapeutic strategies in the age of personalized medicine. In this study, we used a piezoelectric inkjet printer to enable analysis of intratumoral heterogeneity in a bladder cancer for the first time. Patient-derived tumor organoids were dissociated into single cell suspension and used as a bioink. The individual cells were precisely allocated into a microwell plate by drop-on-demand inkjet printing without any additive or treatment, followed by culturing into organoids for further analysis. The sizes and morphologies of the organoids were observed, so as the expression of proliferation and apoptotic markers. The tumor organoids also showed heterogeneous responses against chemotherapeutic agent. Further, we quantified mRNA expression levels of representative luminal and basal genes in both type of tumor organoids. These results verify the heterogeneous expression of various genes among individual organoids. This study demonstrates that the fully automated inkjet printing technique can be used as an effective tool to sort cells for evaluating intratumoral heterogeneity.

Testing of Schistosoma mansoni Vaccine Targets.

Vaccination has greatly reduced the burden of human diseases caused by infectious pathogens. Systematic development of vaccine targets requires established protocols to assess immunogenicity and efficacy of such vaccine candidates. Using a leading schistosomiasis vaccine candidate, Sm-p80, as an example, we describe standardized approaches for testing the immunogenicity and efficacy of Schistosoma mansoni vaccine targets. Unlike other parasite systems in which sterile immunity is required, the goal of S. mansoni vaccine targets is overall reduction in morbidity. Methods related to the parasitological parameters described in this chapter allow for the testing of the prophylactic (reduction in adult worm burden), anti-pathology (liver and intestine egg retention), and transmission blocking (fecal egg expulsion and egg hatching rates) efficacies for the vaccine target. The RNA sequencing approaches provide basis for identification of molecular signatures predictive of desirable outcomes for schistosomiasis vaccines.

Epigenetic regulation of mammalian Hedgehog signaling to the stroma determines the molecular subtype of bladder cancer.

In bladder, loss of mammalian Sonic Hedgehog (Shh) accompanies progression to invasive urothelial carcinoma, but the molecular mechanisms underlying this cancer-initiating event are poorly defined. Here, we show that loss of Shh results from hypermethylation of the CpG shore of the Shh gene, and that inhibition of DNA methylation increases Shh expression to halt the initiation of murine urothelial carcinoma at the early stage of progression. In full-fledged tumors, pharmacologic augmentation of Hedgehog (Hh) pathway activity impedes tumor growth, and this cancer-restraining effect of Hh signaling is mediated by the stromal response to Shh signals, which stimulates subtype conversion of basal to luminal-like urothelial carcinoma. Our findings thus provide a basis to develop subtype-specific strategies for the management of human bladder cancer.

Sm-p80-based schistosomiasis vaccine: double-blind preclinical trial in baboons demonstrates comprehensive prophylactic and parasite transmission-blocking efficacy.

Schistosomiasis is of public health importance to an estimated one billion people in 79 countries. A vaccine is urgently needed. Here, we report the results of four independent, double-blind studies of an Sm-p80-based vaccine in baboons. The vaccine exhibited potent prophylactic efficacy against transmission of Schistosoma mansoni infection and was associated with significantly less egg-induced pathology, compared with unvaccinated control animals. Specifically, the vaccine resulted in a 93.45% reduction of pathology-producing female worms and significantly resolved the major clinical manifestations of hepatic/intestinal schistosomiasis by reducing the tissue egg-load by 89.95%. A 35-fold decrease in fecal egg excretion in vaccinated animals, combined with an 81.51% reduction in hatching of eggs into the snail-infective stage (miracidia), demonstrates the parasite transmission-blocking potential of the vaccine. Substantially higher Sm-p80 expression in female worms and Sm-p80-specific antibodies in vaccinated baboons appear to play an important role in vaccine-mediated protection. Preliminary analyses of RNA sequencing revealed distinct molecular signatures of vaccine-induced effects in baboon immune effector cells. This study provides comprehensive evidence for the effectiveness of an Sm-p80-based vaccine for schistosomiasis.

Schistosoma mansoni antigen Sm-p80: prophylactic efficacy using TLR4 agonist vaccine adjuvant glucopyranosyl lipid A-Alum in murine and non-human primate models.

Sm-p80, the large subunit of Schistosoma mansoni calpain, is a leading candidate for a schistosomiasis vaccine. The prophylactic and antifecundity efficacy of Sm-p80 has been tested in three animal models (mouse, hamster and baboon) using a multitude of vaccine formulations and approaches. In our continual effort to enhance the vaccine efficacy, in this study, we have utilized the adjuvant, synthetic hexa-acylated lipid A derivative, glucopyranosyl lipid A (GLA) formulated in aluminum (GLA-Alum) with recombinant Sm-p80. The rSm-p80+GLA-Alum immunization regimen provided 33.33%-53.13% reduction in worm burden in the mouse model and 38% worm burden reduction in vaccinated baboons. Robust Sm-p80-specific immunoglobulin (Ig)G, IgG1, IgG2a and IgM responses were observed in all immunized animals. The rSm-p80+GLA-Alum coadministration induced a mix of T-helper (Th) cells (Th1, Th2 and Th17) responses as determined via the release of interleukin (IL)-2, IL-4, IL-18, IL-21, IL-22 and interferon-γ.

Sm-p80-based vaccine trial in baboons: efficacy when mimicking natural conditions of chronic disease, praziquantel therapy, immunization, and Schistosoma mansoni re-encounter.

Sm-p80-based vaccine efficacy for Schistosoma mansoni was evaluated in a baboon model of infection and disease. The study was designed to replicate a human vaccine implementation scenario for endemic regions in which vaccine would be administered following drug treatment of infected individuals. In our study, the Sm-p80-based vaccine reduced principal pathology producing hepatic egg burdens by 38.0% and egg load in small and large intestines by 72.2% and 49.4%, respectively, in baboons. Notably, hatching rates of eggs recovered from liver and small and large intestine of vaccinated animals were significantly reduced, by 60.4%, 48.6%, and 82.3%, respectively. Observed reduction in egg maturation/hatching rates was supported by immunofluorescence and confocal microscopy showing unique differences in Sm-p80 expression in worms of both sexes and matured eggs. Vaccinated baboons had a 64.5% reduction in urine schistosome circulating anodic antigen, a parameter that reflects worm numbers/health status in infected hosts. Preliminary analyses of RNA sequencing revealed unique genes and canonical pathways associated with establishment of chronic disease, praziquantel-mediated parasite killing, and Sm-p80-mediated protection in vaccinated baboons. Overall, our study demonstrated efficacy of the Sm-p80 vaccine and provides insight into some of the epistatic interactions associated with protection.

Expression of PGC1α in glioblastoma multiforme patients.

Peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) is a key modulator of mitochondrial biogenesis. It is a coactivator of multiple transcription factors and regulates metabolic processes. However, little is known about the expression and function of PGC1α in glioblastoma multiforme (GBM), the most prevalent and invasive type of brain tumor. The purpose of the present study was to investigate the biological function, localization and expression of PGC1α in GBM. It was observed that PGC1α expression is increased in the tumor cells, and a higher level of expression was observed in the mitochondria. Bioinformatics analyses identified that metabolic and mitochondrial genes were highly expressed in GBM cells, with a high PGC1α mRNA expression. Notably, mitochondrial function-associated genes were highly expressed in cells alongside high PGC1α expression. Collectively, the results of the present study indicate that PGC1α is associated with mitochondrial dysfunction in GBM and may have a role in tumor pathogenesis and progression.