Metabolic dysfunction-associated steatotic liver disease (MASLD) is a major public health issue, with a recent estimated global prevalence of 30%(1). The pathogenesis of MASLD is complex, multifactorial, and incompletely understood(2). MicroRNAs (miRNAs) are small non-coding RNAs that typically inhibit gene expression as post-transcriptional regulators(3). MiRNAs are increasingly recognised for their potential as diagnostic biomarkers and therapeutic targets in liver diseases(3). The aim of this research was to consider the role of miRNAs in MASLD pathogenesis and their regulation by diet.

Liver RNA samples were extracted from male C57BL/6J mice (n = 6/group) fed for 8 weeks either a chow diet (CD) or 60% fat (high fat diet, HFD) and sent for RNA sequencing. The raw data obtained from next-generation sequencing (Novogene, Cambridge) were passed through a quality control pipeline. Differential expression analysis, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, and target predictions were performed in the R environment. Enriched GO terms were summarised and clustered by online tool Revigo. Rich factor (RF) was used to describe the intensity of dysregulated pathways, calculated as the ratio of differentially expressed genes (DEGs) to the total genes included in each pathway. MicroRNAs that targeted the DEGs were identified using two validated miRNA-mRNA interaction databases, Tarbase and miRTarbase.

A total of 1087 DEGs were found in HFD fed mice relative to CD (adjusted P<0.05) and their biological functions were examined using GO and KEGG enrichment analyses. In aggregate, significantly enriched components and functions included: small molecule metabolic processes, collagen-containing extracellular matrix and lipoprotein particles, and oxidoreductase activity. Circadian rhythm was the most enriched pathway (RF = 0.29) among 12 enriched KEGG pathways, which also included fatty acid degradation (RF = 0.21), PPAR signalling (RF = 0.16), retinol (RF = 0.17) and cholesterol metabolism (RF = 0.22) pathways. Indeed, out of the 34 genes that comprise the KEGG circadian rhythm pathway, 10 (29%) were found dysregulated by HFD, including the critical. Period genes (Per2, Per3). Further exploration in human found hsa-miR-133a-3p targets PER2 and PER3 via examination of validated miRNA-mRNA interaction databases. In addition to circadian rhythm, hsa-miR-133a-3p was found to target genes that function in macroautophagy, cold-induced thermogenesis, and insulin response pathways.

Peripheral clocks of circadian rhythm (liver, pancreas, adipose tissue and muscle) are regulated by miRNAs that contribute to the pathogenesis of MASLD through disruption of lipid metabolism and the insulin response. Future work includes the confirmation of miR-133a-3p expression and the related pathways in mouse liver and human primary hepatocytes.

We investigated experimentally the settling behaviour of vertically aligned spherical particles within various quiescent media at different release frequencies. The particles had a diameter of $d = 4$ mm and density of $\rho _s = 2200$ kg m$^{-3}$, and were released near the free surface of water, ethanol, a G60 water–glycerine mixture (60 % glycerine by weight) and oil media at frequencies of $f_P = 4$, 6 and 8 Hz, thereby allowing study of Galileo numbers, $Ga \in [16, 976]$. Particle tracking velocimetry quantified the motion of nearly 800 particles in a 600 mm high tank, and particle image velocimetry examined flow patterns around the particles. Results revealed that the centre of mass of the particle trajectories exhibited preferential in-plane motions, with significant lateral dispersion and large $Ga$ in water and ethanol, and nearly vertical paths with low $Ga$ in the G60 mixture and oil media. Varying degrees of particle separation resulted in higher terminal velocities than for a single particle. Hence, particle drag decreased in all cases, with the oil medium showing the highest drag reduction under the closest particle separation, reaching up to nearly 70 % of that for the single particle. The vertical and lateral pair dispersions, $R^2_z$ and $R^2_L$, exhibited ballistic scaling, with dependences on the initial separation, $r_0$, and the type of medium. With large $Ga$, $R^2_z$ displayed a ballistic regime followed by a slower rate, whereas with small $Ga$, $R^2_z$ maintained a consistent ballistic regime throughout settling. Finally, normalized $R^2_z$ demonstrated distinct scaling (exponent 2/3 and 1) dependent on the normalized initial separation and $Ga$.

In zebrafish, a punctate band of F-actin is reported to develop in the external yolk syncytial layer (E-YSL) during the latter part of epiboly in zebrafish embryos. Here, electron microscopy (EM) and fluorescence confocal microscopy were conducted to investigate dynamic changes in the E-YSL membrane during epiboly. Using scanning EM, we report that the surface of the E-YSL is highly convoluted, consisting of a complex interwoven network of branching membrane surface microvilli-like protrusions. The region of membrane surface protrusions was relatively wide at 30% epiboly but narrowed as epiboly progressed. This narrowing was coincident with the formation of the punctate actin band. We also demonstrated using immunogold transmission EM that actin clusters were localized at the membrane surface mainly within the protrusions as well as in deeper locations of the E-YSL. Furthermore, during the latter part of epiboly, the punctate actin band was coincident with a region of highly dynamic endocytosis. Treatment with cytochalasin B led to the disruption of the punctate actin band and the membrane surface protrusions, as well as the attenuation of endocytosis. Together, our data suggest that, in the E-YSL, the region encompassing the membrane surface protrusions and its associated punctate actin band are likely to be an integral part of the localized endocytosis, which is important for the progression of epiboly in zebrafish embryos.

An emergent volume electron microscopy technique called cryogenic serial plasma focused ion beam milling scanning electron microscopy (pFIB/SEM) can decipher complex biological structures by building a three-dimensional picture of biological samples at mesoscale resolution. This is achieved by collecting consecutive SEM images after successive rounds of FIB milling that expose a new surface after each milling step. Due to instrumental limitations, some image processing is necessary before 3D visualization and analysis of the data is possible. SEM images are affected by noise, drift, and charging effects, that can make precise 3D reconstruction of biological features difficult. This article presents Okapi-EM, an open-source napari plugin developed to process and analyze cryogenic serial pFIB/SEM images. Okapi-EM enables automated image registration of slices, evaluation of image quality metrics specific to pFIB-SEM imaging, and mitigation of charging artifacts. Implementation of Okapi-EM within the napari framework ensures that the tools are both user- and developer-friendly, through provision of a graphical user interface and access to Python programming.

From 2016–2019, dry bulb onions were the suspected cause of three multistate outbreaks in the United States. We investigated a large multistate outbreak of Salmonella Newport infections that caused illnesses in both the United States and Canada in 2020. Epidemiologic, laboratory and traceback investigations were conducted to determine the source of the infections, and data were shared among U.S. and Canadian public health officials. We identified 1127 U.S. illnesses from 48 states with illness onset dates ranging from 19 June to 11 September 2020. Sixty-six per cent of ill people reported consuming red onions in the week before illness onset. Thirty-five illness sub-clusters were identified during the investigation and seventy-four per cent of sub-clusters served red onions to customers during the exposure period. Traceback for the source of onions in illness sub-clusters identified a common onion grower in Bakersfield, CA as the source of red onions, and onions were recalled at this time. Although other strains of Salmonella Newport were identified in environmental samples collected at the Bakersfield, CA grower, extensive environmental and product testing did not yield the outbreak strain. This was the third largest U.S. foodborne Salmonella outbreak in the last 30 years. It is the first U.S. multistate outbreak with a confirmed link to dry bulb onions, and it was nearly 10-fold larger than prior outbreaks with a suspected link to onions. This outbreak is notable for its size and scope, as well as the international data sharing that led to implication of red onions as the primary cause of the outbreak. Although an environmental assessment at the grower identified several factors that likely contributed to the outbreak, no main reason was identified. The expedient identification of the outbreak vehicle and response of multiple public health agencies allowed for recall and removal of product from the marketplace, and rapid messaging to both the public and industry on actions to protect consumers; these features contributed to a decrease in cases and expeditious conclusion of the outbreak.

The great demographic pressure brings tremendous volume of beef demand. The key to solve this problem is the growth and development of Chinese cattle. In order to find molecular markers conducive to the growth and development of Chinese cattle, sequencing was used to determine the position of copy number variations (CNVs), bioinformatics analysis was used to predict the function of ZNF146 gene, real-time fluorescent quantitative polymerase chain reaction (qPCR) was used for CNV genotyping and one-way analysis of variance was used for association analysis. The results showed that there exists CNV in Chr 18: 47225201-47229600 (5.0.1 version) of ZNF146 gene through the early sequencing results in the laboratory and predicted ZNF146 gene was expressed in liver, skeletal muscle and breast cells, and was amplified or overexpressed in pancreatic cancer, which promoted the development of tumour through bioinformatics. Therefore, it is predicted that ZNF146 gene affects the proliferation of muscle cells, and then affects the growth and development of cattle. Furthermore, CNV genotyping of ZNF146 gene was three types (deletion type, normal type and duplication type) by Real-time fluorescent quantitative PCR (qPCR). The association analysis results showed that ZNF146-CNV was significantly correlated with rump length of Qinchuan cattle, hucklebone width of Jiaxian red cattle and heart girth of Yunling cattle. From the above results, ZNF146-CNV had a significant effect on growth traits, which provided an important candidate molecular marker for growth and development of Chinese cattle.

Background: Despite a higher prevalence of traumatic spinal cord injury (TSCI) amongst Canadian Indigenous peoples, there is a paucity of studies focused on Indigenous TSCI. We present the first Canada-wide study comparing TSCI amongst Canadian Indigenous and non-Indigenous peoples. Methods: This study is a retrospective analysis of prospectively-collected TSCI data from the Rick Hansen Spinal Cord Injury Registry (RHSCIR) from 2004-2019. We divided participants into Indigenous and non-Indigenous cohorts and compared them with respect to demographics, injury mechanism, level, severity, and outcomes. Results: Compared with non-Indigenous patients, Indigenous patients were younger, more female, less likely to have higher education, and less likely to be employed. The mechanism of injury was more likely due to assault or transportation-related trauma in the Indigenous group. The length of stay for Indigenous patients was longer. Indigenous patients were more likely to be discharged to a rural setting, less likely to be discharged home, and more likely to be unemployed following injury. Conclusions: Our results suggest that more resources need to be dedicated for transitioning Indigenous patients sustaining a TSCI to community living and for supporting these patients in their home communities. A focus on resources and infrastructure for Indigenous patients by engagement with Indigenous communities is needed.

Background: A correlation between the nerve root sedimentation sign (SedSign) and neurogenic claudication has been demonstrated; though it did not account for leg-dominant pain. This study analyzed the utility of SedSign to diagnose leg-dominant pain using validated classification systems. Methods: We retrospectively reviewed prospective data from 367 patients with back or leg pain collected between January 1, 2012 to May 31, 2018. Baseline characteristics included SSPc (Saskatchewan Spine Pathway classification), Oswestry disability index (ODI), visual analogue pain scores (VAS), and EuroQol Group 5-Dimension Self-Report (EQ5D). Inter- and intra-rater reliability for SedSign was 73% and 91%. Results: SedSign was positive in 111 (30.2%) and negative in 256 (69.8%) patients. Univariate analysis showed a correlation between SedSign and age, male sex, ODI, EQ5D, cross-sectional area (CSA) of stenosis, antero-posterior diameter of stenosis, and leg-dominant pain; negative SedSign was correlated with back-dominant pain. Multivariate analysis revealed an association between SedSign and age, male sex, CSA stenosis, and ODI walking distance. The sensitivity, specificity, positive and negative predictive values of SedSign for leg-dominant pain were 33.5%, 83.2%, 77.0%, and 57.3%. Conclusions: SedSign has high specificity but low sensitivity for leg-dominant pain. Despite a similar correlation between SedSign and neurogenic claudication or sciatica, significance was lost on multivariate analysis.

OBJECTIVES/GOALS: Current approaches to drug development for the aggressive triple negative breast cancer rely on current 2D and 3D in vitro models which have limited capabilities. We have developed a translational microphysiological system that can maintain the human breast microenvironment to capture the complex interaction with the tumor microenvironment. METHODS/STUDY POPULATION: Three different TNBC cell lines were seeded in BC-MPS: MDA-MB-231 parental cell line, MDA-MB-231wiht the gene, LKB1 overexpressed, which is a tumor suppressor, and MDA-MB-231 with the enzyme, ERK5, an enzyme associated with increased metastasis and drug resistance, knocked out. These three TNBC cell lines were cultured in a standard 2D 96-well plate and in BC-MPS. Time-lapse videos were taken to track cellular mobility. RNA-sequencing was performed to compare different expression levels of various cancer related genes of the cell lines cultured in standard 2D and BC-MPS. RESULTS/ANTICIPATED RESULTS: The LKB1 overexpressed MDA-MB-231 and the ERK5-ko MDA-MB-231 cell lines are expected to have decreased mobility compared to the parental cells. The cell lines are expected to have increased expression of cancer related genes when cultured in BC-MPS than when cultured in standard 2D due to the presence of human breast tissue. DISCUSSION/SIGNIFICANCE: BC-MPS is a promising new translational MPS that facilitates studying long term interactions between real human breast tissue and cancer cells. The BC-MPS systems ability to support the growth of established cell lines has been demonstrated. Future studies will focus on developing the model for personalized medicine.

This article examines the origins and dynamics of an extraordinary wave of protests in Hong Kong in 2019–2020. Despite lacking visible political opportunities and organizational resources, the protest movement drew resilient, mass participation unparalleled in the city's history and much of the world. Drawing from original on-site surveys and online datasets, we conceptualize the Anti-Extradition Law Amendment Bill Movement as a form of “total mobilization from below.” The totality of the mobilization depended on a set of interactive mechanisms: abeyant civil society networks concealed after the 2014 Umbrella Movement were activated by threats over extradition and institutional decay, whereas affective ties developed through conflicts and mutual assistance were amplified by digital communication. The movement's characteristics in terms of protest scale, mobilizing structure, use of alternative spaces, and group solidarity are examined. The spasmodic moments of mobilization are explained by a nexus of network building that took place in an unreceptive environment and at a critical juncture. The roles of threats and emotions in mass mobilizations are also analysed.