Systematic assessment of long-read RNA-seq methods for transcript identification and quantification.

The Long-read RNA-Seq Genome Annotation Assessment Project Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. Using different protocols and sequencing platforms, the consortium generated over 427 million long-read sequences from complementary DNA and direct RNA datasets, encompassing human, mouse and manatee species. Developers utilized these data to address challenges in transcript isoform detection, quantification and de novo transcript detection. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. Incorporating additional orthogonal data and replicate samples is advised when aiming to detect rare and novel transcripts or using reference-free approaches. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.

Pbx1-dependent control of VMC differentiation kinetics underlies gross renal vascular patterning.

The architecture of an organ's vascular bed subserves its physiological function and metabolic demands. However, the mechanisms underlying gross vascular patterning remain elusive. Using intravital dye labeling and 3D imaging, we discovered that systems-level vascular patterning in the kidney is dependent on the kinetics of vascular mural cell (VMC) differentiation. Conditional ablation of the TALE transcription factor Pbx1 in renal VMC progenitors in the mouse led to the premature upregulation of PDGFRß, a master initiator of VMC-blood vessel association. This precocious VMC differentiation resulted in nonproductive angiogenesis, abnormal renal arterial tree patterning and neonatal death consistent with kidney dysfunction. Notably, we establish that Pbx1 directly represses Pdgfrb, and demonstrate that decreased Pdgfrb dosage in conditional Pbx1 mutants substantially rescues vascular patterning defects and neonatal survival. These findings identify, for the first time, an in vivo transcriptional regulator of PDGFRß, and reveal a previously unappreciated role for VMCs in systems-level vascular patterning.

Biomimetic Human Lung Alveolar Interstitium Chip with Extended Longevity.

Determining the mechanistic causes of lung diseases, developing new treatments thereof, and assessing toxicity whether from chemical exposures or engineered nanomaterials would benefit significantly from a preclinical human lung alveolar interstitium model of physiological relevance. The existing preclinical models have limitations because they fail to replicate the key anatomical and physiological characteristics of human alveoli. Thus, a human lung alveolar interstitium chip was developed to imitate key alveolar microenvironmental factors including an electrospun nanofibrous membrane as the analogue of the basement membrane for co-culture of epithelial cells with fibroblasts embedded in 3D collagenous gels, physiologically relevant interstitial matrix stiffness, interstitial fluid flow, and 3D breathing-like mechanical stretch. The biomimetic chip substantially improved the epithelial barrier function compared to transwell models. Moreover, the chip having a gel made of a collagen I-fibrin blend as the interstitial matrix sustained the interstitium integrity and further enhanced the epithelial barrier, resulting in a longevity that extended beyond eight weeks. The assessment of multiwalled carbon nanotube toxicity on the chip was in line with the animal study.

Nanofibrous Membrane Promotes and Sustains Vascular Endothelial Barrier Function.

The vascular endothelium serves as a physical barrier between the circulating blood and surrounding tissue and acts as a critical regulator of various physiological processes. In vitro models involving vasculature rely on the maintenance of the endothelial barrier function. In this study, we fabricated 2D aligned nanofibrous membranes with distinct pore sizes via electrospinning and investigated the effect of membrane pore size on endothelial barrier function. Our results demonstrated that the use of the nanofibrous membranes promoted the formation of a tight vascular endothelium and sustained barrier function for over one month in comparison with conventional transwell setups. Moreover, the examination of the nucleocytoplasmic localization of yes-associated protein (YAP) in the endothelial cells indicated that nanofibrous membrane promoted YAP expression and its nuclear localization, critical to endothelial barrier function. Furthermore, the comparison of permeability between random and aligned nanofibrous membranes underscored the importance of pore size in preserving barrier function. Our findings offer a valuable strategy for creating more physiologically relevant in vitro vascular models and contribute to the understanding of endothelial barrier formation and maintenance mechanisms.

Systematic assessment of long-read RNA-seq methods for transcript identification and quantification.

The Long-read RNA-Seq Genome Annotation Assessment Project (LRGASP) Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. The consortium generated over 427 million long-read sequences from cDNA and direct RNA datasets, encompassing human, mouse, and manatee species, using different protocols and sequencing platforms. These data were utilized by developers to address challenges in transcript isoform detection and quantification, as well as de novo transcript isoform identification. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. When aiming to detect rare and novel transcripts or when using reference-free approaches, incorporating additional orthogonal data and replicate samples are advised. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.

A pilot study of lay health worker outreach and colorectal cancer screening among Chinese Americans.

The research team recruited eight Chinese American (seven females, one male) lay health workers (LHWs). They received 12 h of training about colorectal cancer (CRC), its screening, and basic health education techniques. Each LHW were asked to recruit ten participants and conduct two educational sessions. Of the 81 participants recruited, 73 had not received colorectal cancer screening. Their mean age was 63.0 years, and 72.6% were women. Knowledge of colorectal cancer, its causes, and its screening increased significantly. Receipt of first colorectal cancer screening test increased from 0.0% at baseline to 55.7% for fecal occult blood tests, 7.1% for sigmoidoscopy, and 7.1% for colonoscopy. LHW outreach is feasible and may be effective in promoting CRC screening among Chinese Americans.