Molecular mechanisms of gating and selectivity in transport channels

Transport channels and pores are of fundamental importance for translocation of molecules across the otherwise-impermeable biological membrane. As a conduit for the passage of material, specific membrane channels must accomplish the task of selective transport of molecules, while preventing unnecessary loss of other cellular material. The nuclear pore complex (NPC), and the mechanosensitive channel of small conductance, MscS are the focus of the research presented in this thesis. NPCs are sole gateways for passage of material across the nuclear envelope of eukaryotic cells. Several unstructured proteins that are rich in phenylaline- glycine motifs (FG-nups) form the central transport channel. Small molecules passively diffuse through the channel but, larger molecules are selectively transported via transport factors (TFs), which apparently interact with the FG-repeats of the nups in the channel. To understand how nups are assembled in the interior of the NPC, assemblies of one kind of nup, starting from different initial states, are investigated. Results suggest nups form different structures in different regions of the central channel. While only molecules of size < 9nm can penetrate through, a limit known for passive diffusion, the resulting structure posed a selectivity barrier for larger molecules that can be penetrated only via TF interactions. Mechanosensitive (MS) channels, bacterial inner-membrane proteins, open and close in response to mechanical stimuli such as changes in membrane tension during osmotic stress. These channels act as safety valves preventing cell lysis upon hypoosmotic cell swelling: the channels open under membrane tension to release osmolytes along with water. MscS, consists, beside the transmembrane channel, of a large cytoplasmic domain (CD) that features a balloon-like, water filled chamber opening to the cytoplasm through seven side pores and a small distal pore. The CD is apparently a molecular sieve covering the channel, that optimizes loss of osmolytes during osmoadaptation. Diffusion theory and MD simulations are employed to explore the transport kinetics of Glu- and K+ as representative osmolytes. A role of a filter is suggested for the CD such that it balances passage of Glu- and K+ osmolytes, to yield a largely neutral efflux, thereby, reducing cell depolarization in the open state that also conserves to a large degree the essential metabolite Glu-

Diversity of underutilized vegetables and fruit in Sri Lanka: Prioritization for collection, conservation, genetic improvement, and promotion

Despite their crucial role in combating hunger, malnutrition, and poverty, many plant species cultivated as fruits, vegetables, roots, and tuber crops remain neglected and underutilized worldwide, and Sri Lanka is no exception. Integrating these crops into farming systems has the potential to create nutrient-dense, climate-resilient, and sustainable agricultural practices. The study titled "Diversity of underutilized vegetables and fruit in Sri Lanka: prioritization for collection, conservation, genetic improvement, and promotion" highlights the significance of underutilized vegetables and fruits in Sri Lanka's agricultural biodiversity. These often-overlooked crops possess unique nutritional and agronomic traits that could benefit both farmers and consumers. However, they have been overshadowed by more commonly cultivated species and varieties. To safeguard the genetic diversity and potential contributions of these underutilized crops to food security and nutrition, the study emphasizes the need for prioritizing their collection and conservation. Preserving their genetic resources enables researchers and farmers to access a broader range of traits, enhancing resilience to environmental challenges. Additionally, the research underscores the importance of genetic improvement efforts to boost the productivity and adaptability of underutilized vegetables and fruits. Breeding programs focused on priority species can lead to the development of new cultivars with desirable traits, such as increased yield, disease resistance, and improved nutritional content. Moreover, promoting underutilized crops is essential to raise awareness among consumers, retailers, and policymakers about their nutritional benefits and economic potential. By creating market demand and integrating these crops into agricultural systems, their sustainable cultivation and utilization become more viable. In summary, recognizing and prioritizing the diversity of underutilized vegetables and fruits in Sri Lanka is crucial. Through strategic collection, conservation, genetic improvement, and promotion efforts, these crops can significantly contribute to enhancing food security, preserving biodiversity, and supporting sustainable agricultural practices in the country

Assembly of Nsp1 nucleoporins provides insight into nuclear pore complex gating.

Nuclear pore complexes (NPCs) form gateways for material transfer across the nuclear envelope of eukaryotic cells. Disordered proteins, rich in phenylalanine-glycine repeat motifs (FG-nups), form the central transport channel. Understanding how nups are arranged in the interior of the NPC may explain how NPC functions as a selectivity filter for transport of large molecules and a sieve-like filter for diffusion of small molecules (<9 nm or 40 kDa). We employed molecular dynamics to model the structures formed by various assemblies of one kind of nup, namely the 609-aa-long FG domain of Nsp1 (Nsp1-FG). The simulations started from different initial conformations and geometrical arrangements of Nsp1-FGs. In all cases Nsp1-FGs collectively formed brush-like structures with bristles made of bundles of 2-27 nups, however, the bundles being cross-linked through single nups leaving one bundle and joining a nearby one. The degree of cross-linking varies with different initial nup conformations and arrangements. Structural analysis reveals that FG-repeats of the nups not only involve formation of bundle structures, but are abundantly present in cross-linking regions where the epitopes of FG-repeats are highly accessible. Large molecules that are assisted by transport factors (TFs) are selectively transported through NPC apparently by binding to FG-nups through populated FG-binding pockets on the TF surface. Therefore, our finding suggests that TFs bind concertedly to multiple FGs in cross-linking regions and break-up the bundles to create wide pores for themselves and their cargoes to pass. In addition, the cross-linking between Nsp1-FG bundles, arising from simulations, is found to set a molecular size limit of <9 nm (40 kDa) for passive diffusion of molecules. Our simulations suggest that the NPC central channel, near the periphery where tethering of nups is dominant, features brush-like moderately cross-linked bundles, but in the central region, where tethering loses its effect, features a sieve-like structure of bundles and frequent cross-links

Evaluation of two main RNA-seq approaches for gene quantification in clinical RNA sequencing: polyA+ selection versus rRNA depletion

Abstract To allow efficient transcript/gene detection, highly abundant ribosomal RNAs (rRNA) are generally removed from total RNA either by positive polyA+ selection or by rRNA depletion (negative selection) before sequencing. Comparisons between the two methods have been carried out by various groups, but the assessments have relied largely on non-clinical samples. In this study, we evaluated these two RNA sequencing approaches using human blood and colon tissue samples. Our analyses showed that rRNA depletion captured more unique transcriptome features, whereas polyA+ selection outperformed rRNA depletion with higher exonic coverage and better accuracy of gene quantification. For blood- and colon-derived RNAs, we found that 220% and 50% more reads, respectively, would have to be sequenced to achieve the same level of exonic coverage in the rRNA depletion method compared with the polyA+ selection method. Therefore, in most cases we strongly recommend polyA+ selection over rRNA depletion for gene quantification in clinical RNA sequencing. Our evaluation revealed that a small number of lncRNAs and small RNAs made up a large fraction of the reads in the rRNA depletion RNA sequencing data. Thus, we recommend that these RNAs are specifically depleted to improve the sequencing depth of the remaining RNAs

80 Biophysical Journal Volume 101 July 2011 80–89 Cytoplasmic Domain Filter Function in the Mechanosensitive Channel of Small Conductance

ABSTRACT Mechanosensitive channels, inner membrane proteins of bacteria, open and close in response to mechanical stimuli such as changes in membrane tension during osmotic stress. In bacteria, these channels act as safety valves preventing cell lysis upon hypoosmotic cell swelling: the channels open under membrane tension to release osmolytes along with water. The mechanosensitive channel of small conductance, MscS, consists, in addition to the transmembrane channel, of a large cytoplasmic domain (CD) that features a balloon-like, water filled chamber opening to the cytoplasm through seven side pores and a small distal pore. The CD is apparently a molecular sieve covering the channel that optimizes loss of osmolytes during osmoadaptation. We employ diffusion theory and molecular dynamics simulations to explore the transport kinetics of Glu and K þ as representative osmolytes. We suggest that the CD indeed acts as a filter that actually balances passage of Glu and K þ, and possibly other positive and negative osmolytes, to yield a largely neutral efflux and, thereby, reduce cell depolarization in the open state and conserve to a large degree the essential metabolite Glu

QuickIsoSeq for Isoform Quantification in Large-Scale RNA Sequencing

RNA-sequencing (RNA-seq) is a powerful technology for transcriptome profiling. While most RNA-seq projects focus on gene-level quantification and analysis, there is growing evidence that most mammalian genes are alternatively spliced to generate different isoforms that can be subsequently translated to protein molecules with diverse or even opposing biological functions. Quantifying the expression levels of these isoforms is key to understanding the genes biological functions in healthy tissues and the progression of diseases. Among open source tools developed for isoform quantification, Salmon, Kallisto, and RSEM are recommended based upon previous systematic evaluation of these tools using both experimental and simulated RNA-seq datasets. However, isoform quantification in practical RNA-seq data analysis needs to deal with many QC issues, such as the abundance of rRNAs in mRNA-seq, the efficiency of globin RNA depletion in whole blood samples, and potential sample swapping. To overcome these practical challenges, QuickIsoSeq was developed for large-scale RNA-seq isoform quantification along with QC. In this chapter, we describe the pipeline and detailed the steps required to deploy and use it to analyze RNA-seq datasets in practice. The QuickIsoSeq package can be downloaded from https://github.com/shanrongzhao/QuickIsoSeq

Bundle thickness distribution.

<p>Bundle thickness is determined by the number of Nsp1-FG chains involved in a bundle. Shown is here the distribution of these numbers for the simulations carried out. The frequencies with which chain numbers arise were averaged for the last 30 ns of the four simulations wild-type_ring, mutant_ring, random_array, and random_bath. Bundles with fewer than ten Nsp1-FG chains favor a mesh-like structure, namely bundles with frequent cross-links, whereas bundles with more than ten Nsp1-FG chains exhibit brush-like structures with relatively few cross-links. Green represents the frequency distribution of Nsp1-FGs for simulation wild-type_ring, red for mutant_ring, cyan for random_array, and purple for random_bath.</p