Exploring the current dynamics of preprints.

Preprints are non-peer-reviewed and publicly available articles for open and transparent research communication. Preprint servers host the submission of such manuscripts, and despite the presence of established preprint servers, their numbers have continued to rise in recent times. A steep increasing pattern in posted preprints and their accommodating servers has been observed over the last decade. In this article, we explored the global trends in the preprint adoption and its involvement in promoting open and transparent research findings across various domains. We further emphasized the importance of preprinting, highlighting its significant impact during the pandemic through effective information sharing, and advocating for its broader integration in scholarly communication.

Structural basis of prostaglandin efflux by MRP4.

Multidrug resistance protein 4 (MRP4) is a broadly expressed ATP-binding cassette transporter that is unique among the MRP subfamily for transporting prostanoids, a group of signaling molecules derived from unsaturated fatty acids. To better understand the basis of the substrate selectivity of MRP4, we used cryogenic-electron microscopy to determine six structures of nanodisc-reconstituted MRP4 at various stages throughout its transport cycle. Substrate-bound structures of MRP4 in complex with PGE1, PGE2 and the sulfonated-sterol DHEA-S reveal a common binding site that accommodates a diverse set of organic anions and suggest an allosteric mechanism for substrate-induced enhancement of MRP4 ATPase activity. Our structure of a catalytically compromised MRP4 mutant bound to ATP-Mg2+ is outward-occluded, a conformation previously unobserved in the MRP subfamily and consistent with an alternating-access transport mechanism. Our study provides insights into the endogenous function of this versatile efflux transporter and establishes a basis for MRP4-targeted drug design.

Nurturing diversity and inclusion in AI in Biomedicine through a virtual summer program for high school students.

Artificial Intelligence (AI) has the power to improve our lives through a wide variety of applications, many of which fall into the healthcare space; however, a lack of diversity is contributing to limitations in how broadly AI can help people. The UCSF AI4ALL program was established in 2019 to address this issue by targeting high school students from underrepresented backgrounds in AI, giving them a chance to learn about AI with a focus on biomedicine, and promoting diversity and inclusion. In 2020, the UCSF AI4ALL three-week program was held entirely online due to the COVID-19 pandemic. Thus, students participated virtually to gain experience with AI, interact with diverse role models in AI, and learn about advancing health through AI. Specifically, they attended lectures in coding and AI, received an in-depth research experience through hands-on projects exploring COVID-19, and engaged in mentoring and personal development sessions with faculty, researchers, industry professionals, and undergraduate and graduate students, many of whom were women and from underrepresented racial and ethnic backgrounds. At the conclusion of the program, the students presented the results of their research projects at the final symposium. Comparison of pre- and post-program survey responses from students demonstrated that after the program, significantly more students were familiar with how to work with data and to evaluate and apply machine learning algorithms. There were also nominally significant increases in the students' knowing people in AI from historically underrepresented groups, feeling confident in discussing AI, and being aware of careers in AI. We found that we were able to engage young students in AI via our online training program and nurture greater diversity in AI. This work can guide AI training programs aspiring to engage and educate students entirely online, and motivate people in AI to strive towards increasing diversity and inclusion in this field.

Meta-Analysis of the Expansion in the Field of Structural Biology of ABC Transporters.

ABC transporters are molecular machines which power the solute transport using ATP hydrolysis. The structural biology of ABC transporters has been exploding for the last few years, and this study explores timelines and trends for various attributes such as structural tools, resolution, fold, sources, and group leaders. This study also evidences the significance of mammalian expression systems, advancements in structural biology tools, and the developing interest of group leaders across the world in the remarkably progressing field. The field started in 2002 and bloomed in 2016, and COVID years were really productive to the field. Specifically, the study explores 337 structures of 58 unique ABC transporters deposited in the PDB database from which P-glycoprotein has the largest number of structures. Approximately, 62% of total structures are determined at the resolution of 3-4 Å and 53% of structures belong to fold IV type. With progressive advancements in the field, the field is shifting from prokaryotic to eukaryotic sources and X-ray crystallography to cryoelectron microscopy. In the nutshell, this study uniquely provides the detailed snapshot of the field of structural biology of ABC transporters with real-time data.

Interaction of Commonly Used Oral Molecular Excipients with P-glycoprotein.

P-glycoprotein (P-gp) plays a critical role in drug oral bioavailability, and modulation of this transporter can alter the safety and/or efficacy profile of substrate drugs. Individual oral molecular excipients that inhibit P-gp function have been considered a mechanism for improving drug absorption, but a systematic evaluation of the interaction of excipients with P-gp is critical for informed selection of optimal formulations of proprietary and generic drug products. A library of 123 oral molecular excipients was screened for their ability to inhibit P-gp in two orthogonal cell-based assays. ß-Cyclodextrin and light green SF yellowish were identified as modest inhibitors of P-gp with IC50 values of 168 µM (95% CI, 118-251 µM) and 204 µM (95% CI, 5.9-1745 µM), respectively. The lack of effect of most of the tested excipients on P-gp transport provides a wide selection of excipients for inclusion in oral formulations with minimal risk of influencing the oral bioavailability of P-gp substrates.

Screening a Library of FDA-Approved and Bioactive Compounds for Antiviral Activity against SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has emerged as a major global health threat. The COVID-19 pandemic has resulted in over 168 million cases and 3.4 million deaths to date, while the number of cases continues to rise. With limited therapeutic options, the identification of safe and effective therapeutics is urgently needed. The repurposing of known clinical compounds holds the potential for rapid identification of drugs effective against SARS-CoV-2. Here, we utilized a library of FDA-approved and well-studied preclinical and clinical compounds to screen for antivirals against SARS-CoV-2 in human pulmonary epithelial cells. We identified 13 compounds that exhibit potent antiviral activity across multiple orthogonal assays. Hits include known antivirals, compounds with anti-inflammatory activity, and compounds targeting host pathways such as kinases and proteases critical for SARS-CoV-2 replication. We identified seven compounds not previously reported to have activity against SARS-CoV-2, including B02, a human RAD51 inhibitor. We further demonstrated that B02 exhibits synergy with remdesivir, the only antiviral approved by the FDA to treat COVID-19, highlighting the potential for combination therapy. Taken together, our comparative compound screening strategy highlights the potential of drug repurposing screens to identify novel starting points for development of effective antiviral mono- or combination therapies to treat COVID-19.

Highlighting membrane protein structure and function: A celebration of the Protein Data Bank.

Biological membranes define the boundaries of cells and compartmentalize the chemical and physical processes required for life. Many biological processes are carried out by proteins embedded in or associated with such membranes. Determination of membrane protein (MP) structures at atomic or near-atomic resolution plays a vital role in elucidating their structural and functional impact in biology. This endeavor has determined 1198 unique MP structures as of early 2021. The value of these structures is expanded greatly by deposition of their three-dimensional (3D) coordinates into the Protein Data Bank (PDB) after the first atomic MP structure was elucidated in 1985. Since then, free access to MP structures facilitates broader and deeper understanding of MPs, which provides crucial new insights into their biological functions. Here we highlight the structural and functional biology of representative MPs and landmarks in the evolution of new technologies, with insights into key developments influenced by the PDB in magnifying their impact.

Transcriptional responses of soybean roots to colonization with the root endophytic fungus Piriformospora indica reveals altered phenylpropanoid and secondary metabolism.

Piriformospora indica, a root endophytic fungus, has been shown to enhance biomass production and confer tolerance to various abiotic and biotic stresses in many plant hosts. A growth chamber experiment of soybean (Glycine max) colonized by P. indica compared to uninoculated control plants showed that the fungus significantly increased shoot dry weight, nutrient content, and rhizobial biomass. RNA-Seq analyses of root tissue showed upregulation of 61 genes and downregulation of 238 genes in colonized plants. Gene Ontology (GO) enrichment analyses demonstrated that upregulated genes were most significantly enriched in GO categories related to lignin biosynthesis and regulation of iron transport and metabolism but also mapped to categories of nutrient acquisition, hormone signaling, and response to drought stress. Metabolic pathway analysis revealed upregulation of genes within the phenylpropanoid and derivative pathways such as biosynthesis of monolignol subunits, flavonoids and flavonols (luteolin and quercetin), and iron scavenging siderophores. Highly enriched downregulated GO categories included heat shock proteins involved in response to heat, high-light intensity, hydrogen peroxide, and several related to plant defense. Overall, these results suggest that soybean maintains an association with this root endosymbiotic fungus that improves plant growth and nutrient acquisition, modulates abiotic stress, and promotes synergistic interactions with rhizobia.

Conformational Dynamics in the Binding-Protein-Independent Mutant of the Escherichia coli Maltose Transporter, MalG511, and Its Interaction with Maltose Binding Protein.

MalG511 is a genetically selected binding-protein-independent mutant of the Escherichia coli maltose transporter MalFGK2, which retains specificity for maltose and shows a high basal ATPase activity in the absence of maltose binding protein (MBP). It shows an intriguing biphasic behavior in maltose transport assays in the presence of MBP, with low levels of MBP stimulating the activity and higher levels (>50 µM) inhibiting the transport activity. Remarkably, the rescuing effect of the MBP suppressor mutant, MBPG13D, turns it into an attractive model for studying regulatory mechanisms in the ABC transporter superfamily. It is hypothesized that the special characteristics of MalG511 result from mutations that shift its equilibrium toward the transition state of MalFGK2. We tested this hypothesis by using site-directed spin labeling in combination with electron paramagnetic resonance spectroscopy, which showed conformational changes in MalG511 and its interaction with MBP and MBPG13D during its catalytic cycle. We found that MalG511 utilizes the same alternate access mechanism as MalFGK2, including all three open, semi-open, and closed states of the MalK dimer, to transport maltose across the membrane. However, the equilibrium of this mutant is shifted toward the semi-open state in its resting state and interacts with MBP with high affinity, providing an explanation for the inhibition of MalG511 by MBP at higher concentrations. In contrast, the mutant binding protein, MBPG13D, interacts with lower affinity and could restore MalG511 to a normal catalytic cycle.

Biochemical characterization of essential cell division proteins FtsX and FtsE that mediate peptidoglycan hydrolysis by PcsB in Streptococcus pneumoniae.

The FtsEX:PcsB complex forms a molecular machine that carries out peptidoglycan (PG) hydrolysis during normal cell division of the major respiratory pathogenic bacterium, Streptococcus pneumoniae (pneumococcus). FtsX is an integral membrane protein and FtsE is a cytoplasmic ATPase that together structurally resemble ABC transporters. Instead of transport, FtsEX transduces signals from the cell division apparatus to stimulate PG hydrolysis by PcsB, which interacts with extracellular domains of FtsX. Structural studies of PcsB and one extracellular domain of FtsX have recently appeared, but little is known about the biochemical properties of the FtsE ATPase or the intact FtsX transducer protein. We report here purifications and characterizations of tagged FtsX and FtsE proteins. Pneumococcal FtsX-GFP-His and FtsX-His could be overexpressed in Escherichia coli without toxicity, and FtsE-His remained soluble during purification. FtsX-His dimerizes in detergent micelles and when reconstituted in phospholipid nanodiscs. FtsE-His binds an ATP analog with an affinity comparable to that of ATPase subunits of ABC transporters, and FtsE-His preparations have a low, detectable ATPase activity. However, attempts to detect complexes of purified FtsX-His, FtsE-His, and PcsB-His or coexpressed tagged FtsX and FtsE were not successful with the constructs and conditions tested so far. In working with nanodiscs, we found that PcsB-His has an affinity for charged phospholipids, mediated partly by interactions with its coiled-coil domain. Together, these findings represent first steps toward reconstituting the FtsEX:PcsB complex biochemically and provide information that may be relevant to the assembly of the complex on the surface of pneumococcal cells.

Nonfouling NTA-PEG-Based TEM Grid Coatings for Selective Capture of Histidine-Tagged Protein Targets from Cell Lysates.

We report the preparation and performance of TEM grids bearing stabilized nonfouling lipid monolayer coatings. These films contain NTA capture ligands of controllable areal density at the distal end of a flexible poly(ethylene glycol) 2000 (PEG2000) spacer to avoid preferred orientation of surface-bound histidine-tagged (His-tag) protein targets. Langmuir-Schaefer deposition at 30 mN/m of mixed monolayers containing two novel synthetic lipids-1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(5-amido-1-carboxypentyl)iminodiacetic acid]polyethylene glycolamide 2000) (NTA-PEG2000-DSPE) and 1,2-(tricosa-10',12'-diynoyl)-sn-glycero-3-phosphoethanolamine-N-(methoxypolyethylene glycolamide 350) (mPEG350-DTPE)-in 1:99 and 5:95 molar ratios prior to treatment with a 5 min, 254 nm light exposure was used for grid fabrication. These conditions were designed to limit nonspecific protein adsorption onto the stabilized lipid coating by favoring the formation of a mPEG350 brush layer below a flexible, mushroom conformation of NTA-PEG2000 at low surface density to enable specific immobilization and random orientation of the protein target on the EM grid. These grids were then used to capture His6-T7 bacteriophage and RplL from cell lysates, as well as purified His8-green fluorescent protein (GFP) and nanodisc solubilized maltose transporter, His6-MalFGK2. Our findings indicate that TEM grid supported, polymerized NTA lipid monolayers are capable of capturing His-tag protein targets in a manner that controls their areal densities, while efficiently blocking nonspecific adsorption and limiting film degradation, even upon prolonged detergent exposure.

Full engagement of liganded maltose-binding protein stabilizes a semi-open ATP-binding cassette dimer in the maltose transporter.

MalFGK2 is an ATP-binding cassette (ABC) transporter that mediates the uptake of maltose/maltodextrins into Escherichia coli. A periplasmic maltose-binding protein (MBP) delivers maltose to the transmembrane subunits (MalFG) and stimulates the ATPase activity of the cytoplasmic nucleotide-binding subunits (MalK dimer). This MBP-stimulated ATPase activity is independent of maltose for purified transporter in detergent micelles. However, when the transporter is reconstituted in membrane bilayers, only the liganded form of MBP efficiently stimulates its activity. To investigate the mechanism of maltose stimulation, electron paramagnetic resonance spectroscopy was used to study the interactions between the transporter and MBP in nanodiscs and in detergent. We found that full engagement of both lobes of maltose-bound MBP unto MalFGK2 is facilitated by nucleotides and stabilizes a semi-open MalK dimer. Maltose-bound MBP promotes the transition to the semi-open state of MalK when the transporter is in the membrane, whereas such regulation does not require maltose in detergent. We suggest that stabilization of the semi-open MalK2 conformation by maltose-bound MBP is key to the coupling of maltose transport to ATP hydrolysis in vivo, because it facilitates the progression of the MalK dimer from the open to the semi-open conformation, from which it can proceed to hydrolyze ATP.

An ecological role of fungal endophytes to ameliorate plants under biotic stress.

It is our consensus that plants survive and flourish in stressed ecosystems because of endosymbiotic organisms that have co-evolved and were essential for their adaptation to changing environments. Some of these microbial components are noncultivable and vertically transmitted from generation to generation. They represent a vast reservoir of heritable DNA that can enhance plant performance in changing environments and add genetic flexibility to adaptation of long-lived plants. If such endophytes can be identified that not only persist in progeny of novel hosts, but can confer benefits in mechanized, agricultural systems, they would be increasingly important in agricultural production and lead to a rapid and economical method of providing novel germplasms of native and crop plants. In the present review, authors advocate the deployment of fungal diversity and its role to overcome the biotic stress in plants. Endophytic fungal association with plants helps it to protect from various pathogen and pests and adapt to survive in harsh biotic and abiotic stress condition.