Native Function Research Articles

A Puccinia striiformis f. sp. tritici Effector with DPBB Domain Suppresses Wheat Defense

Wheat (Triticum aestivum L.) is a primary crop globally. Among the numerous pathogens affecting wheat production, Puccinia striiformis f. sp. tritici (Pst) is a significant biotic stress agent and poses a major threat to world food security by causing stripe rust or yellow rust disease. Understanding the molecular basis of plant–pathogen interactions is crucial for developing new means of disease management. It is well established that the effector proteins play a pivotal role in pathogenesis. Therefore, studying effector proteins has become an important area of research in plant biology. Our previous work identified differentially expressed candidate secretory effector proteins of stripe rust based on transcriptome sequencing data from susceptible wheat (Avocet S) and resistant wheat (Avocet YR10) infected with Pst. Among the secreted effector proteins, PSTG_14090 contained an ancient double-psi beta-barrel (DPBB) fold, which is conserved in the rare lipoprotein A (RlpA) superfamily. This study investigated the role of PSTG_14090 in plant immune responses, which encodes a protein, here referred to as Pst-DPBB, having 131 amino acids with a predicted signal peptide (SP) of 19 amino acids at the N-terminal end, and the DNA sequence of this effector is highly conserved among different stripe rust races. qRT-PCR analysis indicated that expression levels are upregulated during the early stages of infection. Subcellular localization studies in Nicotiana benthamiana leaves and wheat protoplasts revealed that it is distributed in the cytoplasm, nucleus, and apoplast. We demonstrated that Pst-DPBB negatively regulates the immune response by functioning in various compartments of the plant cells. Based on Co-IP and structural predictions and putative interaction analyses by AlphaFold 3, we propose the probable biological function(s). Pst-DPBB behaves as a papain inhibitor of wheat cysteine protease; Pst-DPBB has high structural homology to kiwellin, which is known to interact with chorismate mutase, suggesting that Pst-DPBB inhibits the native function of the host chorismate mutase involved in salicylic acid synthesis. The DPBB fold is also known to interact with DNA and RNA, which may suggest its possible role in regulating the host gene expression.

Unified approaches in transition metal catalyzed C(sp3)-H functionalization: recent advances and mechanistic aspects.

In organic synthesis, C(sp3)-H functionalization is a revolutionary method that allows direct alteration of unactivated C-H bonds. It can obviate the need for pre-functionalization and provides access to streamlined and atom economical routes for the synthesis of complex molecules starting from simple starting materials. Many strategies have evolved, such as photoredox catalysis, organocatalysis, non-directed C-H activation, transiently directed C-H activation, and native functionality directed C-H activation. Together these advances have reinforced the importance of C(sp3)-H functionalization in synthetic chemistry. C(sp3)-H functionalization has direct applications in pharmacology, agrochemicals, and materials science, demonstrating its ability to transform synthetic approaches by creating new retrosynthetic disconnections and boost the efficiency of chemical processes. This review aims to provide an overview of current state of C(sp3)-H functionalization, focusing more on recent breakthroughs and associated mechanistic insights.

Human cathelicidin LL-37 rapidly disrupted colonic epithelial integrity.

The intestinal barrier, held together by epithelial cells and intercellular tight junction (TJ) proteins, prevents the penetration of microbial pathogens. Concurrently, intestinal epithelial cells secrete antimicrobial peptides, including cathelicidin. Cathelicidin has direct antibacterial and immunomodulatory functions, although its role in intestinal integrity remains elusive. In this study, we demonstrate that direct stimulation of human colonic epithelial (T84) cells with human cathelicidin, LL-37, resulted in a rapid and transient increase in epithelial cell permeability. This increased permeability was associated with the TJ proteins occludin and claudin-2 degradation, mediated by these specific proteins' endocytosis and lysosomal degradation. While murine cathelicidin (CRAMP) failed to modify T84 cell permeability, LL-37 degraded TJ proteins in murine rectal epithelial (CMT-93) cells. The stimulus of (CMT-93) cells with LL-37 aggravated the cell permeability and furthered TJ degradation provoked by the intestinal pathogen, attaching/effacing (A/E) Citrobacter rodentium (C. rodentium). The number of C. rodentium that colonized CMT-93 cells was not severely impacted by the presence of LL-37. While a temporary disruption of tight junctions by LL-37 may lead to a 'leaky gut,' this study demonstrates that LL-37 increases epithelial cell permeability by degrading TJ proteins occludin and claudin-2 through endocytosis and lysosomal degradation. These immunomodulatory actions occurring at concentrations lower than those microbicidal uncover a new guise for cathelicidin modulating the epithelial barrier against A/E pathogens. Recognizing native cathelicidin's functions in a specified disease setting (e.g., colitis) will help establish it as an anti-infectious immunomodulator.

Homocysteine Metabolites, Endothelial Dysfunction, and Cardiovascular Disease.

Atherosclerosis is accompanied by inflammation that underlies cardiovascular disease (CVD) and its vascular manifestations, including acute stroke, myocardial infarction, and peripheral artery disease, the leading causes of morbidity/mortality worldwide. The monolayer of endothelial cells formed on the luminal surface of arteries and veins regulates vascular tone and permeability, which supports vascular homeostasis. Endothelial dysfunction, the first step in the development of atherosclerosis, is caused by mechanical and biochemical factors that disrupt vascular homeostasis and induce inflammation. Together with increased plasma levels of low-density lipoprotein (LDL), diabetes, hypertension, cigarette smoking, infectious microorganisms, and genetic factors, epidemiological studies established that dysregulated metabolism of homocysteine (Hcy) causing hyperhomocysteinemia (HHcy) is associated with CVD. Patients with severe HHcy exhibit severe CVD and die prematurely due to vascular complications. Biochemically, HHcy is characterized by elevated levels of Hcy and related metabolites such as Hcy-thiolactone and N-Hcy-protein, seen in genetic and nutritional deficiencies in Hcy metabolism in humans and animals. The only known source of Hcy in humans is methionine released in the gut from dietary protein. Hcy is generated from S-adenosylhomocysteine (AdoHcy) and metabolized to cystathionine by cystathionine β-synthase (CBS) and to Hcy-thiolactone by methionyl-tRNA synthetase. Hcy-thiolactone, a chemically reactive thioester, modifies protein lysine residues, generating N-homocysteinylated (N-Hcy)-protein. N-Hcy-proteins lose their normal native function and become cytotoxic, autoimmunogenic, proinflammatory, prothrombotic, and proatherogenic. Accumulating evidence, discussed in this review, shows that these Hcy metabolites can promote endothelial dysfunction, CVD, and stroke in humans by inducing pro-atherogenic changes in gene expression, upregulating mTOR signaling, and inhibiting autophagy through epigenetic mechanisms involving specific microRNAs, histone demethylase PHF8, and methylated histone H4K20me1. Clinical studies, also discussed in this review, show that cystathionine and Hcy-thiolactone are associated with myocardial infarction and ischemic stroke by influencing blood clotting. These findings contribute to our understanding of the complex mechanisms underlying endothelial dysfunction, atherosclerosis, CVD, and stroke and identify potential targets for therapeutic intervention.

Conformational Engineering of Flexible Protein Fragments on the Surface of Different Nanoparticles: The Surface-Atom Mobility Rules.

As a newly emerging technology, conformational engineering (CE) has been gradually displaying the power of producing protein-like nanoparticles (NPs) by tuning flexible protein fragments into their original native conformation on NPs. But apparently, not all types of NPs can serve as scaffolds for CE. To expedite the CE technology on a broader variety of NPs, the essential characteristic of NPs as scaffolds for CE needs to be identified. Herein, we investigate the potential of two distinct types of NPs as scaffolds for CE: CdSe/ZnS quantum dots (QDs), an ionic compound NP, and palladium NPs (PdNPs), a metal NP. The results demonstrate that while QDs cannot support the restoration of the native conformation and function of the complementary-determining region (CDR) fragments of antibodies, PdNPs can. The notably disparate outcomes unequivocally show that the mobility of the surface atoms/adatoms of the NPs or the mobility of the conjugating bonds to the NPs is essential for CE, which allows the conjugated peptides to undergo a conformational change from their initial random conformation to their most stable native conformation under the constraints mimicking the native long-range interactions in the original proteins. This discovery opens the door for CE on more NPs in the future.

'Ship-in-a-Bottle' Integration of pH-Sensitive 3D Proteinaceous Meshes into Microfluidic Channels.

Microfluidic sensors incorporated onto chips allow sensor miniaturization and high-throughput analyses for point-of-care or non-clinical analytical tools. Three-dimensional (3D) printing based on femtosecond laser direct writing (fs-LDW) is useful for creating 3D microstructures with high spatial resolution because the structures are printed in 3D space along a designated laser light path. High-performance biochips can be fabricated using the 'ship-in-a-bottle' integration technique, in which functional microcomponents or biomimetic structures are embedded inside closed microchannels using fs-LDW. Solutions containing protein biomacromolecules as a precursor can be used to fabricate microstructures that retain their native protein functions. Here, we demonstrate the ship-in-a-bottle integration of pure 3D proteinaceous microstructures that exhibit pH sensitivity. We fabricated proteinaceous mesh structures with gap sizes of 10 and 5 μm. The sizes of these gaps changed when exposed to physiological buffers ranging from pH of 4 to 10. The size of the gaps in the mesh can be shrunk and expanded repeatedly by changing the pH of the surrounding buffer. Fs-LDW enables the construction of microscopic proteinaceous meshes that exhibit dynamic functions such as pH sensing and might find applications for filtering particles in microfluidic channels.

The chemical ecology and physiological functions of type I polyketide natural products: the emerging picture.

Covering: up to 2024.For many years, the value of complex polyketides lay in their medical properties, including their antibiotic and antifungal activities, with little consideration paid to their native functions. However, more recent evidence gathered from the study of inter-organismal interactions has revealed the influence of these metabolites upon the ecological adaptation and distribution of their hosts, as well as their modes of communication. The increasing number of sequenced genomes and associated transcriptomes has also unveiled the widespread occurrence of the underlying biosynthetic enzymes across all kingdoms of life, and the important contributions they make to physiological events specific to each organism. This review depicts the diversity of roles fulfilled by type I polyketides, particularly in light of studies carried out during the last decade, providing an initial overall picture of their diverse functions.

Purification and Ultramicroscopic Observation of the Influenza A Virus Ribonucleoprotein Complex.

Influenza A virus (IAV) has an eight-segmented, single-stranded, negative-sense viral genomic RNA (vRNA). Each vRNA strand associates with nucleoproteins and an RNA-dependent RNA polymerase complex to form a viral ribonucleoprotein (vRNP) complex. IAV vRNPs adopt a flexible double-helical configuration that varies in length. Although the transcription and replication of vRNA take place in the context of vRNPs, the precise structural conformation of vRNPs during RNA synthesis remains partially elucidated. To unravel the intricate ultrastructure of the vRNP, it is necessary to purify it while preserving its native functionality. Herein, we introduce a comprehensive protocol for the purification of IAV vRNPs using glycerol gradient ultracentrifugation. Furthermore, we provide a method for the high-speed atomic force microscopy observation of vRNPs during viral RNA synthesis.

Fabrication and applications of biofunctional collagen biomaterials in tissue engineering.

Collagen is extensively used in tissue engineering for various organ tissue regeneration due to the main component of human organ extracellular matrix (ECM) and their inherent nature bioactivity. Collagen various types naturally exist in different organ ECMs. Collagen fabricated with natural ECM mimics architecture, composition and mechanical properties for various organ tissue regeneration. Collagen fabrication with organ-specific biofunctionality facilitated organ tissue engineering as compared to unmodified collagen biomaterials. Collagen biofunctionality improved by subjecting collagen to synthesis, fibers and surface modifications, and blending with other components. Furthermore, collagen is loaded with bioactive molecules, growth factors, drugs and cells also enhancing the biofunctionality of collagen biomaterials. In this review, we will explore the recent advancements in biofunctional collagen biomaterials fabrication with organ-specific biofunctionality in tissue engineering to resolve various organ tissue engineering issues and regeneration challenges. Biofunctional collagen biomaterials stimulate microenvironments inside and around the implants to excellently regulate cellular activities, differentiate cells into organ native cells, enhanced ECM production and remodeling to regenerate organ tissues with native structure, function and maturation. This review critically explored biofunctional collagen biomaterials fabrication in resolving various organ tissue engineering issues and regeneration challenges, and opening new directions of biofunctional collagen biomaterials fabrication, design and applications.

A 3D-printed multi-compartment organ-on-chip platform with a tubing-free pump models communication with the lymph node.

Multi-organ-on-chip systems (MOOCs) have the potential to mimic communication between organ systems and reveal mechanisms of health and disease. However, many existing MOOCs are challenging for non-experts to implement due to complex tubing, electronics, or pump mechanisms. In addition, few MOOCs have incorporated immune organs such as the lymph node (LN), limiting their applicability to model critical events such as vaccination. Here we developed a 3D-printed, user-friendly device and companion tubing-free impeller pump with the capacity to co-culture two or more tissue samples, including a LN, under a recirculating common media. Native tissue structure and immune function were incorporated by maintaining slices of murine LN tissue ex vivo in 3D-printed mesh supports for at least 24 h. In a two-compartment model of a LN and an upstream injection site in mock tissue, vaccination of the multi-compartment chip was similar to in vivo vaccination in terms of locations of antigen accumulation and acute changes in activation markers and gene expression in the LN. We anticipate that in the future, this flexible platform will enable models of multi-organ immune responses throughout the body.

Donor Strand Complementation and Calcium Ion Coordination Drive the Chaperone-free Polymerization of Archaeal Cannulae.

Cannulae are tubular protein filaments that accumulate on the extracellular surface of the hyperthermophilic archaeon Pyrodictium abyssi during cell division. Cannulae have been postulated to act as a primitive extracellular matrix through which cells could communicate or exchange material, although their native biological function remains obscure. Here, we report cryoEM structural analyses of ex vivo cannulae and of in vitro protein assemblies derived from recombinant cannula-like proteins. Three-dimensional reconstructions of P. abyssi cannulae revealed that the structural interactions between protomers in the native and recombinant filaments were based on donor strand complementation, a form of non-covalent polymerization in which a donor β-strand from one subunit is inserted into an acceptor groove in a β-sheet of a neighboring subunit. Donor strand complementation in cannulae is reinforced through calcium ion coordination at the interfaces between structural subunits in the respective assemblies. While donor strand complementation occurs during the assembly of chaperone-usher pili, this process requires the participation of accessory proteins that are localized in the outer membrane. In contrast, we demonstrate that calcium ions can induce assembly of cannulae in the absence of other co-factors. Crystallographic analysis of a recombinant cannula-like protein monomer provided evidence that calcium ion binding primes the precursor for donor strand invasion through unblocking of the acceptor groove. Bioinformatic analysis suggested that structurally homologous cannula-like proteins occurred within the genomes of other hyperthermophilic archaea and were encompassed within the TasA superfamily of biomatrix proteins. CryoEM structural analyses of tubular filaments derived from in vitro assembly of a recombinant cannula-like protein from an uncultured Hyperthermus species revealed a common mode of assembly to the Pyrodictium cannulae, in which donor strand complementation and calcium ion binding stabilized longitudinal and lateral assembly in tubular 2D sheets.

Temporary Nanoencapsulation of Human Intestinal Organoids Using Silk Ionomers.

Human intestinal organoids (HIOs) are vital for modeling intestinal development, disease, and therapeutic tissue regeneration. However, their susceptibility to stress, immunological attack, and environmental fluctuations limits their utility in research and therapeutic applications. This study evaluated the effectiveness of temporary silk protein-based layer-by-layer (LbL) nanoencapsulation technique to enhance the viability and functions of HIOs against common biomedical stressors, without compromising their native functions. Cell viability and differentiation capacity are assessed, finding that nanoencapsulation significantly improved HIO survival under the various environmental perturbations studied without compromising cellular functionality. Post-stress exposures, the encapsulated HIOs still successfully differentiated into essential intestinal cell types such as enterocytes, goblet cells, enteroendocrine cells, and Paneth cells. Moreover, the silk nanocoatings effectively protected against environmental stressors such as ultraviolet (UV) light exposure, protease degradation, antibody binding, and cytokine-induced inflammation. This nanoencapsulation technique shows promise for advancing HIO applications in disease modeling, drug testing, and potential transplantation therapies.

The co-chaperone DNAJA2 buffers proteasomal degradation of cytosolic proteins with missense mutations.

Mutations can disrupt the native function of protein by causing misfolding, which is generally handled by an intricate protein quality control network. To better understand the triaging mechanisms for misfolded cytosolic proteins, we screened a human mutation library to identify a panel of unstable mutations. The degradation of these mutated cytosolic proteins is largely dependent on the ubiquitin proteasome system. Using BioID proximity labelling, we found that the co-chaperones DNAJA1 and DNAJA2 are key interactors with one of the mutated proteins. Notably, the absence of DNAJA2 increases the turnover of the mutant but not the wild-type protein. Our work indicates that specific missense mutations in cytosolic proteins can promote enhanced interactions with molecular chaperones. Assessment of the broader panel of cytosolic mutant proteins shows that the co-chaperone DNAJA2 exhibits two distinct behaviours - acting to stabilize a wide array of cytosolic proteins, including wild-type variants, and to specifically 'buffer' some mutant proteins to reduce their turnover. Our work illustrates how distinct elements of the protein homeostasis network are utilized in the presence of a cytosolic misfolded protein.

Restoration of physiologic loading after engineered disc implantation mitigates immobilization-induced facet joint and paraspinal muscle degeneration.

Intervertebral disc degeneration is commonly associated with back and neck pain, and standard surgical treatments do not restore spine function. Replacement of the degenerative disc with a living, tissue-engineered construct has the potential to restore normal structure and function to the spine. Toward this goal, our group developed endplate-modified disc-like angle-ply structures (eDAPS) that recapitulate the native structure and function of the disc. While our initial large animal studies utilized rigid internal fixation of the eDAPS implanted level to ensure retention of the eDAPS, chronic immobilization does not restore full function and is detrimental to the spinal motion segment. The purpose of this study was to utilize a goat cervical disc replacement model coupled with finite element modeling of goat cervical motion segments to investigate the effects of remobilization (removal of fixation) on the eDAPS, the facet joints and the adjacent paraspinal muscle. Our results demonstrated that chronic immobilization caused notable degeneration of the facet joints and paraspinal muscles adjacent to eDAPS implants. Remobilization improved eDAPS composition and integration and mitigated, but did not fully reverse, facet joint osteoarthritis and paraspinal muscle atrophy and fibrosis. Finite element modeling revealed that these changes were likely due to reduced range of motion and reduced facet loading, highlighting the importance of maintaining normal spine biomechanical function with any tissue engineered disc replacement. STATEMENT OF SIGNIFICANCE: : Back and neck pain are ubiquitous in modern society, and the gold standard surgical treatment of spinal fusion limits patient function. This study advances our understanding of the response of the spinal motion segment to tissue engineered disc replacement with provisional fixation in a large animal model, further advancing the clinical translation of this technology.

Characterization of a widespread sugar phosphate-processing bacterial microcompartment

Many prokaryotes form Bacterial Microcompartments (BMCs) that encapsulate segments of specialized metabolic pathways to enhance catalysis. The various functions of metabolosomes, catabolic BMCs, are dictated by the signature enzyme that processes initial substrates of the confined pathway. The components and native functions of several metabolosomes have been experimentally characterized; however one of the most prevalent across all bacteria has yet to be studied. Sugar Phosphate Utilizing (SPU) BMC loci encode enzymes predicted to be involved in sugar phosphate metabolism. The SPU genetic loci are found in organisms occupying habitats ranging from soils to hot springs, highlighting the ubiquity of the SPU BMC. We bioinformatically characterized seven SPU subtypes, all which contain an enzyme unique to SPU BMCs, a deoxyribose 5-phosphate aldolase (DERA). Here, we define the fundamental characteristics of SPU BMCs and have expressed, purified, and characterized a set of SPU core enzymes. These include a protein-protein complex formed between a SPU BMC DERA and a predicted ribose 5-phosphate isomerase. Further, we show that the SPU BMC DERA is catalytically active and propose that it acts as the universal signature enzyme for the SPU BMC, with implications for fundamental understanding and biotechnological applications of SPU BMCs.

Abstract 4123371: Hyperoxia is not a significant predictor of ICU mortality and is likely a surrogate parameter for reduced native cardiac function in patients supported with VA-ECMO

Intro: Hyperoxia has been associated with adverse outcomes in VA-ECMO patients. Whether hyperoxia represents a mechanism of mortality or is a surrogate for worse cardiac function is unknown. Aim: To determine patient characteristics, lab values, and hemodynamic parameters associated with increased mortality in VA-ECMO patients. Methods: Single-center retrospective cohort study at UCSF in VA-ECMO patients (2020-2023). Patients were categorized into 3 groups: Dead, Advanced Therapy-Free Survivors, and Advanced Therapy Survivors (survived to ICU discharge with LVAD or heart transplant). Wilcox rank sum and two sample proportion tests were performed. In-hospital mortality was evaluated using multiple logistic regression. Results: Of 179 patients, 87 died and 92 survived to ICU discharge. Of the survivors, 22 received advanced therapies. Compared to Advanced Therapy-Free Survivors, Dead patients had higher PaO2 levels (297[95% CI,164-400], p = 0.01). Dead and Advanced Therapy Survivors groups had similarly high PaO2 levels, but the latter group showed less acidosis and were more likely to have HFrEF (59%; 13/22; p = 0.02). ECMO support and VIS scores were similar. The multivariate model showed PaO2 is not associated with mortality (adjusted OR, 1.002 [95% CI, 0.99-1.004]; p = 0.131). Conclusion: Hyperoxia was associated with both death and advanced therapies, but did not predict mortality in a multivariate model. Advanced therapy survivors and patients that died were more likely to have HFrEF, suggesting that high PaO2 at cannulation reflects a proximal mixing cloud due to weak native cardiac function. Future work will analyze ECHO parameters to examine the association between hyperoxia and reduced cardiac output in VA-ECMO patients.

Biologic Augmentation of Rotator Cuff Repair: Current Concepts Review.

Rotator cuff tears are common in an aging population. Thus far, primary repairs have shown high re-tear rates suggesting the need for improved healing modalities. Current augmentations of rotator cuff repairs include synthetic and biological scaffolds, surgical bone marrow venting, and infusing the repair with a variety of stem cells and growth factors aimed at restoring the native cellular structure and function of the repaired tissue. This current concepts review discusses the anatomy, physical presentation, diagnosis, and treatment of rotator cuff tears; biological adjuvants for rotator cuff repairs; and the current literature on outcomes after biologically augmented rotator cuff repairs. [Orthopedics. 2024;47(6):e282-e286.].

ASSESSING THE ECOLOGICAL AND MICROBIAL IMPACTS OF SHIP BALLAST WATER DISCHARGE IN GLOBAL SHIPPING

The global shipping industry, responsible for 90% of world trade, plays a pivotal role in facilitating economic growth. However, its impact on marine environments, notably through pollution from ballast water discharges, poses environmental challenges. The discharge of ballast water emerges as a significant threat, introducing non-native species and pathogens into new ecosystems, leading to ecological and economic consequences. The study aims to understand the impact of these invasive species on native ecosystems, biodiversity, ecosystem function, and potential economic implications. Ten vessels from diverse regions were sampled, revealing zero total coliform counts and Escheria coli presence, indicating relatively clean ballast water. Microbiological analysis highlighted variations in heterotrophic counts across samples with zero total coliform counts and Escheria coli. The study identified the presence of pathogenic bacteria, including Bacillus sp., Vibro cholera, and Staphylococcus aureus. The study highlights the potential for the spread of invasive species through ballast water discharge, necessitating strategies to mitigate negative impacts.

Residual Native Kidney Function after Kidney Transplant (KT): Unusual Complications

Residual Native Kidney Function after Kidney Transplant (KT): Unusual Complications

A Case of Recovery of Native Kidney Function Years after Simultaneous Liver-Kidney Transplant

A Case of Recovery of Native Kidney Function Years after Simultaneous Liver-Kidney Transplant