Reduced expression of lipoxygenase genes improves flour processing quality in soft wheat.

Lipoxygenases (Loxs) are dioxygenases that play an important role in plant growth and defense. Loxs affect flour processing quality in common wheat (Triticum aestivum). We conducted a genome-wide association study (GWAS) that identified 306 significant single-nucleotide polymorphisms (SNPs) related to Lox activity in Chinese wheat accessions. Among them, a novel lipoxygenase-encoding (Lpx) gene, TaLpx-B4, was detected on chromosome 3B in a biparental population. Analysis of mutant wheat lines induced using ethyl methanesulfonate confirmed the role of TaLpx-B4 in modulating Lox activity. A phylogenetic tree of various plant Lpx genes indicated the predominance of the 9-Lpx type in common wheat. Further analysis revealed conserved intron number, exon length, and motif number in the TaLpx gene family. GWAS, linkage mapping, and gene annotation collectively showed that 14 out of 29 annotated TaLpx genes played a critical role in regulating Lox activity in the Chinese wheat accessions. Transgenic wheat grains with knockdown of Lpx family genes by RNAi showed significantly lower Lox activity than the wild type. One TaLpx-RNAi line had significantly reduced starch content and dough stability, and thus possessed relatively superior biscuit quality in soft wheat. Further analysis of the transcriptome, lipid components, and other metabolites revealed that knockdown of TaLpx genes significantly increased biscuit quality via changes in unsaturated fatty acid content as well as in starch, sucrose, and galactose metabolism. Our results provide new insights into the role of the TaLpx gene family that will be beneficial in improving soft wheat flour quality.

The species-spanning family of LPX-motif harbouring effector proteins.

The delivery of effector proteins into infected eukaryotic cells represents a key virulence feature of many microbial pathogens in order to derail essential cellular processes and effectively counter the host defence system. Although bacterial effectors are truly numerous and exhibit a wide range of biochemical activities, commonalities in terms of protein structure and function shared by many bacterial pathogens exist. Recent progress has shed light on a species-spanning family of bacterial effectors containing an LPX repeat motif as a subtype of the leucine-rich repeat superfamily, partially combined with a novel E3 ubiquitin ligase domain. This review highlights the immunomodulatory effects of LPX effector proteins, with particular emphasis on the exploitation of the host ubiquitin system.

Bacterial LPX motif-harboring virulence factors constitute a species-spanning family of cell-penetrating effectors.

Effector proteins are key virulence factors of pathogenic bacteria that target and subvert the functions of essential host defense mechanisms. Typically, these proteins are delivered into infected host cells via the type III secretion system (T3SS). Recently, however, several effector proteins have been found to enter host cells in a T3SS-independent manner thereby widening the potential range of these virulence factors. Prototypes of such bacteria-derived cell-penetrating effectors (CPEs) are the Yersinia enterocolitica-derived YopM as well as the Salmonella typhimurium effector SspH1. Here, we investigated specifically the group of bacterial LPX effector proteins comprising the Shigella IpaH proteins, which constitute a subtype of the leucine-rich repeat protein family and share significant homologies in sequence and structure. With particular emphasis on the Shigella-effector IpaH9.8, uptake into eukaryotic cell lines was shown. Recombinant IpaH9.8 (rIpaH9.8) is internalized via endocytic mechanisms and follows the endo-lysosomal pathway before escaping into the cytosol. The N-terminal alpha-helical domain of IpaH9.8 was identified as the protein transduction domain required for its CPE ability as well as for being able to deliver other proteinaceous cargo. rIpaH9.8 is functional as an ubiquitin E3 ligase and targets NEMO for poly-ubiquitination upon cell penetration. Strikingly, we could also detect other recombinant LPX effector proteins from Shigella and Salmonella intracellularly when applied to eukaryotic cells. In this study, we provide further evidence for the general concept of T3SS-independent translocation by identifying novel cell-penetrating features of these LPX effectors revealing an abundant species-spanning family of CPE.

Lpx1p links glucose-induced calcium signaling and plasma membrane H+-ATPase activation in Saccharomyces cerevisiae cells.

In yeast, as in other eukaryotes, calcium plays an essential role in signaling transduction to regulate different processes. Many pieces of evidence suggest that glucose-induced activation of plasma membrane H+-ATPase, essential for yeast physiology, is related to calcium signaling. Until now, no protein that could be regulated by calcium in this context has been identified. Lpx1p, a serine-protease that is also involved in the glucose-induced activation of the plasma membrane H+-ATPase, could be a candidate to respond to intracellular calcium signaling involved in this process. In this work, by using different approaches, we obtained many pieces of evidence suggesting that the requirement of calcium signaling for activation of the plasma membrane H+-ATPase is due to its requirement for activation of Lpx1p. According to the current model, activation of Lpx1p would cause hydrolysis of an acetylated tubulin that maintains the plasma membrane H+-ATPase in an inactive state. Therefore, after its activation, Lpx1p would hydrolyze the acetylated tubulin making the plasma membrane H+-ATPase accessible for phosphorylation by at least one protein kinase.

Lipid profiling of lipoprotein X: Implications for dyslipidemia in cholestasis.

Lipoprotein X (Lp-X) is an abnormal lipoprotein that may typically be formed in intra- and extrahepatic cholestasis and potentially interfere with lipid analysis in the routine lab. To gain insight into lipid class and species composition, Lp-X, LDL and HDL from cholestatic and control serum samples were subjected to mass spectrometric analysis including phospholipids (PL), sphingolipids, free cholesterol (FC), cholesteryl esters (CE) and bile acids. Our analysis of Lp-X revealed a content of 46% FC, 49% PL with 34% phosphatidylcholine (PC) as main PL component. The lipid species pattern of Lp-X showed remarkable high fractions of mono-unsaturated species including PC 32:1 and PC 34:1 and phosphatidylethanolamine (PE) 32:1 and 34:1. LDL and HDL lipid composition in the same specimens strongly reflected the lipid composition of Lp-X with increased PC 32:1, PC 34:1, PE 32:1, PE 34:1 and FC accompanied by decreased CE compared to controls. Comparison of Lp-X and biliary lipid composition clearly indicates that Lp-X does not originate from a sole release of bile lipids. Moreover, these data present evidence for increased hepatic fatty acid and PL synthesis which may represent a reaction to high hepatic FC level observed during cholestasis.

Genomic investigation unveils colistin resistance mechanism in carbapenem-resistant Acinetobacter baumannii clinical isolates.

Colistin resistance in Acinetobacter baumannii is mediated by multiple mechanisms. Recently, mutations within pmrABC two-component system and overexpression of eptA gene due to upstream insertion of ISAba1 have been shown to play a major role. Thus, the aim of our study is to characterize colistin resistance mechanisms among the clinical isolates of A. baumannii in India. A total of 207 clinical isolates of A. baumannii collected from 2016 to 2019 were included in this study. Mutations within lipid A biosynthesis and pmrABC genes were characterized by whole-genome shotgun sequencing. Twenty-eight complete genomes were further characterized by hybrid assembly approach to study insertional inactivation of lpx genes and the association of ISAba1-eptA. Several single point mutations (SNPs), like M12I in pmrA, A138T and A444V in pmrB, and E117K in lpxD, were identified. We are the first to report two novel SNPs (T7I and V383I) in the pmrC gene. Among the five colistin-resistant A. baumannii isolates where complete genome was available, the analysis showed that three of the five isolates had ISAba1 insertion upstream of eptA. No mcr genes were identified among the isolates. We mapped the SNPs on the respective protein structures to understand the effect on the protein activity. We found that majority of the SNPs had little effect on the putative protein function; however, some SNPs might destabilize the local structure. Our study highlights the diversity of colistin resistance mechanisms occurring in A. baumannii, and ISAba1-driven eptA overexpression is responsible for colistin resistance among the Indian isolates.IMPORTANCEAcinetobacter baumannii is a Gram-negative, emerging and opportunistic bacterial pathogen that is often associated with a wide range of nosocomial infections. The treatment of these infections is hindered by increase in the occurrence of A. baumannii strains that are resistant to most of the existing antibiotics. The current drug of choice to treat the infection caused by A. baumannii is colistin, but unfortunately, the bacteria started to show resistance to the last-resort antibiotic. The loss of lipopolysaccharides and mutations in lipid A biosynthesis genes are the main reasons for the colistin resistance. The present study characterized 207 A. baumannii clinical isolates and constructed complete genomes of 28 isolates to recognize the mechanisms of colistin resistance. We showed the mutations in the colistin-resistant variants within genes essential for lipid A biosynthesis and that cause these isolates to lose the ability to produce lipopolysaccharides.

Longitudinal analysis of clinical and laboratory biomarkers in a patient with familial lecithin: cholesterol acyltransferase deficiency (FLD) and accelerated eGFR decline: A case study.

Familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is an ultra-rare autosomal recessive disease characterized by very low high-density lipoprotein cholesterol (HDL-C) levels, corneal opacity, anemia, and progressive renal disease. The rate and severity of renal disease are variable across FLD patients and the biomarkers and risk factors for disease progression are poorly understood. Here we report a 30 year-long comparative analysis of the clinical and laboratory biomarkers in an FLD patient with accelerated renal decline, who underwent two kidney and one liver transplantations. Results show that elevated triglyceride and non-HDL-C levels may promote the formation of LpX and accelerate renal function decline, whereas markers of anemia may be early predictors. Conversely, corneal opacity progresses at a steady rate and does not correlate with lipid, hematologic, or renal biomarkers. Our study suggests that monitoring of markers of anemia may aid the early detection and timely management of kidney disease with conservative therapies. Furthermore, it suggests that controlling hypercholesterolemia and hypertriglyceridemia may help improve renal disease prognosis.

The relationship of cytotoxic and genotoxic damage with blood aluminum levels and oxidative stress induced by this metal in common carp (Cyprinus carpio) erythrocytes.

Aluminum is one of the most abundant elements in nature and is used in diverse industrial processes. As a result, it contaminates aquatic ecosystems, inducing damage on associated biota. In fish, it has been observed to induce hypoxia, hypercapnia, metabolic acidosis and respiratory arrest. Although there is little information on Al-induced cytotoxicity and DNA damage, this type of studies are essential in order to identify the mechanisms of action of this metal. The cytotoxic and genotoxic effects induced by Al on common carp (Cyprinus carpio) erythrocytes were determined in specimens exposed to 0.05, 120 and 239mgAlL(-1) in static exposure systems. Blood samples were taken at 12, 24, 48, 72 and 96h, erythrocytes were separated, and the following were evaluated: frequency of micronuclei and frequency of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells, blood Al levels, lipid peroxidation, protein carbonyl content, and activity of the antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase. The results show that tested aluminum concentrations produces oxidative stress (increase in lipid peroxidation degree and oxidized proteins content, as well as decrease in antioxidant enzymes activity) and induced higher frequencies of micronuclei and TUNEL-positive cells, so this metal can be considered as a cytotoxic and genotoxic agent for erythrocytes of common carp.

Hypercholesterolemia due to lipoprotein-X manifesting as pseudohyponatremia in a patient with cholestasis.

Pseudohyponatremia is an often misdiagnosed condition that needs to be managed by addressing the underlying cause. Treatment of hyponatremic patients with intravenous fluids without ruling out pseudohyponatremia may aggravate a patient's hyponatremia and result in adverse outcomes. In a patient whose sodium is deteriorating, it is critical to diagnose pseudohyponatremia early in the course and acquire necessary consultations, even if the patient is asymptomatic. We discuss a case of a man in his 20s with a history of liver transplantation who presented with unexplained dangerously low sodium while being asymptomatic. The case illustrates an uncommon cause of pseudohyponatremia due to lipoprotein-X hypercholesterolemia in a patient with cholestatic liver disease.

Abnormal Lipoproteins Trigger Oxidative Stress-Mediated Apoptosis of Renal Cells in LCAT Deficiency.

Familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is a rare genetic disease caused by the loss of function mutations in the LCAT gene. LCAT deficiency is characterized by an abnormal lipoprotein profile with severe reduction in plasma levels of high-density lipoprotein (HDL) cholesterol and the accumulation of lipoprotein X (LpX). Renal failure is the major cause of morbidity and mortality in FLD patients; the pathogenesis of renal disease is only partly understood, but abnormalities in the lipoprotein profile could play a role in disease onset and progression. Serum and lipoprotein fractions from LCAT deficient carriers and controls were tested for renal toxicity on podocytes and tubular cells, and the underlying mechanisms were investigated at the cellular level. Both LpX and HDL from LCAT-deficient carriers triggered oxidative stress in renal cells, which culminated in cell apoptosis. These effects are partly explained by lipoprotein enrichment in unesterified cholesterol and ceramides, especially in the HDL fraction. Thus, alterations in lipoprotein composition could explain some of the nephrotoxic effects of LCAT deficient lipoproteins on podocytes and tubular cells.

Colistin Resistance in Acinetobacter baumannii: Molecular Mechanisms and Epidemiology.

Acinetobacter baumannii is recognized as a clinically significant pathogen causing a wide spectrum of nosocomial infections. Colistin was considered a last-resort antibiotic for the treatment of infections caused by multidrug-resistant A. baumannii. Since the reintroduction of colistin, a number of mechanisms of colistin resistance in A. baumannii have been reported, including complete loss of LPS by inactivation of the biosynthetic pathway, modifications of target LPS driven by the addition of phosphoethanolamine (PEtN) moieties to lipid A mediated by the chromosomal pmrCAB operon and eptA gene-encoded enzymes or plasmid-encoded mcr genes and efflux of colistin from the cell. In addition to resistance to colistin, widespread heteroresistance is another feature of A. baumannii that leads to colistin treatment failure. This review aims to present a critical assessment of relevant published (>50 experimental papers) up-to-date knowledge on the molecular mechanisms of colistin resistance in A. baumannii with a detailed review of implicated mutations and the global distribution of colistin-resistant strains.

Inhibiting collagen I production and tumor cell colonization in the lung via miR-29a-3p loading of exosome-/liposome-based nanovesicles.

The lung is one of the most common sites for cancer metastasis. Collagens in the lung provide a permissive microenvironment that supports the colonization and outgrowth of disseminated tumor cells. Therefore, down-regulating the production of collagens may contribute to the inhibition of lung metastasis. It has been suggested that miR-29 exhibits effective anti-fibrotic activity by negatively regulating the expression of collagens. Indeed, our clinical lung tumor data shows that miR-29a-3p expression negatively correlates with collagen I expression in lung tumors and positively correlates with patients' outcomes. However, suitable carriers need to be selected to deliver this therapeutic miRNA to the lungs. In this study, we found that the chemotherapy drug cisplatin facilitated miR-29a-3p accumulation in the exosomes of lung tumor cells, and this type of exosomes exhibited a specific lung-targeting effect and promising collagen down-regulation. To scale up the preparation and simplify the delivery system, we designed a lung-targeting liposomal nanovesicle (by adjusting the molar ratio of DOTAP/cholesterol-miRNAs to 4:1) to carry miR-29a-3p and mimic the exosomes. This liposomal nanovesicle delivery system significantly down-regulated collagen I secretion by lung fibroblasts in vivo, thus alleviating the establishment of a pro-metastatic environment for circulating lung tumor cells.

When LDL Cholesterol Is Not LDL Cholesterol: LpX, A Clinical Lesson.

LpX is a lipoprotein formed in cholestatic conditions and often erroneously reported as LDL-C. A low ApoB level can support the diagnosis of LpX. Treatment should not automatically focus on lowering serum lipid levels, but primarily on resolving the cause of cholestasis. (Level of Difficulty: Advanced.).

Lipoprotein-X and Lipoprotein-Z Induced Hyperviscosity Syndrome in the Setting of Cholestatic Liver Failure.

We describe a case referred for worsening hypercholesterolemia in the setting of atorvastatin and fenofibrate-induced liver injury. The patient reported neurological complaints attributed to hyperviscosity syndrome (induced by lipoprotein-X and lipoprotein-Z). Hepatic recovery was associated with reduction of whole blood viscosity and amelioration of neurological symptoms. (Level of Difficulty: Advanced.).

The NIH Lipo-COVID Study: A Pilot NMR Investigation of Lipoprotein Subfractions and Other Metabolites in Patients with Severe COVID-19.

A complex interplay exists between plasma lipoproteins and inflammation, as evidenced from studies on atherosclerosis. Alterations in plasma lipoprotein levels in the context of infectious diseases, particularly respiratory viral infections, such as SARS-CoV-2, have become of great interest in recent years, due to their potential utility as prognostic markers. Patients with severe COVID-19 have been reported to have low levels of total cholesterol, HDL-cholesterol, and LDL-cholesterol, but elevated levels of triglycerides. However, a detailed characterization of the particle counts and sizes of the different plasma lipoproteins in patients with COVID-19 has yet to be reported. In this pilot study, NMR spectroscopy was used to characterize lipoprotein particle numbers and sizes, and various metabolites, in 32 patients with severe COVID-19 admitted to the intensive care unit. Our study revealed markedly reduced HDL particle (HDL-P) numbers at presentation, especially low numbers of small HDL-P (S-HDL-P), and high counts of triglyceride-rich lipoprotein particle (TRL-P), particularly the very small and small TRL subfractions. Moreover, patients with severe COVID-19 were found to have remarkably elevated GlycA levels, and elevated levels of branched-chain amino acids and beta-hydroxybutyrate. Finally, we detected elevated levels of lipoproteins X and Z in most participants, which are distinct markers of hepatic dysfunction, and that was a novel finding.

A liposomal RNA vaccine inducing neoantigen-specific CD4+ T cells augments the antitumor activity of local radiotherapy in mice.

Antigen-encoding, lipoplex-formulated RNA (RNA-LPX) enables systemic delivery to lymphoid compartments and selective expression in resident antigen-presenting cells. We report here that the rejection of CT26 tumors, mediated by local radiotherapy (LRT), is further augmented in a CD8+ T cell-dependent manner by an RNA-LPX vaccine that encodes CD4+ T cell-recognized neoantigens (CD4 neoantigen vaccine). Whereas CD8+ T cells induced by LRT alone were primarily directed against the immunodominant gp70 antigen, mice treated with LRT plus the CD4 neoantigen vaccine rejected gp70-negative tumors and were protected from rechallenge with these tumors, indicating a potent poly-antigenic CD8+ T cell response and T cell memory. In the spleens of CD4 neoantigen-vaccinated mice, we found a high number of activated, poly-functional, Th1-like CD4+ T cells against ME1, the immunodominant CD4 neoantigen within the poly-neoantigen vaccine. LRT itself strongly increased CD8+ T cell numbers and clonal expansion. However, tumor infiltrates of mice treated with CD4 neoantigen vaccine/LRT, as compared to LRT alone, displayed a higher fraction of activated gp70-specific CD8+ T cells, lower PD-1/LAG-3 expression and contained ME1-specific IFNγ+ CD4+ T cells capable of providing cognate help. CD4 neoantigen vaccine/LRT treatment followed by anti-CTLA-4 antibody therapy further enhanced the efficacy with complete remission of gp70-negative CT26 tumors and survival of all mice. Our data highlight the power of combining synergistic modes of action and warrants further exploration of the presented treatment schema.

Discrepancies in susceptibility testing to colistin in Acinetobacter baumannii: The influence of slow growth and heteroresistance.

The increasing use of polymyxins as last-resort drugs for managing infections by Acinetobacter baumannii has led to the emergence of resistance. This study aimed to determine the resistance mechanisms in Acinetobacter baumannii isolates with colistin MIC ≥ 4 mg/L and to relate the mechanisms of resistance with the difficulties in detecting them. Absolute agreement among the different methodologies (Phoenix automatized system, broth and agar dilution, and a rapid colorimetric test) in the 140 colistin-susceptible isolates was observed; whereas in the 25 resistant isolates, the performance varied according to the colistin MIC value. Most of the discrepancies (irrespective of the methodology that was used) were observed in isolates with an MIC value close to the breakpoint. The number of errors in each method in the resistant isolates was as follows: rapid test, four of 25 (16%); agar dilution, eight of 25 (32%); Phoenix system, 13 of 25 (52%) and its manual reading at 24 h, eight of 25 (32%). Categorical errors were detected in 13 isolates: slow growth was the main reason in five isolates, whereas in the remaining eight isolates, slow growth was detected together with a low proportion of colistin-resistant subpopulations and the colistin MIC value was close to the breakpoint value. To understand the probable reason for the observed MIC values, sequencing of genes associated with colistin resistance was performed. Mutations at lpxA, lpxC, and pmrB genes were detected and it was observed that isolates carrying mutations in lpxC presented slow growth at killing curves.

HPV16 RNA-LPX vaccine mediates complete regression of aggressively growing HPV-positive mouse tumors and establishes protective T cell memory.

HPV16 infections are associated with a variety of cancers and there is compelling evidence that the transforming activity of HPV16 critically depends on the expression of the viral oncoproteins E6 and E7. Therapeutic cancer vaccines capable of generating durable and specific immunity against these HPV16 antigens hold great promise to achieve long-term disease control. Here we show in mice that HPV16 E7 RNA-LPX, an intravenously administered cancer vaccine based on immuno-pharmacologically optimized antigen-encoding mRNA, efficiently primes and expands antigen-specific effector and memory CD8+ T cells. HPV-positive TC-1 and C3 tumors of immunized mice are heavily infiltrated with activated immune cells and HPV16-specific T cells and are polarized towards a proinflammatory, cytotoxic and less immune-suppressive contexture. E7 RNA-LPX immunization mediates complete and durable remission of progressing tumors. Circulating memory T cells are highly cytotoxic and protect from tumor rechallenge. Moreover, E7 RNA-LPX immunization sensitizes anti-PD-L1 refractory tumors to checkpoint blockade. In conclusion, our data highlight the potential of HPV16 RNA-LPX for the treatment of HPV-driven cancers.

Lp8 is potentially associated with partial lecithin:cholesterol acyltransferase deficiency in a patient with primary biliary cirrhosis.

BACKGROUND: The significance of Lp8, that is, abnormal lipoprotein(s) detected in fraction 8 by combined high-performance liquid chromatography/gel filtration column in patients with familial lecithin:cholesterol acyltransferase (LCAT) syndrome, in relation to the severity of LCAT deficiency has not been analyzed. OBJECTIVE: We have studied Lp8 in a patient with primary biliary cirrhosis. METHODS: Plasma lipoproteins were analyzed using high-performance liquid chromatography/gel filtration column in the course of treatment of a 47-year-old female patient with primary biliary cirrhosis. RESULTS: Electrophoretic lipoprotein analyses showed massive accumulation of abnormal ß- and preß-lipoproteins with a minor lipoprotein fraction at a position near the cathode corresponding to Lp-X, on day A (status: hypercholesterolemia, LCAT activity undetectable). Chromatographic lipoprotein subfraction analysis revealed free cholesterol- and phospholipid-rich lipoproteins in fractions 1-6, corresponding to chylomicrons and very low-density lipoprotein, and phospholipid- and triglyceride-rich lipoproteins with increased free cholesterol, that is, Lp8, in fractions 7-9 (corresponding to low-density lipoprotein). On day B, after additional treatment for 7 months (status: almost normolipidemia, decreased LCAT activity), although the abnormal lipoprotein and the lipoproteins in fractions 1-6, were drastically decreased, the presence of Lp8 persisted. CONCLUSIONS: Lp8 likely is a minor abnormal lipoprotein fraction in patients with mildly decreased secondary LCAT activity, as well as with severely reduced primary LCAT activity.

Lipopolysaccharide transport to the cell surface: biosynthesis and extraction from the inner membrane.

The cell surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS). The network of charges and sugars provided by the dense packing of LPS molecules in the outer leaflet of the outer membrane interferes with the entry of hydrophobic compounds into the cell, including many antibiotics. In addition, LPS can be recognized by the immune system and plays a crucial role in many interactions between bacteria and their animal hosts. LPS is synthesized in the inner membrane of Gram-negative bacteria, so it must be transported across their cell envelope to assemble at the cell surface. Over the past two decades, much of the research on LPS biogenesis has focused on the discovery and understanding of Lpt, a multi-protein complex that spans the cell envelope and functions to transport LPS from the inner membrane to the outer membrane. This paper focuses on the early steps of the transport of LPS by the Lpt machinery: the extraction of LPS from the inner membrane. The accompanying paper (May JM, Sherman DJ, Simpson BW, Ruiz N, Kahne D. 2015 Phil. Trans. R. Soc. B 370, 20150027. (doi:10.1098/rstb.2015.0027)) describes the subsequent steps as LPS travels through the periplasm and the outer membrane to its final destination at the cell surface.