Necroptosis contributes to the intestinal toxicity of deoxynivalenol and is mediated by methyltransferase SETDB1.

Deoxynivalenol (DON) is a secondary metabolite produced by fungi, which causes serious health issues worldwide due to its widespread presence in human and animal diets. Necroptosis is a newly proposed cell death mode and has been proposed as a potential mechanism of intestinal disease. This study aimed to investigate the role of necroptosis in intestinal damage caused by DON exposure. Piglets were fed diets with or without 4 mg/kg DON for 3 weeks or given a gavage of 2 mg/kg BW DON or sterile saline to investigate the effects of chronic or acute DON exposure on the gut, respectively. IPEC-1 cells were challenged with different concentrations of DON to investigate the effect of DON exposure on the intestinal epithelial cells (IECs) in vitro. Subsequently, the inhibitors of necroptosis were used to treat cells or piglets prior to DON challenge. Chronic and acute DON exposure both caused morphological damage, reduction of disaccharidase activity, decrease of tight junction protein expression, inflammation of the small intestine, and necroptosis of intestinal epithelial cells in piglets. Necroptosis was also detected when IPEC-1 cell damage was induced by DON in vitro. The suppression of necroptosis in IPEC-1 cells by inhibitors (necrostatin-1 (Nec-1), GSK'872, or GW806742X) alleviated cell death, the decrease of tight junction protein expression, oxidative stress, and the inflammatory response induced by DON. Furthermore, pre-treatment with Nec-1 in piglets was also observed to protect the intestine against DON-induced enterotoxicity. Additionally, the expression of histone methyltransferase SETDB1 was abnormally downregulated upon chronic and acute DON exposure in piglets, and necroptosis was activated in IPEC-1 cells due to knockout of SETDB1. Collectively, these results demonstrate that necroptosis of IECs is a mechanism of DON-induced enterotoxicity and SETDB1 mediates necroptosis upon DON exposure in IECs, suggesting the potential for targeted inhibition of necroptosis to alleviate mycotoxin-induced enterotoxicity and intestinal disease.

Quercetin Ameliorates Deoxynivalenol-Induced Intestinal Injury and Barrier Dysfunction Associated with Inhibiting Necroptosis Signaling Pathway in Weaned Pigs.

Quercetin (Que) is a flavonol compound found in plants, which has a variety of biological activities. Necroptosis, a special form of programmed cell death, plays a vital role in the development of many gastrointestinal diseases. This study aimed to explore whether Que could attenuate the intestinal injury and barrier dysfunction of piglets after deoxynivalenol (DON) exposure through modulating the necroptosis signaling pathway. Firstly, twenty-four weaned piglets were used in a 2 × 2 factorial design and the main factors, including Que (basal diet or diet supplemented with 100 mg/kg Que) and DON exposure (control feed or feed contaminated with 4 mg/kg DON). After feeding for 21 d, piglets were killed for samples. Next, the intestinal porcine epithelial cell line (IPEC-1) was pretreated with or without Que (10 µmol/mL) in the presence or absence of a DON challenge (0.5 µg/mL). Dietary Que increased the body weight, average daily gain, and average daily feed intake (p < 0.05) through the trial. Que supplementation improved the villus height, and enhanced the intestinal barrier function (p < 0.05) indicated by the higher protein expression of occludin and claudin-1 (p < 0.05) in the jejunum of the weaned piglets after DON exposure. Dietary Que also down-regulated the protein abundance of total receptor interacting protein kinase 1 (t-RIP1), phosphorylated RIP1 (p-RIP1), p-RIP3, total mixed lineage kinase domain-like protein (t-MLKL), and p-MLKL (p < 0.05) in piglets after DON exposure. Moreover, Que pretreatment increased the cell viability and decreased the lactate dehydrogenase (LDH) activity (p < 0.05) in the supernatant of IPEC-1 cells after DON challenge. Que treatment also improved the epithelial barrier function indicated by a higher transepithelial electrical resistance (TEER) (p < 0.001), lower fluorescein isothiocyanate-labeled dextran (FD4) flux (p < 0.001), and better distribution of occludin and claudin-1 (p < 0.05) after DON challenge. Additionally, pretreatment with Que also inhibited the protein abundance of t-RIP1, p-RIP1, t-RIP3, p-RIP3, t-MLKL, and p-MLKL (p < 0.05) in IPEC-1 cells after DON challenge. In general, our data suggest that Que can ameliorate DON-induced intestinal injury and barrier dysfunction associated with suppressing the necroptosis signaling pathway.

Necroptosis Contributes to LPS-Induced Activation of the Hypothalamic-Pituitary-Adrenal Axis in a Piglet Model.

Stressors cause activation of the hypothalamic-pituitary-adrenal (HPA) axis and a systemic inflammatory response. As a newly proposed cell death manner in recent years, necroptosis occurs in a variety of tissue damage and inflammation. However, the role of necroptosis in HPA axis activation remains to be elucidated. The aim of this study was to investigate the occurrence of necroptosis and its role in HPA activation in a porcine stress model induced by Escherichia coli lipopolysaccharide (LPS). Several typical stress behaviors like fever, anorexia, shivering and vomiting were observed in piglets after LPS injection. HPA axis was activated as shown by increased plasma cortisol concentration and mRNA expression of pituitary corticotropin-releasing hormone receptor 1 (CRHR1) and adrenal steroidogenic acute regulatory protein (StAR). The mRNA expression of tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß) and IL-6 in the hypothalamus, pituitary gland and adrenal gland was elevated by LPS, accompanied by the activation of necroptosis indicated by higher mRNA expression of necroptosis signals including receptor-interacting protein kinase (RIP) 1, RIP3, and phosphorylated mixed-lineage kinase domain-like protein (MLKL). Furthermore, necrostatin-1 (Nec-1), an inhibitor of necroptosis, inhibited necroptosis indicated by decreased mRNA levels of RIP1, RIP3, MLKL, and phosphoglycerate mutase family member 5 (PGAM5) in the hypothalamus, pituitary gland and adrenal gland. Nec-1 also decreased the mRNA expression of TNF-α and IL-ß and inhibited the activation of the HPA axis indicated by lower plasma cortisol concentration and mRNA expression of adrenal type 2 melanocortin receptor (MC2R) and StAR. These findings suggest that necroptosis is present and contributes to HPA axis activation induced by LPS. These findings provide a potential possibility for necroptosis as an intervention target for alleviating HPA axis activation and stress responses.

High-Voltage FDS of Thermally Aged XLPE Cable and Its Correlation with Physicochemical Properties.

This paper aims to investigate the influence of thermal aging on a crosslinked polyethylene (XLPE) cable, and the relationships between the macroscopical high-voltage dielectric and the microscopical physicochemical properties are also elucidated. To better simulate thermal aging under working condition, the medium-voltage-level cable is subjected to accelerated inner thermal aging for different aging times. Then, high-voltage frequency domain spectroscopy (FDS) (cable sample) and analyses of microscopic physical and chemical properties (sampling from the cable), including Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and elongation at the break (EAB), are conducted at different cable aging stages. The dielectric test results show that after a certain aging time, the high-voltage FDS curves of the cable have layered characteristics, and this phenomenon is more obvious as the aging degree increases. Moreover, the slope and the integral of the high-voltage FDS curves rise with aging time. The mechanism is deduced by the physicochemical results that thermo-oxidative aging results in increasing polar groups and dislocation defects in the crystal region, which leads to the above phenomenon. On the one hand, the appearance of polar groups increases the density of the dipole. On the other hand, the destruction of the crystal region increases the probability and amplitude of dipole reversal. In addition, the breaking of molecular bonds and the increase in the amorphous phase also reduce the rigidity of the XLPE molecular main chain. The above factors lead to obvious delamination and larger dielectric parameters of the thermally aged cable. Finally, according to the experimental results, an on-site diagnosis method of cable insulation thermal aging based on high-voltage FDS is discussed.

Integrated analysis of lncRNA and mRNA for the apoptosis of porcine ovarian granulosa cells after polyphenol resveratrol treatment.

Resveratrol (RES) is a non-flavonoid polyphenol compound that can be involved in follicular development and ovulation. However, the mechanism by which resveratrol regulates the apoptosis of porcine ovarian granulosa cells (POGCs) through long non-coding RNA (lncRNA) is poorly understood. We generated POGCs models of different doses of RES (0, 25, 50, 75, and 100 µM). It was observed that the cell viability was the highest in the 50 µM group, and the highest apoptosis rates were recorded in the 100 µM group. Therefore, a control group (n = 3, 0 µM RES group), a low RES group (n = 3, 50 µM RES group), and a high RES group (n = 3, 100 µM RES group) of POGCs were created for next RNA sequencing. Gene Ontology (GO) indicated that differentially expressed lncRNAs associated with apoptotic process were highly enriched. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of lncRNA target genes found that the Wnt signaling pathway and PI3K-Akt signaling pathway were both enriched. Furthermore, we constructed lncRNA-mRNA networks related to Metabolic and Cell Apoptosis, respectively. In the networks, five key-lncRNAs were screened, which may play a significant role in the process of POGCs metabolism and apoptosis. Furthermore, we focused on the function of a lnc-GAM (lncRNA associated with Granulosa cells Apoptosis and Metabolism) and verified that lnc-GAM could influence cell apoptosis in POGCs development by affecting the mRNA expression of apoptosis-related markers, and also affects the secretion of steroid hormones and related genes expression in POGCs cultured in vitro. Our study provides seminal data and important new insights into the regulation of reproductive mechanisms in porcine and other female mammals.

[Analysis of quality difference based on Astragalus membranaceus var. mongholicus in genuine region].

This work is to establish the fingerprint of Astragalus membranaceus var. mongholicus by HPLC-ELSD method, and to analyze the simulated wildness degree of A. membranaceus var. mongholicus in the genuine region of Inner Mongolia, Ningxia and Gansu. Compared with wild A. membranaceus var. mongholicus, the quality differences of A. membranaceus var. mongholicus in the genuine region were analyzed by identification of chromatographic peaks and similarity evaluation, cluster analysis(CA), principal components analysis(PCA) and orthogonal partial least squares discriminant analysis(OPLS-DA). HPLC fingerprints of A. membranaceus var. mongholicus in different genuine regions are established. The qualitative analysis of mass spectrometry identified 18 components. The similarity evaluation shows that the similarity of 32 batches of A. membranaceus var. mongholicus samples was 0.688-0.993. Among them, the similarity of samples in Shanxi, Inner Mongolia, Ningxia is 0.688-0.993, 0.835-0.989, 0.934-0.988, respectively and the similarity of samples in Gansu is 0.729-0.876 except No. 25 sample. The results of CA show that the samples of A. membranaceus var. mongholicus can be grouped into four categories according to the production area except the No. 11 and No. 25 samples. The results of PCA indicate that 32 batches of A. membranaceus var. mongholicus samples can be clustered according to quality and origin, and the quality of A. membranaceus var. mongholicus in Inner Mongolia is the closest to the wild breed. The results of OPLS-DA indicate that there are six components that can distinguish the wild and domestic A. membranaceus var. mongholicus, which are malonylastragaloside Ⅰ, astragaloside Ⅰ, calycosin-7-O-ß-D-glycoside-6″-O-malonate, calycosin-7-O-ß-D-glycoside, formononetin-7-O-ß-D-glycoside-6″-O-malonate, and astrapterocarpan-3-O-ß-D-glycoside-6″-O-malonate. The established method can be used to analyze differences between A. membranaceus var. mongholicus origin and planting environment, and can provide references for the protection and replacement of wild A. membranaceus var. mongholicus resources, and the cultivation, processing and production of A. membranaceus var. mongholicus.

Sequential treatment with aT19 cells generates memory CAR-T cells and prolongs the lifespan of Raji-B-NDG mice.

Treatment with chimeric antigen receptor (CAR)-modified T cells targeting CD19 has proved successful in patients with relapsed/refractory B cell malignancies. However, long-term follow-up indicates that remission in a substantial proportion of patients is not sustainable. Most patients that experience recurrence have tumors and lost the CAR-T cells. To maintain the activity of CAR-T cells, Raji-B-NDG mice were treated sequentially with CAR-T-19 cells and homologous cells expressing human CD19 to promote expansion of CAR-T cells. Sequential treatment of mice with CAR-T-19 cells followed by Raji tumor cells led to marked prolongation of survival. The best case scenario after sequential treatment was a survival time of more than 200 days; the average survival time of mice in the non-sequential treatment group was 80 days. We treated mice with autologous CD19-modified T cells after initial treatment with CAR-T-19 cells. The overall survival and recurrence-free survival times of mice receiving sequential treatment were significantly longer. The percentages of CAR+ T cells in peripheral blood increased. Sequential therapy with autologous CAR-T-19 and aT19 cells provides a new strategy for generating memory CAR-T cells, which may lead ultimately to increased clinical efficacy.

Generation of Transgene-Free Maize Male Sterile Lines Using the CRISPR/Cas9 System.

Male sterility (MS) provides a useful breeding tool to harness hybrid vigor for hybrid seed production. It is necessary to generate new male sterile mutant lines for the development of hybrid seed production technology. The CRISPR/Cas9 technology is well suited for targeting genomes to generate male sterile mutants. In this study, we artificially synthesized Streptococcus pyogenes Cas9 gene with biased codons of maize. A CRISPR/Cas9 vector targeting the MS8 gene of maize was constructed and transformed into maize using an Agrobacterium-mediated method, and eight T0 independent transgenic lines were generated. Sequencing results showed that MS8 genes in these T0 transgenic lines were not mutated. However, we detected mutations in the MS8 gene in F1 and F2 progenies of the transgenic line H17. A potential off-target site sequence which had a single nucleotide that was different from the target was also mutated in the F2 progeny of the transgenic line H17. Mutation in the MS8 gene and the male sterile phenotype could be stably inherited by the next generation in a Mendelian fashion. Transgene-free ms8 male sterile plants were obtained by screening the F2 generation of male sterile plants, and the MS phenotype could be introduced into other elite inbred lines for hybrid production.

Differential Effect of Food Sanitizers on Formation of Viable but Nonculturable Salmonella enterica in Poultry.

A method for microscopic enumeration of viable Salmonella enterica in meat samples was developed by using the LIVE/DEAD BacLight kit technology. A two-step centrifugation and wash process was developed to clean the samples from food and chemical impurities that might otherwise interfere with the appropriate staining reactions. The accuracy of the BacLight kit-based viability assessments was confirmed with various validation tests that were conducted by following the manufacturer's instructions. For the biocide challenge tests, chicken parts each bearing around 8.5 log of S. enterica were sprayed with common food sanitizers such as 1,3-dibromo-5,5-dimethylhydantoin (DBDMH), lactic acid (LA), and peracetic acid (PAA). The log reduction (LR) of S. enterica for each test biocide was evaluated by microscopic and conventional culture plate methods. The results show that both LA and PAA treatments generated a greater number of microscopic counts compared with the corresponding plate counts with differences being around half a log. This discrepancy is believed to occur when cells enter a so-called viable but nonculturable (VBNC) state, and to our knowledge, this is the first report documenting the presence of VBNC in PAA- and LA-treated food samples. In contrast, the BacLight-based viable counts were comparable to the culture-based enumerations of all DBDMH-treated samples. Therefore, we concluded that DBDMH-treated meat did not contain significant VBNC populations of S. enterica. A detailed description of our spray system, the dye validation, and the treatment reproducibility are also provided in this work.

Increasing RpoS expression causes cell death in Borrelia burgdorferi.

RpoS, one of the two alternative σ factors in Borrelia burgdorferi, is tightly controlled by multiple regulators and, in turn, determines expression of many critical virulence factors. Here we show that increasing RpoS expression causes cell death. The immediate effect of increasing RpoS expression was to promote bacterial division and as a consequence result in a rapid increase in cell number before causing bacterial death. No DNA fragmentation or degradation was observed during this induced cell death. Cryo-electron microscopy showed induced cells first formed blebs, which were eventually released from dying cells. Apparently blebbing initiated cell disintegration leading to cell death. These findings led us to hypothesize that increasing RpoS expression triggers intracellular programs and/or pathways that cause spirochete death. The potential biological significance of induced cell death may help B. burgdorferi regulate its population to maintain its life cycle in nature.

RpoS regulates essential virulence factors remaining to be identified in Borrelia burgdorferi.

BACKGROUND: Since the RpoN-RpoS regulatory network was revealed in the Lyme disease spirochete Borrelia burgdorferi a decade ago, both upstream and downstream of the pathway have been intensively investigated. While significant progress has been made into understanding of how the network is regulated, most notably, discovering a relationship of the network with Rrp2 and BosR, only three crucial virulence factors, including outer surface protein C (OspC) and decorin-binding proteins (Dbps) A and B, are associated with the pathway. Moreover, for more than 10 years no single RpoS-controlled gene has been found to be critical for infection, raising a question about whether additional RpoS-dependent virulence factors remain to be identified. METHODOLOGY/PRINCIPAL FINDINGS: The rpoS gene was deleted in B. burgdorferi; resulting mutants were modified to constitutively express all the known virulence factors, OspC, DbpA and DbpB. This genetic modification was unable to restore the rpoS mutant with infectivity. CONCLUSIONS/SIGNIFICANCE: The inability to restore the rpoS mutant with infectivity by simultaneously over-expressing all the three virulence factors allows us to conclude RpoS also regulates essential genes that remain to be identified in B. burgdorferi.

BB0250 of Borrelia burgdorferi is a conserved and essential inner membrane protein required for cell division.

The gene bb0250 of Borrelia burgdorferi is a homolog of the dedA family, encoding integral inner membrane proteins that are present in nearly all species of bacteria. To date, no precise function has been attributed to any dedA gene. Unlike many bacterial species, such as Escherichia coli, which has eight dedA genes, B. burgdorferi possesses only one, annotated bb0250, providing a unique opportunity to investigate the functions of the dedA family. Here, we show that bb0250 is able to restore normal growth and cell division to a temperature-sensitive E. coli mutant with simultaneous deletions of two dedA genes, yqjA and yghB, and encodes a protein that localizes to the inner membrane of E. coli. The bb0250 gene could be deleted from B. burgdorferi only after introduction of a promoterless bb0250 under the control of an inducible lac promoter, indicating that it is an essential gene in this organism. Growth of the mutant in the absence of isopropyl-ß-d-thiogalactopyranoside resulted in cell death, preceded by cell division defects characterized by elongated cells and membrane bulges, demonstrating that bb0250 is required for proper cell division and envelope integrity. Finally, we show that BB0250 depletion leads to imbalanced membrane phospholipid composition in borrelia. These results demonstrate a strong conservation of function of the dedA gene family across diverse species of Gram-negative bacteria and a requirement for this protein family for normal membrane lipid composition and cell division.

Two regulatory elements required for enhancing ospA expression in Borrelia burgdorferi grown in vitro but repressing its expression during mammalian infection.

During cycling between the tick vector and a mammal, the Lyme disease spirochaete Borrelia burgdorferi must coordinate expression of outer-surface proteins (Osps) A and B to quickly respond to environmental changes. The pathogen abundantly produces OspA/B in the tick, but represses their expression during mammalian infection. This paper reports a regulatory structure, consisting of two sequences flanking the ospAB promoter, that is required for enhancing ospA expression in B. burgdorferi grown in vitro, but repressing its expression during murine infection. Deletion or replacement of either the upstream or downstream sequence of the ospAB promoter caused a significant decrease in ospA expression in vitro, but a dramatic increase during murine infection. Fusion of either sequence with the flaB reporter promoter led to increased expression of an ospA reporter gene in vitro, but a decrease in the murine host. Furthermore, simultaneous fusion of both sequences with the reporter promoter showed a synergistic effect in enhancing expression of the ospA reporter in vitro, but repressing its expression during murine infection. Taken together, the results demonstrate that the regulatory structure functions oppositely in the two different environments and potentially provides B. burgdorferi with a molecular mechanism to quickly adapt to the distinct environments during its enzootic life cycle.

Outer surface protein C is a dissemination-facilitating factor of Borrelia burgdorferi during mammalian infection.

BACKGROUND: The Lyme disease spirochete Borrelia burgdorferi dramatically upregulates outer surface protein C (OspC) in response to fresh bloodmeal during transmission from the tick vector to a mammal, and abundantly produces the antigen during early infection. As OspC is an effective immune target, to evade the immune system B. burgdorferi downregulates the antigen once the anti-OspC humoral response has developed, suggesting an important role for OspC during early infection. METHODOLOGY/PRINCIPAL FINDINGS: In this study, a borrelial mutant producing an OspC antigen with a 5-amino-acid deletion was generated. The deletion didn't significantly increase the 50% infectious dose or reduce the tissue bacterial burden during infection of the murine host, indicating that the truncated OspC can effectively protect B. burgdorferi against innate elimination. However, the deletion greatly impaired the ability of B. burgdorferi to disseminate to remote tissues after inoculation into mice. CONCLUSIONS/SIGNIFICANCE: The study indicates that OspC plays an important role in dissemination of B. burgdorferi during mammalian infection.

Modification of Borrelia burgdorferi to overproduce OspA or VlsE alters its infectious behaviour.

The surface lipoproteins of the Lyme disease spirochaete Borrelia burgdorferi directly interact with tissue microenvironments during mammalian infection, and thus potentially affect various aspects of infection. To investigate the influence of surface antigen synthesis on infectious behaviour, B. burgdorferi was modified to constitutively produce the well-characterized surface lipoproteins OspA and invariant VlsE. Although increasing OspA or VlsE production did not significantly affect synthesis of other surface lipoproteins or spirochaetal growth in vitro, overexpressing vlsE resulted in increased ospA but decreased ospC expression, and overexpressing ospA led to decreased ospC and vlsE expression in severe combined immunodeficient (SCID) mice. Increasing the expression of either ospA or vlsE did not alter the ID(50), but affected spirochaetal dissemination and significantly reduced tissue spirochaete loads in SCID mice. In immunocompetent mice, increased vlsE expression resulted in quick clearance of infection, while constitutive ospA expression led to a substantial ID(50) increase and severely impaired dissemination. Furthermore, B. burgdorferi with constitutive ospA expression persisted in the skin tissue but was cleared from both heart and joints of chronically infected immunocompetent mice. Taken together, the study indicates that increasing production of OspA or invariant VlsE influences lipoprotein gene expression in the murine host and alters the infectious behaviour of B. burgdorferi.

Common and unique contributions of decorin-binding proteins A and B to the overall virulence of Borrelia burgdorferi.

As an extracellular bacterium, the Lyme disease spirochete Borrelia burgdorferi resides primarily in the extracellular matrix and connective tissues and between host cells during mammalian infection, where decorin and glycosaminoglycans are abundantly found, so its interactions with these host ligands potentially affect various aspects of infection. Decorin-binding proteins (Dbps) A and B, encoded by a 2-gene operon, are outer surface lipoproteins with similar molecular weights and share approximately 40% identity, and both bind decorin and glycosaminoglycans. To investigate how DbpA and DbpB contribute differently to the overall virulence of B. burgdorferi, a dbpAB mutant was modified to overproduce the adhesins. Overproduction of either DbpA or DbpB resulted in restoration of the infectivity of the mutant to the control level, measured by 50% infectious dose (ID(50)), indicating that the two virulence factors are interchangeable in this regard. Overproduction of DbpA also allowed the mutant to disseminate to some but not all distal tissues slightly slower than the control, but the mutant with DbpB overproduction showed severely impaired dissemination to all tissues that were analyzed. The mutant with DbpA overproduction colonized all tissues, albeit generating bacterial loads significantly lower than the control in heart and joint, while the mutant overproducing DbpB remained severely defective in heart colonization and registered bacterial loads substantially lower than the control in joint. Taken together, the study indicated that DbpA and DbpB play a similar role in contribution to infectivity as measured by ID(50) value but contribute differently to dissemination and tissue colonization.

Verification and dissection of the ospC operator by using flaB promoter as a reporter in Borrelia burgdorferi.

The Lyme disease spirochete Borrelia burgdorferi must repress expression of outer surface protein C (OspC) to effectively evade specific humoral immunity and to establish persistent infection. This ability largely relies upon a regulatory element, the only operator that has been reported in spirochetal bacteria. Immediately upstream of the ospC promoter, two sets of inverted repeats (IRs) constitute small and large palindromes, in which the right IR of the large palindrome contains the left IR of the small one, and may collectively function as the ospC operator. In the study, the large palindrome with or without the small IR was fused with an flaB promoter, which was used to drive expression of a promoterless ospC copy as a reporter gene, and introduced into OspC-deficient B. burgdorferi. The presence of the large palindrome alone significantly reduced ospC expression driven by the fused flaB promoter in the joint tissue of severe combined immunodeficiency (SCID) mice, and rescued spirochetes from elimination by passively transferred OspC antibody in infected SCID mice and specific immune responses elicited in immunocompetent mice, confirming a function of the IRs as an operator. Inclusion of the small IR further enhanced the ability of the large palindrome to reduce the activity of the fused flaB promoter, indicating that the small IR is a part of the operator. Taken together, the study led to successful verification and dissection of the ospC operator.

Essential protective role attributed to the surface lipoproteins of Borrelia burgdorferi against innate defences.

To initiate infection, a microbial pathogen must be able to evade innate immunity. Here we show that the Lyme disease spirochete Borrelia burgdorferi depends on its surface lipoproteins for protection against innate defences. The deficiency for OspC, an abundantly expressed surface lipoprotein during early infection, led to quick clearance of B. burgdorferi after inoculation into the skin of SCID mice. Increasing expression of any of the four randomly chosen surface lipoproteins, OspA, OspE, VlsE or DbpA, fully protected the ospC mutant from elimination from the skin tissue of SCID mice; moreover, increased OspA, OspE or VlsE expression allowed the mutant to cause disseminated infection and restored the ability to effectively colonize both joint and skin tissues, albeit the dissemination process was much slower than that of the mutant restored with OspC expression. When the ospC mutant was modified to express OspA under control of the ospC regulatory elements, it registered only a slight increase in the 50% infectious dose than the control in SCID mice but a dramatic increase in immunocompetent mice. Taken together, the study demonstrated that the surface lipoproteins provide B. burgdorferi with an essential protective function against host innate elimination.

Changing transcriptional initiation sites and alternative 5'- and 3'-splice site selection of the first intron deploys Arabidopsis protein isoaspartyl methyltransferase2 variants to different subcellular compartments.

Arabidopsis thaliana (L.) Heynh. possesses two PROTEIN-L-ISOASPARTATE METHYLTRANSFERASE (PIMT) genes encoding enzymes (EC 2.1.1.77) capable of converting uncoded l-isoaspartyl residues, arising spontaneously at l-asparaginyl and l-aspartyl sites in proteins, to l-aspartate. PIMT2 produces at least eight transcripts by using four transcriptional initiation sites (TIS; resulting in three different initiating methionines) and both 5'- and 3'-alternative splice site selection of the first intron. The transcripts produce mature proteins capable of converting l-isoaspartate to l-aspartate in small peptide substrates. PIMT:GFP fusion proteins generated a detectable signal in the nucleus. However, whether the protein was also detectable in the cytoplasm, endo-membrane system, chloroplasts, and/or mitochondria, depended on the transcript from which it was produced. On-blot-methylation of proteins, prior to the completion of germination, indicated that cruciferin subunits contain isoaspartate. The implications of using transcriptional mechanisms to expand a single gene's repertoire to protein variants capable of entry into the cell's various compartments are discussed in light of PIMT's presumed role in repairing the proteome.

Both decorin-binding proteins A and B are critical for the overall virulence of Borrelia burgdorferi.

Both decorin-binding proteins (DbpA and DbpB) of the Lyme disease spirochete Borrelia burgdorferi bind decorin and glycosaminoglycans, two important building blocks of proteoglycans that are abundantly found in the extracellular matrix (ECM) and connective tissues as well as on cell surfaces of mammals. As an extracellular pathogen, B. burgdorferi resides primarily in the ECM and connective tissues and between host cells during mammalian infection. The interactions of B. burgdorferi with these host ligands mediated by DbpA and DbpB potentially influence various aspects of infection. Here, we show that both DbpA and DbpB are critical for the overall virulence of B. burgdorferi in the murine host. Disruption of the dbpBA locus led to nearly a 10(4)-fold increase in the 50% infectious dose (ID50). Complementation of the mutant with either dbpA or dbpB reduced the ID50 from over 10(4) to roughly 10(3) organisms. Deletion of the dbpBA locus affected colonization in all tissues of infected mice. The lack of dbpA alone precluded the pathogen from colonizing the heart tissue, and B. burgdorferi deficient for DbpB was recovered only from 42% of the heart specimens of infected mice. Although B. burgdorferi lacking either dbpA or dbpB was consistently grown from joint specimens of almost all infected mice, it generated bacterial loads significantly lower than the control. The deficiency in either DbpA or DbpB did not reduce the bacterial load in skin, but lack of both significantly did. Taken together, the study results indicate that neither DbpA nor DbpB is essential for mammalian infection but that both are critical for the overall virulence of B. burgdorferi.