Fecal microbiota changes in NZB/W F1 mice after induction of lupus disease.

The association between the gut microbiota and the development of lupus is unclear. We investigated alterations in the gut microbiota after induction of lupus in a murine model using viral peptide of human cytomegalovirus (HCMV). Three treatment arms for the animals were prepared: intraperitoneal injection of HCMVpp65 peptide, adjuvant alone, and PBS injection. Feces were collected before and after lupus induction biweekly for 16S rRNA sequencing. HCMVpp65 peptide immunization induced lupus-like effects, with higher levels of anti-dsDNA antibodies, creatinine, proteinuria, and glomerular damage, compared with mice treated with nothing or adjuvant only. The Simpson diversity value was higher in mice injected with HCMVpp65 peptide, but there was no difference in ACE or Chao1 among the three groups. Statistical analysis of metagenomic profiles showed a higher abundance of various families (Saccharimonadaceae, Marinifiaceae, and Desulfovibrionaceae) and genera (Candidatus Saccharimonas, Roseburia, Odoribacter, and Desulfovibrio) in HCMVpp65 peptide-treated mice. Significant correlations between increased abundances of related genera (Candidatus Saccharimonas, Roseburia, Odoribacter, and Desulfovibrio) and HCMVpp65 peptide immunization-induced lupus-like effects were observed. This study provides insight into the changes in the gut microbiota after lupus onset in a murine model.

Cytomegalovirus-Associated Autoantibody against TAF9 Protein in Patients with Systemic Lupus Erythematosus.

Background: Evidence indicates a causal link between cytomegalovirus (CMV) infection and the triggering of systemic lupus erythematosus (SLE). Animal studies have revealed that CMV phosphoprotein 65 (pp65) induces autoantibodies against nuclear materials and causes the autoantibody attack of glomeruli. IgG eluted from the glomeruli of CMVpp65-peptide-immunized mice exhibited cross-reactivity against dsDNA and TATA-box-binding protein associated factor 9 (TAF9). Whether the elevation of anti-TAF9 IgG is associated with anti-CMV reactivity in human lupus remains unclear. Methods: The sera from patients with rheumatic diseases, including ankylosing spondylitis (AS), gout, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and Sjögren syndrome (SS) were examined using ELISA for antibodies of CMV, CMVpp65, and TAF9. Results: In total, 83.8% of the rheumatic patients had acquired CMV infections. The SLE patients had a high prevalence of anti-CMV IgM. The highest seropositivity rates for anti-HCMVpp65 and anti-TAF9 IgG were observed in the SLE patients. Purified anti-CMVpp65 IgG from CMVpp65/TAF9 dual-positive SLE sera reacted to both TAF9 and dsDNA. An increased prevalence of proteinuria and low hemoglobin levels were found in CMV IgG- and CMVpp65 IgG-positive SLE patients. Conclusions: This observation suggests that immunity to CMVpp65 is associated with cross-reactivity with TAF9 and dsDNA and that it is involved in the development of clinical manifestations in SLE.

Impact of urate-lowering drugs on the progression and recovery from chronic kidney disease among gout patients.

BACKGROUND: This study investigates the association between exposure to urate-lowering drugs (ULDs) and progression and recovery from chronic kidney disease (CKD). METHODS: We identified 5860 incident gout patients at Chang Gung Memorial Hospital from 2012 to 2015. Propensity score (PS)-weighted Cox proportional hazards model was used to estimate hazard ratios (HRs) for CKD progression and improvement. A separate analysis was conducted to assess the HR for CKD progression and CKD recovery among those with worsening CKD. RESULTS: The incidence of CKD progression among allopurinol, febuxostat and uricosuric agent users were 1.98, 1.88 and 1.64 per 1000 person-days. Compared with allopurinol users, the PS-weighted HR (95% confidence intervals [CIs]) was 1.77 (0.85-1.76) for febuxostat users and 1.37 (0.71-1.37) for uricosuric agent users for CKD progression and 1.43 (1.26-1.62) for febuxostat users and 1.00 (0.88-1.14) for uricosuric agent users for CKD improvement. Compared to allopurinol users, the HRs for CKD progression were 1.14 (0.80-1.66) for febuxostat users and 0.92 (0.67-1.31) for uricosuric agent users. Among 741 patients who had CKD progression, the incidence of CKD recovery was 1.33, 6.21 and 3.53 per 1000 person-days for allopurinol, febuxostat and uricosuric agent users. The HRs (95% CIs) for recovery in febuxostat and uricosuric agent users were 2.17 (1.40-3.47) and 1.80 (1.20-2.83) compared to allopurinol users. CONCLUSIONS: CKD progression and recovery are common in gout patients using ULDs. Febuxostat and benzbromarone were associated with a similar risk of CKD progression with allopurinol, which has a poorer recovery compared with other ULDs.

The Arabidopsis heat-intolerant 5 (hit5)/enhanced response to aba 1 (era1) mutant reveals the crucial role of protein farnesylation in plant responses to heat stress.

Protein farnesylation is a post-translational modification known to regulate abscisic acid (ABA)-mediated drought tolerance in plants. However, it is unclear whether and to what extent protein farnesylation affects plant tolerance to high-temperature conditions. The Arabidopsis heat-intolerant 5 (hit5) mutant was isolated because it was thermosensitive to prolonged heat incubation at 37°C for 4 d but thermotolerant to sudden heat shock at 44°C for 40 min. Map-based cloning revealed that HIT5 encodes the ß-subunit of the protein farnesyltransferase. hit5 was crossed with the aba-insensitive 3 (abi3) mutant, the aba-deficient 3 (aba3) mutant, and the heat shock protein 101 (hsp101) mutant, to characterize the HIT5-mediated heat stress response. hit5/abi3 and hit5/aba3 double mutants had the same temperature-dependent phenotypes as hit5. Additionally, exogenous supplementation of neither ABA nor the ABA synthesis inhibitor fluridone altered the temperature-dependent phenotypes of hit5. The hit5/hsp101 double mutant was still sensitive to prolonged heat incubation, yet its ability to tolerate sudden heat shock was lost. The results suggest that protein farnesylation either positively or negatively affects the ability of plants to survive heat stress, depending on the intensity and duration of high-temperature exposure, in an ABA-independent manner. HSP101 is involved in the hit5-derived heat shock tolerance phenotype.

Arabidopsis HIT4, a regulator involved in heat-triggered reorganization of chromatin and release of transcriptional gene silencing, relocates from chromocenters to the nucleolus in response to heat stress.

Arabidopsis HIT4 is known to mediate heat-induced decondensation of chromocenters and release from transcriptional gene silencing (TGS) with no change in the level of DNA methylation. It is unclear whether HIT4 and MOM1, a well-known DNA methylation-independent transcriptional silencer, have overlapping regulatory functions. A hit4-1/mom1 double mutant strain was generated. Its nuclear morphology and TGS state were compared with those of wild-type, hit4-1, and mom1 plants. Fluorescent protein tagging was employed to track the fates of HIT4, hit4-1 and MOM1 in vivo under heat stress. HIT4- and MOM1-mediated TGS were distinguishable. Both HIT4 and MOM1 were localized normally to chromocenters. Under heat stress, HIT4 relocated to the nucleolus, whereas MOM1 dispersed with the chromocenters. hit4-1 was able to relocate to the nucleolus under heat stress, but its relocation was insufficient to trigger the decompaction of chromocenters. The hypersensitivity to heat associated with the impaired reactivation of TGS in hit4-1 was not alleviated by mom1-induced release from TGS. HIT4 delineates a novel and MOM1-independent TGS regulation pathway. The involvement of a currently unidentified component that links HIT4 relocation and the large-scale reorganization of chromatin, and which is essential for heat tolerance in plants is hypothesized.

Arabidopsis HIT4 encodes a novel chromocentre-localized protein involved in the heat reactivation of transcriptionally silent loci and is essential for heat tolerance in plants.

The Arabidopsis mutant heat-intolerant 4-1 (hit4-1) was isolated from an ethyl methanesulphonate-mutagenized M2 population on the basis of its inability to withstand prolonged heat stress (4 days at 37°C). Further characterization indicated that hit4-1 was impaired specifically in terms of basal but not acquired thermotolerance. Map-based cloning revealed that the HIT4 gene encoded a plant-specific protein for which the molecular function has yet to be studied. To investigate the cellular role of HIT4 and hence elucidate better its protective function in heat tolerance in plants, a GFP-HIT4 reporter construct was created for a protoplast transient expression assay. Results showed that fluorescently tagged HIT4 was localized to the chromocentre, a condensed heterochromatin domain that harbours repetitive elements for which transcription is normally suppressed by transcriptional gene silencing (TGS). DAPI-staining analysis and FISH with a probe that targeted centromeric repeats showed that heat-induced chromocentre decondensation was inhibited in nuclei of hit4-1 subjected to direct heat treatment, but not in those that were allowed to acquire thermotolerance. Moreover, heat reactivation of various TGS loci, regardless of whether they were endogenous or transgenic, or existed as a single copy or as repeats, was found to be attenuated in hit4-1. Meanwhile, the levels of transcripts of heat shock protein genes in response to heat stress were similar in both hit4-1 and wild-type plants. Collectively, these results demonstrated that HIT4 defines a new TGS regulator that acts at the level of heterochromatin organization and is essential for basal thermotolerance in plants.

The Arabidopsis hit1-1 mutant has a plasma membrane profile distinct from that of wild-type plants at optimal growing temperature.

High temperatures alter the physical properties of the plasma membrane and cause loss of function in the embedded proteins. Effective membrane and protein recycling through intracellular vesicular traffic is vital to maintain the structural and functional integrity of the plasma membrane under heat stress. However, in this regard, little experimental data is available. Our characterization of the Arabidopsis hit1-1 mutant, linking a subunit of a vesicle tethering complex to plasma membrane thermostability, provided valuable information to this end. We further dissected the effect of the hit1-1 mutation on plasma membrane properties and found that even at optimal growth temperature (23 °C), the hit1-1 mutant exhibited a plasma membrane protein profile distinct from that of wild-type plants. This result implies that the hit1-1 mutation essentially alters vesicle trafficking and results in changes in the plasma membrane components under non-stress conditions. Such changes do not affect normal plant growth and development, but is significant for plant survival under heat stress.

Involvement of the Arabidopsis HIT1/AtVPS53 tethering protein homologue in the acclimation of the plasma membrane to heat stress.

Arabidopsis thaliana hit1-1 is a heat-intolerant mutant. The HIT1 gene encodes a protein that is homologous to yeast Vps53p, which is a subunit of the Golgi-associated retrograde protein (GARP) complex that is involved in retrograde membrane trafficking to the Golgi. To investigate the correlation between the cellular role of HIT1 and its protective function in heat tolerance in plants, it was verified that HIT1 was co-localized with AtVPS52 and AtVPS54, the other putative subunits of GARP, in the Golgi and post-Golgi compartments in Arabidopsis protoplasts. A bimolecular fluorescence complementation assay showed that HIT1 interacted with AtVPS52 and AtVPS54, which indicated their assembly into a protein complex in vivo. Under heat stress conditions, the plasma membrane of hit1-1 was less stable than that of the wild type, as determined by an electrolyte leakage assay, and enhanced leakage occurred before peroxidation injury to the membrane. In addition, the ability of hit1-1 to survive heat stress was not influenced by exposure to light, which suggested that the heat intolerance of hit-1 was a direct outcome of reduced membrane thermostability rather than heat-induced oxidative stress. Furthermore, hit1-1 was sensitive to the duration (sustained high temperature stress at 37 °C for 3 d) but not the intensity (heat shock at 44 °C for 30 min) of exposure to heat. Collectively, these results imply that HIT1 functions in the membrane trafficking that is involved in the thermal adaptation of the plasma membrane for tolerance to long-term heat stress in plants.

Isolation and characterization of the Arabidopsis heat-intolerant 2 (hit2) mutant reveal the essential role of the nuclear export receptor EXPORTIN1A (XPO1A) in plant heat tolerance.

*The Arabidopsis heat-intolerant 2 (hit2) mutant was isolated on the basis of its impaired ability to withstand moderate heat stress (37 degrees C). Determination of the genetic mutation that underlies the hit2 thermosensitive phenotype allowed better understanding of the mechanisms by which plants cope with heat stress. *Genetic analysis revealed that hit2 is a single recessive mutation. Map-based cloning was used to identify the hit2 locus. The response of hit2 to other types of heat stress was also investigated to characterize the protective role of HIT2. *hit2 was defective in basal but not in acquired thermotolerance. hit2 was sensitive to methyl viologen-induced oxidative stress, and the survival of hit2 seedlings in response to heat stress was affected by light conditions. The mutated locus was located at the EXPORTIN1A (XPO1A) gene, which encodes a nuclear transport receptor. Two T-DNA insertion lines, xpo1a-1 and xpo1a-3, exhibited the same phenotypes as hit2. *The results provide evidence that Arabidopsis XPO1A is dispensable for normal plant growth and development but is essential for thermotolerance, in part by mediating the protection of plants against heat-induced oxidative stress.

Mutation in a homolog of yeast Vps53p accounts for the heat and osmotic hypersensitive phenotypes in Arabidopsis hit1-1 mutant.

Previously, the growth of Arabidopsis hit1-1 (heat-intolerant) mutant was found to be inhibited by both heat and water stress (Wu et al. in J Plant Physiol 157:543-547, 2000). In order to determine the genetic mutation underlying the hit1-1 phenotype, map-based cloning of HIT1 gene was conducted. Transformation of the hit1-1 mutant with a HIT1 cDNA clone reverts the mutant to the heat tolerance phenotype, confirming the identity of HIT1. Sequence analysis revealed the HIT1 gene encodes a protein of 829 amino acid residues and is homologous to yeast (Saccharomyces cerevisiae) Vps53p protein. The yeast Vps53p protein has been shown to be a tethering factor that associates with Vps52p and Vps54p in a complex formation involved in the retrograde trafficking of vesicles to the late Golgi. An Arabidopsis homolog of yeast Vps52p has previously been identified and mutation of either the homolog or HIT1 by T-DNA insertion resulted in a male-specific transmission defect. The growth of yeast vps53Delta null mutant also shows reduced thermotolerance, and expression of HIT1 in this mutant can partially complement the defect, supporting the possibility of a conserved biological function for Vps53p and HIT1. Collectively, the hit1-1 is the first mutant in higher plant linking a homolog of the vesicle tethering factor to both heat and osmotic stress tolerance.