DAT, deacylating autotransporter toxin, from Bordetella parapertussis demyristoylates Gαi GTPases and contributes to cough.

The pathogenic bacteria Bordetella pertussis and Bordetella parapertussis cause pertussis (whooping cough) and pertussis-like disease, respectively, both of which are characterized by paroxysmal coughing. We previously reported that pertussis toxin (PTx), which inactivates heterotrimeric GTPases of the Gi family through ADP-ribosylation of their α subunits, causes coughing in combination with Vag8 and lipid A in B. pertussis infection. In contrast, the mechanism of cough induced by B. parapertussis, which produces Vag8 and lipopolysaccharide (LPS) containing lipid A, but not PTx, remained to be elucidated. Here, we show that a toxin we named deacylating autotransporter toxin (DAT) of B. parapertussis inactivates heterotrimeric Gi GTPases through demyristoylation of their α subunits and contributes to cough production along with Vag8 and LPS. These results indicate that DAT plays a role in B. parapertussis infection in place of PTx.

Dual roles of cellular communication network factor 6 (CCN6) in the invasion and metastasis of oral cancer cells to bone via binding to BMP2 and RANKL.

The acquisition of motility via epithelial-mesenchymal transition (EMT) and osteoclast induction are essential for the invasion and metastasis of oral squamous cell carcinoma (OSCC) to bone. However, the molecule suppressing both EMT and osteoclastogenesis is still unknown. In this study, we found that cellular communication network factor 6 (CCN6) was less produced in a human OSCC cell line, HSC-3 with mesenchymal phenotype, than in HSC-2 cells without it. Notably, CCN6 interacted with bone morphogenetic protein 2 (BMP2) and suppressed the cell migration of HSC-3 cells stimulated by BMP2. Moreover, knockdown of CCN6 in HSC-2 cells led to the promotion of EMT and enhanced the effect of transforming growth factor-ß (TGF-ß) on the promotion of EMT. Furthermore, CCN6 combined with BMP2 suppressed EMT. These results suggest that CCN6 strongly suppresses EMT in cooperation with BMP2 and TGF-ß. Interestingly, CCN6 combined with BMP2 increased the gene expression of receptor activator of nuclear factor-κB ligand (RANKL) in HSC-2 and HSC-3 cells. Additionally, CCN6 interacted with RANKL, and CCN6 combined with RANKL suppressed RANKL-induced osteoclast formation. In metastatic lesions, increasing BMP2 due to the bone destruction led to interference with binding of CCN6 to RANKL, which results in the promotion of bone metastasis of OSCC cells due to continuous osteoclastogenesis. These findings suggest that CCN6 plays dual roles in the suppression of EMT and in the promotion of bone destruction of OSCC in primary and metastatic lesions, respectively, through cooperation with BMP2 and interference with RANKL.

Immunization with autotransporter Vag8 prevents coughing induced by Bordetella pertussis infection in mice.

Bordetella pertussis causes pertussis, which is characterized by paroxysmal coughing. This disease is generally prevented through vaccination; however, the number of pertussis cases is increasing worldwide despite high vaccination coverage. We previously reported that an autotransporter of B. pertussis, virulence-associated gene 8 (Vag8), causes coughing in combination with pertussis toxin and lipooligosaccharide. Here, we show that immunization with Vag8 protected mice from coughing after B. pertussis infection and enhanced the efficacy of a current pertussis vaccine containing pertussis toxoid against the cough. Our findings indicate that Vag8 could be a vaccine antigen to prevent pertussis cough.

Quality of life in glaucoma.

BACKGROUND: An essential goal of glaucoma management is to prevent a patient's visual impairment that compromises their health-related quality of life (QOL). The disease itself, in addition to the medical or surgical treatment, can have a large impact on one's life. We aim to briefly review and evaluate aspects of QOL in glaucoma. METHODS: The PubMed database was utilized for the literature examination of this review. Keywords that were searched included glaucoma, quality of life, vision-related QOL (VRQOL), quality of life questionnaire, and glaucoma therapy. RESULTS: The main topics identified and analyzed during the literature review stages include factors affecting VRQOL, the assessment of VRQOL using questionnaires, QOL in early and severe glaucoma, glaucoma and activities of daily living, glaucoma treatments, and new advances in clinically assessing QOL. The study findings indicate a relationship between the deterioration of visual field and the quality of life. The investigation shows that visual loss can result in a range of daily life challenges, which include compromised mental health status and difficulties with driving, reading, and recognizing people. CONCLUSION: Glaucoma-induced visual field loss can significantly impact different aspects of patients' life, and several methods exist for evaluating changes in quality of life. Quality of life assessments have their limitations as they are subjective. As potential future steps, we suggest exploring technological advancements such as virtual reality to improve patient care and outcomes.

Impact of Smoking on Visual Field Progression in a Long-term Clinical Follow-up.

PURPOSE: To investigate the effect of smoking on rates of progressive visual field (VF) damage over time in glaucoma. DESIGN: Retrospective cohort study. PARTICIPANTS: Five hundred eleven eyes of 354 patients with glaucoma followed up from multicenter glaucoma registries. METHODS: In this longitudinal study, 354 patients with primary open-angle glaucoma with a minimum of 3 years of follow-up and 5 VF tests were enrolled from the Diagnostic Innovations in Glaucoma Study and the African Descent and Glaucoma Evaluation Study. Univariate and multivariate linear mixed models were used to investigate the effects of smoking on rates of 24-2 VF mean deviation loss. Visual field progression was defined using pointwise linear and significant negative VF mean deviation loss. Logistic regression was used to identify baseline factors and whether different levels of smoking intensity were associated with VF progression. Kaplan-Meier survival analysis and the log-rank test were used to compare the cumulative risk ratio of progression between smoker and never smoker groups. MAIN OUTCOME MEASURES: Visual field progression. RESULTS: Five hundred eleven eyes of 354 patients were included over the median follow-up of 12.5 years. Median baseline age was 64.8 years. Of the 354 patients, 124 (35%) were Black, and 149 (42.1%) and 168 (59.8%) had reported a history of smoking or alcohol consumption, respectively. In a multivariate model, higher smoking intensity was associated with faster VF loss (coefficient, -0.05 decibels (dB)/year per 10 pack-years; 95% confidence interval [CI], -0.08 to -0.01 dB/year per 10 pack-years; P = 0.010). Developing VF progression in eyes of heavy smokers (≥ 20 pack-years) was 2.2 times more than in eyes of patients without smoking history (odds ratio, 2.21; 95% CI, 1.02-4.76; P = 0.044). Statistically significant differences were found between heavy smokers (≥ 20 pack-years) and never smokers by Kaplan-Meier analysis (P = 0.011, log-rank test). CONCLUSIONS: Heavy smokers are more likely to sustain VF loss in eyes with glaucoma. The prospective longitudinal design of this study supports the hypothesis that levels of smoking may be a significant predictor for glaucoma progression. Additionally, this information can be used for clinically relevant tobacco prevention and intervention messages.

Intraocular pressure-lowering efficacy and ocular safety of Rho-kinase inhibitor in glaucoma: a meta-analysis and systematic review of prospective randomized trials.

PURPOSE: To evaluate the intraocular pressure (IOP)-reducing efficacy and safety of Rho-kinase inhibitor (RKI). METHODS: Published studies in PubMed and EMBASE were searched on March 20, 2021. Study selection and data extraction were performed according to PRISMA. Meta-analysis of the IOP-lowering effect was performed with the bivariate random-effects model, with studies categorized into 2 classes: RKI versus placebo and RKI versus another medication. The main outcome was the difference in IOP reduction between RKI and non-RKI groups. Subgroup analysis of adjunctive RKI efficacy and additional review of its major ocular adverse events (AE) were also performed. RESULTS: Ten (2.6%) out of 391 studies were retrieved. In the RKI versus placebo class, RKI showed greater IOP reduction after 4-8 weeks (mean difference = - 1.69 mmHg [- 2.22, - 1.16], P < 0.001). In the RKI versus another medication class, IOP reduction by RKI was noninferior to timolol 0.5% twice-daily after 4-8 weeks (mean difference = 0.39 mmHg [0.01, 0.76], P = 0.043) and 12 weeks (mean difference = 0.48 mmHg [0.11, 0.85]; P = 0.011). In the subgroup analysis, the mean difference in IOP reduction by adjunctive RKI and placebo was - 1.42 mmHg (P < 0.001). The most common ocular AE of RKI was conjunctival hyperemia (19-65%), followed by conjunctival hemorrhage (6-20%) and cornea verticillata (13-26%). CONCLUSIONS: With a treatment duration of 1-3 months, RKI showed effective IOP reduction noninferior to timolol as monotherapy and as adjunctive therapy. Our results suggested RKI be a reliable IOP control medication; however, its higher incidence of some ocular complications should be attended to.

Molecular and Genetic Interactions between CCN2 and CCN3 behind Their Yin-Yang Collaboration.

Cellular communication network factor (CCN) 2 and 3 are the members of the CCN family that conduct the harmonized development of a variety of tissues and organs under interaction with multiple biomolecules in the microenvironment. Despite their striking structural similarities, these two members show contrastive molecular functions as well as temporospatial emergence in living tissues. Typically, CCN2 promotes cell growth, whereas CCN3 restrains it. Where CCN2 is produced, CCN3 disappears. Nevertheless, these two proteins collaborate together to execute their mission in a yin-yang fashion. The apparent functional counteractions of CCN2 and CCN3 can be ascribed to their direct molecular interaction and interference over the cofactors that are shared by the two. Recent studies have revealed the mutual negative regulation systems between CCN2 and CCN3. Moreover, the simultaneous and bidirectional regulatory system of CCN2 and CCN3 is also being clarified. It is of particular note that these regulations were found to be closely associated with glycolysis, a fundamental procedure of energy metabolism. Here, the molecular interplay and metabolic gene regulation that enable the yin-yang collaboration of CCN2 and CCN3 typically found in cartilage development/regeneration and fibrosis are described.

Fibroblast Growth Factors and Cellular Communication Network Factors: Intimate Interplay by the Founding Members in Cartilage.

Fibroblast growth factors (FGFs) constitute a large family of signaling molecules that act in an autocrine/paracrine, endocrine, or intracrine manner, whereas the cellular communication network factors (CCN) family is composed of six members that manipulate extracellular signaling networks. FGFs and CCNs are structurally and functionally distinct, except for the common characteristics as matricellular proteins. Both play significant roles in the development of a variety of tissues and organs, including the skeletal system. In vertebrates, most of the skeletal parts are formed and grow through a process designated endochondral ossification, in which chondrocytes play the central role. The growth plate cartilage is the place where endochondral ossification occurs, and articular cartilage is left to support the locomotive function of joints. Several FGFs, including FGF-2, one of the founding members of this family, and all of the CCNs represented by CCN2, which is required for proper skeletal development, can be found therein. Research over a decade has revealed direct binding of CCN2 to FGFs and FGF receptors (FGFRs), which occasionally affect the biological outcome via FGF signaling. Moreover, a recent study uncovered an integrated regulation of FGF and CCN genes by FGF signaling. In this review, after a brief introduction of these two families, molecular and genetic interactions between CCN and FGF family members in cartilage, and their biological effects, are summarized. The molecular interplay represents the mutual involvement of the other in their molecular functions, leading to collaboration between CCN2 and FGFs during skeletal development.

RFX1-mediated CCN3 induction that may support chondrocyte survival under starved conditions.

Cellular communication network factor (CCN) family members are multifunctional matricellular proteins that manipulate and integrate extracellular signals. In our previous studies investigating the role of CCN family members in cellular metabolism, we found three members that might be under the regulation of energy metabolism. In this study, we confirmed that CCN2 and CCN3 are the only members that are tightly regulated by glycolysis in human chondrocytic cells. Interestingly, CCN3 was induced under a variety of impaired glycolytic conditions. This CCN3 induction was also observed in two breast cancer cell lines with a distinct phenotype, suggesting a basic role of CCN3 in cellular metabolism. Reporter gene assays indicated a transcriptional regulation mediated by an enhancer in the proximal promoter region. As a result of analyses in silico, we specified regulatory factor binding to the X-box 1 (RFX1) as a candidate that mediated the transcriptional activation by impaired glycolysis. Indeed, the inhibition of glycolysis induced the expression of RFX1, and RFX1 silencing nullified the CCN3 induction by impaired glycolysis. Subsequent experiments with an anti-CCN3 antibody indicated that CCN3 supported the survival of chondrocytes under impaired glycolysis. Consistent with these findings in vitro, abundant CCN3 production by chondrocytes in the deep zones of developing epiphysial cartilage, which are located far away from the synovial fluid, was confirmed in vivo. Our present study uncovered that RFX1 is the mediator that enables CCN3 induction upon cellular starvation, which may eventually assist chondrocytes in retaining their viability, even when there is an energy supply shortage.

Effect of Angiotensin II on Chondrocyte Degeneration and Protection via Differential Usage of Angiotensin II Receptors.

The renin-angiotensin system (RAS) controls not only systemic functions, such as blood pressure, but also local tissue-specific events. Previous studies have shown that angiotensin II receptor type 1 (AT1R) and type 2 (AT2R), two RAS components, are expressed in chondrocytes. However, the angiotensin II (ANG II) effects exerted through these receptors on chondrocyte metabolism are not fully understood. In this study, we investigated the effects of ANG II and AT1R blockade on chondrocyte proliferation and differentiation. Firstly, we observed that ANG II significantly suppressed cell proliferation and glycosaminoglycan content in rat chondrocytic RCS cells. Additionally, ANG II decreased CCN2, which is an anabolic factor for chondrocytes, via increased MMP9. In Agtr1a-deficient RCS cells generated by the CRISPR-Cas9 system, Ccn2 and Aggrecan (Acan) expression increased. Losartan, an AT1R antagonist, blocked the ANG II-induced decrease in CCN2 production and Acan expression in RCS cells. These findings suggest that AT1R blockade reduces ANG II-induced chondrocyte degeneration. Interestingly, AT1R-positive cells, which were localized on the surface of the articular cartilage of 7-month-old mice expanded throughout the articular cartilage with aging. These findings suggest that ANG II regulates age-related cartilage degeneration through the ANG II-AT1R axis.

Suppression of adipocyte differentiation by low-intensity pulsed ultrasound via inhibition of insulin signaling and promotion of CCN family protein 2.

Adipocyte differentiation is regulated by several transcription factors such as the CCAAT/enhancer-binding proteins (C/EBPs) and peroxisome proliferator-activated receptor-γ (PPARγ). Here, we demonstrate that low-intensity pulsed ultrasound (LIPUS) suppressed differentiation into mature adipocytes via multiple signaling pathways. When C3H10T1/2, a mesenchymal stem cell line, was treated with LIPUS (3.0 MHz, 60 mW/cm2 ) for 20 minutes once a day for 4 days during adipogenesis, and both the number of lipid droplets and the gene expression of PPARγ and C/EBPα were significantly decreased. Furthermore, LIPUS treatment decreased the phosphorylation of the insulin receptor and also that of Akt and ERK1/2, which are located downstream of this receptor. Next, we showed that LIPUS suppressed the gene expression of angiotensinogen (AGT), which is an adipokine produced by mature adipocytes, as well as that of angiotensin-converting enzyme 1 (ACE1) and angiotensin receptor type 1 (AT1 R) during adipogenesis of pre-adipogenic 3T3-L1 cells. Next, the translocation of Yes-associated protein (YAP) into the nucleus of 3T3-L1 cells was promoted by LIPUS, leading to upregulation of CCN family protein 2 (CCN2), a cellular communication network factor. Moreover, forced expression of CCN2 in 3T3-L1 cells decreased PPARγ gene expression, but it did not increase alkaline phosphatase and osterix gene expression. Finally, gene silencing of CCN2 in C3H10T1/2 cells diminished the effect of LIPUS on the gene expression of PPARγ and C/EBPα. These findings suggest that LIPUS suppressed adipogenesis through inhibition of insulin signaling and decreased PPARγ expression via increased CCN2 production, resulting in a possible decrease of mature adipocytes.

Endogenous endophthalmitis caused by group B streptococcus; case reports and review of 35 reported cases.

BACKGROUND: Group B streptococcus (GBS), a gram-positive coccus that occasionally causes neonatal sepsis or invasive infection in the elderly, has been considered a rare cause of endogenous bacterial endophthalmitis (EBE). However, the number of invasive GBS infections is increasing, particularly in elderly patients with underlying conditions such as diabetes mellitus (DM), cardiovascular disease and cancer. We report 6 cases of EBE caused by GBS and review the literature. METHODS: Retrospective case series and literature review. RESULTS: In the current case series, 6 eyes of 6 patients developed EBE caused by GBS. The average age was 73.5 years. The focus of infection included the urinary tract, cellulitis, arthritis, peritonitis, catheter-associated infection and endocarditis. Four patients had DM. While all 6 strains were sensitive to ß-lactams (penicillins and cephems), 4 strains were resistant to levofloxacin (no data for 1 isolate). Each case was treated with the systemic antibiotic to which the individual strain was sensitive. All cases showed poor visual acuity at presentation (decimal visual acuity: less than 0.03). Vitrectomy with intravitreal antibiotics injection was performed in 4 cases. Visual acuity recovered in 4 cases and did not recover in 2 cases, even after vitrectomy. The literature review of 53 eyes of 41 patients revealed that 60% of eyes finally lost all vision, and death occurred in 2 cases. Initial visual acuity of less than counting fingers was associated with a final outcome of lost vision. Of 41 patients, 13 (32%) had DM as an underlying medical condition. The most common extra-ocular infection focus was endocarditis (37%). CONCLUSIONS: DM is common in patients with EBE caused by GBS. While the 4 cases in the current report had a relatively good visual acuity outcome, despite poor initial visual acuity, the literature review indicated that EBE caused by GBS is generally a severe condition with a poor prognosis. The current study also indicates the importance of considering the possibility of endocarditis on encountering EBE caused by GBS.

Retrotransposons Manipulating Mammalian Skeletal Development in Chondrocytes.

Retrotransposons are genetic elements that copy and paste themselves in the host genome through transcription, reverse-transcription, and integration processes. Along with their proliferation in the genome, retrotransposons inevitably modify host genes around the integration sites, and occasionally create novel genes. Even now, a number of retrotransposons are still actively editing our genomes. As such, their profound role in the evolution of mammalian genomes is obvious; thus, their contribution to mammalian skeletal evolution and development is also unquestionable. In mammals, most of the skeletal parts are formed and grown through a process entitled endochondral ossification, in which chondrocytes play central roles. In this review, current knowledge on the evolutional, physiological, and pathological roles of retrotransposons in mammalian chondrocyte differentiation and cartilage development is summarized. The possible biological impact of these mobile genetic elements in the future is also discussed.

Roles of Interaction between CCN2 and Rab14 in Aggrecan Production by Chondrocytes.

To identify proteins that cooperate with cellular communication network factor 2 (CCN2), we carried out GAL4-based yeast two-hybrid screening using a cDNA library derived from the chondrocytic cell line HCS-2/8. Rab14 GTPase (Rab14) polypeptide was selected as a CCN2-interactive protein. The interaction between CCN2 and Rab14 in HCS-2/8 cells was confirmed using the in situ proximity ligation assay. We also found that CCN2 interacted with Rab14 through its IGFBP-like domain among the four domains in CCN2 protein. To detect the colocalization between CCN2 and Rab14 in the cells in detail, CCN2, wild-type Rab14 (Rab14WT), a constitutive active form (Rab14CA), and a dominant negative form (Rab14DN) of Rab14 were overexpressed in monkey kidney-tissue derived COS7 cells. Ectopically overexpressed Rab14 showed a diffuse cytosolic distribution in COS7 cells; however, when Rab14WT was overexpressed with CCN2, the Rab14WT distribution changed to dots that were evenly distributed within the cytosol, and both Rab14 and CCN2 showed clear colocalization. When Rab14CA was overexpressed with CCN2, Rab14CA and CCN2 also showed good localization as dots, but their distribution was more widespread within cytosol. The coexpression of Rab14DN and CCN2 also showed a dotted codistribution but was more concentrated in the perinuclear area. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed that the reduction in RAB14 or CCN2 mRNA by their respective siRNA significantly enhanced the expression of ER stress markers, BIP and CHOP mRNA in HCS-2/8 chondrocytic cells, suggesting that ER and Golgi stress were induced by the inhibition of membrane vesicle transfer via the suppression of CCN2 or Rab14. Moreover, to study the effect of the interaction between CCN2 and its interactive protein Rab14 on proteoglycan synthesis, we overexpressed Rab14WT or Rab14CA or Rab14DN in HCS-2/8 cells and found that the overexpression of Rab14DN decreased the extracellular proteoglycan accumulation more than the overexpression of Rab14WT/CA did in the chondrocytic cells. These results suggest that intracellular CCN2 is associated with Rab14 on proteoglycan-containing vesicles during their transport from the Golgi apparatus to endosomes in chondrocytes and that this association may play a role in proteoglycan secretion by chondrocytes.

Physiological role of urothelial cancer-associated one long noncoding RNA in human skeletogenic cell differentiation.

A vast number of long-noncoding RNAs (lncRNA) are found expressed in human cells, which RNAs have been developed along with human evolution. However, the physiological functions of these lncRNAs remain mostly unknown. In the present study, we for the first time uncovered the fact that one of such lncRNAs plays a significant role in the differentiation of chondrocytes and, possibly, of osteoblasts differentiated from mesenchymal stem cells, which cells eventually construct the human skeleton. The urothelial cancer-associated 1 (UCA1) lncRNA is known to be associated with several human malignancies. Firstly, we confirmed that UCA1 was expressed in normal human chondrocytes, as well as in a human chondrocytic cell line; whereas it was not detected in human bone marrow mesenchymal stem cells (hBMSCs). Of note, although UCA1 expression was undetectable in hBMSCs, it was markedly induced along with the differentiation toward chondrocytes, suggesting its critical role in chondrogenesis. Consistent with this finding, silencing of the UCA1 gene significantly repressed the expression of chondrogenic genes in human chondrocytic cells. UCA1 gene silencing and hyper-expression also had a significant impact on the osteoblastic phenotype in a human cell line. Finally, forced expression of UCA1 in a murine chondrocyte precursor, which did not possess a UCA1 gene, overdrove its differentiation into chondrocytes. These results indicate a physiological and important role of this lncRNA in the skeletal development of humans, who require more sustained endochondral ossification and osteogenesis than do smaller vertebrates.

Analysis of Ca2+ response of osteocyte network by three-dimensional time-lapse imaging in living bone.

Osteocytes form a three-dimensional (3D) cellular network within the mineralized bone matrix. The cellular network has important roles in mechanosensation and mechanotransduction related to bone homeostasis. We visualized the embedded osteocyte network in chick calvariae and observed the flow-induced Ca2+ signaling in osteocytes using 3D time-lapse imaging. In response to the flow, intracellular Ca2+ ([Ca2+]i) significantly increased in developmentally mature osteocytes in comparison with young osteocytes in the bone matrix. To investigate the differences in response between young and developmentally mature osteocytes in detail, we evaluated the expression of osteocyte-related genes using the osteocyte-like cell line MLO-Y4, which was 3D-cultured within type I collagen gels. We found that the c-Fos, Cx43, Panx3, Col1a1, and OCN mRNA levels significantly increased on day 15 in comparison with day 7. These findings indicate that developmentally mature osteocytes are more responsive to mechanical stress than young osteocytes and have important functions in bone formation and remodeling.

Isolates and antibiotic susceptibilities of endogenous bacterial endophthalmitis: A retrospective multicenter study in Japan.

Endogenous bacterial endophthalmitis, also called metastatic endophthalmitis, is a rare bacterial endophthalmitis derived from distant infectious foci via the bloodstream. This infection can potentially cause not only severe visual disturbance, but also loss of the eyeball or death, as most patients are immunocompromised. This retrospective Japanese multicenter study analyzed 32 eyes in 25 definitive cases. Twelve patients (48.0%) had diabetes mellitus. Typical ocular findings were vitreous haze (87.5%), cells in the anterior chambers (62.5%) and retinal infiltrates (50.0%). Elevated body temperature (64.0%), high serum C-reactive protein (96.0%) and leukocytosis (52.0%) were also frequently observed. Culture positivity rates for intraocular fluid were higher in the vitreous (62.5%) versus aqueous humor (28.6%). High positivity rates were also observed for blood (57.1%) and central venous catheters (100%). The most common pathogen was Staphylococcus aureus (10 cases), including methicillin-resistant S. aureus (4 cases). The next most common pathogen was Klebsiella pneumoniae (7 cases), which was highly associated with liver abscess. Compared to a previous 1991 national multicenter study, there has been a fourfold increase in the ratio of S. aureus. Antibiotic susceptibility tests revealed that all Gram-positives were susceptible to vancomycin and all Gram-negatives were susceptible to third-generation cephalosporins, imipenem/cilastatin, gentamycin and levofloxacin. Prognostic factors influencing poor visual outcome included poor initial visual acuity (p < 0.01), K. pneumoniae (p = 0.027) and gram-negative bacteria (p = 0.014) as the causative bacteria. Intravitreal antibiotic injection in combination with vancomycin and ceftazidime may be applicable for use as part of the standard treatment regimen for EBE.

Characterization of the cryptic plasmid pOfk55 from Legionella pneumophila and construction of a pOfk55-derived shuttle vector.

In this study, a cryptic plasmid pOfk55 from Legionella pneumophila was isolated and characterized. pOfk55 comprised 2584bp with a GC content of 37.3% and contained three putative open reading frames (ORFs). orf1 encoded a protein of 195 amino acids and the putative protein shared 39% sequence identity with a putative plasmid replication protein RepL. ORF1 was needed for replication in L. pneumophila but pOfk55 did not replicate in Escherichia coli. orf2 and orf3 encoded putative hypothetical proteins of 114 amino acids and 78 amino acids, respectively, but the functions of the putative proteins ORF2 and OFR3 are not clear. The transfer mechanism for pOfk55 was independent on the type IVB secretion system in the original host. A L. pneumophila-E. coli shuttle vector, pNT562 (5058bp, KmR), was constructed by In-Fusion Cloning of pOfk55 with a kanamycin-resistance gene from pUTmini-Tn5Km and the origin of replication from pBluescript SK(+) (pNT561). Multiple cloning sites from pBluescript SK(+) as well as the tac promoter region and lacI gene from pAM239-GFP were inserted into pNT561 to construct pNT562. The transformation efficiency of pNT562 in L. pneumophila strains ranged from 1.6×101 to 1.0×105CFU/ng. The relative number of pNT562 was estimated at 5.7±1.0 copies and 73.6% of cells maintained the plasmid after 1week in liquid culture without kanamycin. A green fluorescent protein (GFP) expression vector, pNT563, was constructed by ligating pNT562 with the gfpmut3 gene from pAM239-GFP. pNT563 was introduced into L. pneumophila Lp02 and E. coli DH5α, and both strains expressed GFP successfully. These results suggest that the shuttle vector is useful for genetic studies in L. pneumophila.

Novel role of CCN3 that maintains the differentiated phenotype of articular cartilage.

Knowledge of the microenvironment of articular cartilage in health and disease is the key to accomplishing fundamental disease-modifying treatments for osteoarthritis. The proteins comprising the CCN Family are matricellular proteins with a remarkable relevance within the context of cartilage metabolism. CCN2 displays a great capability for regenerating articular cartilage, and CCN3 has been shown to activate the expression of genes related to articular chondrocytes and to repress genes related to endochondral ossification in epiphyseal chondrocytes. Moreover, mice lacking CCN3 protein have been shown to display ostearthritic changes in their knee articular cartilage. In this study, we employed a monoiodoacetic acid (MIA)-induced osteoarthritic model to investigate whether osteoarthritic changes in the cartilage are reciprocally accompanied by CCN3 down-regulation and an inducible overexpression system to evaluate the effects of CCN3 on articular chondrocytes in vitro. Finally, we also investigated the effects of exogenous CCN3 in vivo during the early stages of MIA-induced osteoarthritis. We discovered that CCN3 is expressed by articular chondrocytes in normal rat knees, whereas it is rapidly down-regulated in osteoarthritic knees. In vitro, we also discovered that CCN3 increases the proteoglycan accumulation, the gene expression of type II collagen, tenascin-C and lubricin, as well as the protein production of tenascin-C and lubricin in articular chondrocytes. In vivo, it was discovered that exogenous CCN3 increased tidemark integrity and produced an increased production of lubricin protein. The potential utility of CCN3 as a future therapeutic agent and possible strategies to improve its therapeutic functions are also discussed.

Roussoella solani causing keratomycosis, with an observed both sexual and asexual morphs.

We describe an 82-year-old male farmer who had diabetes mellitus with no history of ocular trauma by soil or plants and who developed a corneal infection due to a fungus. The organism was identified as Roussoella solani based on both the morphological characteristics and phylogenetic analysis using LSU and ITS nrDNA sequences. The sexual stage of R. solani is described and illustrated for the first time. The patient was treated successfully with a combination of topical and systemic voriconazole and micafungin. This case is the first report of keratomycosis caused by R. solani.