A comparative phenotypic and genomic analysis of methicillin-resistant Staphylococcus aureus ST45 isolates from cellulitis and from osteomyelitis in Taiwan.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) sequence type (ST) 45 is a globally disseminated MRSA lineage. Herein, we investigated whether MRSA ST45 isolates from cellulitis and from osteomyelitis display distinctive phenotypic and genomic characteristics. METHODS: A total of 15 MRSA ST45 isolates from cellulitis (CL-MRSAs; n = 6) or osteomyelitis (OM-MRSAs; n = 9) were collected in a Taiwan hospital. These MRSA ST45 isolates were characterized for their antimicrobial susceptibility, biofilm-forming ability, cellular infectivity in vitro, and pathogenicity in vivo. Four CL-MRSA and six OM-MRSA ST45 isolates were selected for whole-genome sequencing (WGS). RESULTS: Antibiotic resistance tests showed that all OM-MRSA ST45 strains, but not CL-MRSA ST45 strains, were resistant to ciprofloxacin, levofloxacin, gentamicin and doxycycline. Compared to the CL-MRSA ST45 isolates, the OM-MRSA ST45 isolates had stronger biofilm-forming ability and cellular infectivity, and caused more severe disease in mice. WGS analysis revealed that these OM-MRSA ST45 isolates carry multiple common mutations or polymorphisms in genes associated with antibiotic resistance and virulence. Moreover, the transposable elements IS256 and IS257R2 were found only in the OM-MRSA ST45 isolates. CONCLUSIONS: The emergence and spread of the highly pathogenic and multidrug-resistant ST45 MRSAs identified from osteomyelitis may pose a serious threat on public health.

Assessment of the (Pro)renin Receptor Protein Expression in Organs.

The (pro)renin receptor ((P)RR) is an essential component of the renin-angiotensin system (RAS) as a specific single-pass transmembrane receptor for prorenin and renin and has now emerged as a multifunctional protein implicated in a wide variety of developmental and physio-pathological processes and pathways. The (P)RR may be of pathological significance in metabolic syndrome. The (P)RR has received much consideration; substantial efforts have been made to understand the localization, regulation, and function of the (P)RR at both a molecular and system level. (P)RR regulation of cell function depends on whether it is intact or cleaved into its constituent forms. Therefore, the present chapter describes immunohistochemical approaches to examine the expression of (P)RR in various organs. It was shown that different molecular forms of (P)RR could be present in different tissue compartments in almost all organs. Among them, the liver has high PRR activity. Our findings could elucidate more detailed distribution of different (P)RR molecular forms in different organs, which could provide useful information to further investigate the pathophysiological mechanisms of the development of various diseases in the future.

Dickkopf-1 Acts as a Profibrotic Mediator in Progressive Chronic Kidney Disease.

Chronic kidney disease (CKD) is a serious public health problem. Due to a high variability in the speed of CKD progression to end-stage renal disease (ESRD) and the critical involvement of Wnt/ß-catenin signaling in CKD, we investigated the role of the Wnt antagonist Dickkopf-1 (DKK1) in CKD progression. Our data revealed that patients with CKD stages 4-5 had higher DKK1 levels in their serum and renal tissues than the control subjects. In an 8-year follow-up, the serum DKK1-high group in the enrolled CKD patients showed a faster progression to ESRD than the serum DKK1-low group. Using a rat model of 5/6 nephrectomy (Nx)-induced CKD, we consistently detected elevated serum levels and renal production of DKK1 in 5/6 Nx rats compared to sham-operated rats. Importantly, the knockdown of the DKK1 levels in the 5/6 Nx rats markedly attenuated the CKD-associated phenotypes. Mechanistically, we demonstrated that the treatment of mouse mesangial cells with recombinant DKK1 protein induced not only the production of multiple fibrogenic proteins, but also the expression of endogenous DKK1. Collectively, our findings suggest that DKK1 acts as a profibrotic mediator in CKD, and elevated levels of serum DKK1 may be an independent predictor of faster disease progression to ESRD in patients with advanced CKD.

The difference in pathogenic bacteria between chronic rhinosinusitis in patients with and without Sjogren's syndrome: a retrospective case-control study.

BACKGROUND: Chronic rhinosinusitis (CRS) affects the quality of life of many people worldwide and can cause comorbidities. Our previous research proved that Sjogren's syndrome (SS) is a predisposing factor for CRS, with a 2.5-fold associated risk. Antibiotics are important in CRS treatment; however, there is a paucity of research on the pathogenic bacteria of SS-CRS in the past. We conducted this study to investigate the pathogenic difference of SS-CRS and non-SS-CRS and aimed to give clinicians references when selecting antibiotics to treat SS-CRS. MATERIALS AND METHODS: A total of 14,678 patients hospitalized for CRS operation from 2004 to 2018 were identified from the Chang Gung Research Database. These CRS cases were classified as either SS-CRS or non-SS-CRS. We analyzed their bacterial distribution by studying the results of the pus cultures performed alongside surgery. RESULTS: The top three facultative anaerobic or aerobic isolated bacteria in the SS-CRS group were coagulase-negative Staphylococcus (CoNS: 34.3%), Pseudomonas aeruginosa (28.6%), methicillin-sensitive Staphylococcus aureus (MSSA: 20%), and Staphylococcus epidermidis (20%). In the non-SS-CRS group, S. epidermidis (29.3%), CoNS (25.7%), and MSSA (14.2%) were identified. The top three anaerobic bacterial genera were Cutibacterium (54.3%), Peptostreptococcus (11.4%), and Fusobacterium (11.4%) in the SS-CRS group and Cutibacterium (53.8%), Peptostreptococcus (25%), and Prevotella (12.9%) in the non-SS-CRS group. CONCLUSIONS: P. aeruginosa is a major pathogen in SS-CRS patients. In addition, physicians should be aware of potential Fusobacterium and antibiotic-resistant bacterial infection in patients with SS-CRS.

Kaposi's Sarcoma-Associated Herpesvirus ORF50 Protein Represses Cellular MDM2 Expression via Suppressing the Sp1- and p53-Mediated Transactivation.

The Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded ORF50 protein is a potent transcriptional activator essential for triggering KSHV lytic reactivation. Despite extensive studies, little is known about whether ORF50 possesses the ability to repress gene expression or has an antagonistic action to cellular transcription factors. Previously, we demonstrated that human oncoprotein MDM2 can promote the degradation of ORF50 protein. Herein, we show that abundant ORF50 expression in cells can conversely downregulate MDM2 expression via repressing both the upstream (P1) and internal (P2) promoters of the MDM2 gene. Deletion analysis of the MDM2 P1 promoter revealed that there were two ORF50-dependent negative response elements located from -102 to -63 and from -39 to +1, which contain Sp1-binding sites. For the MDM2 P2 promoter, the ORF50-dependent negative response element was identified in the region from -110 to -25, which is coincident with the location of two known p53-binding sites. Importantly, we further demonstrated that overexpression of Sp1 or p53 in cells indeed upregulated MDM2 expression; however, coexpression with ORF50 protein remarkably reduced the Sp1- or p53-mediated MDM2 upregulation. Collectively, our findings propose a reciprocal negative regulation between ORF50 and MDM2 and uncover that ORF50 decreases MDM2 expression through repressing Sp1- and p53-mediated transactivation.

Sp3 Transcription Factor Cooperates with the Kaposi's Sarcoma-Associated Herpesvirus ORF50 Protein To Synergistically Activate Specific Viral and Cellular Gene Promoters.

The Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded open reading frame 50 (ORF50) protein is the key transactivator responsible for the latent-to-lytic switch. Here, we investigated the transcriptional activation of the ORF56 gene (encoding a primase protein) by ORF50 and successfully identified an ORF50-responsive element located in the promoter region between positions -97 and -44 (designated 56p-RE). This 56p-RE element contains a noncanonical RBP-Jκ-binding sequence and a nonconsensus Sp1/Sp3-binding sequence. Electrophoretic mobility shift assays revealed that RBP-Jκ, Sp3, and ORF50 could form stable complexes on the 56p-RE element. Importantly, transient-reporter analysis showed that Sp3, but not RBP-Jκ or Sp1, acts in synergy with ORF50 to activate the 56p-RE-containing reporter construct, and the synergy mainly depends on the Sp1/Sp3-binding region of the 56p-RE element. Sequence similarity searches revealed that the promoters for ORF21 (thymidine kinase), ORF60 (ribonucleotide reductase, small subunit), and cellular interleukin-10 (IL-10) contain a sequence motif similar to the Sp1/Sp3-binding region of the 56p-RE element, and we found that these promoters could also be synergistically activated by ORF50 and Sp3 via the conserved motifs. Noteworthily, the conversion of the Sp1/Sp3-binding sequence of the 56p-RE element into a consensus high-affinity Sp-binding sequence completely lost the synergistic response to ORF50 and Sp3. Moreover, transcriptional synergy could not be detected through other ORF50-responsive elements from the viral PAN, K12, ORF57, and K6 promoters. Collectively, the results of our study demonstrate that ORF50 and Sp3 can act in synergy on the transcription of specific gene promoters, and we find a novel conserved cis-acting motif in these promoters essential for transcriptional synergy.IMPORTANCE Despite the critical role of ORF50 in the KSHV latent-to-lytic switch, the molecular mechanism by which ORF50 activates its downstream target genes, especially those that encode the viral DNA replication enzymes, is not yet fully understood. Here, we find that ORF50 can cooperate with Sp3 to synergistically activate promoters of the viral ORF56 (primase), ORF21 (thymidine kinase), and ORF60 (ribonucleotide reductase) genes via similar Sp1/Sp3-binding motifs. Additionally, the same synergistic effect can be seen on the promoter of the cellular IL-10 gene. Overall, our data reveal an important role for Sp3 in ORF50-mediated transactivation, and we propose a new subclass of ORF50-responsive elements in viral and cellular promoters.

Patients with comorbid rheumatoid arthritis are predisposed to peritonsillar abscess: real-world evidence.

PURPOSE: The peritonsillar abscess (PTA)-rheumatoid arthritis (RA) association remains unclear. Here, the effects of RA on PTA incidence and prognosis are elucidated. METHODS: We compared PTA incidence and prognosis of 30,706 RFCIP-registered patients with RA (RA cohort) with matched individuals without RA from another database of 1 million randomly selected people representing Taiwan's population (non-RA cohort). RESULTS: The RA cohort had significantly higher PTA incidence [incidence rate ratio (IRR) (95% CI) 1.73 (1.10-2.71), P = 0.017) and cumulative incidence (P = 0.016, Kaplan-Meier curves). Cox regression analyses demonstrated RA cohort to have an estimated 1.72-fold increased PTA risk (95% CI 1.09-2.69, P = 0.019). PTA was more likely within the first 5 years of RA diagnosis (for < 1, 1-5, and ≥ 5 postdiagnosis years, IRRs: 2.67, 2.31, and 1.10, respectively, and P = 0.063, 0.021, and 0.794, respectively; average onset duration: 4.3 ± 3.3 years after RA diagnosis). PTA increased length of hospital stay significantly and risk of complication with deep neck infection nonsignificantly [6.5 ± 4.5 vs 4.6 ± 2.8 days (P = 0.045) and 18.52% vs 7.81% (P = 0.155), respectively]. Moreover, RA-cohort patients not receiving RA therapy exhibited 5.06-fold higher PTA risk than those receiving RA-related therapy (95% CI 1.75-14.62, P = 0.003). CONCLUSIONS: In patients with RA, PTA incidence is the highest within 5 years of RA diagnosis, and RA therapy is essential for reducing PTA risk. LEVEL OF EVIDENCE: 4.

The Epstein-Barr Virus Lytic Protein BMLF1 Induces Upregulation of GRP78 Expression through ATF6 Activation.

The unfolded protein response (UPR) is an intracellular signaling pathway essential for alleviating the endoplasmic reticulum (ER) stress. To support the productive infection, many viruses are known to use different strategies to manipulate the UPR signaling network. However, it remains largely unclear whether the UPR signaling pathways are modulated in the lytic cycle of Epstein-Barr virus (EBV), a widely distributed human pathogen. Herein, we show that the expression of GRP78, a central UPR regulator, is up-regulated during the EBV lytic cycle. Our data further revealed that knockdown of GRP78 in EBV-infected cell lines did not substantially affect lytic gene expression; however, GRP78 knockdown in these cells markedly reduced the production of virus particles. Importantly, we identified that the early lytic protein BMLF1 is the key regulator critically contributing to the activation of the grp78 gene promoter. Mechanistically, we found that BMLF1 can trigger the proteolytic cleavage and activation of the UPR senor ATF6, which then transcriptionally activates the grp78 promoter through the ER stress response elements. Our findings therefore provide evidence for the connection between the EBV lytic cycle and the UPR, and implicate that the BMLF1-mediated ATF6 activation may play critical roles in EBV lytic replication.

Biodegradable Stent with mTOR Inhibitor-Eluting Reduces Progression of Ureteral Stricture.

In this study, we investigated the effect of mTOR inhibitor (mTORi) drug-eluting biodegradable stent (DE stent), a putative restenosis-inhibiting device for coronary artery, on thermal-injury-related ureteral stricture in rabbits. In vitro evaluation confirmed the dose-dependent effect of mTORi, i.e., rapamycin, on fibrotic markers in ureteral component cell lines. Upper ureteral fibrosis was induced by ureteral thermal injury in open surgery, which was followed by insertion of biodegradable stents, with or without rapamycin drug-eluting. Immunohistochemistry and Western blotting were performed 4 weeks after the operation to determine gross anatomy changes, collagen deposition, expression of epithelial-mesenchymal transition markers, including Smad, α-SMA, and SNAI 1. Ureteral thermal injury resulted in severe ipsilateral hydronephrosis. The levels of type III collagen, Smad, α-SMA, and SNAI 1 were increased 28 days after ureteral thermal injury. Treatment with mTORi-eluting biodegradable stents significantly attenuated thermal injury-induced urinary tract obstruction and reduced the level of fibrosis proteins, i.e., type III collagen. TGF-ß and EMT signaling pathway markers, Smad and SNAI 1, were significantly modified in DE stent-treated thermal-injury-related ureteral stricture rabbits. These results suggested that intra-ureteral administration of rapamycin by DE stent provides modification of fibrosis signaling pathway, and inhibiting mTOR may result in fibrotic process change.

GMI, an Immunomodulatory Peptide from Ganoderma microsporum, Restrains Periprosthetic Joint Infections via Modulating the Functions of Myeloid-Derived Suppressor Cells and Effector T Cells.

Periprosthetic joint infections (PJIs) caused by Staphylococcus aureus infection are difficult to treat due to antibiotic resistance. It is known that the biofilms from methicillin-resistant S. aureus (MRSA) promote expansion of myeloid-derived suppressor cells (MDSCs) to suppress T-cell proliferation and benefit bacterial infections. This study finds that GMI, a fungal immunomodulatory peptide isolated from Ganoderma microsporum, suppresses MDSC expansion to promote the proliferation of cytotoxic T cells. The enhancement is likely attributed to increased expression of IL-6 and TNF-α and reduction in ROS expression. Similar beneficial effects of GMI on the suppression of MDSC expansion and IL-6 expression are also observed in the whole blood and reduces the accumulation of MDSCs in the infected bone region in a mouse PJI infection model. This study shows that GMI is potentially useful for treating S. aureus-induced PJIs.

Beneficial Effects of Inflammatory Cytokine-Targeting Aptamers in an Animal Model of Chronic Prostatitis.

Non-bacterial prostatitis is an inflammatory disease that is difficult to treat. Oligonucleotide aptamers are well known for their stability and flexibility in conjugating various inflammatory molecules. In this study, we investigated the effects of inflammatory cytokine-targeting aptamers (ICTA), putative neutralizers of TNF-alpha and IL-1 beta activation, on local carrageenan-induced prostate inflammation, allodynia, and hyperalgesia in rats. In vitro evaluation confirmed the binding capability of ICTA. Intraprostatic injection of carrageenan or control vehicle was performed in six-week-old rats, and ICTA (150 µg) or vehicle was administered in the prostate along with carrageenan injection. The von Frey filament test was performed to determine mechanical allodynia, and prostate inflammation was examined seven days after drug administration. Local carrageenan administration resulted in a reduction of the tactile threshold. The levels of mononuclear cell infiltration, pro-inflammatory cytokine interleukin-1 beta (b), caspase-1 (casp-1), and Nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain containing proteins 1 and 3 (NALP1 and NALP3) in the prostate of rats were increased seven days after carrageenan injection. Treatment with ICTA significantly attenuated the carrageenan-induced hyperalgesia and reduced the elevated levels of proteins including TNF-a and IL-1b in the rats. Apoptosis markers, B-cell lymphoma 2-associated X protein (Bax) and caspase-3, were elevated in ICTA-treated Chronic pelvic pain syndrome (CPPS) rats. These results suggest that ICTA provides protection against local carrageenan-induced enhanced pain sensitivity, and that the neutralization of proinflammatory cytokines may result in inflammatory cell apoptosis.

Subcellular Localization and Functional Characterization of GII.4 Norovirus-Encoded NTPase.

The genotype II.4 (GII.4) variants of human noroviruses (HuNVs) are recognized as the major agent of global gastroenteritis outbreaks. Due to the lack of an efficient cell culture system for HuNV propagation, the exact roles of HuNV-encoded nonstructural proteins (including Nterm, NTPase, P22, VPg, Pro, and RdRp) in viral replication or pathogenesis have not yet been fully understood. Here, we report the molecular characterization of the GII.4 HuNV-encoded NTPase (designated GII-NTPase). Results from our studies showed that GII-NTPase forms vesicular or nonvesicular textures in the cell cytoplasm, and the nonvesicular fraction of GII-NTPase significantly localizes to the endoplasmic reticulum (ER) or mitochondria. Deletion analysis revealed that the N-terminal 179-amino-acid (aa) region of GII-NTPase is required for vesicle formation and for ER colocalization, whereas the C-terminal region is involved in mitochondrial colocalization. In particular, two mitochondrion-targeting domains were identified in the C-terminal region of GII-NTPase which perfectly colocalized with mitochondria when the N-terminal region of GII-NTPase was deleted. However, the corresponding C-terminal portions of NTPase derived from the GI HuNV did not show mitochondrial colocalization. We also found that GII-NTPase physically interacts with itself as well as with Nterm and P22, but not VPg, Pro, and RdRp, in cells. The Nterm- and P22-interacting region was mapped to the N-terminal 179-aa region of GII-NTPase, whereas the self-assembly of GII-NTPase could be achieved via a head-to-head, tail-to-tail, or head-to-tail configuration. More importantly, we demonstrate that GII-NTPase possesses a proapoptotic activity, which can be further enhanced by coexpression with Nterm or P22.IMPORTANCE Despite the importance of human norovirus GII.4 variants in global gastroenteritis outbreaks, the basic biological functions of the viral nonstructural proteins in cells remain rarely investigated. In this report, we focus our studies on characteristics of the GII.4 norovirus-encoded NTPase (GII-NTPase). We unexpectedly find that GII-NTPase can perfectly colocalize with mitochondria after its N-terminal region is deleted. However, such a phenomenon is not observed for NTPase encoded by a GI strain. We further reveal that the N-terminal 179-aa region of GII-NTPase is sufficient to mediate (i) vesicle formation, (ii) ER colocalization, (iii) the interaction with two other nonstructural proteins, including Nterm and P22, (iv) the formation of homodimers or homo-oligomers, and (v) the induction of cell apoptosis. Taken together, our findings emphasize that the virus-encoded NTPase must have multiple activities during viral replication or pathogenesis; however, these activities may vary somewhat among different genogroups.

Particulate matter exposure aggravates osteoarthritis severity.

Several diseases have been linked to particulate matter (PM) exposure. Outdoor activities, such as road running or jogging, are popular aerobic exercises due to few participatory limitations. Osteoarthritis (OA) is a progressive degenerative joint disease, usually observed at age 40, and not noticed before pain or diagnosis. Although exercise has health benefits, it is unclear whether outdoor jogging in higher PM (standard reference material 1649b, SRM 1649b) concentration environments could affect OA development or severity. Hence, a PM exposure monosodium iodoacetate (MIA)-induced OA animal jogged model was established for investigation. Results showed that high doses of PM (5 mg) significantly increased pro-inflammatory factors such as tumor necrosis factor α (TNF-α), interleukin (IL)-1ß, and IL-6, and M1 macrophages in the lung region, also obtained in systemic IL-6 and TNF-α expressions in this MIA-OA rat model. Moreover, levels of osteocalcin, cartilage oligomeric matrix protein (COMP), and N-telopeptides of type I collagen were especially influenced in MIA+PM groups. Morphological and structural changes of the knee joint were detected by micro-computed tomography images (micro-CT) and immunohistochemistry. MIA + PM rats exhibited severe bone density decrease, cartilage wear, and structure damages, accompanied by lower levels of physical activity, than the sham group and groups receiving MIA or PM alone. The findings suggest that the severity of OA could be promoted by PM exposure with a PM concentration effect via systemic inflammatory mechanisms. To the best of our knowledge, this is the first study to provide direct effects of PM exposure on OA severity.

Effects of the NEDD8-Activating Enzyme Inhibitor MLN4924 on Lytic Reactivation of Kaposi's Sarcoma-Associated Herpesvirus.

The switch of Kaposi's sarcoma-associated herpesvirus (KSHV) from latency to lytic replication is a key event for viral dissemination and pathogenesis. MLN4924, a novel neddylation inhibitor, reportedly causes the onset of KSHV reactivation but impairs later phases of the viral lytic program in infected cells. Thus far, the molecular mechanism involved in the modulation of the KSHV lytic cycle by MLN4924 is not yet fully understood. Here, we confirmed that treatment of different KSHV-infected primary effusion lymphoma (PEL) cell lines with MLN4924 substantially induces viral lytic protein expression. Due to the key role of the virally encoded ORF50 protein in the latent-to-lytic switch, we investigated its transcriptional regulation by MLN4924. We found that MLN4924 activates the ORF50 promoter (ORF50p) in KSHV-positive cells (but not in KSHV-negative cells), and the RBP-Jκ-binding elements within the promoter are critically required for MLN4924 responsiveness. In KSHV-negative cells, reactivation of the ORF50 promoter by MLN4924 requires the presence of the latency-associated nuclear antigen (LANA). Under such a condition, LANA acts as a repressor to block the ORF50p activity, whereas MLN4924 treatment relieves LANA-mediated repression. Importantly, we showed that LANA is a neddylated protein and can be deneddylated by MLN4924. On the other hand, we revealed that MLN4924 exhibits concentration-dependent biphasic effects on 12-O-tetradecanoylphorbol-13-acetate (TPA)- or sodium butyrate (SB)-induced viral reactivation in PEL cell lines. In other words, low concentrations of MLN4924 promote activation of TPA- or SB-mediated viral reactivation, whereas high concentrations of MLN4924, conversely, inhibit the progression of TPA- or SB-mediated viral lytic program.IMPORTANCE MLN4924 is a neddylation (NEDD8 modification) inhibitor, which currently acts as an anti-cancer drug in clinical trials. Although MLN4924 has been reported to trigger KSHV reactivation, many aspects regarding the action of MLN4924 in regulating the KSHV lytic cycle are not fully understood. Since the KSHV ORF50 protein is the key regulator of viral lytic reactivation, we focus on its transcriptional regulation by MLN4924. We here show that activation of the ORF50 gene by MLN4924 involves the relief of LANA-mediated transcriptional repression. Importantly, we find that LANA is a neddylated protein. To our knowledge, this is the first report showing that neddylation occurs in viral proteins. Additionally, we provide evidence that different concentrations of MLN4924 have opposite effects on TPA-mediated or SB-mediated KSHV lytic cycle activation. Therefore, in clinical application, we propose that MLN4924 needs to be used with caution in combination therapy to treat KSHV-positive subjects.

Regulation of the Abundance of Kaposi's Sarcoma-Associated Herpesvirus ORF50 Protein by Oncoprotein MDM2.

The switch between latency and the lytic cycle of Kaposi's sarcoma-associated herpesvirus (KSHV) is controlled by the expression of virally encoded ORF50 protein. Thus far, the regulatory mechanism underlying the protein stability of ORF50 is unknown. Our earlier studies have demonstrated that a protein abundance regulatory signal (PARS) at the ORF50 C-terminal region modulates its protein abundance. The PARS region consists of PARS-I (aa 490-535) and PARS-II (aa 590-650), and mutations in either component result in abundant expression of ORF50. Here, we show that ORF50 protein is polyubiquitinated and its abundance is controlled through the proteasomal degradation pathway. The PARS-I motif mainly functions as a nuclear localization signal in the control of ORF50 abundance, whereas the PARS-II motif is required for the binding of ubiquitin enzymes in the nucleus. We find that human oncoprotein MDM2, an ubiquitin E3 ligase, is capable of interacting with ORF50 and promoting ORF50 degradation in cells. The interaction domains between both proteins are mapped to the PARS region of ORF50 and the N-terminal 220-aa region of MDM2. Additionally, we identify lysine residues at positions 152 and 154 in the N-terminal domain of ORF50 critically involved in MDM2-mediated downregulation of ORF50 levels. Within KSHV-infected cells, the levels of MDM2 were greatly reduced during viral lytic cycle and genetic knockdown of MDM2 in these cells favored the enhancement of ORF50 expression, supporting that MDM2 is a negative regulator of ORF50 expression. Collectively, the study elucidates the regulatory mechanism of ORF50 stability and implicates that MDM2 may have a significant role in the maintenance of viral latency by lowering basal level of ORF50.

Small P particles formed by the Taiwan-native norovirus P domain overexpressed in Komagataella pastoris.

The protrusion (P) domain of the major structural protein VP1 of norovirus (NoV) is critical for the host's immune response and receptor binding. Most heterologous P domains expressed in Escherichia coli or Komagataella pastoris (formally known as Pichia pastoris) form P particles consisting of 24 P monomers formed through intermolecular contact in the P regions and an end-linked cysteine tag. The small P particle is only found in P domains with terminal modifications. In this study, the NoV P domain of the most predominant NoV strain GII.4 isolated from Taiwan was expressed in K. pastoris. A high yield of NoV P was obtained using the high-cell density fermentation process in K. pastoris. A large amount of the small P particles and the trimer and dimer complexes formed by 12, 6, and 2 P monomers were observed in both the expression of the NoV P-His and P containing cysteine tag at the N-terminus. Dynamic light scattering and transmission electron microscopy analysis of the purified NoV P-His and P revealed that most of these small P particles are triangle-, square-, and ring-shaped with a diameter of 14-15 nm. The binding ability of purified NoV P-His and P to human histo-blood group antigen was confirmed by a saliva-binding assay. Without terminal modification, small P particles were formed in our study. The amino acid sequence analysis showed only four different amino acids (residue 84, 119, 136, and 313) between the P domain in this study and other investigated GII.4 strains suggesting that these amino acids might play an important role in the P particle formation. The small P particles formed by the Taiwan-native norovirus P domain overexpressed in K. pastoris may provide further information for morphogenesis studies and vaccine development.

Glomerular mesangial cell and podocyte injuries in diabetic nephropathy.

Diabetic nephropathy is one of the leading causes of end-stage renal disease and creates heavy healthcare burdens globally. Dysfunction of mesangial cells and podocytes contributes to diabetic nephropathy. Dysregulation of signaling involved in renal development and regeneration may cause diabetic kidney damages. Growing evidences suggest the importance of dysregulated dickkopf-1 (DKK1)/Wnt/ ß-catenin signaling pathways in the pathogenesis of diabetic glomerular injuries. The inhibition of Wnt signaling in injured mesangial cells is likely attributed to the high glucose-induced Ras/Rac1 dependent superoxide formation. When DKK1, the cellular inhibitor of Wnt signaling, binds to the Kremen-2 receptor, depositions of extracellular matrix increase in the mesangium of diabetic kidneys. Additionally, reactivation of Notch-1 signaling has been implicated in podocytopathy during diabetic proteinuria development. Knocking down Notch-1 alleviates vascular endothelial growth factor (VEGF) expression, nephrin repression and proteinuria in diabetic kidneys. It is also found that epigenetic modulations by histone deacetylase 4 (HDAC4) and miR-29a could lead to diabetic nephropathy. High glucose increases the expression of HDAC4, which causes deacetylation with subsequent ubiquitination of nephrin. Overexpression of miR-29a in diabetic transgenic mice would decrease the expression of HDAC4 and stabilize nephrin. Surprisingly, reprogramming or reactivation of signaling involved in renal development or regeneration often brings about diabetic glomerular sclerosis in mesangial cells and podocytes. Better knowledge about modifications of embryonic stem cell signaling will have a chance to implement strategically focused pharmacological research programs aiming to the development of new drugs for diabetic kidney injuries.

A common polymorphism decreases LRP1 mRNA stability and is associated with increased plasma factor VIII levels.

The low-density lipoprotein receptor-related protein 1 (LRP1) gene is associated with increased levels of plasma factor VIII (FVIII). We aimed to explore eight functional genetic LRP1 variants for their potential roles in regulating FVIII levels and acute ischemic stroke (AIS). This genetic association study enrolled 192 patients with AIS and 134 controls. There were no significant differences in the genetic frequency of the eight functional single-nucleotide polymorphisms (SNPs) between the control and AIS groups. However, while analyzing the association between the eight SNPs and plasma FVIII levels, subjects with T/T genotype of rs1800137 (vs. CC+CT) were found to be associated with higher FVIII levels (23.5IU/dL; 95% confidence interval, 7.4-39.5IU/dL; P=0.0044) after adjusting for age, gender, estimated glomerular filtration rate, O blood type, inflammatory state, and body mass index. An analysis of the mRNA stability and abundance was designed and performed using minigene system transfected into HepG2 cells to assess the possible differences in mRNA stabilities between rs1800137 CC (rs1800137C) and TT (rs1800137T) genotypes. Site-directed mutagenesis revealed that rs1800137T accounts for the observed decrease in mRNA stability. The SNP rs1800137, located in exon 8, has been identified as an exon-splicing enhancer in silico. However, alternative splicing of LRP1 without inclusion of exon 8 was not identified. In transfected HepG2 cells, cycloheximide slowed down the degradation of the rs1800137T-containing minigene. These results demonstrate that synonymous SNP rs1800137 can lead to increased plasma FVIII levels due to decreased mRNA stability via translation-dependent mRNA degradation associated with codon optimality.

Transpapillary biliary biopsy for malignant biliary strictures: comparison between cholangiocarcinoma and pancreatic cancer.

BACKGROUND: Tissue sampling for biliary stricture is important for differential diagnosis and further treatment. This study aims to assess the differences of transpapillary biliary biopsy for malignant biliary strictures between cholangiocarcinoma and pancreatic cancer. METHODS: From January 2010 to December 2013, we retrospectively studied 79 patients who suffered from biliary strictures and received transpapillary forceps biopsy after sphincterotomy for tissue sampling. The diagnostic sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of forceps biopsy were calculated in all cases for both cholangiocarcinoma and pancreatic cancer patients. Possible factors that distinguish malignant strictures from benign strictures and which could affect the accuracy of tissue sampling were analyzed. RESULTS: There are 65 malignant and 14 benign biliary stricture patients enrolled. The malignant group has a significantly higher serum bilirubin level than the benign group, but age, clinical presentation, level of serum carcinoembryonic antigen (CEA), carbohydrate antigen (CA) 19-9, and alkaline phosphatase are not. The sensitivity, specificity, PPV, and NPV of forceps biopsy for biliary stricture are 53.85, 100, 100, and 31.82%, respectively. The cholangiocarcinoma group has a higher sensitivity (73.53 versus 29.17%, p < 0.001), older age, lower CA 19-9 level, and more common hepatic duct strictures than the pancreatic group. The age, serum CEA, CA 19-9 and the alkaline phosphatase level, serum bilirubin level >10 mg/dL, tissue sampling ≧3 are not significant factors affecting diagnostic accuracy in forceps biopsy for pancreatobiliary strictures. There is neither major bleeding nor perforation in our study. CONCLUSIONS: Transpapillary forceps biopsy of biliary strictures after sphincterotomy for tissue sampling is safe and a significantly higher sensitive method in cholangiocarcinoma but not in pancreatic cancer.

Treatment of critically sized femoral defects with recombinant BMP-2 delivered by a modified mPEG-PLGA biodegradable thermosensitive hydrogel.

BACKGROUND: Reconstruction of a segmental fracture with massive bone loss is still a challenge for orthopaedic surgeons. The aim of our study was to develop a suitable biodegradable thermosensitive hydrogel system as a carrier for bone morphogenetic protein (BMP)-2 delivery in the treatment of critical-sized femoral defects. METHODS: A block copolymer composed of monomethoxypoly(ethylene glycol) (mPEG), poly(lactic-co-glycolic acid) (PLGA) and 2, 2'-Bis (2-oxazolin) (Box) was synthesized by ring opening polymerization. The synthesized block copolymer was characterized by (1)H-NMR spectroscopy and gel permeation chromatography (GPC). Different biophysical and biochemical properties of the synthesized copolymer, including temperature-induced structure changes, degradation rate, pH changes during hydrolytic degradation, cell toxicity, and the release profile of BMP-2, were also evaluated and/or were compared with those of a well-characterized mPEG-PLGA copolymer. In animal testing, rabbits (n = 36) that received critically sized (10 mm) femoral defects were divided into 6 groups. These experimental groups included an untreated group, autograft, and groups treated with the synthesized copolymer carrying different concentrations of BMP-2 (0, 5, 10, and 20 µg/ml). Bone repair was evaluated using X-ray radiography, histological staining, micro-computed tomography (µCT), biomarker examination and biomechanical testing in a 12-week treatment period. RESULTS: A new thermosensitive mPEG-PLGA/Box/mPEG-PLGA block copolymer, or named as BOX copolymer, was successfully prepared. Compared to the reported mPEG-PLGA in vitro, the prepared BOX copolymer at the same weight percent concentrations exhibited wider temperature ranges of gelation, slower degradation rates, higher the pH values, as well as less cytotoxicity. Furthermore, the BMP-2 release from BOX hydrogel exhibited a near-linear release profile in vitro. In animal experiments, treatment of critical-sized bony defects with 25 wt% BOX hydrogel carrying BMP-2 effectively promoted fracture healing during the 12-week trial period and higher concentrations of BMP-2 treatment correlated with better bone quality. Most importantly, clinical outcome and bone healing in the BOX-hydrogel group with 20 µg/ml BMP-2 were nearly equivalent to those in the autograft group in a 12-week treatment course. CONCLUSION: These data support that the use of BOX hydrogel (25 wt%) as a drug delivery system is a promising method in the treatment of large bone defects.