Dynamic Effects of the Third Generation Bisphosphonate of Risedronate on Rat Osteoporotic Fractures for Clinical Usage Guidance.

OBJECTIVE: To better understand the risks of bisphosphonates in order to develop guidance for appropriate clinical usage, to compared femoral fracture healing at different time points and to explore the effects of Residronate on fracture healing. METHODS: Osteoporosis model was achieved by ovariectomy surgery, followed by surgical incision of left femoral shaft 4 weeks after ovariectomy surgery. Three days after fracture surgery, risedronateor saline was fed by intragastric administration. X ray examination was used to check the callus formation, Bone Mineral Density (BMD), Bone Mineral Content (BMC), biomechanical, imaging and micromorphological of bone tissue as well as the trabecular bone parameters were all examined. The femoral pathology tissue of each rat was used to analyze trabecular bone parameters, including trabecular bone volume/tissue volume (Tb. BV/TV), bone surface to tissue volume ratio (BS/TV), trabecular bone mineral density (Tb. BMD), trabecular bone number (Tb. N), trabecular bone thickness (Tb. Th) and small bone Trabecular bone space (Tb. Sp). RESULTS: Via X-ray and pathologically, risedronate treatment promoted the callus forming at the fracture site during the following 6 weeks after osteoporotic fracture by X-ray (P < 0.01), increased the local bone mineral density (P < 0.01), and accelerated the fracture healing during the first 3 weeks (P <0.01), but delayed facture healing in the later 3 weeks (P < 0.01). Risedronate increased the bone continuity of fracture at 7th week, but this phenomenon was not found at the 10th week (P < 0.01). Delayed fracture healing occurred locally at the fracture site. At 7th week, Risedronate may promote cartilage cells proliferating at fracture site, increase the dense of bone trabeculae and the connection of bone trabeculae, thicken the bone cortex showing better fracture healing than OPF-Saline groups (P < 0.01). However, these parameter did not continue during the 7th and 10th weeks. Comparing the first and the later 3 weeks, the rats in group Osteoporotic Fracture-Risedronate (OPF-RD) accelerated the local fracture healing in the first 3 weeks but not in the last 3 weeks, which is consistent for the BMD and BMC among each group (P < 0.05). Through evaluation of bone mineral density and bone mineral content, risedronate dramatically increased the BMD at the fracture site and resulted in reduction of BMC by risedronate at the fracture site (P < 0.05) among each group still exist, indicating dramatic (P < 0.05). Through load testing, Risedronate increased the structural strength and mechanical indexes of the new callus (P < 0.01). CONCLUSION: Risedronate can improve the structural strength and mechanical index of newborn callus. Longer than 7 weeks usage of third generation bisphosphonate of risedronate does not contribute to osteoporotic fracture.

Revelation of AbfR in regulation of mismatch repair and energy metabolism in S. epidermidis by integrated proteomic and metabolomic analysis.

Staphylococcus epidermidis is a common causative of nosocomial infections associated with indwelling medical devices. To date, the mechanisms of the pathogenicity and drug resistance of S. epidermidis have not been clearly elucidated. AbfR has been previously identified as an oxidation-sensing regulator that regulates bacterial aggregation and biofilm formation by responding to oxidative stress in S. epidermidis; however, the regulatory pathways of AbfR are underexplored. In this study, we investigated the oxidation-sensing regulatory mechanism of AbfR using TMT10-plex labelling quantitative proteomic and untargeted metabolomic approaches. Integrated analysis of two omics datasets indicated that abfR depletion influenced nucleic acid metabolism and activated the DNA mismatch repair pathway. In addition, several energy-related metabolic pathways, including tricarboxylic acid (TCA) cycle, glycolysis, and arginine metabolism, were remarkably impacted by the deletion of abfR. This study revealed the regulatory networks of the transcription factor AbfR from a multi-omics view and demonstrated that AbfR played a broad role in not only mismatch repair but also energy metabolism, enabling S. epidermidis to constantly sense and adapt to environmental stress. SIGNIFICANCE: Staphylococcus epidermidis has emerged as a major nosocomial infection causing pathogen. AbfR, a transcription factor of S. epidermidis, plays an important role in oxidative stress, cell aggregation, and biofilm formation; however, the regulatory mechanism of AbfR is unknown. Using proteomic and metabolomic approaches, this study unveils the global regulatory networks of AbfR, and demonstrates that AbfR not only regulates the DNA mismatch repair pathway by an oxidation sensing mechanism but also affects energy metabolism. This study expands the body of knowledge related to regulatory transcription factors in staphylococci and lays a foundation for future research on clinical infections caused by S. epidermidis.

Heterogeneous immunogenomic features and distinct escape mechanisms in multifocal hepatocellular carcinoma.

BACKGROUND & AIMS: The presence of multifocal tumors, developed either from intrahepatic metastasis (IM) or multicentric occurrence (MO), is a distinct feature of hepatocellular carcinoma (HCC). Immunogenomic characterization of multifocal HCC is important for understanding immune escape in different lesions and developing immunotherapy. METHODS: We combined whole-exome/transcriptome sequencing, multiplex immunostaining, immunopeptidomes, T cell receptor (TCR) sequencing and bioinformatic analyses of 47 tumors from 15 patients with HCC and multifocal lesions. RESULTS: IM and MO demonstrated distinct clonal architecture, mutational spectrum and genetic susceptibility. The immune microenvironment also displayed spatiotemporal heterogeneity, such as less T cell and more M2 macrophage infiltration in IM and higher expression of inhibitory immune checkpoints in MO. Similar to mutational profiles, shared neoantigens and TCR repertoires among tumors from the same patients were abundant in IM but scarce in MO. Combining neoantigen prediction and immunopeptidomes identified T cell-specific neoepitopes and achieved a high verification rate in vitro. Immunoediting mainly occurred in MO but not IM, due to the relatively low immune infiltration. Loss of heterozygosity of human leukocyte antigen (HLA) alleles, identified in 17% of multifocal HCC, hampered the ability of major histocompatibility complex to present neoantigens, especially in IM. An integrated analysis of Immunoscore, immunoediting, TCR clonality and HLA loss of heterozygosity in each tumor could stratify patients into 2 groups based on whether they have a high or low risk of recurrence (p = 0.038). CONCLUSION: Our study comprehensively characterized the genetic structure, neoepitope landscape, T cell profile and immunoediting status that collectively shape tumor evolution and could be used to optimize personalized immunotherapies for multifocal HCC. LAY SUMMARY: Immunogenomic features of multifocal hepatocellular carcinoma (HCC) are important for understanding immune-escape mechanisms and developing more effective immunotherapy. Herein, comprehensive immunogenomic characterization showed that diverse genomic structures within multifocal HCC would leave footprints on the immune landscape. Only a few tumors were under the control of immunosurveillance, while others evaded the immune system through multiple mechanisms that led to poor prognosis. Our study revealed heterogeneous immunogenomic landscapes and immune-constrained tumor evolution, the understanding of which could be used to optimize personalized immunotherapies for multifocal HCC.

Identification of two novel mutations in the ATP7B gene that cause Wilson's disease.

BACKGROUND: Wilson's disease is an autosomal recessive disorder characterized by liver disease and/or neurologic deficits due to copper accumulation and is caused by pathogenic mutations in the ATP7B gene. DATA SOURCES: Two unrelated Chinese patients born to nonconsanguineous parents who were diagnosed with earlyonset Wilson's disease. DNA sequencing and bioinformation analysis were conducted. RESULTS: We have identified four mutations in two family trios, of which two were novel, namely, c. 3028A>G (p. K1010E) and c.3992T>G (p.Y1331X), in each patient. CONCLUSIONS: Gene testing is playing an important role in diagnosis of Wilson's disease. The early-onset of Wilson's disease is apparently not associated with P-ATPase domain in the ATP7B protein. Our findings further widen the spectrum of mutations involving the ATP7B gene.

Knockdown of DDX46 inhibits proliferation and induces apoptosis in esophageal squamous cell carcinoma cells.

Esophageal squamous cell carcinoma (ESCC) is the most common type of esophageal carcinoma and remains the leading cause of cancer-related death worldwide. DEAD-box RNA helicases play critical roles in cellular metabolism and in many cases have been implicated in cellular proliferation and neoplastic transformation. DDX46 belongs to DEAD-box helicase family, the expression pattern of DDX46 in ESCC tissues and the biologic role in ESCC progression have not been implicated previously. In this study, DDX46 expression in human ESCC and adjacent normal tissues were explored using immunohistochemistry, and ESCC cell lines compared with normal esophageal epithelium cell were quantified using real­time PCR. Next, lentivirus-mediated RNA interference was applied to silence DDX46 in TE-1 and Eca-109 cells. Cell growth was monitored using high content screening. Cell viability was measured by MTT assay. Cell colony-forming capacity was measured by colony formation assay. Cell cycle progression and apoptosis were determined by flow cytometry. Further, the stress and apoptosis signaling antibody array kit was used to detect the changes of signaling molecules in TE-1 cells after DDX46 knockdown. We found that DDX46 was significantly upregulated in ESCC tissues and cells compared with normal tissues and cells. DDX46 knockdown led to decreased proliferation and increased apoptosis in TE-1 and Eca-109 cells. Moreover, DDX46 silencing resulted in apoptotic induction via decreased phosphorylation of Akt and IκBα, as well as negative regulation of NF-κB signaling. In conclusion, these results demonstrate that DDX46 knockdown inhibited cell growth, and induced apoptosis, suggest that DDX46 is critical for ESCC cells proliferation. In addition, this study provides a foundation for further study into the clinical potential diagnosis and novel therapeutic target for ESCC.

[Mutation analysis of phenylalanine hydroxylase gene in 55 patients with phenylketonuria from Hebei province].

OBJECTIVE: To identify the pathogenic mutations of phenylalanine hydroxylase gene (PAH) in patients with phenylketonuria (PKU) from Hebei Province. METHODS: Genomic DNA was extracted from 55 unrelated PKU patients from September 2007 to July 2009. All PAH exons and exon-intron junctions were amplified by polymerase chain reaction (PCR) and sequenced. Multiplex ligation-dependent probe amplifications (MLPA) was performed to detect the deletions or duplications of PAH. Gap-PCR was used to determine the breakpoints of large deletions. RESULTS: Among them, 108 mutant alleles (98.2%) were found. All PAH exons with the exceptions of exons 9 and 13 were affected. A total of 41 different mutations were detected, including missense (n = 24), nonsense (n = 7), splicing (n = 7), small deletion (n = 1) and large deletion (n = 2). Among them, 4 missense mutations (p.Pro147Leu, p.Gly289Arg, p.Phe392Ser, p.Ile421Thr) and 2 large deletions (-4163_-406del and -1932_+3402del) were novel. The most common mutations were p.Arg243Gln (12.7%), c.611A > G (11.8%) and c.1197A > T (9.1%). CONCLUSION: The mutations of PKU patients with from Hebei Province are scattered throughout the PAH gene. Most of them are of single nucleotide substitutions, but large deletions are not rare.

[Effect of extract of ginkgo bilboa leaf on early diabetic nephropathy].

OBJECTIVE: To study the effect of extract of Ginkgo bilboa leaf (EGB) on renal lesions of early diabetic nephropathy (DN). METHODS: Sixty-eight patients with early DN were randomly divided into two groups, the control group (34 patients) and the treated group (34 patients). Patients in both groups received conventional therapy, while additional 9.6 mg EGB was orally taken by patients in the treated group, three times per day. The therapeutic course for both groups was three months. Indexes such as urinary microalbumin (mALB), alpha1-microglobulin (alpha1-MG), immunoglobulin (IgG), transferrin (TF), retinal binding protein (RBP) and N-acety-beta-D-glucosaminidase (NAG) before and after treatment between the two groups were compared respectively. RESULTS: Compared with before treatment, mALB, alpha1-MG, IgG, TF, RBP and NAG obviously decreased with significant difference in the treated group after treatment (P < 0.05). However, no significant decrease in the above-mentioned indexes in the control group (P > 0.05). CONCLUSION: EGB has the protective action on early DN.