Florid reactive periostitis of the clavicle: A case report and literature review.

RATIONALE: Florid reactive periostitis (FRP), a rare reactive bone lesion, typically presents in the short tubular bones of the extremities, with infrequent occurrences in the long tubular bones. This report discusses a unique case of FRP in the clavicle, managed through comprehensive lesion debridement and bone grafting, yielding positive results over a 3-year duration. PATIENT CONCERN: A 25-year-old male presented with a discernible mass at the left sternal end of the clavicle, discovered incidentally 2 weeks prior. The patient exhibited no clinical signs of inflammation, pain, sinus tract, or suppuration. DIAGNOSIS: Initial pathological examination of the local excision suggested benign lesions, although malignancy could not be ruled out. A definitive diagnosis of clavicular FRP was reached post complete lesion resection, with supporting evidence from postoperative pathology, imaging, and clinical symptoms. INTERVENTION: The left clavicle was reconstructed through an open surgical procedure involving total mass removal and ipsilateral extraction of an iliac bone of suitable dimensions. This was implanted into the clavicular bone defect and internally fixed with a plate. OUTCOMES: Three years of consecutive follow-up revealed no recurrence of hyperplasia, absence of mass or tenderness at the left sternal end of the clavicle, and unimpaired function of adjacent joints. LESSONS: The primary clinical challenge with FRP is its diagnosis. While pathological diagnosis remains crucial, it is also important to incorporate imaging and clinical symptoms for a comprehensive assessment. Complete mass excision may offer specific benefits in distinguishing FRP from its malignant counterparts.

Natural biopolymer scaffold for meniscus tissue engineering.

Meniscal injuries caused by trauma, degeneration, osteoarthritis, or other diseases always result in severe joint pain and motor dysfunction. Due to the unique anatomy of the human meniscus, the damaged meniscus lacks the ability to repair itself. Moreover, current clinical treatments for meniscal injuries, including meniscal suturing or resection, have significant limitations and drawbacks. With developments in tissue engineering, biopolymer scaffolds have shown promise in meniscal injury repair. They act as templates for tissue repair and regeneration, interacting with surrounding cells and providing structural support for newly formed meniscal tissue. Biomaterials offer tremendous advantages in terms of biocompatibility, bioactivity, and modifiable mechanical and degradation kinetics. In this study, the preparation and composition of meniscal biopolymer scaffolds, as well as their properties, are summarized. The current status of research and future research prospects for meniscal biopolymer scaffolds are reviewed in terms of collagen, silk, hyaluronic acid, chitosan, and extracellular matrix (ECM) materials. Overall, such a comprehensive summary provides constructive suggestions for the development of meniscal biopolymer scaffolds in tissue engineering.

Glycolysis Rate-Limiting Enzymes: Novel Potential Regulators of Rheumatoid Arthritis Pathogenesis.

Rheumatoid arthritis (RA) is a classic autoimmune disease characterized by uncontrolled synovial proliferation, pannus formation, cartilage injury, and bone destruction. The specific pathogenesis of RA, a chronic inflammatory disease, remains unclear. However, both key glycolysis rate-limiting enzymes, hexokinase-II (HK-II), phosphofructokinase-1 (PFK-1), and pyruvate kinase M2 (PKM2), as well as indirect rate-limiting enzymes, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), are thought to participate in the pathogenesis of RA. In here, we review the latest literature on the pathogenesis of RA, introduce the pathophysiological characteristics of HK-II, PFK-1/PFKFB3, and PKM2 and their expression characteristics in this autoimmune disease, and systematically assess the association between the glycolytic rate-limiting enzymes and RA from a molecular level. Moreover, we highlight HK-II, PFK-1/PFKFB3, and PKM2 as potential targets for the clinical treatment of RA. There is great potential to develop new anti-rheumatic therapies through safe inhibition or overexpression of glycolysis rate-limiting enzymes.

In vitro and in vivo Effect of Antimicrobial Agent Combinations Against Carbapenem-Resistant Klebsiella pneumoniae with Different Resistance Mechanisms in China.

OBJECTIVE: This study aimed to evaluate the in vitro and in vivo effects of different combinations of antimicrobial agents against carbapenemase-producing and non-producing Klebsiella pneumoniae from China. METHODS: A checkerboard assay of meropenem (MEM), amikacin (AK), tigecycline (TGC), colistin (COL) and their combinations was carried out against 58 clinical carbapenem-resistant K. pneumoniae (CRKp) isolates, including 11 carbapenemase-non-producing K. pneumoniae isolates and 21 isolates producing KPC-2 enzyme, 11 NDM-1, 13 IMP, one VIM-1 and one OXA-48. The checkerboard assay was analyzed by the fractional inhibitory concentration index (FICI). A time-kill assay and Galleria mellonella infection model were conducted to evaluate the in vitro and in vivo effects of the four drugs alone and in combination. RESULTS: In the checkerboard assay, TGC+AK and MEM+AK combinations showed the highest synergistic effect against KPC-2 and NDM-1 carbapenemase-producing isolates, with synergy+partial synergy (defined as FICI <1) rates of 76.2% and 71.4% against KPC-2 producers, and 54.5% and 81.8% against NDM-1 producers. TGC+AK and MEM+COL combinations showed the highest rate of synergistic effect against IMP-producing isolates. Against carbapenemase-non-producing isolates, TGC+COL and TGC+AK combinations showed the highest rate of synergy effect (63.6% and 54.5%). MEM+AK showed a synergistic effect against one VIM-1 producer (FICI=0.31) and an additivite effect (FICI=1) against one OXA-48 producer. In the time-kill assay, COL+AK, COL+TGC, COL+MEM and AK+TGC showed good synergistic effects against the KPC-2-producing isolate D16. COL+MEM and COL+TGC combinations showed good effects against the NDM-1-producing isolate L13 and IMP-4-producing isolate L34. Against the carbapenemase-non-producing isolate Y105, MEM+TGC and COL+AK showed high synergistic effects, with log10CFU/mL decreases of 6.2 and 5.5 compared to the most active single drug. In the G. mellonella survival assay, MEM-based combinations had relatively high survival rates, especially when combined with colistin, against KPC-2 producers (90% survival rate) and with amikacin against metallo-beta-lactamase producers (95-100% survival rate). CONCLUSION: Our study suggests that different antimicrobial agent combinations should be considered against CRKp infections with different resistance mechanisms.

Antimicrobial Susceptibility and Virulence of mcr-1-Positive Enterobacteriaceae in China, a Multicenter Longitudinal Epidemiological Study.

This study was to investigate the prevalence of mcr-1-positive Enterobacteriaceae (MPE) in intra-abdominal infections (IAIs), urinary tract infections (UTIs), and lower respiratory tract infections (LRTIs) in China. A total of 6,401 Enterobacteriaceae isolates were collected consecutively from IAI, UTI, and LRTI patients in 19 hospitals across mainland China during 2014-2016. MPE isolates were screened by PCR detection for the mcr gene. The resistance profiles were tested by antimicrobial susceptibility test. All MPE isolates were characterized by pulsed-field gel electrophoresis (PFGE), multi-locus-sequence typing, O and H serotyping, and whole-genome sequencing. Among the 6,401 Enterobacteriaceae isolates, 17 Escherichia coli strains (0.27%) were positive for the mcr-1 gene. The MPE prevalence rates in IAI, UTI, and LRTI patients were 0.34% (12/3502), 0.23% (5/2154), and 0% (0/745), respectively. The minimum inhibition concentrations (MICs) of colistin against 3 isolates were of 0.5-2 mg/L, and 4-8 mg/L against other 14 isolates. All the 17 isolates were susceptible to meropenem, imipenem, tigecycline, and ceftazidime/avibactam. The 17 MPE isolates belonged to 14 different ST types, and those that belonged to the same STs were not clonal by PFGE. The mcr-1-harboring plasmid of ten MPE isolates could transfer to the recipients by conjugation and the colistin MICs of the transconjugants ranged from 0.5 to 8 mg/L. Mcr-1-carrying plasmids from the 17 MPE isolates could be grouped into four clusters, including 8 IncX4 type, 4 IncI2 type, 4 IncHI2A type, and 1 p0111 type. Multiple-drug resistance genes and virulence genes were detected. In conclusion, the prevalence of MPE in IAI, UTI, and LRTI were low in China, and no clonal transmission was identified in our study. Most MPE isolates exhibited low-level colistin resistance. However, our study indicated that MPE isolates always carried a variety of drug resistance and virulence genes, which should be paid more attention.

Identification of the integrin-binding site on coagulation factor VIIa required for proangiogenic PAR2 signaling.

The tissue factor (TF) pathway serves both hemostasis and cell signaling, but how cells control these divergent functions of TF remains incompletely understood. TF is the receptor and scaffold of coagulation proteases cleaving protease-activated receptor 2 (PAR2) that plays pivotal roles in angiogenesis and tumor development. Here we demonstrate that coagulation factor VIIa (FVIIa) elicits TF cytoplasmic domain-dependent proangiogenic cell signaling independent of the alternative PAR2 activator matriptase. We identify a Lys-Gly-Glu (KGE) integrin-binding motif in the FVIIa protease domain that is required for association of the TF-FVIIa complex with the active conformer of integrin ß1. A point mutation in this motif markedly reduces TF-FVIIa association with integrins, attenuates integrin translocation into early endosomes, and reduces delayed mitogen-activated protein kinase phosphorylation required for the induction of proangiogenic cytokines. Pharmacologic or genetic blockade of the small GTPase ADP-ribosylation factor 6 (arf6) that regulates integrin trafficking increases availability of TF-FVIIa with procoagulant activity on the cell surface, while inhibiting TF-FVIIa signaling that leads to proangiogenic cytokine expression and tumor cell migration. These experiments delineate the structural basis for the crosstalk of the TF-FVIIa complex with integrin trafficking and suggest a crucial role for endosomal PAR2 signaling in pathways of tissue repair and tumor biology.

Targeting cancer-specific glycans by cyclic peptide lectinomimics.

The transformation from normal to malignant phenotype in human cancers is associated with aberrant cell-surface glycosylation. Thus, targeting glycosylation changes in cancer is likely to provide not only better insight into the roles of carbohydrates in biological systems, but also facilitate the development of new molecular probes for bioanalytical and biomedical applications. In the reported study, we have synthesized lectinomimics based on odorranalectin 1; the smallest lectin-like cyclic peptide isolated from the frog Odorrana grahami skin, and assessed the ability of these peptides to bind specific carbohydrates on molecular and cellular levels. In addition, we have shown that the disulfide bond found in 1 can be replaced with a lactam bridge. However, the orientation of the lactam bridge, peptides 2 and 3, influenced cyclic peptide's conformation and thus these peptides' ability to bind carbohydrates. Naturally occurring 1 and its analog 3 that adopt similar conformation in water bind preferentially L-fucose, and to a lesser degree D-galactose and N-acetyl-D-galactosamine, typically found within the mucin O-glycan core structures. In cell-based assays, peptides 1 and 3 showed a similar binding profile to Aleuria aurantia lectin and these two peptides inhibited the migration of metastatic breast cancer cell lines in a Transwell assay. Altogether, the reported data demonstrate the feasibility of designing lectinomimics based on cyclic peptides.

Local adipocytes enable estrogen-dependent breast cancer growth: Role of leptin and aromatase.

The importance of the microenvironment in breast cancer growth and progression is becoming increasingly clear. Adipocytes are abundant in the mammary microenvironment, and recent studies show that adipocytes produce endocrine, inflammatory, and angiogenic factors that have tremendous potential to affect adjacent breast cancer cells. Yet, the extent to which local adipocyte function contributes to the pathogenesis of breast cancer is largely unexplored. Here we describe a unique animal model to study interactions between adipocytes and breast cancer cells in the tumor microenvironment. Our results suggest that local interactions between adipocytes and tumor cells are sufficient to promote the growth of hormone-dependent breast cancer. We also demonstrate that leptin signaling in adipocytes induces aromatase expression, expected to result in higher estrogen in the microenvironment thus enabling mammary tumorigenesis.

Control of mTORC1 signaling by the Opitz syndrome protein MID1.

Mutations in the MID1 gene are causally linked to X-linked Opitz BBB/G syndrome (OS), a congenital disorder that primarily affects the formation of diverse ventral midline structures. The MID1 protein has been shown to function as an E3 ligase targeting the catalytic subunit of protein phosphatase 2A (PP2A-C) for ubiquitin-mediated degradation. However, the molecular pathways downstream of the MID1/PP2A axis that are dysregulated in OS and that translate dysfunctional MID1 and elevated levels of PP2A-C into the OS phenotype are poorly understood. Here, we show that perturbations in MID1/PP2A affect mTORC1 signaling. Increased PP2A levels, resulting from proteasome inhibition or depletion of MID1, lead to disruption of the mTOR/Raptor complex and down-regulated mTORC1 signaling. Congruously, cells derived from OS patients that carry MID1 mutations exhibit decreased mTORC1 formation, S6K1 phosphorylation, cell size, and cap-dependent translation, all of which is rescued by expression of wild-type MID1 or an activated mTOR allele. Our findings define mTORC1 signaling as a downstream pathway regulated by the MID1/PP2A axis, suggesting that mTORC1 plays a key role in OS pathogenesis.

REDD1, an inhibitor of mTOR signalling, is regulated by the CUL4A-DDB1 ubiquitin ligase.

The cellular response to hypoxia involves several signalling pathways that mediate adaptation and survival. REDD1 (regulated in development and DNA damage responses 1), a hypoxia-inducible factor-1 target gene, has a crucial role in inhibiting mammalian target of rapamycin complex 1 (mTORC1) signalling during hypoxic stress. However, little is known about the signalling pathways and post-translational modifications that regulate REDD1 function. Here, we show that REDD1 is subject to ubiquitin-mediated degradation mediated by the CUL4A-DDB1-ROC1-beta-TRCP E3 ligase complex and through the activity of glycogen synthase kinase 3beta. Furthermore, REDD1 degradation is crucially required for the restoration of mTOR signalling as cells recover from hypoxic stress. Our findings define a mechanism underlying REDD1 degradation and its importance for regulating mTOR signalling.

The ATR-mediated S phase checkpoint prevents rereplication in mammalian cells when licensing control is disrupted.

DNA replication in eukaryotic cells is tightly controlled by a licensing mechanism, ensuring that each origin fires once and only once per cell cycle. We demonstrate that the ataxia telangiectasia and Rad3 related (ATR)-mediated S phase checkpoint acts as a surveillance mechanism to prevent rereplication. Thus, disruption of licensing control will not induce significant rereplication in mammalian cells when the ATR checkpoint is intact. We also demonstrate that single-stranded DNA (ssDNA) is the initial signal that activates the checkpoint when licensing control is compromised in mammalian cells. We demonstrate that uncontrolled DNA unwinding by minichromosome maintenance proteins upon Cdt1 overexpression is an important mechanism that leads to ssDNA accumulation and checkpoint activation. Furthermore, we show that replication protein A 2 and retinoblastoma protein are both downstream targets for ATR that are important for the inhibition of DNA rereplication. We reveal the molecular mechanisms by which the ATR-mediated S phase checkpoint pathway prevents DNA rereplication and thus significantly improve our understanding of how rereplication is prevented in mammalian cells.

The Mre11/Rad50/Nbs1 complex plays an important role in the prevention of DNA rereplication in mammalian cells.

The Mre11/Nbs1/Rad50 complex (MRN) plays multiple roles in the maintenance of genome stability, including repair of double-stranded breaks (DSBs) and activation of the S-phase checkpoint. Here we demonstrate that MRN is required for the prevention of DNA rereplication in mammalian cells. DNA replication is strictly regulated by licensing control so that the genome is replicated once and only once per cell cycle. Inactivation of Nbs1 or Mre11 leads to a substantial increase of DNA rereplication induced by overexpression of the licensing factor Cdt1. Our studies reveal that multiple mechanisms are likely involved in the MRN-mediated suppression of rereplication. First, both Mre11 and Nbs1 are required for facilitating ATR activation when Cdt1 is overexpressed, which in turn suppresses rereplication. Second, Cdt1 overexpression induces ATR-mediated phosphorylation of Nbs1 at Ser343 and this phosphorylation depends on the FHA and BRCT domains of Nbs1. Mutations at Ser343 or in the FHA and BRCT domains lead to more severe rereplication when Cdt1 is overexpressed. Third, the interaction of the Mre11 complex with RPA is important for the suppression of rereplication. This suggests that modulating RPA activity via a direct interaction of MRN is likely one of the effector mechanisms to suppress rereplication. Moreover, we demonstrate that MRN is also required for preventing the accumulation of DSBs when rereplication is induced. Therefore, our studies suggest new roles of MRN in the maintenance of genome stability through preventing rereplication and rereplication-associated DSBs when licensing control is compromised.

The Mre11 complex mediates the S-phase checkpoint through an interaction with replication protein A.

The Mre11/Rad50/Nbs1 complex (MRN) plays an essential role in the S-phase checkpoint. Cells derived from patients with Nijmegen breakage syndrome and ataxia telangiectasia-like disorder undergo radioresistant DNA synthesis (RDS), failing to suppress DNA replication in response to ionizing radiation (IR). How MRN affects DNA replication to control the S-phase checkpoint, however, remains unclear. We demonstrate that MRN directly interacts with replication protein A (RPA) in unperturbed cells and that the interaction is regulated by cyclin-dependent kinases. We also show that this interaction is needed for MRN to correctly localize to replication centers. Abolishing the interaction of Mre11 with RPA leads to pronounced RDS without affecting phosphorylation of Nbs1 or SMC1 following IR. Moreover, MRN is recruited to sites at or adjacent to replication origins by RPA and acts there to inhibit new origin firing upon IR. These studies suggest a direct role of MRN at origin-proximal sites to control DNA replication initiation in response to DNA damage, thereby providing an important mechanism underlying the intra-S-phase checkpoint in mammalian cells.

Cyclin-dependent kinases phosphorylate human Cdt1 and induce its degradation.

Eukaryotic cells tightly control DNA replication so that replication origins fire only once during S phase within the same cell cycle. Cell cycle-regulated degradation of the replication licensing factor Cdt1 plays important roles in preventing more than one round of DNA replication per cell cycle. We have previously shown that the SCF(Skp2)-mediated ubiquitination pathway plays an important role in Cdt1 degradation. In this study, we demonstrate that human Cdt1 is a substrate of Cdk2 and Cdk4 both in vivo and in vitro. Overexpression of cyclin-dependent kinase inhibitors such as p21 and p27 dramatically suppresses the phosphorylation of Cdt1, disrupts the interaction of Cdt1 with the F-box protein Skp2, and blocks the degradation of Cdt1. Further analysis reveals that Cdt1 interacts with cyclin/cyclin-dependent kinase (Cdk) complexes through a cyclin/Cdk binding consensus site, located at the N terminus of Cdt1. A Cdt1 mutant carrying four amino acid substitutions at the Cdk binding site dramatically reduces associations with cyclin/Cdk complexes. This mutant is not phosphorylated, fails to bind Skp2 and is more stable than wild-type Cdt1. These data suggest that cyclin/Cdk-mediated Cdt1 phosphorylation is required for the association of Cdt1 with the SCF(Skp2) ubiquitin ligase and thus is important for the cell cycle dependent degradation of Cdt1 in mammalian cells.

Negative regulation of FAK signaling by SOCS proteins.

Focal adhesion kinase (FAK) becomes activated upon integrin-mediated cell adhesion and controls cellular responses to the engagement of integrins, including cell migration and survival. We show here that a coordinated signaling by integrins and growth factor receptors induces expression of suppressor of cytokine signaling-3 (SOCS-3) and subsequent interaction between endogenous FAK and SOCS-3 proteins in 3T3 fibroblasts. Cotransfection studies demonstrated that SOCS-3, and also SOCS-1, interact with FAK in a FAK-Y397-dependent manner, and that both the Src homology 2 (SH2) and the kinase inhibitory region (KIR) domains of the SOCS proteins contribute to FAK binding. SOCS-1 and SOCS-3 were found to inhibit FAK-associated kinase activity in vitro and tyrosine phosphorylation of FAK in cells. The SOCS proteins also promoted polyubiquitination and degradation of FAK in a SOCS box-dependent manner and inhibited FAK-dependent signaling events, such as cell motility on fibronectin. These studies suggest a negative role of SOCS proteins in FAK signaling, and for a previously unidentified regulatory mechanism for FAK function.