Application of aptamer-based viral detection in animals.

Viral infections are common causes of diseases in animals and appropriate methods are increasingly being required to detect viral pathogens in animals. In this regard, similar to antigen- -antibody interactions, aptamers have high affinity and specificity for their respective target molecules, and can be selected using the Systematic Evolution of Ligands by EXponential enrichment (SELEX) technique. Recently, significant progress has been made in the development of aptamer selection and aptamer-based sensors for viral detection, and here we review some of the recent advances in aptamer-based detection of viral infections in animals. This review will serve as a comprehensive resource for aptamer-based strategies in viral diagnostics.

[Observation on the curative effect of fibula transverse transport technique in the treatment of diabetic foot ulcer].

To investigate the clinical effect of fibula transverse transport technique in the treatment of diabetic foot ulcer. Nine patients (7 males and 2 females) with diabetic foot ulcer were treated with fibula transverse transport technique from September 2017 to September 2020. The mean age was (55±9) years (ranged from 43 to 66 years). In terms of Wagner classification, 2 cases were in grade 2, 5 cases were in grade 3, and the other 2 cases were in grade 4. All of the cases involved ischemic ulcers or ischemic-nerve ulcers. The transcutaneous oxygen pressure (TcPO2) of the dorsal foot of the affected limb was measured, with the ulcer healing and TcPO2 changes recorded at follow-up. All the 9 patients were followed up for (23±12) months (3 to 38 months). It was found that all patients with foot ulcers were cured within (4.2±1.9) months (2 to 8 months), and all the patients obtained limb salvage. Besides, there was no serious complications occurred, such as skin necrosis and needle tract infection. Before the operation, the TcPO2 of the affected foot was (28.6±3.8) mmHg(1 mmHg=0.133 kPa), while it was (35.0±5.6) mmHg three months after operation (P<0.05). The technique of fibula transverse transport can effectively improve the microcirculation function of diabetic foot, and it can promote the healing of the ulcer with few complications.

[Clinical effects of free thinned deep inferior epigastric artery perforator flap in repairing extensive soft tissue defects in extremities].

Objective: To explore the feasibility and clinical effects of using free thinned deep inferior epigastric artery perforator flap to repair extensive soft tissue defects in extremities. Methods: From April 2010 to January 2014, 12 patients with extensive soft tissue defect in extremities after trauma, including 10 males and 2 females, aged 21 to 48 years, 6 patients with defect in the back of wrist and 6 patients with defect in ankle were admitted to the Department of Bone Microsurgery of Xi'an Honghui hospital. After debridement, the size of soft tissue defect ranged from 15.0 cm×4.5 cm to 28.0 cm×11.0 cm. The free thinned deep inferior epigastric artery perforator flap was designed, cut and transferred for reconstruction, with size of 15.0 cm×5.0 cm to 29.0 cm×12.0 cm. The flap thickness ranged from 4.0 to 6.5 cm before defatting, and was 0.6 to 0.9 cm after defatting. All the donor sites of flaps were closed directly by suturing. The flap survival and the appearance and function of flap and donor site were observed during follow-up. Results: All the flaps survived smoothly after surgery. During follow-up of 10 to 42 months, the flaps showed no bloat in appearance, no further flap revision or defatting procedures were required, the distance of static 2-point discrimination was 11 to 17 mm (14.5 mm on average). The abdominal function of patients was not affected, and no postoperative abdomen hernia or ulceration was noted. Conclusions: The free thinned deep inferior epigastric artery perforator flap is thin and suitable for repairing extensive soft tissue defects in extremities with very good outcomes.

Association of the Abl tyrosine kinase with the Trk nerve growth factor receptor.

Nerve growth factor (NGF) initiates the majority of its biological effects by promoting the dimerization and activation of the tyrosine kinase receptor TrkA. In addition to rapid increases in the phosphorylation of phosphatidylinositol 3'-kinase (PI 3-kinase) and phospholipase C-gamma and increased ras activity, phosphorylation of c-Crk and paxillin proteins has been observed upon TrkA activation. The c-Abl tyrosine kinase is involved in the control of the axonal cytoskeleton and is known to interact with c-Crk proteins. Here we have tested the possibility that TrkA receptors might form an association with the c-Abl protein. After transfection in 293T cells, TrkA and c-Abl kinases could be coimmunoprecipitated. This interaction did not require TrkA receptors to be autophosphorylated. Mapping analysis indicated that the region of c-Abl association was confined to the juxtamembrane region of TrkA. The interaction of c-Abl with TrkA was also observed in differentiated pheochromocytoma PC12 cells. These results suggest that c-Abl may be recruited to the NGF receptor complex and be involved in regulating specific phosphorylation events that occur during neuronal differentiation.

p53 deficiency increases transformation by v-Abl and rescues the ability of a C-terminally truncated v-Abl mutant to induce pre-B lymphoma in vivo.

Abelson murine leukemia virus (A-MuLV) is an acute transforming retrovirus that preferentially transforms early B-lineage cells both in vivo and in vitro. Its transforming protein, v-Abl, is a tyrosine kinase related to v-Src but containing an extended C-terminal domain. Many mutations affecting the C-terminal portion of the molecule block the pre-B-transforming activity of v-Abl without affecting the fibroblast-transforming ability. In this study we have determined the abilities of both wild-type and C-terminally truncated (p90) forms of v-Abl to transform cells from p53(-/-) mice. Lack of p53 increases the susceptibility of bone marrow cells to transformation by v-Abl by a factor of more than 7 but does not alter v-Abl's preference for B220(+) IgM(-) pre-B cells. p53-deficient mice have earlier tumor onset, more rapid tumor progression, and decreased survival time following A-MuLV infection, but all of the tumors are pre-B lymphomas. Thus, p53-dependent pathways inhibit v-Abl transformation but play no role in conferring preferential transformation of pre-B cells. Surprisingly, the C-terminally truncated form of v-Abl (p90) transforms pre-B cells very efficiently in mice lacking p53, thus demonstrating that the C terminus of v-Abl does not determine preB tropism but is necessary to overcome p53-dependent inhibition of transformation.

Cytoskeletal protein PSTPIP1 directs the PEST-type protein tyrosine phosphatase to the c-Abl kinase to mediate Abl dephosphorylation.

A search for c-Abl interacting proteins resulted in the recovery of PSTPIP1, originally identified as a binding protein of the PEST-type protein tyrosine phosphatases (PTP). PSTPIP1 was phosphorylated by c-Abl, and growth factor-induced PSTPIP1 phosphorylation was diminished in Abl null fibroblasts. PSTPIP1 was able to bridge c-Abl to the PEST-type PTPs. Several experiments suggest that the PEST-type PTPs negatively regulate c-Abl activity: c-Abl was hyperphosphorylated in PTP-PEST-deficient cells; disruption of the c-Abl-PSTPIP1-PEST-type PTP ternary complex by overexpression of PSTPIP1 mutants increased c-Abl phosphotyrosine content; and PDGF-induced c-Abl kinase activation was prolonged in PTP-PEST-deficient cells. Dephosphorylation of c-Abl by PEST-type PTP represents a novel mechanism by which c-Abl activity is regulated.

Characterization of a novel member of the DOK family that binds and modulates Abl signaling.

A novel member of the p62(dok) family of proteins, termed DOKL, is described. DOKL contains features of intracellular signaling molecules, including an N-terminal PH (pleckstrin homology) domain, a central PTB (phosphotyrosine binding) domain, and a C-terminal domain with multiple potential tyrosine phosphorylation sites and proline-rich regions, which might serve as docking sites for SH2- and SH3-containing proteins. The DOKL gene is predominantly expressed in bone marrow, spleen, and lung, although low-level expression of the RNA can also be detected in other tissues. DOKL and p62(dok) bind through their PTB domains to the Abelson tyrosine kinase in a kinase-dependent manner in both yeast and mammalian cells. DOKL is phosphorylated by the Abl tyrosine kinase in vivo. In contrast to p62(dok), DOKL lacks YxxP motifs in the C terminus and does not bind to Ras GTPase-activating protein (RasGAP) upon phosphorylation. Overexpression of DOKL, but not p62(dok), suppresses v-Abl-induced mitogen-activated protein (MAP) kinase activation but has no effect on constitutively activated Ras- and epidermal growth factor-induced MAP kinase activation. The inhibitory effect requires the PTB domain of DOKL. Finally, overexpression of DOKL in NIH 3T3 cells inhibits the transforming activity of v-Abl. These results suggest that DOKL may modulate Abl function.

c-Abl-induced apoptosis, but not cell cycle arrest, requires mitogen-activated protein kinase kinase 6 activation.

c-Abl is a ubiquitously expressed protein tyrosine kinase activated by DNA damage and implicated in two responses: cell cycle arrest and apoptosis. The downstream pathways by which c-Abl induces these responses remain unclear. We examined the effect of overexpression of c-Abl on the activation of mitogen-activated protein kinase pathways and found that overexpression of c-Abl selectively stimulated p38, while having no effect on c-Jun N-terminal kinase or on extracellular signal-regulated kinase. c-Abl-induced p38 activation was primarily mediated by mitogen-activated protein kinase kinase (MKK)6. A C-terminal truncation mutant of c-Abl showed no activity for stimulating p38 and MKK6, while a kinase-deficient c-Abl mutant still retained a residual activity. We tested different forms of c-Abl for their ability to induce apoptosis and found that apoptosis induction correlated with the activation of the MKK6-p38 kinase pathway. Importantly, dominant-negative MKK6, but not dominant-negative MKK3 or p38, blocked c-Abl-induced apoptosis. Because overexpression of p38 blocks cell cycle G(1)/S transition, we also tested whether the MKK6-p38 pathway is required for c-Abl-induced cell cycle arrest, and we found that neither MKK6 nor p38 dominant-negative mutants could relieve c-Abl-induced cell cycle arrest. Finally, DNA damage-induced MKK6 and p38 activation was diminished in c-Abl null fibroblasts. Our study suggests that c-Abl is required for DNA damage-induced MKK6 and p38 activation, and that activation of MKK6 by c-Abl is required for c-Abl-induced apoptosis but not c-Abl-induced cell cycle arrest.

Radiation-induced assembly of Rad51 and Rad52 recombination complex requires ATM and c-Abl.

Cells from individuals with the recessive cancer-prone disorder ataxia telangiectasia (A-T) are hypersensitive to ionizing radiation (I-R). ATM (mutated in A-T) is a protein kinase whose activity is stimulated by I-R. c-Abl, a nonreceptor tyrosine kinase, interacts with ATM and is activated by ATM following I-R. Rad51 is a homologue of bacterial RecA protein required for DNA recombination and repair. Here we demonstrate that there is an I-R-induced Rad51 tyrosine phosphorylation, and this induction is dependent on both ATM and c-Abl. ATM, c-Abl, and Rad51 can be co-immunoprecipitated from cell extracts. Consistent with the physical interaction, c-Abl phosphorylates Rad51 in vitro and in vivo. In assays using purified components, phosphorylation of Rad51 by c-Abl enhances complex formation between Rad51 and Rad52, which cooperates with Rad51 in recombination and repair. After I-R, an increase in association between Rad51 and Rad52 occurs in wild-type cells but not in cells with mutations that compromise ATM or c-Abl. Our data suggest signaling mediated through ATM, and c-Abl is required for the correct post-translational modification of Rad51, which is critical for the assembly of Rad51 repair protein complex following I-R.

Inhibition of v-Abl transformation by p53 and p19ARF.

Tumorigenesis is a multistep process that involves the activation of oncogenes and the inactivation of tumor suppressor genes. The transforming activity of the v-Abl oncogene of Abelson murine leukemia virus (A-MuLV) in immortal cell lines has been well studied, while the effects of v-Abl in primary fibroblasts are less clear. Here we show that v-Abl causes cell cycle arrest in primary mouse embryonic fibroblasts (MEFs) and elevated levels of both p53 and the cyclin-dependent kinase inhibitor p21Cip. p53-/- or p19ARF-/- MEFs were resistant to v-Abl-induced cell cycle arrest. Although wild-type MEFs were resistant to v-Abl transforming activity, p53-/- or p19ARF-/- MEFs were susceptible. The results indicate that loss of p19ARF and p53 function plays an important role during the transformation of primary cells by v-Abl. We suggest that although v-Abl is a potent oncogene, its full potential transforming activity cannot be realized until the ARF-, and p53-dependent growth inhibitory pathway is disabled. We also show that p53 is not the mediator of v-Abl toxicity in immortal fibroblasts and does not determine the susceptibility of immortal fibroblasts to v-Abl transformation.

Cell cycle-regulated transcription of the CLB2 gene is dependent on Mcm1 and a ternary complex factor.

Clb2 is the major B-type mitotic cyclin required for entry into mitosis in the budding yeast Saccharomyces cerevisiae. We showed that accumulation of CLB2 transcripts in G2 cells is controlled at the transcriptional level and identified a 55-bp upstream activating sequence (UAS) containing an Mcm1 binding site as being necessary and sufficient for cell cycle regulation. Sequences within the cell cycle-regulated UAS were shown to bind Mcm1 in vitro, and mutation which abolished Mcm1-dependent DNA binding activity eliminated cell cycle-regulated transcription in vivo. A second protein with no autonomous DNA binding activity was also recruited into Mcm1-UAS complexes, generating a ternary complex. A point mutation in the CLB2 UAS which blocked ternary complex formation, but still allowed Mcm1 to bind, resulted in loss of cell cycle regulation in vivo, suggesting that the ternary complex factor is also important in control of CLB2 transcription. We discuss the possibility that the CLB2 gene is coregulated with other genes known to be regulated with the same periodicity and suggest that Mcm1 and the ternary complex factor may coordinately regulate several other G2-regulated transcripts.