Mutational Analysis of Highly Conserved Residues in the Phage PhiC31 Integrase Reveals Key Amino Acids Necessary for the DNA Recombination

Background: Amino acid sequence alignment of phage phiC31 integrase with the serine recombinases family revealed highly conserved regions outside the catalytic domain. Until now, no system mutational or biochemical studies have been carried out to assess the roles of these conserved residues in the recombinaton of phiC31 integrase. Methodology/Principal Findings: To determine the functional roles of these conserved residues, a series of conserved residues were targeted by site-directed mutagenesis. Out of the 17 mutants, 11 mutants showed impaired or no recombination ability, as analyzed by recombination assay both in vivo and in vitro. Results of DNA binding activity assays showed that mutants (R18A, I141A, L143A,E153A, I432A and V571A) exhibited a great decrease in DNA binding affinity, and mutants (G182A/F183A, C374A, C376A/G377A, Y393A and V566A) had completely lost their ability to bind to the specific target DNA attB as compared with wild-type protein. Further analysis of mutants (R18A, I141A, L143A and E153A) synapse and cleavage showed that these mutants were blocked in recombination at the stage of strand cleavage. Conclusions/Significance: This data reveals that some of the highly conserved residues both in the N-terminus and C-terminus region of phiC31 integrase, play vital roles in the substrate binding and cleavage. The cysteine-rich motif and th

Evaluation of fMRI activation in post-stroke patients with movement disorders after repetitive transcranial magnetic stimulation: a scoping review

BackgroundMovement disorders are one of the most common stroke residual effects, which cause a major stress on their families and society. Repetitive transcranial magnetic stimulation (rTMS) could change neuroplasticity, which has been suggested as an alternative rehabilitative treatment for enhancing stroke recovery. Functional magnetic resonance imaging (fMRI) is a promising tool to explore neural mechanisms underlying rTMS intervention.ObjectOur primary goal is to better understand the neuroplastic mechanisms of rTMS in stroke rehabilitation, this paper provides a scoping review of recent studies, which investigate the alteration of brain activity using fMRI after the application of rTMS over the primary motor area (M1) in movement disorders patients after stroke.MethodThe database PubMed, Embase, Web of Science, WanFang Chinese database, ZhiWang Chinese database from establishment of each database until December 2022 were included. Two researchers reviewed the study, collected the information and the relevant characteristic extracted to a summary table. Two researchers also assessed the quality of literature with the Downs and Black criteria. When the two researchers unable to reach an agreement, a third researcher would have been consulted.ResultsSeven hundred and eleven studies in all were discovered in the databases, and nine were finally enrolled. They were of good quality or fair quality. The literature mainly involved the therapeutic effect and imaging mechanisms of rTMS on improving movement disorders after stroke. In all of them, there was improvement of the motor function post-rTMS treatment. Both high-frequency rTMS (HF-rTMS) and low-frequency rTMS (LF-rTMS) can induce increased functional connectivity, which may not directly correspond to the impact of rTMS on the activation of the stimulated brain areas. Comparing real rTMS with sham group, the neuroplastic effect of real rTMS can lead to better functional connectivity in the brain network in assisting stroke recovery.ConclusionrTMS allows the excitation and synchronization of neural activity, promotes the reorganization of brain function, and achieves the motor function recovery. fMRI can observe the influence of rTMS on brain networks and reveal the neuroplasticity mechanism of post-stroke rehabilitation. The scoping review helps us to put forward a series of recommendations that might guide future researchers exploring the effect of motor stroke treatments on brain connectivity

The calpain system is associated with survival of breast cancer patients with large but operable inflammatory and non-inflammatory tumours treated with neoadjuvant chemotherapy

The calpains are a family of intracellular cysteine proteases that function in a variety of important cellular functions, including cell signalling, motility, apoptosis and survival. In early invasive breast cancer expression of calpain-1, calpain-2 and their inhibitor, calpastatin, have been associated with clinical outcome and clinicopathological factors. The expression of calpain-1, calpain-2 and calpastatin was determined using immunohistochemistry on core biopsy samples, in a cohort of large but operable inflammatory and non-inflammatory primary breast cancer patients treated with neoadjuvant chemotherapy. Information on treatment and prognostic variables together with long-term clinical follow-up was available for these patients. Diagnostic pre-chemotherapy core biopsy samples and surgically excised specimens were available for analysis. Expression of calpastatin, calpain-1 or calpain-2 in the core biopsies was not associated with breast cancer specific survival in the total patient cohort; however, in patients with non-inflammatory breast cancer, high calpastatin expression was significantly associated with adverse breast cancer-specific survival (P=0.035), as was low calpain-2 expression (P=0.031). Low calpastatin expression was significantly associated with adverse breast cancer-specific survival of the inflammatory breast cancer patients (P=0.020), as was low calpain-1 expression (P=0.003). In conclusion, high calpain-2 and low calpastatin expression is associated with improved breast cancer-specific survival in non-inflammatory large but operable primary breast cancer treated with neoadjuvant chemotherapy. In inflammatory cases, high calpain-1 and high calpastatin expression is associated with improved breast cancer-specific survival. Determining the expression of these proteins may be of clinical relevance. Further validation, in multi-centre cohorts of breast cancer patients treated with neoadjuvant chemotherapy, is warranted

Effects of WeChat platform-based continuing care on self-management and quality of life in patients with arthritis: A quasi-experimental study

Objective To assess the effects of WeChat platform-based continuing care for arthritis on patients’ self-management, self-efficiency, quality of life (QoL), and medication compliance. Methods A study was conducted on arthritis patients recruited between December 2017 and February 2018 and divided into two groups. The intervention group received continuing care from the WeChat platform and regular follow-ups, while the control group only received regular follow-ups. The outcomes in both groups were assessed using questionnaires twice: before the study (T0) and eight weeks after T0 (T1), which consists of the evaluation of self-management, QoL, self-efficacy, and medication compliance. Results There were 23 people in each of the intervention and control groups completed two outcome measures. At eight weeks, participants in the intervention group showed an improvement in psychological QoL, cognitive symptom management, and self-efficacy, compared to the control group (QoL scores: mean difference in change between groups was 12.29, 95% CI: 4.51, 20.07, p  < 0.001; cognitive symptom management: mean difference in change between groups was 0.65, 95% CI: 0.24, 1.05, p  < 0.001; self-efficacy: mean difference in change between groups was 0.69, 95% CI: 0.14, 1.24, p  < 0.05). Self-management, self-efficacy, and psychological quality of life were significantly improved in the intervention group before and after the intervention ( p  < 0.05). Conclusion Using the WeChat platform for continuing care is useful in improving the psychological state, self-efficacy, and self-management ability of patients with arthritis. The study is relevant to Clinical Practice

Assay of DNA binding activity of mutant phi C31 integrases.

<p>A fixed amount (16 fmol) of a 41 bp biotin -labeled <i>attB</i> fragment (A) or a 50 bp biotin -labeled <i>attP</i> fragment (B) was incubated with a similar amount of purified wild -type and mutant phi C31 in binding buffer at 30°C for 30 min. Results were analyzed by polyacrylamide gel electrophoresis (5% polyacrylamide, 1x Tris-borate-EDTA). Arrows show the positions of free probe (F) and the DNA binding complexes (I, II and III).</p

Recombination assay of the purified wild-type and mutant phiC31 integrases <i>in vitro</i>.

<p>(A) Schematic representation of functional phiC31 integrase assay. The linear 4.9 kb DNA substrate (PB-L) containing correct <i>attB</i> and <i>attP</i> sites was treated with wild-type or mutants phiC31 integrase, resulting in irreversibly produce a circular pBCSK molecule (or pBC-<i>attL</i>) and a 1.4 kb linear <i>attR</i> fragment (<i>attR</i>-L). (B) A 0.04 µM linear DNA substrate (PB-L) was treated with a 0.1 µM purified wild-type or mutant phiC31 integrase in recombination buffer at 30°C for 30 min and then stopped at 75°C for 10 min. The recombination reaction products were separated by electrophoresis on a 1% agarose gel. Arrows show the positions of circular molecule (solid line arrows), and a linear <i>attR</i> fragment (dotted line arrows). Wt is wild-type.</p

Sequence alignment of eight members including phiC31 integrase within large serine integrase family.

<p>The conserved residuals are shown in colour. Positions mutated in this study are indicated by asterisks. Sequence alignment was accomplished by using VNTI 9.0.</p

Recombination assay of phiC31 mutants in <i>E. coli</i>.

<p>Each mutant plasmid and its report plasmid were co-transformed into Top10 bacteria, respectively. The bacteria were then plated to form colonies on double antibiotic selecting agar plates containing X-Gal. When mutant recombinases still have their recombination ability, this recombination event consequently produces white colonies (A). When mutant integrase have lost or reduced recombination ability, It is observed that mixture of white and blue colonies (B) or only blue coloneies (C) on the plates. To further verify site-specific recombination at molecular level, the white colonies were picked, and plasmid DNA was subjected to PCR with specific primers that would amplify a 400-bp product only from the recombinant. DNA from 5 white colonies was subjected to PCR with specific primers, and a 400-bp product got amplified (D). The sequence of this PCR product contained the predicted chimeric <i>attL</i> site, comprising an <i>attP</i> arm (left) and <i>attB</i> arm (right) around the core AA dinucleotides (gray ellipse).</p

Targeted residues and DNA sequence of mutagenic primers of phiC31 integrase.

a<p>Numbers refer to positions in the wild-type 613 amino acid protein.</p>b<p>Relation to a collection of 30 serine recombinases (<a href="http://www.blackwellscience.com/products/journals/suppmat/mole/mole2891/mmi2891sm.htm" target="_blank">http://www.blackwellscience.com/products/journals/suppmat/mole/mole2891/mmi2891sm.htm</a>).</p><p>Mutated bases in the oligonucleotides are shown in bold.</p