Timed material self-assembly controlled by circadian clock proteins.

Active biological molecules present a powerful, yet largely untapped, opportunity to impart autonomous regulation to materials. Because these systems can function robustly to regulate when and where chemical reactions occur, they have the ability to bring complex, life-like behavior to synthetic materials. Here, we achieve this design feat by using functionalized circadian clock proteins, KaiB and KaiC, to engineer time-dependent crosslinking of colloids. The resulting material self-assembles with programmable kinetics, producing macroscopic changes in material properties, via molecular assembly of KaiB-KaiC complexes. We show that colloid crosslinking depends strictly on the phosphorylation state of KaiC, with kinetics that are synced with KaiB-KaiC complexing. Our microscopic image analyses and computational models indicate that the stability of colloidal super-structures depends sensitively on the number of Kai complexes per colloid connection. Consistent with our model predictions, a high concentration stabilizes the material against dissolution after a robust self-assembly phase, while a low concentration allows circadian oscillation of material structure. This work introduces the concept of harnessing biological timers to control synthetic materials; and, more generally, opens the door to using protein-based reaction networks to endow synthetic systems with life-like functional properties.

The Disruption of Mage-11 Gene via CRISPR/Cas9 Method Induced Apoptosis in the in vitro Model of Prostate Cancer.

BACKGROUNDS AND AIMS: Prostate cancer is the most common malignant cancer among men and is the second deadliest cancer in men after lung cancer. Understanding the molecular mechanisms involved in development and progression of prostate cancer is essential to improve both diagnostic and therapeutic strategies in this regard. In addition, using novel gene therapy-based methods for treatment of cancers has gotten increasing attention during the recent years. Accordingly, this study was aimed to evaluate the inhibitory effect of MAGE-A11 gene, as an important oncogene involved in the pathophysiology of prostate cancer invitro model. The study was also aimed to evaluate the downstream genes related to MAGE-A11. MATERIALS AND METHODS: First, MAGE-A11 gene was knocked out in PC-3 cell line using "Clustered regularly interspaced short palindromic repeats" (CRISPR)/ "CRISPR-associated genes 9" (CRISPR/Cas9) method. Next, the expression levels of MAGE-A11, survivin and Ribonucleotide Reductase Small Subunit M2 (RRM2) genes were determined by quantitative polymerase chain reaction (qPCR) technique. The levels of proliferation and apoptosis were also analyzed in PC-3 cells using CCK-8 and Annexin V-PE/7-AAD assays. RESULTS: The results showed that the disruption of MAGE-A11 by CRISPR/Cas9 method significantly decreased proliferation (P< 0.0001) and enhanced apoptosis (P< 0.05) in PC-3 cells compared to control group. Moreover, the disruption of MAGE-A11 significantly down regulated the expression levels of survivin and RRM2 genes (P< 0.05). CONCLUSION: Our results demonstrated that knocking out MAGE-11 gene by CRISPR/CAS9 technique could efficiently inhibit cell proliferation and induce apoptosis in PC3 cells. Survivin and RRM2 genes might also participated in these processes.

An Easy-to-Use Platform for Reporting COVID-19 Patients by Private Offices and Clinics Without IT Support: A Pilot Study.

BACKGROUND: It is feasible to collect data rapidly and online using IT solutions. OBJECTIVES: To present a data collection platform for COVID-19 suspected patients in private offices and clinics without a standard software. METHODS: The proposed system for collecting and sharing data of patients with respiratory symptoms was designed to be simple to use, without the need for special technology, and with proper security to authenticate reporters. RESULTS: Two methods were developed to collect data from private physicians and offices. Finally, the data collected by both approaches is integrated and provided to primary healthcare staff to arrange appropriate healthcare measures. CONCLUSION: Our platform can provide an easy-to-use case reporting system for private physicians.

The effects of supplemented sericin on in vitro maturation and preimplantation development of mouse embryos: An experimental study.

BACKGROUND: Mouse embryo culture condition is an essential part of transgenic, reproductive and developmental biology laboratories. Mouse embryonic culture media may have a high risk of serum contamination with pathogens. OBJECTIVE: To investigate the effect of sericin as an embryo culture medium supplement on in vitro maturation (IVM), in vitro fertilization (IVF), and development of the preimplantation embryo in mice. MATERIALS AND METHODS: The effects of sericin at three concentrations (subgroups) of 0.1%, 0.5%, and 1% as a medium supplement on IVM, IVF, and in vitro development of mouse embryos were separately investigated and compared with a sericin-free (control) group. The cumulative effect of the three concentrations was evaluated for IVM + in vitro development and IVF + in vitro development as follow-up groups. RESULTS: In the IVM group, compared to the control group, the number of oocysts reaching the MII stage was significantly higher when 1% sericin was used (161/208 = 77.4%). No significant results were observed in the IVF and in vitro development groups with different concentrations of sericin compared to the control group. Among the follow-up groups, in the IVM + in vitro development group, the number of oocytes was higher after passing the IVM and IVF and reaching the blastocysts stage when 1% sericin was used, compared with other sericin subgroups. A significant difference was also noted when compared with the control group (p = 0.048). The IVF + in vitro development study group, on the other hand, did not show any significant relationship. CONCLUSION: It can be concluded that 1% sericin can be used as a supplement in mouse embryo cultures to improve the IVM rate. Also, based on the findings, sericin appears to be an effective supplement which can have a positive effect on the development of embryos derived from IVM.

Contribution of toll-like receptor 2 and nicotinamide adenine dinucleotide phosphate oxidase to the trimethylamine N-oxide-induced inflammatory reactions in U937-derived macrophages.

BACKGROUND: Trimethylamine N-oxide (TMAO) is emerging as a new generation of metabolites related to the activation of inflammatory reactions in the macrophages during atherosclerosis. Stress-activation of cell surface toll-like receptors (TLRs) as well as nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOX) is also assumed to be involved in TMAO-induced inflammatory reaction in the macrophages. To elucidate the possible contribution of TLRs and NOX to the mentioned signaling pathway, we aimed to simultaneously evaluate the expression level of TLR2, TLR6, and NOX2 in TMAO-treated macrophages. METHODS: 2.5 × 106 cells of U937-derived macrophages were treated in triplicates with different concentrations (37.5, 75, 150, and 300 µM) of TMAO for 24 hours. The cells were also treated with tunicamycin (TUN), as a positive control of stress. Normal control group (CTR) cells received no treatment. The viability of treated cells was checked by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole (MTT) assay. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was also used to evaluate the relative expression (fold change) of TLR2, TLR6, and NOX2 at messenger ribonucleic acid (mRNA) levels. One-way analysis of variance (ANOVA) with post-hoc Dunnett's test was performed to compare every mean with that of the control. RESULTS: No cell death occurred because of treatments. Dose of 300 µM of TMAO significantly increased the relative expression of both TLR2 and NOX2 compared to the CTR cells (P < 0.001 for both). The elevation of TLR6 was not statistically significant in all groups of TMAO-treated cells (P > 0.050). CONCLUSION: Our results provide documentation supporting contribution of TLR2 and NOX2 to previously described inflammatory reactions induced by TMAO in macrophages. In addition, they may clarify the proatherogenic role of TMAO in foam cell formation as well as abnormal activation of macrophages during atherosclerosis.

The potential role of HLA-G in the pathogenesis of HBV infection: Immunosuppressive or immunoprotective?

The non-classical human leukocyte antigens (HLA)-G could be generally considered as a potent tolerogenic molecule, which modulates immune responses. HLA-G due to the immunosuppressive properties may play an important role in the pathogenesis of infections related to the liver. HLA-G may display two distinct activities in the pathological conditions so that it could be protective in the autoimmune and inflammatory diseases or could be suppressive of the immune system in the infections or cancers. HLA-G might be used as a novel therapeutic target for liver diseases in the future. Indeed, new therapeutic agents targeting HLA-G expression or antibodies which block HLA-G activity are being developed and tested. However, further consideration of the HLA-G function in liver disease is required. This review aims to summarize the role of HLA-G in the liver of patients with HBV infection.

Actin and microtubule crosslinkers tune mobility and control co-localization in a composite cytoskeletal network.

Actin and microtubule filaments, with their auxiliary proteins, enable the cytoskeleton to carry out vital processes in the cell by tuning the organizational and mechanical properties of the network. Despite their critical importance and interactions in cells, we are only beginning to uncover information about the composite network. The challenge is due to the high complexity of combining actin, microtubules, and their hundreds of known associated proteins. Here, we use fluorescence microscopy, fluctuation, and cross-correlation analysis to examine the role of actin and microtubules in the presence of an antiparallel microtubule crosslinker, MAP65, and a generic, strong actin crosslinker, biotin-NeutrAvidin. For a fixed ratio of actin and microtubule filaments, we vary the amount of each crosslinker and measure the organization and fluctuations of the filaments. We find that the microtubule crosslinker plays the principle role in the organization of the system, while, actin crosslinking dictates the mobility of the filaments. We have previously demonstrated that the fluctuations of filaments are related to the mechanics, implying that actin crosslinking controls the mechanical properties of the network, independent of the microtubule-driven re-organization.

Non-monotonic dependence of stiffness on actin crosslinking in cytoskeleton composites.

The cytoskeleton is able to precisely tune its structure and mechanics through interactions between semiflexible actin filaments, rigid microtubules and a suite of crosslinker proteins. However, the role that each of these components, as well as the interactions between them, plays in the dynamics of the composite cytoskeleton remains an open question. Here, we use optical tweezers microrheology and fluorescence confocal microscopy to reveal the surprising ways in which actin crosslinking tunes the viscoelasticity and mobility of actin-microtubule composites from steady-state to the highly nonlinear regime. While previous studies have shown that increasing crosslinking in actin networks increases elasticity and stiffness, we instead find that composite stiffness displays a striking non-monotonic dependence on actin crosslinking - first increasing then decreasing to a response similar to or even lower than un-linked composites. We further show that actin crosslinking has an unexpectedly strong impact on the mobility of microtubules; and it is in fact the microtubule mobility - dictated by crosslinker-driven rearrangements of actin filaments - that controls composite stiffness. This result is at odds with conventional thought that actin mobility drives cytoskeleton mechanics. More generally, our results demonstrate that - when crosslinking composite materials to confer strength and resilience - more is not always better.

Varying crosslinking motifs drive the mesoscale mechanics of actin-microtubule composites.

The cytoskeleton precisely tunes its mechanics by altering interactions between semiflexible actin filaments, rigid microtubules, and crosslinking proteins. We use optical tweezers microrheology and confocal microscopy to characterize how varying crosslinking motifs impact the mesoscale mechanics and mobility of actin-microtubule composites. We show that, upon subtle changes in crosslinking patterns, composites can exhibit two distinct classes of force response - primarily elastic versus more viscous. For example, a composite in which actin and microtubules are crosslinked to each other but not to themselves is markedly more elastic than one in which both filaments are independently crosslinked. Notably, this distinction only emerges at mesoscopic scales in response to nonlinear forcing, whereas varying crosslinking motifs have little impact on the microscale mechanics and mobility. Our unexpected scale-dependent results not only inform the physics underlying key cytoskeleton processes and structures, but, more generally, provide valuable perspective to materials engineering endeavors focused on polymer composites.

Forearm as a valuable source of vascularized bone graft for the distal humerus.

BACKGROUND: The nonunion of a distal humeral fracture is a challenging complication. Our aim was to assess the results of the application of pedicled bone flap to treat this problem. METHODS: There were 6 men and 3 women with delayed union or nonunion of the distal humerus treated by 10 procedures of rigid fixation plus radial bone forearm or posterior interosseous bone flaps. A locking plate was applied for fixation in 4 patients and a pin and plate for 5 patients. The olecranon osteotomy was performed in 6 elbows. RESULTS: Patients were a mean age of 49.2 years (range, 31-70 years). The average interval between the trauma and the index operation was 19.2 months (range, 3-49 months). Eight elbows showed union within an average of 3.75 months (range, 3-6 months) in the column where the bony flap was placed. The radial forearm bone flap failed to unite the nonunion of the lateral column of 1 elbow, and additional tension banding was required. Two elbows did not heal at the medial column when the radial forearm bone flap was located on the lateral column. For 1 elbow, another posterior interosseous bone flap was applied on the medial side in another session. By a mean follow-up of 37.3 months (range, 24-79 months), the mean of the visual analog scale demonstrated significant improvement from 6.44 postoperatively to 2.22 (P < .001). The Mayo Elbow Performance Score and 11-Item version of the Disabilities of the Arm, Shoulder and Hand score also showed clinically and statistically significant improvements. CONCLUSION: Vascularized bone flaps could be considered as an option to facilitate healing of distal humerus delayed union or nonunion.

Treatment of Nonunion of Forearm Bones Using Radial Forearm Bone Flap.

BACKGROUND: Treatment of forearm fracture nonunion is challenging. Several surgical techniques for the treatment of forearm nonunion have been reported. OBJECTIVES: The aim of this prospective study was to evaluate the effects of the radial forearm bone graft technique in cases with forearm nonunion. PATIENTS AND METHODS: We treated 7 patients with forearm nonunions via radial forearm bone flap and internal plate fixation. On the preoperative examination and last follow-up, the quick disabilities of the arm, shoulder, and hand (Q-DASH) score, elbow, forearm and wrist arches of motion and grip force were measured. At the final follow-up, radiographic union and patient satisfaction and return to work were assessed. RESULTS: The mean follow-up duration was 34 ± 23 months. The Q-DASH scores improved from 70 ± 6 points preoperatively to 13 ± 15 points at final follow-up (P < 0.01). The preopetative range of motion (ROM) was 101 ± 26 degrees; whereas the final forearm ROM was 140 ± 17 (P = 0.01). All nonunions united uneventfully (range 3-6 months). At the last follow-up, the grip force was 83% of uninjured extremity and all patients were satisfied with the outcome of the operation. CONCLUSIONS: Radial forearm bone flap is a safe, useful and novel technique in cases with forearm nonunion.

Low Concentrations of Flavonoid - Rich Fraction of Shallot Extract Induce Delayed - Type Hypersensitivity and TH1 Cytokine IFNγ Expression in BALB/c Mice.

Flavonoids are potentially immunomodulatory factors and it may be inferred that these phytochemicals contribute to immunomodulatory properties of the Allium family. In the present study, we investigated the potential mechanism underlying the immunomodulatory effect of shallot and its ethyl acetate (EA) fraction as flavonoid-rich sources. Ex vivo, effects of a hydroalcoholic extract of shallot, its fractions and quercetin on lymphocyte viability were evaluated. The proliferative effects of the fractions were examined using naive mouse lymphocytes to determine the fraction with highest impact/ activity. In addition, in a mouse model, both delayed- type hypersensitivity (DTH) responses and production of a key cytokine (interferon [IFN]-ᵧ) were evaluated. Both the shallot extract and its fractions inhibited lymphocytes cell growth and survival in a concentration- dependent manner. The findings also showed that the extract and especially the ethyl acetate (EA) fraction could induce lymphocyte proliferation. The evaluation of the extract and its EA fraction on DTH responses indicated that both caused a significant increase in DTH response. Furthermore, they triggered significant increases in IFNγ and decreases in interleukin (IL)-4 production by splenic mononuclear cells. Because of the significant immunomodulatory activity displayed in these studies, it is plausible that shallot could have a potential use as an immunomodulatory agent in clinical settings.

Treatment of nonunion of the forearm bones with posterior interosseous bone flap.

BACKGROUND: Reconstructions of forearm fracture nonunions are frequently complex. A few studies that help guide the treatment of forearm nonunion have been reported. We offer a novel surgical technique to treat nonunion of the forearm using a regional vascularized bone graft. METHODS: Four females and 5 males ranging from 27 to 74 years of age with 7 ulna and 2 radius nonunions were surgically treated by pedicle grafting with a posterior interosseous bone flap (PIBF) and internal fixation with a plate. There were no cases with extensive soft tissue damage or infection. Pre- and postoperatively (mean 21 months), all patients were assessed by radiographs and for function by the Disabilities of the Arm, Shoulder and Hand (DASH) score. RESULTS: Function of the upper extremity was improved in all patients. DASH scores improved from 61.2 points pre-operatively to 12 points at final follow-up. All nonunions were united uneventfully. CONCLUSIONS: Following debridement of the necrotic tissue, the bone defect can be filled with a vascularized graft from posterior interosseous pedicles. Pedicled PIBF is a safe and useful novel technique in cases of atrophic or hypertrophic nonunion of the middle third of the radius or proximal two-thirds of the ulna.