Metabolic reprogramming-driven homologous recombination and TCA cycle dysregulation contribute to poor prognoses in lung adenocarcinoma.

Increasing evidence has shown that homologous recombination (HR) and metabolic reprogramming are essential for cellular homeostasis. These two processes are independent as well as closely intertwined. Nevertheless, they have rarely been reported in lung adenocarcinoma (LUAD). We analysed the genomic, immune microenvironment and metabolic microenvironment features under different HR activity states. Using cell cycle, EDU and cell invasion assays, we determined the impacts of si-SHFM1 on the LUAD cell cycle, proliferation and invasion. The levels of isocitrate dehydrogenase (IDH) and α-ketoglutarate dehydrogenase (α-KGDH) were determined by ELISA in the NC and si-SHFM1 groups of A549 cells. Finally, cell samples were used to extract metabolites for HPIC-MS/MS to analyse central carbon metabolism. We found that high HR activity was associated with a poor prognosis in LUAD, and HR was an independent prognostic factor for TCGA-LUAD patients. Moreover, LUAD samples with a high HR activity presented low immune infiltration levels, a high degree of genomic instability, a good response status to immune checkpoint blockade therapy and a high degree of drug sensitivity. The si-SHFM1 group presented a significantly higher proportion of cells in the G0/G1 phase, lower levels of DNA replication, and significantly lower levels of cell migration and both TCA enzymes. Our current results indicated that there is a strong correlation between HR and the TCA cycle in LUAD. The TCA cycle can promote SHFM1-mediated HR in LUAD, raising their activities, which can finally result in a poor prognosis and impair immunotherapeutic efficacy.

Selenoprotein synthesis is not induced by hepatotoxic drugs.

Background and Aims: Many of the proteins that contain the amino acid selenocysteine are required for optimal defense against cellular stress. As such, one might expect selenoprotein synthesis to persist or be induced upon cellular insult. Because selenocysteine is incorporated by a complex post-transcriptional mechanism, monitoring the transcription of selenoprotein genes is not adequate to understand the regulation of selenoprotein synthesis. We aimed to determine whether selenoprotein synthesis is regulated by the induction of hepatotoxic stress. Methods: We used hepatotropic clinically relevant drugs to evaluate the regulation of selenoprotein synthesis in human hepatocarcinoma cells. Results: We found that two drugs, benzbromarone and sorafenib, caused significant inhibition of selenoprotein synthesis. However, the loss of selenoprotein expression was not specific as total protein synthesis was similarly down-regulated only by benzbromarone and sorafenib. Conclusions: These results allow us to conclude that these hepatotoxins do not induce or preserve selenoprotein synthesis as a protective mechanism. Highlights: The treatment of liver cells with hepatotoxic and hepatotropic compounds does not result in increased synthesis of selenoproteins.Compounds that induced the canonical oxidative stress response that features NRF2 activation eliminated selenoprotein synthesis.The downregulation of selenoproteins was accompanied by general inhibition of protein synthesis.

Electrical aligned polyurethane nerve guidance conduit modulates macrophage polarization and facilitates immunoregulatory peripheral nerve regeneration.

Biomaterials can modulate the local immune microenvironments to promote peripheral nerve regeneration. Inspired by the spatial orderly distribution and endogenous electric field of nerve fibers, we aimed to investigate the synergistic effects of electrical and topological cues on immune microenvironments of peripheral nerve regeneration. Nerve guidance conduits (NGCs) with aligned electrospun nanofibers were fabricated using a polyurethane copolymer containing a conductive aniline trimer and degradable L-lysine (PUAT). In vitro experiments showed that the aligned PUAT (A-PUAT) membranes promoted the recruitment of macrophages and induced their polarization towards the pro-healing M2 phenotype, which subsequently facilitated the migration and myelination of Schwann cells. Furthermore, NGCs fabricated from A-PUAT increased the proportion of pro-healing macrophages and improved peripheral nerve regeneration in a rat model of sciatic nerve injury. In conclusion, this study demonstrated the potential application of NGCs in peripheral nerve regeneration from an immunomodulatory perspective and revealed A-PUAT as a clinically-actionable strategy for peripheral nerve injury.

Ultra-High Frequency Self-Focusing Ultrasonic Sensors With Half-Concave Geometry for Visualization of Mouse Brain Atrophy.

Ultra-high frequency (>100 MHz) acoustic waves feature biocompatibility and high sensitivity and allow biomedical imaging and acoustic tweezers. Primarily, excellent spatial resolution and broad bandwidth at ultra-high frequency is the goal for pathological research and cell selection at the cellular level. Here, we propose an efficient approach to visualize mouse brain atrophy by self-focused ultrasonic sensors at ultra-high frequency with ultra-broad bandwidth. The numerical models of geometry and theoretically predicted acoustic parameters for half-concave piezoelectric elements are calculated by the differential method, which agrees with measured results (lateral resolution: 24 µm, and bandwidth: 115% at -6 dB). Compared with the brain slices of 2-month-old mouse, the atrophy visualization of the 6-month-old mouse brain was realized by C-mode imaging with an acoustic microscopy system, which is a potential prospect for diagnosis and treatment of Alzheimer's disease (AD) combined with neuroscience. Meanwhile, the acoustic properties of the brain slices were quantitatively measured by the acoustic microscopy. These encouraging results demonstrate the promising application for high-resolution imaging in vitro biological tissue with ultra-high frequency self-focusing ultrasonic sensors.

Bredigite-Based Bioactive Nerve Guidance Conduit for Pro-Healing Macrophage Polarization and Peripheral Nerve Regeneration.

Structural and functional healing of peripheral nerves damaged by trauma or chronic disease remain major clinical challenges, requiring the development of an effective nerve guidance conduit (NGC). The present study investigates a NGC fabrication strategy based on bredigite (BRT, Ca7MgSi4O16) bioceramic for the treatment of peripheral nerve injury. Here, BRT bioceramic shows good biocompatibility and sustainable release of Ca2+, Mg2+, and Si4+ ions. Both BRT extracts and BRT-incorporating electrospun membranes promote the proliferation and myelination potential of RSC96 cells, as well as accelerate vascular formation by human umbilical vein endothelial cells. Notably, BRT facilitates RAW 264.7 cell polarization to the pro-healing phenotype under LPS-induced inflammatory stimulation. More importantly, the macrophages activated by BRT in turn promote RSC96 cell migration and remyelination. In a rat sciatic nerve defect model, improved electrophysiological performance and alleviated gastrocnemius muscle atrophy are observed at 12 weeks post-implantation. Further experiments verify that BRT-loaded NGC facilitates axonal regrowth and revascularization with high M2-like macrophage infiltration. These findings support the beneficial effects of BRT for creating a pro-healing immune microenvironment and orchestrating multicellular processes associated with functional nerve regeneration, indicating the potential of rationally engineered bioceramics as safe, effective, and economical materials for peripheral nerve repair.

Activation of N-Methyl-D-aspartate receptor contributed to the ultrasonic modulation of neurons in vitro.

Ultrasound stimulation is increasingly used to investigate brain function and treat brain diseases due to its high level of safety and precise spatiotemporal resolution. Therefore, it is crucial to understand the underlying mechanisms involved in ultrasound brain stimulation. In this study, we investigate the role of NMDA receptors in mediating the effects of ultrasound on primary hippocampal neurons in mice. Our results show that ultrasound alone can activate heterologous NMDA receptor subunits, including NR1A, NR2A, and NR2B, in 293T cells, as well as endogenous NMDA receptors in primary neurons. This activation leads to an influx of calcium and an increase in nuclear c-Fos expression in primary neurons that have not been pre-treated with an NMDA receptor inhibitor. In conclusion, our findings demonstrate that NMDA receptors contribute to neuronal activation by ultrasound stimulation in vitro, providing insight into the molecular mechanisms of ultrasound neuromodulation and a new mediator for the sonogenetics technique.

Decellularized periosteum promotes guided bone regeneration via manipulation of macrophage polarization.

Periosteum has shown potential as an effective barrier membrane for guided bone regeneration (GBR). However, if recognized as a "foreign body," insertion of a barrier membrane in GBR treatment will inevitably alter the local immune microenvironment and subsequently influence bone regeneration. The aim of this investigation was to fabricate decellularized periosteum (DP) and investigate its immunomodulatory properties in GBR. DP was successfully fabricated from periosteum from the mini-pig cranium. In vitro experiments indicated that the DP scaffold modulated macrophage polarization toward a pro-regenerative M2 phenotype, which in turn facilitated migration and osteogenic differentiation of bone marrow-derived mesenchymal stem cells. A rat GBR model with a cranial critical-size defect was established, and our in vivo experiment confirmed the beneficial effects of DP on the local immune microenvironment and bone regeneration. Collectively, the findings of this study indicate that the prepared DP possesses immunomodulatory properties and represents a promising barrier membrane for GBR procedures.

Li-Mg-Si bioceramics provide a dynamic immuno-modulatory and repair-supportive microenvironment for peripheral nerve regeneration.

Biomaterials can modulate the local immune and repair-supportive microenvironments to promote peripheral nerve regeneration. Inorganic bioceramics have been widely used for regulating tissue regeneration and local immune response. However, little is known on whether inorganic bioceramics can have potential for enhancing peripheral nerve regeneration and what are the mechanisms underlying their actions. Here, the inorganic lithium-magnesium-silicon (Li-Mg-Si, LMS) bioceramics containing scaffolds are fabricated and characterized. The LMS-containing scaffolds had no cytotoxicity against rat Schwann cells (SCs), but promoted their migration and differentiation towards a remyelination state by up-regulating the expression of neurotrophic factors in a ß-catenin-dependent manner. Furthermore, using single cell-sequencing, we showed that LMS-containing scaffolds promoted macrophage polarization towards the pro-regenerative M2-like cells, which subsequently facilitated the migration and differentiation of SCs. Moreover, implantation with the LMS-containing nerve guidance conduits (NGCs) increased the frequency of M2-like macrophage infiltration and enhanced nerve regeneration and motor functional recovery in a rat model of sciatic nerve injury. Collectively, these findings indicated that the inorganic LMS bioceramics offered a potential strategy for enhancing peripheral nerve regeneration by modulating the immune microenvironment and promoting SCs remyelination.

The mechanosensitive ion channel Piezo1 contributes to ultrasound neuromodulation.

Transcranial low-intensity ultrasound is a promising neuromodulation modality, with the advantages of noninvasiveness, deep penetration, and high spatiotemporal accuracy. However, the underlying biological mechanism of ultrasonic neuromodulation remains unclear, hindering the development of efficacious treatments. Here, the well-known Piezo1 was studied through a conditional knockout mouse model as a major mediator for ultrasound neuromodulation ex vivo and in vivo. We showed that Piezo1 knockout (P1KO) in the right motor cortex of mice significantly reduced ultrasound-induced neuronal calcium responses, limb movement, and muscle electromyogram (EMG) responses. We also detected higher Piezo1 expression in the central amygdala (CEA), which was found to be more sensitive to ultrasound stimulation than the cortex was. Knocking out the Piezo1 in CEA neurons showed a significant reduction of response under ultrasound stimulation, while knocking out astrocytic Piezo1 showed no-obvious changes in neuronal responses. Additionally, we excluded an auditory confound by monitoring auditory cortical activation and using smooth waveform ultrasound with randomized parameters to stimulate P1KO ipsilateral and contralateral regions of the same brain and recording evoked movement in the corresponding limb. Thus, we demonstrate that Piezo1 is functionally expressed in different brain regions and that it is an important mediator of ultrasound neuromodulation in the brain, laying the ground for further mechanistic studies of ultrasound.

Surgical Management for Vertical Maxillary Excess.

Orthognathic surgery is an effective approach to correct vertical maxillary excess (VME), which is a common maxillofacial deformity and exhibits excessive vertical development of maxilla. This review summarizes different clinical features of total, anterior and posterior VME, as well as corresponding surgical managements guided by preoperative computer-assisted surgical planning. The virtual simulation will do favor to the final determination of individual surgical plans to achieve satisfactory outcomes. Finally, a typical clinical case will be presented to demonstrate the surgical management of VME.

Potassium channel-related genes are a novel prognostic signature for the tumor microenvironment of renal clear cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) accounts for 80% of renal cell carcinomas (RCCs), and its morbidity and prognosis are unfavorable. Surgical resection is the first-line treatment for ccRCC, but the oncogenesis of ccRCC is very complex. With the development of high-throughput sequencing technology, it is necessary to analyze the transcriptome to determine more effective treatment methods. The tumor microenvironment (TME) is composed of tumor cells, various immune-infiltrating cells, fibroblasts, many cytokines, and catalysts. It is a complex system with a dynamic balance that plays an essential role in tumor growth, invasion, and metastasis. Previous studies have confirmed that potassium channels can affect the immune system, especially T lymphocytes that require potassium channel activation. However, the effect of potassium channels on the TME of ccRCC remains to be studied. Therefore, this study aims to construct a prognostic signature for ccRCC patients based on potassium ion channel-related genes (PCRGs), assess patient risk scores, and divide patients into high- and low-risk groups based on the cutoff value. In addition, we investigated whether there were differences in immune cell infiltration, immune activator expression, somatic mutations, and chemotherapeutic responses between the high- and low-risk groups. Our results demonstrate that the PCRG signature can accurately assess patient prognosis and the tumor microenvironment and predict chemotherapeutic responses. In summary, the PCRG signature could serve as an auxiliary tool for the precision treatment of ccRCC.

Photonic Nanojet-Mediated Optogenetics.

Optogenetics has become a widely used technique in neuroscience research, capable of controlling neuronal activity with high spatiotemporal precision and cell-type specificity. Expressing exogenous opsins in the selected cells can induce neuronal activation upon light irradiation, and the activation depends on the power of incident light. However, high optical power can also lead to off-target neuronal activation or even cell damage. Limiting the incident power, but enhancing power distribution to the targeted neurons, can improve optogenetic efficiency and reduce off-target effects. Here, the use of optical lenses made of polystyrene microspheres is demonstrated to achieve effective focusing of the incident light of relatively low power to neighboring neurons via photonic jets. The presence of microspheres significantly localizes and enhances the power density to the target neurons both in vitro and ex vivo, resulting in increased inward current and evoked action potentials. In vivo results show optogenetic stimulation with microspheres that can evoke significantly more motor behavior and neuronal activation at lowered power density. In all, a proof-of-concept of a strategy is demonstrated to increase the efficacy of optogenetic neuromodulation using pulses of reduced optical power.

Mutational analysis reveals potential phosphorylation sites in eukaryotic elongation factor 1A that are important for its activity.

Previous studies have suggested that phosphorylation of translation elongation factor 1A (eEF1A) can alter its function, and large-scale phospho-proteomic analyses in Saccharomyces cerevisiae have identified 14 eEF1A residues phosphorylated under various conditions. Here, a series of eEF1A mutations at these proposed sites were created and the effects on eEF1A activity were analyzed. The eEF1A-S53D and eEF1A-T430D phosphomimetic mutant strains were inviable, while corresponding alanine mutants survived but displayed defects in growth and protein synthesis. The activity of an eEF1A-S289D mutant was significantly reduced in the absence of the guanine nucleotide exchange factor eEF1Bα and could be restored by an exchange-deficient form of the protein, suggesting that eEF1Bα promotes eEF1A activity by a mechanism other than nucleotide exchange. Our data show that several of the phosphorylation sites identified by high-throughput analysis are critical for eEF1A function.

Upper Airway Changes After Mandibular Setback and/or Advancement Genioplasty in Obese Patients.

PURPOSE: Orthognathic surgeries, such as bilateral sagittal split ramus osteotomy (BSSO) and genioplasty, can influence the pharyngeal airway space (PAS) and this has been supported by previous studies. The purpose of this study was to assess changes of the PAS in patients with a high body mass index (BMI) likely to have narrow airways before and after setback BSSO with or without advancement genioplasty surgery by 3-dimensional computed tomography. MATERIALS AND METHODS: Thirty-five adults with a BMI of at least 24.0 kg/m2 were treated from 2010 to 2016. Samples were grouped mandibular setback (group A; n = 11), advancement genioplasty (group B; n = 12), and mandibular setback plus advancement genioplasty (group C; n = 12). Computed tomograms were obtained 1 week preoperatively (T0), 1 week postoperatively (T1), and at least 1 year postoperatively (T2). The area of the posterior nasal spine and posterior plane (PPA), the soft palate plane (SPA), the plane of the most posterior point of the tongue base (PTA), the plane of the root of the epiglottis (EA), and the volumes of the palatopharyngeal part (VP), oropharyngeal part (VO), glossopharyngeal part (VG), and laryngeal part (VL) were measured and compared within groups using analysis of variance. The P value was set at .05. RESULTS: In group A, all results showed statistically significant differences (P < .05) from T0 to T2 except for VO, VG, VL, SPA, PTA, and EA. In group B, VO, VG, VL, SPA, PTA, and EA showed statistically significant increases (P < .05) from T0 to T2. The hyoid at T2 showed significant advancement compared with T0 (P < .05). In group C, there were statistically significant decreases (P < .05) from T0 to T1 for VG, VL, PTA, and EA. CONCLUSION: In adults with a high BMI, mandibular setback BSSO could decrease the PAS, whereas advancement genioplasty could enlarge the PAS, after surgery. Therefore, undergoing advancement genioplasty concurrently with mandibular setback BSSO could help in lessening the negative effects of a PAS decrease.

Histopathologic comparison of condylar hyperplasia and condylar osteochondroma by using different staining methods.

OBJECTIVE: The objectives of this study were to investigate the application and differential diagnostic value of safranin O staining, safranin-fast green staining, and Runt-related transcription factor 2 (Runx2) immunohistochemistry with regard to condylar hyperplasia and condylar osteochondroma. STUDY DESIGN: Histopathologic presence was evaluated by using hematoxylin and eosin staining, safranin O staining, safranin-fast green staining, and immunohistochemistry of Runx2 in postoperative specimens of normal condyle (control), condylar hyperplasia, and condylar osteochondroma. RESULTS: Safranin O staining clearly highlighted the tissue structure of the condylar cartilage, especially the hypertrophic layer. The safranin-fast green method showed a contrast in staining between cartilage and subchondral cancellous bone in the condyle specimens. Both methods were better than hematoxylin and eosin staining for morphologically distinguishing condylar hyperplasia and condylar osteochondroma. The expression of Runx2 in condylar hyperplasia was significantly greater than that in condylar osteochondroma. CONCLUSIONS: This study indicated that safranin O staining and safranin-fast green staining are effective staining methods to differentiate between condylar hyperplasia and condylar osteochondroma. Immunohistochemistry findings suggested that Runx2 is valuable in the differential diagnosis of these two diseases.

SEMA3A suspended in matrigel improves titanium implant fixation in ovariectomized rats.

The aim of this study was to evaluate the effect of SEMA3A released from matrigel on implant fixation in ovariectomized (OVX) rats. Sixty female rats were subjected to bilateral ovariectomy. Twelve weeks later, rats were randomly divided into three groups according to implants they accepted: (1) Control, implants with distilled water; (2) Matrigel, implants with matrigel coating; (3) Matrigel + SEMA3A, implants with coating of SEMA3A suspended in matrigel. Implants were inserted in metaphysis of proximal tibiae in all animas bilaterally. In vitro release of SEMA3A was tested using enzyme linked immunosorbent assay. In vitro release of SEMA3A was detectable during the first 10 days, and a burst release of was observed during the first 3 days. No significant difference was observed between Control and Matrigel group. The protective effects of SEMA3A in matrigel on peri-implant bone, implant osseointegration and fixation was confirmed. Compared to matrigel alone, SEMA3A suspended in matrigel increased percent bone volume by 88.7% and 83.3% (p < 0.01), bone-to-implant contact ratio by 148.9% (p < 0.01), and 24.8% (p < 0.05), the maximal push-out force by 149.3% and 209.2% (p < 0.01) at 4 and 8 weeks after implant insertion, respectively. Surface modification with SEMA3A suspended in matrigel improved implant osseointegration and fixation in the proximal tibiae of OVX rats. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2060-2065, 2017.

Promotion of regulatory T cell induction by immunomodulatory herbal medicine licorice and its two constituents.

Regulatory T cells (Treg) play a critical role to control immune responses and to prevent autoimmunity, thus selective increase of Treg cells in vivo has broad therapeutic implications for autoimmune and inflammatory diseases. Licorice is a well-known herbal medicine used worldwide for over thousands of years, and accumulating evidence has shown its immunomodulatory potential. However, it is not clear whether licorice could regulate the induction and function of Treg cells. Here we found licorice extract could promote Treg cell induction, and then we used a rational approach to isolate its functional fractions and constituents. The results showed that two constituents, isoliquiritigenin and naringenin, promoted Treg cell induction both in vitro and in vivo. The effective fractions and two constituents of licorice also enhanced immune suppression of Treg cells, and they further reduced severity of DSS-induced colitis in mice. This study suggested that promotion of regulatory T cell induction could be an underlying mechanism of the historically and widely used herbal medicine licorice, providing its two effective molecules against autoimmune and inflammatory diseases.

Eukaryotic polyribosome profile analysis.

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase or the elongation phase by a variety of cellular stresses such as amino acid starvation or growth factor withdrawal. Alternatively, translation of individual mRNAs can be regulated by mRNA localization or the presence of cognate microRNAs. Studies of protein synthesis frequently utilize polyribosome analysis to shed light on the mechanisms of translation regulation or defects in protein synthesis. In this assay, mRNA/ribosome complexes are isolated from eukaryotic cells. A sucrose density gradient separates mRNAs bound to multiple ribosomes known as polyribosomes from mRNAs bound to a single ribosome or monosome. Fractionation of the gradients allows isolation and quantification of the different ribosomal populations and their associated mRNAs or proteins. Differences in the ratio of polyribosomes to monosomes under defined conditions can be indicative of defects in either translation initiation or elongation/termination. Examination of the mRNAs present in the polyribosome fractions can reveal whether the cohort of individual mRNAs being translated changes with experimental conditions. In addition, ribosome assembly can be monitored by analysis of the small and large ribosomal subunit peaks which are also separated by the gradient. In this video, we present a method for the preparation of crude ribosomal extracts from yeast cells, separation of the extract by sucrose gradient and interpretation of the results. This procedure is readily adaptable to mammalian cells.