Silk Fibroin-Based Tough Hydrogels with Strong Underwater Adhesion for Fast Hemostasis and Wound Sealing.

Rapid and strong adhesion of hydrogel adhesives is required for instant wound closure and hemostasis. However, in situ hydrogel formation and sufficient adhesion at target tissue sites in biological environments are severely compromised by the presence of blood and body fluids. In this work, an underwater adhesive hydrogel (named SHCa) is fabricated with rapid in situ gelation, enhanced mechanical toughness, and robust underwater adhesion. The SHCa can undergo rapid UV irradiation-induced gelation under water within 5 s and adhere firmly to underwater surfaces for 6 months. The synergistic effects of crystalline ß-sheet structures and dynamic energy-dissipating mechanisms enhance the mechanical toughness and cohesion, supporting the balance between adhesion and cohesion in wet environments. Importantly, the SHCa can achieve rapid in situ gelation and robust underwater adhesion at various tissue surfaces in highly dynamic fluid environments, substantially outperforming the commercially available tissue adhesives. The lap shear adhesion strength and wound closure strength of SHCa on blood-covered substrates are 7.24 and 12.68 times higher than those of cyanoacrylate glue, respectively. Its fast hemostasis and wound sealing performance are further demonstrated in in vivo animal models. The proposed hydrogel with strong underwater adhesion provides an effective tool for fast wound closure and hemostasis.

Stepwise versus Concerted: Theoretical Insights into the Stereoselectivity in Aryl Imine Formation Assisted by Acid and Water.

The wide applications of the aryl Schiff base require extensive understanding of the mechanism of its formation, which remains unclear. In this work, the detailed formation mechanisms between benzaldehyde and aniline or 4-(9-anthryl) ethynyl aniline were investigated at the CCSD(T)//B3LYP level, and the influence of water molecules and acid catalysis and the stereoselectivity were addressed. The results show that the participation of explicit water molecules greatly accelerates the reactions by alleviating the ring tension of the transition states, and acid catalysis strongly favors the imine formation and provides driving force for the forward reaction. In acidic conditions, both N-protonated carbinolamine formations and imine formations are achieved under mild conditions with the assistance of water molecules, and the proton transfer is more advanced than the C-N and C═N bond formation, which is in good agreement with the experimental observations. In contrast, under neutral conditions, even with the assistance of two water molecules, the reaction is hard to take place at room temperature owing to the high Gibbs free energy barriers with the proton transfer and the C-N or C═N bond formation concerted. The analysis of stereoselectivity shows that the formation of trans imine is both kinetically and thermodynamically more favorable than the cis one under the acidic condition with the assistance of water molecules, and the presence of conjugated substituent 4-(9-anthryl) ethynyl of aniline marginally raises the energy barriers. This work provides a systematic view of the mechanism for the formation of aryl imine and is expected to offer insights for the control of the dynamic covalent chemistry and the synthesis of covalent organic frameworks.

Does joint-sparing tumor resection jeopardize oncologic and functional outcomes in non-metastatic high-grade osteosarcoma around the knee?

BACKGROUND: We previously reported joint-sparing tumor resection for osteosarcoma with epiphyseal involvement in which transepiphyseal osteotomy went through the in situ ablated epiphysis. However, we do not know whether this is a safe approach when compared with joint-sacrificed tumor resection. Our objective was to compare oncologic and functional outcomes between patients who underwent joint preservation (JP) and joint replacement (JR) tumor resection. Furthermore, we identified the risk factors of local recurrence, metastasis and survival. METHODS: Eighty-nine patients with non-metastatic high-grade osteosarcoma around the knee were treated with limb-salvage surgery (JP in 47 and JR in 42). Age, gender, tumor location, pathologic fracture, plain radiographic pattern, limb diameter change, perivascular space alteration, surgical margin, local recurrence, metastasis, death, and the Musculoskeletal Tumor Society (MSTS)-93 scores were extracted from the records. Univariate analysis was performed to compare oncologic and functional outcomes. Binary logistic and cox regression models were used to identify predicted factors for local recurrence, metastasis, and survival. RESULTS: Local recurrence, metastasis and overall survival were similar in the JP and JR group (p = 0.3; p = 0.211; p = 0.143). Major complications and limb survival were also similar in the JR and JP group (p = 0.14; p = 0.181). The MSTS score of 27.06 ± 1.77 in the JP group was higher than that of 25.88 ± 1.79 in the JR group (p = 0.005). The marginal margin of soft tissue compared with a wide margin was the only independent predictor of local recurrence (p = 0.006). Limb diameter increase and perivascular fat plane disappearance during neoadjuvant chemotherapy were independent predictors for metastasis (p = 0.002; p = 0.000) and worse survival (p = 0.000; p = 0.001). CONCLUSIONS: Joint-sparing tumor resection with the ablative bone margin offers advantage of native joint preservation with favorable functional outcomes while not jeopardizing oncologic outcomes compared with joint-sacrificed tumor resection. Surgeon should strive to obtain adequate soft tissue surgical margin decreasing risk of local recurrence. Novel drug regimens might be reasonable options for patients with obvious limb diameter increase and perivascular fat disappearance during chemotherapy.

Is the Capanna Technique a Reliable Method for Revision Surgery after Failure of Previous Limb-Salvage Surgery?

BACKGROUND: Reconstruction of a massive bone defect caused by previous failed limb-salvage surgery in patients with bone sarcoma is challenging. Many procedures have been used, but they all have their inherent disadvantages. The Capanna technique has demonstrated good functional outcomes and a low incidence of complications in primary reconstructive surgery of massive bone defect. However, few studies have focused on its usage in revision surgery after failed primary limb-salvage surgery. METHODS: Between June 2011 and January 2017, 13 patients underwent revision surgery with the Capanna technique for reconstruction of a secondary segmental bone defect caused by a previous failed surgical procedure. The demographics, operating procedures, graft union, functional outcomes, oncologic outcomes, and postoperative complications of each patient were recorded. RESULTS: The current study investigated 13 patients. The rate of limb salvage was 100 %. Bone union was achieved for all patients during a mean time of 8.54 ± 2.15 months (range 4-11 months) at the fibula-host bone junction and 14.92 ± 2.33 months (range 12-21 months) at the allograft-host bone junction. The postoperative complications included wound healing issues and internal fixation loosening. Allograft fracture, nonunion, and infection were not observed. All the patients achieved good functional outcomes, with a Musculoskeletal Tumor Society (MSTS) score of 0.86 ± 0.03 at the latest follow-up visit. CONCLUSIONS: The Capanna technique is a reliable alternative method for revision reconstruction of a segmental bone defect caused by a previous failed surgical procedure. LEVEL OF EVIDENCE: Level IV, therapeutic study.

Disruption of Gprasp2 down-regulates Hedgehog signaling and leads to apoptosis in auditory cells.

GPRASP2 is implicated in nervous system diseases, tumors and immune inflammation. In our previous study, G protein-coupled receptor associated sorting protein 2 (GPRASP2) was identified as a novel causal gene for X-linked recessive syndromic hearing loss (SHL). However, the role of GPRASP2 in auditory function has not been elucidated. The Gprasp2-knockout (KO) mouse HEI-OC1 auditory cells were constructed using CRISPR/Cas9-mediated gene editing. RNA-sequencing (RNA-seq) was used to investigate the differentially expressed genes (DEGs) and DEGs-enriched signaling pathways, which was verified by Western blot. Flow cytometry assay was used to examine cell apoptosis. The cytological pathology was evaluated by laser scanning confocal microscopy (LSCM) and transmission electron microscopy (TEM). Mitochondrial damage was observed in Gprasp2-KO HEI-OC1 cells. RNA-seq analysis suggested that Gprasp2-KO was implicated in the apoptosis process, which could be mediated by Hedgehog (Hh) signaling pathway. The key molecules in Hh signaling pathway (Smo, Gli1, Gli2) were detected to be down-regulated in Gprasp2-KO HEI-OC1 cells. The differential expression of apoptosis molecules (Bcl2, Bax, Caspase-3/cleaved-Caspase-3) indicated that Gprasp2-KO induced apoptosis in HEI-OC1 cells. The treatment of smoothened agonist (Purmorphamine, PUR) activated the Hh-Gli signaling pathway and reduced apoptosis in Gprasp2-KO HEI-OC1 cells. This study revealed that Gprasp2-disruption inhibited Hh signaling pathway and led to cell apoptosis in HEI-OC1 cells, which might provide the potential molecular mechanism of GPRASP2 mutation associated with human SHL.

Use of Vascularized Fibular Epiphyseal Transfer with Massive Bone Allograft for Proximal Humeral Reconstruction in Children with Bone Sarcoma.

BACKGROUND: The vascularized fibula epiphyseal transfer provides a reconstructive option for longitudinal growth after oncologic resection of the proximal humerus in pediatric patients. However, postoperative fractures and poor shoulder function are common. The purpose of this review was to introduce a composite approach in oncologic reconstruction of the proximal humerus and assess its clinical outcomes. METHODS: We retrospectively investigated five children (3 osteosarcoma and 2 Ewing's sarcoma) who underwent biological reconstruction with combination of vascularized fibula epiphyseal transfer and massive bone allograft after oncologic resection of the proximal humerus. The mean follow-up was 46.8 months. RESULTS: All patients were alive at the last follow-up. There was no graft fracture, hardware failure, or infection. The mean time of osseous union was 2.9 months at fibula-humerus junction and 6.2 months at allograft-humerus junction. Hypertrophy and axial growth were evident in all, except one patient who has avascular necrosis of the fibula head. The mean hypertrophy index was 51.5%, and the mean growth was 4.4 mm per annum. The mean arm discrepancy was 4.6 cm. All reconstruction was in situ with the average abduction of 113° and forward flexion of 69°. The mean Musculoskeletal Tumor Society (MSTS) score was 85.4% at the final follow-up. All patients experienced dropped foot and resolved spontaneously. CONCLUSIONS: The combination of vascularized fibula epiphyseal transfer with massive allograft bone provides a reliable oncologic reconstruction of proximal humerus in children. It not only offers the ability of longitudinal growth, hypertrophy, and osseous union but also diminishes reconstructive complications and improves shoulder function. LEVEL OF EVIDENCE: Therapeutic Level IV.

Insights into the functional divergence of the haloacid dehalogenase superfamily from phosphomonoesterase to inorganic pyrophosphatase.

The evolution of enzyme catalytic structures and mechanisms has drawn increasing attention. In this study, we investigate the functional divergence from phosphomonoesterase to inorganic pyrophosphatase in the haloacid dehalogenase (HAD) superfamily. In this study, a series of models was constructed, and calculations were performed by using density functional theory with the B3LYP functional. The calculations suggest that in most HAD members, the active-site structure is unstable due to the binding of the substrate inorganic pyrophosphate (PPi), and reactions involving PPi cannot be catalyzed. In BT2127, which is a unique member of the HAD superfamily, the Mg2+-coordinating residues Asn172 and Glu47 play a role in stabilizing the active-site structure to adapt to the substrate PPi by providing much stronger coordination interactions with the Mg2+ ion. The calculation results suggest that Asn172 and Glu47 are crucial in the evolution of the inorganic pyrophosphatase activity in the HAD superfamily. Our study provides definitive chemical insight into the functional divergence of the HAD superfamily, and helps in understanding the evolution of enzyme catalytic structures and mechanisms.

Self-Assembled FRET Nanoprobe with Metal-Organic Framework As a Scaffold for Ratiometric Detection of Hypochlorous Acid.

Metal-organic framework (MOF) has been extensively explored in a number of fields due to its diverse properties. In this work, we demonstrated the potential of MOF in the establishment of a self-assembled fluorescence resonance energy transfer (FRET) system for developing ratiometric fluorescent nanoprobe. For this purpose, zeolitic imidazolate framework-8 (ZIF-8) was selected as a MOF model to entrap carbon dot (CD) and curcumin (CCM) during its self-assembly, which produces CD/CCM@ZIF-8. Benefiting from the confinement effect of ZIF-8, the loaded CD and CCM can be brought in close proximity for energy transfer to occur. Under optimal conditions, a high FRET efficiency of 68.7% can be obtained. Importantly, compared with traditional FRET systems, the fabrication process of CD/CCM@ZIF-8 is much more simple and straightforward, which does not involve the elaborate design and complicated synthesis of molecular linkers. However, in the presence of hypochlorous acid (HClO), the FRET process from CD to CCM will be disrupted, rendering CD/CCM@ZIF-8 to display a ratiometric response to HClO. This finding led to a method for ratiometric fluorescent detection of HClO with a detection limit of 67 nM and excellent selectivity over other reactive oxygen species. We believe that this study can give a new insight into the rational design and application of FRET-based nanoprobes.

Comparative transcriptome analysis of auditory OC-1 cells and zebrafish inner ear tissues in the absence of human OSBPL2 orthologues.

In our previous study, Oxysterol-binding protein-related protein 2 (OSBPL2) was first identified as a new deafness-causative gene contribute to non-syndromic hearing loss. However, the underlying mechanism of OSBPL2-induced hearing loss remains unknown. Here, we used hearing-specific cells and tissues OC-1 cells and zebrafish inner ear tissues as models to identify common transcriptome changes in genes and pathways in the absence of human OSBPL2 orthologues by RNA-seq analysis. In total, 2112 differentially expressed genes (DEGs) were identified between wild-type (WT) and Osbpl2-/- OC-1 cells, and 877 DEGs were identified between WT and osbpl2b-/- zebrafish inner ear tissues. Functional annotation implicated Osbpl2/osbpl2b in lipid metabolism, cell adhesion and the extracellular matrix in both OC-1 cells and zebrafish inner ear tissues. Protein-protein interaction (PPI) analysis indicated that Osbpl2/osbpl2b were also involved in ubiquitination. Further experiments showed that Osbpl2-/- OC-1 cells exhibited an abnormal focal adhesion morphology characterized by inhibited FAK activity and impaired cell adhesion. In conclusion, we identified novel pathways modulated by OSBPL2 orthologues, providing new insight into the mechanism of hearing loss induced by OSBPL2 deficiency.

OSBPL2 deficiency upregulate SQLE expression increasing intracellular cholesterol and cholesteryl ester by AMPK/SP1 and SREBF2 signalling pathway.

Previous studies have shown that oxysterol binding protein like 2 (OSBPL2) knockdown is closely related to cholesterol metabolism. However, whether there is a direct relation between OSBPL2 and cholesterol synthesis is unknown. This study explored the mechanism of OSBPL2 deficiency in the upregulation of squalene epoxidase (SQLE) and the subsequent accumulation of intracellular cholesterol and cholesteryl ester. Here, we constructed an OSBPL2-deleted HeLa cell line using CRISPR/Cas9 technology, screened differentially expressed genes and examined the transcriptional regulation of SQLE using a dual-luciferase reporter gene. RNA-seq analysis showed that SQLE was upregulated significantly and the dual luciferase reporter gene assay revealed that two new functional transcription factor binding sites of Sp1 transcription factor (SP1) and sterol regulatory element-binding transcription factor 2 (SREBF2) in the SQLE promoter participated in the SQLE transcription and expression. In addition, we also observed that OSBPL2 deletion inhibited the AMPK signalling pathway and that the inhibition of AMPK signalling promoted SP1 and SREBF2 entry into the nuclear to upregulate SQLE expression. Therefore, these data support that OSBPL2 deficiency upregulates SQLE expression and increases the accumulation of cholesterol and cholesteryl ester by suppressing AMPK signalling, which provides new evidence of the connection between OSBPL2 and cholesterol synthesis.

Asymmetric Difluoroboron Quinazolinone-Pyridine Dyes with Large Stokes Shift: High Emission Efficiencies Both in Solution and in the Solid State.

Boron difluoroquinazolinone-pyridine (BODIQPy) and substituted derivatives have been designed and synthesized. Strong emission with high fluorescence quantum yield both in solution (up to 0.99) and in solid state (up to 0.60) was achieved in these asymmetric BODIQPys. For 6-halogen substituted BODIQPys, the heavy atomic effect of bromine and iodine was suppressed in solution due to the strong electron-withdrawing ability of the BODIQPy core. As a result, 6-iodine substituted BODIQPy shows the unusual fluorescence quantum yields (>0.70) in dichloromethane and tetrahydrofuran. In the solid state, the asymmetric structure induced a unique dimeric structure, and efficient luminescence was also observed. The formation of halogen bonds between carbonyl oxygen and bromine or iodine regulated the stacking mode and enhanced intermolecular interaction, resulting in a decline in the fluorescence quantum yield. In addition, a large Stokes shift was also achieved in these asymmetric BODIQPys.

GPRASP2, a novel causative gene mutated in an X-linked recessive syndromic hearing loss.

BACKGROUND: A substantial amount of nuclear genes have been identified to be implicated in genetic hearing loss, while X-linked hearing loss is genetically heterogeneous and relatively infrequent. OBJECTIVE: To identify the causative gene mutation in a five-generation Chinese family with an X-linked recessive syndromic hearing loss (SHL). METHODS: Targeted X-chromosome exome sequencing was conducted, and cosegregation analysis was performed in the members of the affected family. The in silico and expression studies were also performed. RESULTS: A 2-bp missense mutation (c.1717_1718GC>AA, p.A573N) in the G protein-coupled receptor associated sorting protein 2 (GPRASP2) gene was identified in four hemizygous male patients and two heterozygous female carriers, which was cosegregated with the clinical phenotypes in this family. In silico analysis supported that this gene mutation is functionally deleterious, and it was detected that homologous Gprasp2 was highly expressed in multiple structures of the mouse cochlea, which suggested that GPRASP2 might be the genetic cause for the described disease phenotypes. CONCLUSIONS: This study presented a novel X-linked SHL combined with unique and unrecognised clinical features, and a missense variation of GPRASP2 was first identified to be implicated in X-linked SHL.

Minimally invasive treatment for osteonecrosis of the femoral head with angioconductive bioceramic rod.

PURPOSE: To describe the rationale, the surgical technique, and the short-term follow-up results of a new minimally invasive treatment of osteonecrosis of the femoral head (ONFH) with an angioconductive bioceramic rod (ABR) implant. METHODS: Sixty-two patients (72 hips) with ARCO stage IIA-IIIC ONFH treated with the minimally invasive ABR from January 2012 to December 2016 were reviewed (17 females, 45 males, mean age 44.49). This technique used the angioconductive properties of the porous implant to repair the necrosis by driving vascularization from the trochanter to the necrotic area. Patients had a mean follow-up period of 26.74 months. The outcomes were evaluated by hip joint survival, radiograph, and the Harris Hip Score (HHS). The complications occurred during the treatment period were recorded. RESULTS: No serious post-operative complications occurred during the treatment. The overall joint survival rate was 90.27%, with seven conversions to THA. Improvements were observed in 23 (31.95%) hips, 24 (33.33%) hips remained stable, and 25 (34.72%) hips had worse results according to the radiographic evaluation. The mean HHS at the end follow-up significantly improved compared to the pre-operative mean HHS (82.27 vs 58.14, p < 0.001). In both radiographic evaluation and HHS, the treatment was more effective on patients beneath 44 years old (p < 0.05); ARCO stage II compared to stage III (p < 0.05); and China-Japan Friendship Hospital (CJFH) type C compared to CJFH type L (p < 0.05). CONCLUSIONS: The minimally invasive treatment of ONFH with ABR showed promising results in delaying or even terminating the progression of the necrosis and improving hip function, especially in younger patients and in the early stages of the disease.

Identification of OSBPL2 as a novel candidate gene for progressive nonsyndromic hearing loss by whole-exome sequencing.

PURPOSE: Various forms of hearing loss have genetic causes, but many of the responsible genes have not yet been identified. Here, we describe a large seven-generation Chinese family with autosomal dominant nonsyndromic hearing loss that has been excluded as being caused by known deafness gene mutations associated with autosomal dominant nonsyndromic hearing loss with the aim of identifying a novel causative gene involved in deafness. METHODS: Whole-exome sequencing was conducted in three affected family members, and cosegregation analysis was performed on other members of the family. RESULTS: Whole-exome sequencing and subsequent segregation analysis identified a heterozygous frameshift mutation (c.153_154delCT, p.Gln53Argfs*100) in the oxysterol binding protein-like 2 (OSBPL2) gene in 25 affected family members. The deletion mutation is predicted to lead to premature truncation of the OSBPL2 protein. Modeling and structure-based analysis support the theory that this gene deletion is functionally deleterious. Our finding was further confirmed by the detection of another missense mutation, a c.583C>A transversion (p.Leu195Met) in exon 7 of OSBPL2, in an additional sporadic case of deafness. CONCLUSION: Based on this study, OSBPL2 was identified as an excellent novel candidate gene for autosomal dominant nonsyndromic hearing loss; this study is the first to implicate OSBPL2 mutations in autosomal dominant nonsyndromic hearing loss.

Targeted genomic capture and massively parallel sequencing to identify novel variants causing Chinese hereditary hearing loss.

BACKGROUND: Hereditary hearing loss is genetically heterogeneous, and hundreds of mutations in than 60 genes are involved in this disease. Therefore, it is difficult to identify the causative gene mutations involved. In this study, we combined targeted genomic capture and massively parallel sequencing (MPS) to address this issue. METHODS: Using targeted genomic capture and MPS, 104 genes and three microRNA regions were selected and simultaneously sequenced in 23 unrelated probands of Chinese families with nonsyndromic hearing loss. The results were validated by Sanger sequencing for all available members of the probands' families. To analyze the possible pathogenic functional effects of the variants, three types of prediction programs (Mutation Taster, PROVEAN and SIFT) were used. A total of 195 healthy Chinese Han individuals were compared as controls to verify the novel causative mutations. RESULTS: Of the 23 probands, six had mutations in DFNA genes [WFS1 (n = 2), COCH, ACTG1, TMC1, and POU4F3] known to cause autosomal dominant nonsyndromic hearing loss. These included one novel in-frame indel mutation, three novel missense mutations and two reported missense mutations. Furthermore, one proband from a family with recessive DFNB carried two monoallelic mutations in the GJB2 and USH2A genes. All of these mutations co-segregated with the hearing loss phenotype in 36 affected individuals from 7 families and were predicted to be pathogenic. CONCLUSIONS: Mutations in uncommon deafness genes contribute to a portion of nonsyndromic deafness cases. In the future, critical gene mutations may be accurately and quickly identified in families with hereditary hearing loss by targeted genomic capture and MPS.

Progress in extracellular vesicle homeostasis as it relates to cardiovascular diseases.

Extracellular vesicles (EVs) are involved in both physiological and pathological processes in many organ systems and are essential in mediating intercellular communication and maintaining organismal homeostasis. It is helpful to propose new strategies for disease treatment by elucidating the mechanisms of EV release and sorting. An increasing number of studies have shown that there is specific homeostasis in EVs, which is helpful for the human body to carry out physiological activities. In contrast, an EV homeostasis im-balance promotes or accelerates disease onset and development. Alternatively, regulating the quality of EVs can maintain homeostasis and even achieve the purpose of treating conditions. An analysis of the role of EV homeostasis in the onset and development of cardiovascular disease is presented in this review. This article also summarizes the methods that regulate EV homeostasis and their application in cardiovascular diseases. In particular, this study focuses on the connection between EV steady states and the cardiovascular system and the potential value of EVs in treating cardiovascular diseases.

Identifying specific TLS-associated genes as potential biomarkers for predicting prognosis and evaluating the efficacy of immunotherapy in soft tissue sarcoma.

Background: The tertiary lymphatic structure (TLS) is an important component of the tumor immune microenvironment and has important significance in patient prognosis and response to immune therapy. However, the underlying mechanism of TLS in soft tissue sarcoma remains unclear. Methods: A total of 256 RNAseq and 7 single-cell sequencing samples were collected from TCGA-SARC and GSE212527 cohorts. Based on published TLS-related gene sets, four TLS scores were established by GSVA algorithm. The immune cell infiltration was calculated via TIMER2.0 and "MCPcounter" algorithms. In addition, the univariate, LASSO, and multivariate-Cox analyses were used to select TLS-related and prognosis-significant hub genes. Single-cell sequencing dataset, clinical immunohistochemical, and cell experiments were utilized to validate the hub genes. Results: In this study, four TLS-related scores were identified, and the total-gene TLS score more accurately reflected the infiltration level of TLS in STS. We further established two hub genes (DUSP9 and TNFSF14) prognosis markers and risk scores associated with soft tissue sarcoma prognosis and immune therapy response. Flow cytometry analysis showed that the amount of CD3, CD8, CD19, and CD11c positive immune cell infiltration in the tumor tissue dedifferentiated liposarcoma patients was significantly higher than that of liposarcoma patients. Cytological experiments showed that soft tissue sarcoma cell lines overexpressing TNFSF14 could inhibit the proliferation and migration of sarcoma cells. Conclusion: This study systematically explored the TLS and related genes from the perspectives of bioinformatics, clinical features and cytology experiments. The total-gene TLS score, risk score and TNFSF14 hub gene may be useful biomarkers for predicting the prognosis and immunotherapy efficacy of soft tissue sarcoma.

Identification and characterization of microRNAs expressed in human breast cancer T-47D cells in response to prolactin treatment by Solexa deep-sequencing technology.

MicroRNAs (miRNAs) are key regulators of gene expression and perform critical roles in various biological processes. To investigate the functional roles of miRNAs in the prolactin receptor (PRLR) signaling pathway in breast cancer, we constructed two small RNA libraries from human breast cancer T-47D cells treated with or without prolactin (PRL). The miRNA expression profiles were systematically screened using Solexa deep-sequencing technology. More than 40 miRNAs were significantly differentially expressed, from which 4 miRNAs were chosen for validation by stem-loop real-time PCR. In addition, 3 novel miRNAs were selected for verification by PCR. Furthermore, upstream miRNA target genes were predicted using different algorithms, GO and KEGG analyses revealed that these targets were highly related to the PRLR signaling pathway. This study provides a reference for elucidating the complex miRNA-mediated regulatory networks of PRL/PRLR signaling pathway that affect breast cancer tumorigenesis and progression.

Genetic mutations of GJB2 and mitochondrial 12S rRNA in nonsyndromic hearing loss in Jiangsu Province of China.

BACKGROUND: Hearing loss is caused by several environmental and genetic factors and the proportion attributed to inherited causes is assumed at 50 ~ 60% . Mutations in GJB2 and mitochondrial DNA (mtDNA) 12S rRNA are the most common molecular etiology for nonsyndromic sensorineural hearing loss (NSHL). The mutation spectra of these genes vary among different ethnic groups. METHODS: To add the molecular etiologic information of hearing loss in the Chinese population, a total of 658 unrelated patients with NSHL from Jiangsu Province of China were selected for mutational screening including GJB2 and mtDNA 12S rRNA genes using PCR and DNA sequencing technology. As for controls, 462 normal-hearing individuals were collected. RESULTS: A total of 9 pathogenic mutations in the GJB2 and 7 pathogenic mutations in the 12S rRNA gene were identified. Of all patients, 70 had monoallelic GJB2 coding region mutation in the heterozygous state, 94 carried two confirmed pathogenic mutations including 79 homozygotes and 15 compound heterozygotes. The 235delC appears to be the most common deafness-causing GJB2 mutation (102/658, 15.50% ). No mutations or variants in the GJB2 exon1 and basal promoter region were found. In these patients, 4 subjects carried the m.1494C > T mutation (0.61% ) and 39 subjects harbored the m.1555A > G mutation (5.93% ) in mtDNA 12S rRNA gene. A novel sequence variant at m.1222A > G in the 12S rRNA gene was identified, which could alter the secondary structure of the 12S rRNA. CONCLUSION: The mutation spectrum and prevalence of GJB2 and mtDNA 12S rRNA genes in Jiangsu population are similar to other areas of China. There are in total 31.46% of the patients with NSHL carry deafness-causing mutation in GJB2 or mtDNA 12S rRNA genes. Mutation in GJB2 gene is the most common factor, mtDNA 12S rRNA also plays an important part in the pathogenesis of hearing loss in Jiangsu Province areas. The m.1222A > G was found to be a new candidate mutation associated with hearing loss. Our results indicated the necessity of genetic screening for mutations of these genes in Jiangsu patients with NSHL.

Maternally transmitted aminoglycoside-induced and non-syndromic hearing loss caused by the 1494C > T mutation in the mitochondrial 12S rRNA gene in two Chinese families.

OBJECTIVE: To explore the molecular genetic characterization of two Chinese families with aminoglycoside-induced and non-syndromic hearing loss (NSHL). DESIGN: Clinical evaluations, sequence analysis of mitochondrial DNA (mtDNA) as well as two nuclear genes TRMU and MTO1 encoding mitochondrial proteins. STUDY SAMPLE: Two Chinese families with aminoglycoside-induced and NSHL. RESULTS: Clinical evaluations revealed incomplete penetrance (28.6% vs. 40.0%) and variable phenotype of hearing losses between two families. When the effect of aminoglycosides was excluded, the penetrances were both 0%. Sequence analysis of mitochondrial genomes showed a homoplasmic 1494C > T mutation in the12S rRNA gene (MT-RNR1) in all maternal relatives, as well as distinct sets of mtDNA polymorphism belonging to Eastern Asian haplogroups D4j and D5a2, respectively. However, none of these mtDNA variants was highly evolutionarily conserved and implicated to have functional significance. No mutations were identified in either TRMU or MTO1 gene. CONCLUSIONS: Mitochondrial 1494C> T mutation is the molecular basis responsible for the NSHL of two families, and the use of aminoglycoside antibiotics can worsen the hearing of the mutation carriers. Our results indicate the importance of a systematic screening for the mitochondrial 1494C > T mutation in Chinese subjects in the prevention of aminoglycoside-induced and non-syndromic hearing loss.