N6-methyladenosine (m6A) and its reader proteins are involved in pre-mRNA processing and play a variety of roles in numerous biological processes. However, much remains to be understood about the regulation of m6A and the function of its specific readers during meiotic processes. Here, this study shows that the potential m6A reader protein hnRNPC is essential for both male and female meiosis in mice. Germ cell-specific knockout of Hnrnpc causes meiotic arrest at pachynema in male mice. Specifically, hnRNPC-deficient males show abnormal meiosis initiation and defective meiotic progression, ultimately leading to meiotic arrest at the pachytene stage. Interestingly, hnRNPC-null females show similar meiotic defects to males. Mechanistically, this study discovers that in male germ cells, hnRNPC works with HuR to directly bind and modulate alternative splicing of meiotic-related genes (e.g., Sycp1, Brca1, and Smc5) in an m6A-dependent manner during spermatogenesis. Collectively, these findings reveal hnRNPC as a critical factor for meiosis and contribute to a mechanistic understanding of the hnRNPC-HuR interaction in alternative splicing of mRNAs during germ cell development.

Teratozoospermia is a significant cause of male infertility, but the pathogenic mechanism of acephalic spermatozoa syndrome (ASS), one of the most severe teratozoospermia, remains elusive. We previously reported Spermatogenesis Associated 6 (SPATA6) as the component of the sperm head-tail coupling apparatus (HTCA) required for normal assembly of the sperm head-tail conjunction, but the underlying molecular mechanism has not been explored. Here, we find that the co-chaperone protein BAG5, expressed in step 9-16 spermatids, is essential for sperm HTCA assembly. BAG5-deficient male mice show abnormal assembly of HTCA, leading to ASS and male infertility, phenocopying SPATA6-deficient mice. In vivo and in vitro experiments demonstrate that SPATA6, cargo transport-related myosin proteins (MYO5A and MYL6) and dynein proteins (DYNLT1, DCTN1, and DNAL1) are misfolded upon BAG5 depletion. Mechanistically, we find that BAG5 forms a complex with HSPA8 and promotes the folding of SPATA6 by enhancing HSPA8's affinity for substrate proteins. Collectively, our findings reveal a novel protein-regulated network in sperm formation in which BAG5 governs the assembly of the HTCA by activating the protein-folding function of HSPA8.

Circulating tumor cells (CTCs) are rare, meaning that current isolation strategies can hardly satisfy efficiency and cell biocompatibility requirements, which hinders clinical applications. In addition, the selected cells require immunofluorescence identification, which is a time-consuming and expensive process. Here, we developed a method to simultaneously separate and identify CTCs by the integration of optical force and fluorescent microspheres. Our method achieved high-purity separation of CTCs without damage through light manipulation and avoided additional immunofluorescence staining procedures, thus achieving rapid identification of sorted cells. White blood cells (WBCs) and CTCs are similar in size and density, which creates difficulties in distinguishing them optically. Therefore, fluorescent PS microspheres with high refractive index (RI) are designed here to capture the CTCs (PS-CTCs) and increase the average index of refraction of PS-CTCs. In optofluidic chips, PS-CTCs were propelled to the collection channel from the sample mixture, under the radiation of light force. Cells from the collection outlet were easily identified under a fluorescence microscope due to the fluorescence signals of PS microspheres. This method provides an approach for the sorting and identification of CTCs, which holds great potential for clinical applications in early diagnosis of disease.

Blood-labyrinth barrier (BLB) disruption plays a crucial role in the development of otitis media. The aims of our study was to explore the role and action mechanism of adenosine in LPS-induced endothelial cells (ECs) damage, which are one of the major principal cell type for blood-labyrinth barrier (BLB), and so as to assess the potential of adenosine to be used in the treatment of BLB disruption in animal experiment. In our study, ECs were treated with LPS to mimic BLB damage in vitro. Our data showed that adenosine at dosage of 1, 10, and 20 μM had no influence on the cell viability of ECs. LPS treatment obviously suppressed the expression of Occludin and Zonula occludens-1 (ZO-1) in ECs, which was partly recused by adenosine treatment. Meantime, LPS-induced increasing in reactive oxygen species (ROS) production and ECs permeability also was rescued by adenosine treatment. However, inhibition the A2A receptor (A2AR) could attenuate the influence of adenosine on LPS-treated ECs, indicating that adenosine alleviated LPS-induced BLB damage by activating A2AR. Moreover, the inhibition of adenosine to LPS-induced inactivation of AMPK/AKT signaling pathway was partly recused by A2AR suppression. In addition, Compound C (an AMPK inhibitor) decreased the expression of Occludin and ZO-1 in ECs following LPS combined with adenosine treatment. In conclusion, adenosine alleviates LPS-induced BLB damage via AMPK/AKT pathway through activation of A2AR. This work suggests that adenosine may be a candidate drug for the treatment of BLB dysfunction-related diseases.

The intense metabolism of cancer cells leads to hypoxia and lack of crucial nutrients in the tumor microenvironment, which hinders the function of immune cells. We designed a biomimetic immune metabolic nanoplatform, in which a type I aggregation-induced emission photosensitizer and a glutamine antagonist are encapsulated into a cancer cell membrane for achieving specific delivery in vivo. This approach greatly satisfies the glucose and glutamine required by T cells, significantly improves the tumor hypoxic environment, enables the reprogramming of tumor and immune cell metabolism, induces immunogenic cell death, promotes dendritic cell maturation, and effectively inhibits tumor proliferation. Strong tumor-specific immune responses are further triggered, and the tumor immune-suppressing microenvironment is modulated, by decreasing the number of immunosuppressive cells. Moreover, subsequent combination with anti-PD-1 is able to generate strong abscopal effects to prevent tumor distant metastasis and provide long-term immune memory against tumor recurrence.

Photothermal therapy (PTT) has presented its inherent application value in cancer treatment. Nevertheless, single-functional photothermal materials cannot meet the precise diagnosis and treatment of cancer. Therefore, it is important to design a nanocomposite that has both high therapeutic efficiency and multimodal imaging capabilities. In our work, a new nanostructure of gold nanorods (AuNRs) with silver shells decorated by BaGdF5:Yb3+,Er3+ nanoparticle was synthesized by a simple way. The biostability of AuNRs is increased by coating with silver shells, and the AuNR@Ag nanoparticles can be used as excellent surface enhanced Raman scattering (SERS) probe. Moreover, the modification of BaGdF5:Yb3+,Er3+ nanoparticles provides the possibility of real-time optical imaging of the tumor area. Under the irradiation of NIR laser, the AuNR@Ag/BaGdF5:Yb3+,Er3+ nanocomposites (NCs) have strong upconversion emission and excellent photothermal conversion efficiency. Meanwhile, the NCs show low cytotoxicity and good biocompatibility in MTT cytotoxicity test. Moreover, the NCs are also exceptional contrast agents for CT imaging. For in vitro photothermal therapy test, NCs show excellent killing efficiency on tumor cells. Therefore, the multifaceted research of AuNR@Ag/BaGdF5:Yb3+,Er3+ multifunctional nanomaterials provides a break for high-efficiency tumor photothermal therapy and multimodal imaging.

The treatment of inner ear disorders always remains a challenge for researchers. The presence of various physiological barriers, primarily the blood-labyrinth barrier (BLB), limits the accessibility of the inner ear and hinders the efficacy of various drug therapies. Yet despite recent advances in the cochlea for repair and regeneration, there are currently no pharmacological or biological interventions for hearing loss. Current research focuses on the localized drug-, gene-, and cell-based therapies. Drug delivery based on nanotechnology represents an innovative strategy to improve inner ear treatments. Materials with specific nanostructures not only exhibit a unique ability to encapsulate and transport therapeutics to the inner ear but also endow specific targeting properties to auditory hair cells as well as the stabilization and sustained drug release. Along with this, some alternative routes, like intratympanic drug delivery, can also offer a better means to access the inner ear without exposure to the BLB. This review discusses a variety of nano-based drug delivery systems to the ear for treating inner ear diseases. The main factors affecting the curative efficacy of nanomaterials are also discussed. With a deeper understanding of the link between these crucial factors and the clinical effect of nanomaterials, it paves the way for the optimization of the therapeutic activity of nanocarriers.

Isolation of circulating tumor cells (CTCs) from patients is a challenge due to the rarity of CTCs. Recently, various platforms to capture and release CTCs for downstream analysis have been developed. However, most of the reported release methods provide external stimuli to release all captured cells, which lead to lack of specificity in the pool of collected cells, and the external stimuli may affect the activity of the released cells. Here, we presented a simple method for single-cell recovery to overcome the shortcomings, which combined the nanostructures with a photocurable hydrogel, chondroitin sulfate methacryloyl (CSMA). In brief, we synthesized gelatin nanoparticles (Gnps) and modified them on flat glass (Gnp substrate) for the specific capture of CTCs. A 405 nm laser was projected onto the selected cells, and then CSMA was cured to encapsulate the selected CTCs. Unselected cells were removed with MMP-9 enzyme solution, and selected CTCs were recovered using a microcapillary. Finally, the photocurable hydrogel-encapsulated cells were analyzed by nucleic acid detection. In addition, the results suggested that the isolation platform showed good biocompatibility and successfully achieved the isolation of selected cells. In summary, our light-induced hydrogel responsive platform holds certain potential for clinical applications.

BACKGROUND:The enlarged vestibular aqueduct (EVA) is the commonest malformation of inner ear accompanied by sensorineural hearing loss in children. Three genes SLC26A4, FOXI1, and KCNJ10 have been associated with EVA, among them SLC26A4 being the most common. Yet, hotspot mutation screening can only diagnose a small number of patients. METHODS:Thus, in this study, we designed a new molecular diagnosis panel for EVA based on multiplex PCR enrichment and next-generation sequencing of the exon and flanking regions of SLC26A4. A total of 112 hearing loss families with EVA were enrolled and the pathogenicity of the rare variants detected was interpreted according to the American College of Medical Genetics and Genomics (ACMG) guidelines. RESULTS:Our results showed that 107/112 (95.54%) families carried SLC26A4 biallelic mutations, 4/112 (3.57%) carried monoallelic variants, and 1/112 (0.89%) had none variant, resulting in a diagnostic rate of 95.54%. A total of 49 different variants were detected in those patients and we classified 30 rare variants as pathogenic/likely pathogenic, of which 13 were not included in the Clinvar database. CONCLUSION:Our diagnostic panel has an increased diagnostic yield with less cost, and the curated list of pathogenic variants in the SLC26A4 gene can be directly used to aid the genetic counseling to patients.

Background: Branchio-oto-renal syndrome (BOR) and branchio-oto syndrome (BOS) are rare autosomal dominant disorders defined by varying combinations of branchial, otic, and renal anomalies. Here, we characterized the clinical features and genetic etiology of BOR/BOS in several Chinese families and then explored the genotypes and phenotypes of BOR/BOS-related genes, as well as the outcomes of auditory rehabilitation in different modalities.Materials and Methods: Probands and all affected family members underwent detailed clinical examinations. Their DNA was subjected to whole-exome sequencing to explore the underlying molecular etiology of BOR/BOS; candidate variants were validated using Sanger sequencing and interpreted in accordance with the American College of Medical Genetics guidelines. In addition, a literature review concerning EYA1 and SIX1 alterations was performed to explore the genotypes and phenotypes of BOR/BOS-related genes.Results: Genetic testing identified the novel deletion (c.1425delC, p(Asp476Thrfs*4); NM_000,503.6), a nonsense variant (c.889C > T, p(Arg297*)), and two splicing variants in the EYA1 gene (c.1050+1G > T and c.1140+1G > A); it also identified one novel missense variant in the SIX1 gene (c.316G > A, p(Val106Met); NM_005,982.4). All cases exhibited a degree of phenotypic variability between or within families. Middle ear surgeries for improving bone-conduction component hearing loss had unsuccessful outcomes; cochlear implantation (CI) contributed to hearing gains.Conclusion: This is the first report of BOR/BOS caused by the SIX1 variant in China. Our findings increase the numbers of known EYA1 and SIX1 variants. They also emphasize the usefulness of genetic testing in the diagnosis and prevention of BOR/BOS while demonstrating that CI for auditory rehabilitation is a feasible option in some BOR/BOS patients.

Nanotechnology has revolutionized the field of biology and medicine in the 21st century.Bone injury incidences during sports activities are common,and they are traditionally treated with allogeneic grafting,a common clinical practice but limited by the quality of the graft and some side effects.Nano-hydroxyapatite(nHA)is considered as an ideal bone graft material owing to its bone-like structure,excellent biological activity,bone conductivity,non-toxicity,and non-immunogenicity.nHA and its composite materials have been found suitable for the adhesion,proliferation,and differentiation of mesenchymal stem cells,which leads to their potential applications in treating the bone injury.In this review,we classified different applications of nHA to explore the role of these materials in bone repair and tendon healing,highlighting the supe-rior characteristics of nanomaterials in the treatment of bone injury,hoping to provide ideas for nHA applied to clinical practices for the treatment of bone injury.

Increasing number of resistant bacteria have emerged with the overuse of antibiotics, which indicates that the bacterial infection has become a global challenge. Furthermore, the pollution of antibiotics to the environment has become a serious threat to public health. It is known that toxins produced by bacteria are the main cause of bacterial infections. Photothermal therapy is an effective antibacterial approach. However, the photothermal reagents cannot eliminate bacterial toxins, and even some anti-bacterial materials are toxic. Here, we synthesized a biomimetic recycled nanoparticle, red blood cell (RBC) membrane-coated Fe3O4 nanoparticles (RBC@Fe3O4), as an antibacterial agent. The RBC@Fe3O4 nanoparticles act as nano-sponges to trap toxins and then kill them all with a photothermal effect. We can describe this process simply as a battle between two armies. Our strategy is to disarm the "enemy" so that we can easily kill the "enemy" who has no power, which results in enhancing the bactericidal efficacy. The toxin of methicillin-resistant Staphylococcus aureus (MRSA) was absorbed by RBC@Fe3O4 in vitro. In addition, in vivo studies proved that the RBC@Fe3O4 nanoparticles confer obvious survival benefits against toxin-induced lethality by absorbing the toxin of MRSA. Furthermore, using a mouse model of MRSA wound infection, the RBC@Fe3O4 nanoparticles with laser irradiation were found to have a superior wound-healing effect. Simultaneously, the RBC@Fe3O4 nanoparticles could be recycled in a simple way without affecting the bactericidal efficacy. The highly biocompatible and recyclable RBC@Fe3O4 biomimetic nanoparticles based on photothermal therapy and bacterial toxin adsorption strategy are promising for treating bacterial infections.

Objective This study aimed to explore the genetic causes of probands who were diagnosed with Waardenburg syndrome (WS) or congenital sensorineural hearing loss. Methods A detailed physical and audiological examinations were carried out to make an accurate diagnosis of 14 patients from seven unrelated families. We performed whole-exome sequencing in probands to detect the potential genetic causes and further validated them by Sanger sequencing in the probands and their family members. Results The genetic causes for all 14 patients with WS or congenital sensorineural hearing loss were identified. A total of seven heterozygous variants including c.1459C > T, c.123del, and c.959-409_1173+3402del of PAX3 gene (NM_181459.4), c.198_262del and c.529_556del of SOX10 gene (NM_006941.4), and c.731G > A and c.970dup of MITF gene (NM_000248.3) were found for the first time. Of these mutations, we had confirmed two (c.1459C > T and c.970dup) are de novo by Sanger sequencing of variants in the probands and their parents. Conclusion We revealed a total of seven novel mutations in PAX3, SOX10, and MITF, which underlie the pathogenesis of WS. The clinical and genetic characterization of these families with WS elucidated high heterogeneity in Chinese patients with WS. This study expands the database of PAX3, SOX10, and MITF mutations and improves our understanding of the causes of WS.

Perivascular-resident macrophage-like melanocytes (PVM/Ms) can upregulate the expression of tight junction-related proteins in endothelial cells (ECs) by secreting pigment epithelial-derived factor (PEDF), and thereby regulate the permeability of the intrastrial fluid–blood barrier critical for maintaining inner ear homeostasis. This study aimed to investigate the effects of long non-coding RNA (lncRNA) Rian on cell growth of PVM/Ms and PVM/Ms regulation of intrastrial fluid–blood barrier integrity mediated by PEDF. Rian was downregulated in the aged cochlea from 12-month-old C57BL/6 mice. Rian overexpression inhibited cell apoptosis and promoted cell viability of hypoxia-injured PVM/Ms as well as increased the concentration and expression of PEDF secreted by PVM/Ms. In contrast, Rian silencing exerted the opposite effects. Furthermore, in a cell co-culture model of ECs and PVM/Ms, Rian overexpression in PVM/Ms increased the expression of the junction-associated proteins in co-cultured ECs, and this effect was abrogated by blockade of PEDF by anti-PEDF in PVM/Ms. Further mechanistical investigation revealed that Rian promoted STAT3 nuclear translocation and activation by binding to FUS, and thereby promoted the secretion of PEDF. Collectively, Rian attenuates PVM/Ms injury and strengthens the ability of PVM/Ms to maintain the integrity of the endothelial barrier by promoting PEDF expression.

Disulfiram (DSF) is an important drug for the treatment and management of alcohol dependency. This drug has been approved by US-FDA, and its activity against the tumor is dependent on copper ion (Cu2+). However, the copper toxicity (caused via external copper) and its intrinsic anfractuous distribution in the human body have adversely suppressed the mechanism of DSF in in vivo. In this study, we aimed to design an injectable hydrogel, as CRC (Cu2+ release controller) for the effective treatment of tumors. The hydrogels of agarose have been used for wrapping of CuCl2, and hierarchical microparticles (HMP) for the generation of CRC system. When the laser irradiations (808 nm) have been provided to the system, light energy is transferred into heat energy, which results in the hydrogel hydrolysis (reversible) due to the overheating effect. This is followed by a reaction with DSF (pre-injected) to suppress tumor progression. Hence, the CRC system brings innovative ideas for designing of a Cu2+ delivery system.

Integrating multiple mechanisms to maximize photothermal conversion efficiency is a significant strategy but remains challenging to construct therapeutic agents toward photothermal tumor treatment. Here, an approach to synthesize asymmetric Bi 2 Se 3 /CdSe‐Au hierarchical nanorods with excellent photothermal conversion is reported. Ag wetting‐layer is firstly grown to help overcome the interfacial lattice mismatch and promote the site‐selective growth of AgCdSe onto one end or side surface of Au nanorods. Subsequently, extraction of Ag + ions out of lattice is observed during cation exchange reaction and epitaxial growth of Bi 2 Se 3 shell. Bi 2 Se 3 /CdSe heterojunction with type‐II band alignment is formed and located at the plasmonic hotspots of Au nanorods, which experiences enhanced light absorption and accelerates the charge separation of photo‐excited carriers. Under excitation of near‐infrared 808 nm laser, the matchstick‐like Bi 2 Se 3 /CdSe‐Au nanorods show an excellent photothermal conversion, with 4.3 times temperature increment ( Δ T ) than that of bare Au nanorods. Moreover, in vitro and in vivo experiments verify them as excellent photothermal therapeutic agents.

Mutations in connexin 30 (Cx30) are known to cause severe congenital hearing impairment; however, the mechanism by which Cx30 mediates homeostasis of endocochlear gap junctions is unclear. We used a gene deletion mouse model to explore the mechanisms of Cx30 in preventing hearing loss. Our results suggest that despite severe loss of the auditory brain-stem response and endocochlear potential at postnatal day 18, Cx30−/− mice only show sporadic loss of the outer hair cells. This inconsistency in the time course and severity of hearing and hair cell losses in Cx30−/− mice might be explained, in part, by an increase in reactive oxygen species generation beginning at postnatal day 10. The expression of oxidative stress genes was increased in Cx30−/− mice in the stria vascularis, spiral ligament, and organ of Corti. Furthermore, Cx30 deficiency caused mitochondrial dysfunction at postnatal day 18, as assessed by decreased ATP levels and decreased expression of mitochondrial complex I proteins, especially in the stria vascularis. Proteomic analysis further identified 444 proteins that were dysregulated in Cx30−/− mice, including several that are involved in mitochondria electron transport, ATP synthesis, or ion transport. Additionally, proapoptotic proteins, including Bax, Bad, and caspase-3, were upregulated at postnatal day 18, providing a molecular basis to explain the loss of hearing that occurs before hair cell loss. Therefore, our results are consistent with an environment of oxidative stress and mitochondrial damage in the cochlea of Cx30−/− mice that is coincident with hearing loss but precedes hair cell loss.

In recent years, carbon dot (CD)-based fluorescent sensors for selective ions or small biomolecules have drawn great attention. In this work, highly fluorescent CDs (QY = 21%) were prepared from 2,3-diamino pyridine as the precursor through a facile solvothermal process. The CDs showed high stability and a green emission in aqueous, and the optimal emission wavelength of CDs is 508 nm under the excitation wavelength of 438 nm. Interestingly, a CDs-based nanoprobe was developed for a selective and sensitive fluorescence quenching response to NO2 - in water, and the quenching mechanism was investigated in the work. Besides, the recovery rates of NO2 - in the range of 98-103.5% were found to be acceptable, indicating that the proposed CDs could be act as potential candidates for determination of nitrite ions in real samples. Meanwhile, the nanoprobe was also successfully employed in a visualization biosensing platform for determination of NO2 - in living cells due to its eminent biocompatibility.

Copper ions (Cu2+) and l-cysteine (l-Cys) in the human body always play critical roles in various physiological processes, while abnormal Cu2+ and l-Cys concentrations in the biological system lead to many diseases. In this manuscript, Si-doped carbon dots (Si-CDs) with near-infrared fluorescence were designed for the detection of Cu2+ and l-Cys through the fluorescence "on-off-on" mode. The carbon dots exhibited not only excellent optical merits including good stability against photobleaching and high chemical stability, but also superior biological compatibility. Interestingly, due to the abundant amino groups distributed on the surface of Si-CDs, they could bind to copper ions to form cupric amine complexes and then quench the fluorescence of Si-CDs due to an electron transfer process. In addition, upon the addition of l-Cys, the FL intensity of Si-CDs could be effectively recovered accompanied with complexation between Cu2+ and the functional groups in l-Cys, due to which Cu2+ was removed from the surface of Si-CDs. Notably, as far as we know, these are the first red-emitting carbon dots for copper ion and l-Cys assays in water samples and human plasma samples. Furthermore, this strategy was successfully applied to the determination of Cu2+ and l-Cys in living systems, demonstrating great practicability in biomedical applications.

BACKGROUND:Treacher Collins syndrome-1 (TCS1; OMIM# 154500) is a rare autosomal dominant disease that is defined by congenital craniofacial dysplasia. Here, we report four sporadic and one familial case of TCS1 in Chinese patients with clinical features presenting as hypoplasia of the zygomatic complex and mandible, downslanting palpebral fissures, coloboma of the lower eyelids, and conductive hearing loss. MATERIALS AND METHODS:Audiological, radiological, and physical examinations were performed. Targeted next-generation sequencing (NGS) was performed to examine the genetics of this disease in five probands, and Sanger sequencing was used to confirm the identified variants. A literature review discusses the pathogenesis, treatment, and prevention of TCS1. RESULTS:We identified a novel insertion of c.939_940insA (p.Gly314Argfs*35; NM_001135243.1), a novel deletion of c.1766delC (p.Pro589Leufs*7), two previously reported insertions of c.1999_2000insC (p.Arg667Profs*31) and c.4218_4219insG (p.Ser1407Valfs*23), and one previously reported deletion of c.4369_4373delAAGAA (p.Lys1457Glufs*12) in the TCOF1 gene. All five cases exhibited a degree of interfamilial and intrafamilial phenotypic variability. A review of the literature revealed no clear evidence of a genotype-phenotype correlation in TCS1. CONCLUSION:Our results expand the variant spectrum of TCOF1 and highlight that NGS is essential for the diagnosis of TCS and that genetic counseling is beneficial for guiding prevention.