Tumor-resident intracellular microbiota promotes metastatic colonization in breast cancer.

Tumor-resident intracellular microbiota is an emerging tumor component that has been documented for a variety of cancer types with unclear biological functions. Here, we explored the functional significance of these intratumor bacteria, primarily using a murine spontaneous breast-tumor model MMTV-PyMT. We found that depletion of intratumor bacteria significantly reduced lung metastasis without affecting primary tumor growth. During metastatic colonization, intratumor bacteria carried by circulating tumor cells promoted host-cell survival by enhancing resistance to fluid shear stress by reorganizing actin cytoskeleton. We further showed that intratumor administration of selected bacteria strains isolated from tumor-resident microbiota promoted metastasis in two murine tumor models with significantly different levels of metastasis potential. Our findings suggest that tumor-resident microbiota, albeit at low biomass, play an important role in promoting cancer metastasis, intervention of which might therefore be worth exploring for advancing oncology care.

Genome-wide systematic survey and analysis of the RNA helicase gene family and their response to abiotic stress in sweetpotato.

Sweetpotato (Ipomoea batatas (L.) Lam.) holds a crucial position as one of the staple foods globally, however, its yields are frequently impacted by environmental stresses. In the realm of plant evolution and the response to abiotic stress, the RNA helicase family assumes a significant role. Despite this importance, a comprehensive understanding of the RNA helicase gene family in sweetpotato has been lacking. Therefore, we conducted a comprehensive genome-wide analysis of the sweetpotato RNA helicase family, encompassing aspects such as chromosome distribution, promoter elements, and motif compositions. This study aims to shed light on the intricate mechanisms underlying the stress responses and evolutionary adaptations in sweetpotato, thereby facilitating the development of strategies for enhancing its resilience and productivity. 300 RNA helicase genes were identified in sweetpotato and categorized into three subfamilies, namely IbDEAD, IbDEAH and IbDExDH. The collinearity relationship between the sweetpotato RNA helicase gene and 8 related homologous genes from other species was explored, providing a reliable foundation for further study of the sweetpotato RNA helicase gene family's evolution. Furthermore, through RNA-Seq analysis and qRT-PCR verification, it was observed that the expression of eight RNA helicase genes exhibited significant responsiveness to four abiotic stresses (cold, drought, heat, and salt) across various tissues of ten different sweetpotato varieties. Sweetpotato transgenic lines overexpressing the RNA helicase gene IbDExDH96 were generated using A.rhizogenes-mediated technology. This approach allowed for the preliminary investigation of the role of sweetpotato RNA helicase genes in the response to cold stress. Notably, the promoters of RNA helicase genes contained numerous cis-acting elements associated with temperature, hormone, and light response, highlighting their crucial role in sweetpotato abiotic stress response.

Systematic identification and expression analysis of bHLH gene family reveal their relevance to abiotic stress response and anthocyanin biosynthesis in sweetpotato.

BACKGROUND: bHLH transcription factors play significant roles in regulating plant growth and development, stress response, and anthocyanin biosynthesis. Sweetpotato is a pivotal food and industry crop, but little information is available on sweetpotato bHLH genes. RESULTS: Herein, 227 putative IbbHLH genes were defined on sweetpotato chromosomes, and fragment duplications were identified as the dominant driving force for IbbHLH expansion. These IbbHLHs were divided into 26 subfamilies through phylogenetic analysis, as supported by further analysis of exon-intron structure and conserved motif composition. The syntenic analysis between IbbHLHs and their orthologs from other plants depicted evolutionary relationships of IbbHLHs. Based on the transcriptome data under salt stress, the expression of 12 IbbHLHs was screened for validation by qRT-PCR, and differential and significant transcriptions under abiotic stress were detected. Moreover, IbbHLH123 and IbbHLH215, which were remarkably upregulated by stress treatments, had obvious transactivation activity in yeasts. Protein interaction detections and yeast two-hybrid assays suggested an intricate interaction correlation between IbbHLHs. Besides, transcriptome screening revealed that multiple IbbHLHs may be closely related to anthocyanin biosynthesis based on the phenotype (purple vs. white tissues), which was confirmed by subsequent qRT-PCR analysis. CONCLUSIONS: These results shed light on the promising functions of sweetpotato IbbHLHs in abiotic stress response and anthocyanin biosynthesis.

The unique sweet potato NAC transcription factor IbNAC3 modulates combined salt and drought stresses.

Plants often simultaneously experience combined stresses rather than a single stress, causing more serious damage, but the underlying mechanisms remain unknown. Here, we identified the stress-induced IbNAC3 from sweet potato (Ipomoea batatas) as a nucleus-localized transcription activator. IbNAC3 contains a unique activation domain whose MKD sequence confers transactivation activities to multiple other TFs and is essential for the activated expression of downstream target genes. Ectopic expression of IbNAC3 conferred tolerance to single and combined salt and drought stresses in Arabidopsis (Arabidopsis thaliana), and a group of NAM, ATAF1/2, and CUC2 (NAC) TFs, including ANAC011, ANAC072, ANAC083, ANAC100, and NAP, interacted with IbNAC3, and the specific domains responsible for each interaction varied. Intriguingly, IbNAC3 repressed the interaction among the five NACs, and knockout or mutation of ANAC011 and ANAC072 dramatically impaired combined stress tolerance. IbNAC3-ANAC072 and IbNAC3-NAP modules synergistically activated the MICROTUBULE-RELATED E3 LIGASE57 (MREL57) gene. Consistently, mutation of MREL57 and overexpression of WAVE-DAM-PENED2-LIKE7, encoding a target protein of MREL57, both remarkably impaired combined stress tolerance. Moreover, transgenic plants displayed abscisic acid (ABA) hyposensitivity by directly promoting the transcription of ENHANCED RESPONSE TO ABA 1, a key negative regulator of ABA signaling. The data unravel the unique IbNAC3 TF functions as a pivotal component in combined stress tolerance by integrating multiple regulatory events and ubiquitin pathways, which is essential for developing high-tolerant plants in natural environments.

Joint equalization of frequency offset and phase noise using two-stage cascaded extended Kalman filter for discrete spectrum 16/64APSK NFDM systems.

For the discrete spectrum nonlinear frequency division multiplexing (DS-NFDM) 16/64 amplitude phase shift keying (APSK) system, the inevitable laser impairments including frequency offset (FO) and carrier phase noise (CPN) would cause different rotations of the received signal constellations. In addition, the combined effect of FO and amplifier spontaneous emission (ASE) noise induces the eigenvalue shift, accordingly the residual channel impairment (RCI) is inevitably yielded. To address the above problems, we deduce the joint impairment model of FO, CPN and RCI, and then propose a joint equalization scheme using two-stage cascaded extended Kalman filter (TSC-EKF) for these impairments. It performs frequency offset compensation in the first stage, subsequently carries out joint equalization of CPN and RCI in the second stage. Meanwhile, the minimum Euclidean distance and phase difference between the received symbols and the ideal 16/64APSK constellations are ingeniously fused to calculate the innovations of TSC-EKF. The effectiveness has been verified by 2 GBaud DS-NFDM 16/64 APSK simulations and DS-NFDM 16APSK transmission experiments. The results demonstrate that when performing the joint equalization of FO, CPN and RCI, the maximum FOE range of TSC-EKF scheme achieves 1.2 and 9.6 times as that of nonlinear frequency domain (NFD) scheme and fast Fourier transform -Like (FFT-Like) scheme, respectively. Furthermore, its maximum LW tolerance reaches 3.3 times as that of the M-th power scheme. Importantly, the complexity of TSC-EKF is 63.4% as that of NFD scheme and on an order of O(N).

Two SEPALLATA MADS-Box Genes, SlMBP21 and SlMADS1, Have Cooperative Functions Required for Sepal Development in Tomato.

MADS-box transcription factors have crucial functions in numerous physiological and biochemical processes during plant growth and development. Previous studies have reported that two MADS-box genes, SlMBP21 and SlMADS1, play important regulatory roles in the sepal development of tomato, respectively. However, the functional relationships between these two genes are still unknown. In order to investigate this, we simultaneously studied these two genes in tomato. Phylogenetic analysis showed that they were classified into the same branch of the SEPALLATA (SEP) clade. qRT-PCR displayed that both SlMBP21 and SlMADS1 transcripts are preferentially accumulated in sepals, and are increased with flower development. During sepal development, SlMBP21 is increased but SlMADS1 is decreased. Using the RNAi, tomato plants with reduced SlMBP21 mRNA generated enlarged and fused sepals, while simultaneous inhibition of SlMBP21 and SlMADS1 led to larger (longer and wider) and fused sepals than that in SlMBP21-RNAi lines. qRT-PCR results exhibited that the transcripts of genes relating to sepal development, ethylene, auxin and cell expansion were dramatically changed in SlMBP21-RNAi sepals, especially in SlMBP21-SlMADS1-RNAi sepals. Yeast two-hybrid assay displayed that SlMBP21 can interact with SlMBP21, SlAP2a, TAGL1 and RIN, and SlMADS1 can interact with SlAP2a and RIN, respectively. In conclusion, SlMBP21 and SlMADS1 cooperatively regulate sepal development in tomato by impacting the expression or activities of other related regulators or via interactions with other regulatory proteins.

Determination of operation parameters for magnesium-air fuel cell to recover nitrogen and phosphorus from wastewater.

Recovering phosphorus (P) and nitrogen (N) from wastewater not only contributes to environmental protection but also aligns with sustainable development goals. This study employed a magnesium-air fuel cell (Mg-O2-FC) to extract P and N from wastewater in the form of struvite (MgNH4·6H2O), based on the removal efficiency of ammonia and phosphate, electricity generation capacity and struvite purity to determine the optimal operation parameters. These parameters included hydraulic retention time (HRT), service life of magnesium sheet, and precipitation discharge frequency. The results showed that the removal efficiency of ammonia from 0 to 4h was 55.99%, and that from 4 to 12h was only 15.74%. The phosphate removal efficiency in the initial cycle was 97.68% but decreased to 63.25% after 24h. The phosphate removal rate in 2 min increased by 145% when the precipitation discharge frequency increased from 4 h/time to 24 h/time. Consequently, the HRT, service life of the magnesium sheet, and precipitation discharge frequency were selected as 4 h, 24 h, and 24 h/time. These optimized conditions provide valuable insights for the practical implementation of Mg-O2-FC in recovering N and P from wastewater.

[Clinical Evaluation of Dural (Spinal) Membrane Repair Materials].

Objective: To select high-quality and cost-effective dural (spinal) membrane repair materials, in order to reduce the cost of consumables procurement, save medical insurance funds, and optimize hospital operation and management. Methods: Taking the BS06B disease group (spinal cord and spinal canal surgery without extremely severe or severe complications and comorbidities, mainly diagnosed as congenital tethered cord syndrome) as an example, a retrospective analysis was conducted on the relevant data of surgical treatment for congenital tethered cord syndrome conducted in our hospital from January 2021 to June 2023. Safety and efficacy indicators in clinical application (incidence of postoperative epidural hemorrhage, incidence of postoperative purulent cerebrospinal meningitis, incidence of cerebrospinal fluid leakage, surgical duration, and postoperative hospital stay) were compared. Results: There was no difference in safety and effectiveness between different brands of dura mater repair materials. Conclusion: For the repair of small incisions in dura mater surgery, high-quality and cost-effective dura mater repair materials can be selected to reduce hospital costs and control expenses for the disease group.

ASE noise mitigation with digital frequency offset loading for discrete spectrum nonlinear frequency division multiplexing systems.

We propose an amplified spontaneous emission (ASE) noise mitigation scheme utilizing digital frequency offset loading (DFO-loading) for discrete spectrum nonlinear frequency division multiplexing (DS-NFDM) systems. Firstly, based on the one-to-one mapping relationship between frequency offsets and eigenvalue positions, the transmitter side encodes 4-bit information onto 16 kinds of different digital frequency offsets. Then, a sliding window-assisted eigenvalue position (SWA-EP) decoding technology is further proposed to substitute the classical channel equalization and carrier phase recovery processes, with the purpose of recovering the original information. The numerical and experimental results demonstrate that, compared with b-coefficient 16 quadrature amplitude modulation (QAM) scheme, Q-factor gains are 2.1 dB under 15 dB optical signal-to-noise ratio (OSNR) and 1.8 dB after 800 km fiber transmission, respectively. Moreover, its complexity is only 0.6% of the b-coefficient scheme. The DFO-loading scheme offers an effective and low-complexity way to mitigate ASE noise of DS-NFDM system.

Identification of the GDP-L-Galactose Phosphorylase Gene as a Candidate for the Regulation of Ascorbic Acid Content in Fruits of Capsicum annuum L.

Ascorbic acid (AsA) is an antioxidant with significant functions in both plants and animals. Despite its importance, there has been limited research on the molecular basis of AsA production in the fruits of Capsicum annuum L. In this study, we used Illumina transcriptome sequencing (RNA-seq) technology to explore the candidate genes involved in AsA biosynthesis in Capsicum annuum L. A total of 8272 differentially expressed genes (DEGs) were identified by the comparative transcriptome analysis. Weighted gene co-expression network analysis identified two co-expressed modules related to the AsA content (purple and light-cyan modules), and eight interested DEGs related to AsA biosynthesis were selected according to gene annotations in the purple and light-cyan modules. Moreover, we found that the gene GDP-L-galactose phosphorylase (GGP) was related to AsA content, and silencing GGP led to a reduction in the AsA content in fruit. These results demonstrated that GGP is an important gene controlling AsA biosynthesis in the fruit of Capsicum annuum L. In addition, we developed capsanthin/capsorubin synthase as the reporter gene for visual analysis of gene function in mature fruit, enabling us to accurately select silenced tissues and analyze the results of silencing. The findings of this study provide the theoretical basis for future research to elucidate AsA biosynthesis in Capsicum annuum L.

Phosphorylation of the LCB1 subunit of Arabidopsis serine palmitoyltransferase stimulates its activity and modulates sphingolipid biosynthesis.

Sphingolipids are the structural components of membrane lipid bilayers and act as signaling molecules in many cellular processes. Serine palmitoyltransferase (SPT) is the first committed and rate-limiting enzyme in the de novo sphingolipids biosynthetic pathway. The core SPT enzyme is a heterodimer consisting of LONG-CHAIN BASE1 (LCB1) and LCB2 subunits. SPT activity is inhibited by orosomucoid proteins and stimulated by small subunits of SPT (ssSPTs). However, whether LCB1 is modified and how such modification might regulate SPT activity have to date been unclear. Here, we show that activation of MITOGEN-ACTIVATED PROTEIN KINASE 3 (MPK3) and MPK6 by upstream MKK9 and treatment with Flg22 (a pathogen-associated molecular pattern) increases SPT activity and induces the accumulation of sphingosine long-chain base t18:0 in Arabidopsis thaliana, with activated MPK3 and MPK6 phosphorylating AtLCB1. Phosphorylation of AtLCB1 strengthened its binding with AtLCB2b, promoted its binding with ssSPTs, and stimulated the formation of higher order oligomeric and active SPT complexes. Our findings therefore suggest a novel regulatory mechanism for SPT activity.

A systematical genome-wide analysis and screening of WRKY transcription factor family engaged in abiotic stress response in sweetpotato.

BACKGROUND: WRKY transcription factors play pivotal roles in regulating plant multiple abiotic stress tolerance, however, a genome-wide systematical analysis of WRKY genes in sweetpotato is still missing. RESULTS: Herein, 84 putative IbWRKYs with WRKY element sequence variants were identified in sweetpotato reference genomes. Fragment duplications, rather than tandem duplications, were shown to play prominent roles in IbWRKY gene expansion. The collinearity analysis between IbWRKYs and the related orthologs from other plants further depicted evolutionary insights into IbWRKYs. Phylogenetic relationships displayed that IbWRKYs were divided into three main groups (I, II and III), with the support of the characteristics of exon-intron structures and conserved protein motifs. The IbWRKY genes, mainly from the group Ib, displayed remarkable and diverse expression profiles under multiple abiotic stress (NaCl, PEG6000, cold and heat) and hormone (ABA, ACC, JA and SA) treatments, which were determined by RNA-seq and qRT-PCR assays, suggesting their potential roles in mediating particular stress responses. Moreover, IbWRKY58L could interact with IbWRKY82 as revealed by yeast two-hybrid based on the protein interaction network screening. And abiotic stress-remarkably induced IbWRKY21L and IbWRKY51 were shown to be localized in the nucleus and had no transactivation activities. CONCLUSION: These results provide valuable insights into sweetpotato IbWRKYs and will lay a foundation for further exploring functions and possible regulatory mechanisms of IbWRKYs in abiotic stress tolerance.

Genome-wide survey and expression analysis of GRAS transcription factor family in sweetpotato provides insights into their potential roles in stress response.

BACKGROUND: The plant-specific GRAS transcription factors play pivotal roles in various adverse environmental conditions. Numerous GRAS genes have been explored and characterized in different plants, however, comprehensive survey on GRASs in sweetpotato is lagging. RESULTS: In this study, 72 putative sweetpotato IbGRAS genes with uneven distribution were isolated on 15 chromosomes and classified into 12 subfamilies supported by gene structures and motif compositions. Moreover, both tandem duplication and segmental duplication events played critical roles in the expansion of sweetpotato GRAS genes, and the collinearity between IbGRAS genes and the related orthologs from nine other plants further depicted evolutionary insights into GRAS gene family. RNA-seq analysis under salt stress and qRT-PCR detection of 12 selected IbGRAS genes demonstrated their significant and varying inductions under multiple abiotic stresses (salt, drought, heat and cold) and hormone treatments (ABA, ACC and JA). Consistently, the promoter regions of IbGRAS genes harbored a series of stress- and hormone-associated cis-acting elements. Among them, IbGRAS71, the potential candidate for breeding tolerant plants, was characterized as having transactivation activity in yeasts, while IbGRAS-2/-4/-9 did not. Moreover, a complex interaction relationship between IbGRASs was observed through the interaction network analysis and yeast two-hybrid assays. CONCLUSIONS: Our results laid a foundation for further functional identifications of IbGRAS genes, and multiple members may serve as potential regulators for molecular breeding of tolerant sweetpotato.

Preparation of a Chitosan/Coal Gasification Slag Composite Membrane and Its Adsorption and Removal of Cr (VI) and RhB in Water.

At present, there are many kinds of pollutants, including dyes and heavy metal ions, in wastewater. It is very important to develop adsorbents that can simultaneously remove heavy metal ions and dyes. In this study, a renewable composite membrane material was synthesized using chitosan and treated coal gasification slag. The Cr (VI) maximum adsorption capacity of the composite membrane was 50.0 mg/L, which was 4.3~8.8% higher than that of the chitosan membrane. For the adsorption of RhB, the removal rate of the chitosan membrane was only approximately 5.0%, but this value could be improved to 95.3% by introducing coal gasification slag. The specific surface area of the chitosan membrane could also be increased 16.2 times by the introduction of coal gasification slag. This is because coal gasification slag could open the nanopores of the chitosan membrane (from 80 µm to 110 µm). Based on the adsorption kinetics and adsorption mechanism analysis, it was found that the adsorption of Cr (VI) occurred mainly through the formation of coordination bonds with the amino groups on the molecular chains of chitosan. Meanwhile, RhB adsorption occurred through the formation of hydrogen bonds with the surface of coal gasification slag. Additionally, coal gasification slag can improve the mechanical properties of the chitosan membrane by 2.2 times, which may facilitate the practical application of the composite membrane. This study provides new insight into the adsorbent design and the resource utilization of coal gasification slag.

Genetic polymorphisms of 66 InDel loci in the Chinese Han population.

Insertion/deletion polymorphism (InDel) genetic markers refer to insertion or deletion of DNA fragments into genomic DNA, which have advantages in the identification of degraded samples. In this study, we independently screened 66 highly polymorphic InDel markers from the dbSNP database to establish a multiplex PCR system for forensic DNA identification using next-generation sequencing system (66-plex InDels). We assessed the population genetic data among 251 Chinese Han population using this system and evaluated their potential forensic application. The results showed that all 66 InDel loci conformed to the Hardy-Weinberg equilibrium (P>0.000 758), and all the pairwise InDel loci were in linkage equilibrium after Bonferroni correction. The mean observed heterozygosity (Ho) was 0.482, the mean expected heterozygosity (He) was 0.483,the mean discrimination power (DP) was 0.612, the mean polymorphism information content (PIC) was 0.365, the total discrimination power (TDP) reached 0.999 999 999 999 999 999 999 999 999 428 18. The cumulative power of exclusion for 66 InDel loci was 0.999 739 in duo cases (CPEduo) and was 0.999 999 999 417 in trios cases (CPEtrio). The results show that the 66 InDel loci have high genetic polymorphisms in the Chinese Han population and can be used independently for forensic DNA identification and paternity testing.

Investigation of absolute dose calibration accuracy for TomoTherapy using real water.

A systematic bias in TomoTherapy output calibration was reported by the Imaging and Radiation Oncology Core Houston (IROC-H) after analyzing intensity-modulated radiation therapy (IMRT) credentialing results from hundreds of TomoTherapy units. Multiple theories were developed to explain this observation. One theory was that the use of a solid water "cheese" phantom instead of real water in the calibration measurement was the culprit. A phantom filled with distilled water was built to investigate whether our TomoTherapy was miscalibrated due to the use of a solid water phantom. A miscalibration of -1.47% was detected on our TomoTherapy unit. It is found that despite following the vendor's updated recommendation on computed tomography (CT) number to density calibration, the cheese phantom was still mapped to a density of 1.028 g/cm3 , rather than the 1.01 g/cm3 value reported in literature. When the density of the cheese phantom was modified to 1.01 g/cm3 in the treatment planning system, the measurement also indicated that our TomoTherapy machine was miscalibrated by -1.52%, agreeing with the real water phantom findings. Our single-institution finding showed that the cheese phantom density assignment can introduce greater than 1% errors in the TomoTherapy absolute dose calibration. It is recommended that the absolute dose calibration for TomoTherapy be performed either in real water or in the cheese phantom with the density in TPS overridden as 1.01 g/cm3 .

Design and Modification of a High-Resolution Optical Interferometer Accelerometer.

The Micro-Opto-Electro-Mechanical Systems (MOEMS) accelerometer is a new type of accelerometer that combines the merits of optical measurement and Micro-Electro-Mechanical Systems (MEMS) to enable high precision, small volume, and anti-electromagnetism disturbance measurement of acceleration, which makes it a promising candidate for inertial navigation and seismic monitoring. This paper proposes a modified micro-grating-based accelerometer and introduces a new design method to characterize the grating interferometer. A MEMS sensor chip with high sensitivity was designed and fabricated, and the processing circuit was modified. The micro-grating interference measurement system was modeled, and the response sensitivity was analyzed. The accelerometer was then built and benchmarked with a commercial seismometer in detail. Compared to the previous prototype in the experiment, the results indicate that the noise floor has an ultra-low self-noise of 15 ng/Hz1/2.

Altitudinal Variation of Metabolites, Mineral Elements and Antioxidant Activities of Rhodiola crenulata (Hook.f. & Thomson) H.Ohba.

Rhodiolacrenulata (Hook.f. & Thomson) H.Ohba is an alpine medicinal plant that can survive in extreme high altitude environments. However, its changes to extreme high altitude are not yet clear. In this study, the response of Rhodiola crenulata to differences in altitude gradients was investigated through chemical, ICP-MS and metabolomic methods. A targeted study of Rhodiola crenulata growing at three vertical altitudes revealed that the contents of seven elements Ca, Sr, B, Mn, Ni, Cu, and Cd, the phenolic components, the ascorbic acid, the ascorbic acid/dehydroascorbate ratio, and the antioxidant capacity were positively correlated with altitude, while the opposite was true for total ascorbic acid content. Furthermore, 1165 metabolites were identified: flavonoids (200), gallic acids (30), phenylpropanoids (237), amino acids (100), free fatty acids and glycerides (56), nucleotides (60), as well as other metabolites (482). The differential metabolite and biomarker analyses suggested that, with an increasing altitude: (1) the shikimic acid-phenylalanine-phenylpropanoids-flavonoids pathway was enhanced, with phenylpropanoids upregulating biomarkers much more than flavonoids; phenylpropanes and phenylmethanes upregulated, and phenylethanes downregulated; the upregulation of quercetin was especially significant in flavonoids; upregulation of condensed tannins and downregulation of hydrolyzed tannins; upregulation of shikimic acids and amino acids including phenylalanine. (2) significant upregulation of free fatty acids and downregulation of glycerides; and (3) upregulation of adenosine phosphates. Our findings provide new insights on the responses of Rhodiola crenulata to extreme high altitude adversity.

Adeno-associated virus vector enables safe and efficient Cas9 activation in neonatal and adult Cas9 knockin murine cochleae.

Adeno-associated virus (AAV)-mediated gene delivery systems have been shown to be effective tools for gene manipulation in the inner ear. For example, hair cells (HCs) and multiple other cell types can be transduced by the local injection of AAVs into the inner ear. However, application of the AAV-mediated CRISPR/Cas9 gene-editing approach to the inner ear in adult mice has not yet been studied. Based on our previous work, we investigated several AAV serotypes in neonatal and adult mice in parallel, and found that AAV8 had the top efficiency to transduce inner HCs. We then tested the ability of Cre-expressing AAV8 to activate Cas9 in floxed-Cas9 knockin mice, and observed significant Cas9 activation in the inner ear of both neonatal and adult animals. Neither the AAV8 virus itself nor the surgical procedures used to deliver it-cochleostomy for neonatal mice and canalostomy for adult mice-caused any damage to HCs or impaired normal hearing. Our studies indicate that the local injection of AAV8-Cre can induce Cas9 activation to perform safe and efficient gene editing in the inner ear, expanding the repertoire of gene-editing tools for regulating gene expression in the inner ear as a part of efforts to rescue genetic hearing loss, initiate regeneration of HCs, or develop gene therapy techniques.

High-throughput deep sequencing reveals the important role that microRNAs play in the salt response in sweet potato (Ipomoea batatas L.).

BACKGROUND: MicroRNAs (miRNAs), a class of small regulatory RNAs, have been proven to play important roles in plant growth, development and stress responses. Sweet potato (Ipomoea batatas L.) is an important food and industrial crop that ranks seventh in staple food production. However, the regulatory mechanism of miRNA-mediated abiotic stress response in sweet potato remains unclear. RESULTS: In this study, we employed deep sequencing to identify both conserved and novel miRNAs from salinity-exposed sweet potato cultivars and its untreated control. Twelve small non-coding RNA libraries from NaCl-free (CK) and NaCl-treated (Na150) sweet potato leaves and roots were constructed for salt-responsive miRNA identification in sweet potatoes. A total of 475 known miRNAs (belonging to 66 miRNA families) and 175 novel miRNAs were identified. Among them, 51 (22 known miRNAs and 29 novel miRNAs) were significantly up-regulated and 76 (61 known miRNAs and 15 novel miRNAs) were significantly down-regulated by salinity stress in sweet potato leaves; 13 (12 known miRNAs and 1 novel miRNAs) were significantly up-regulated and 9 (7 known miRNAs and 2 novel miRNAs) were significantly down-regulated in sweet potato roots. Furthermore, 636 target genes of 314 miRNAs were validated by degradome sequencing. Deep sequencing results confirmed by qRT-PCR experiments indicated that the expression of most miRNAs exhibit a negative correlation with the expression of their targets under salt stress. CONCLUSIONS: This study provides insights into the regulatory mechanism of miRNA-mediated salt response and molecular breeding of sweet potatoes though miRNA manipulation.