Eureka lemon zinc finger protein ClDOF3.4 interacts with citrus yellow vein clearing virus coat protein to inhibit viral infection

Citrus yellow vein clearing virus(CYVCV)is a new citrus virus that has become an important factor restricting the development of China’s citrus industry,and the CYVCV coat protein(CP)is associated with viral pathogenicity.In this study,the Eureka lemon zinc finger protein(ZFP)ClDOF3.4 was shown to interact with CYVCV CP in vivo and in vitro.Transient expression of ClDOF3.4 in Eureka lemon induced the expression of salicylic acid(SA)-related and hypersensitive response marker genes,and triggered a reactive oxygen species burst,ion leakage necrosis,and the accumulation of free SA.Furthermore,the CYVCV titer in ClDOF3.4 transgenic Eureka lemon plants was approximately 69.4%that in control plants 6 mon after inoculation,with only mild leaf chlorotic spots observed in those transgenic plants.Taken together,the results indicate that ClDOF3.4 not only interacts with CP but also induces an immune response in Eureka lemon by inducing the SA pathways.This is the first report that ZFP is involved in the immune response of a citrus viral disease,which provides a basis for further study of the molecular mechanism of CYVCV infection.

Tissue optical clearing enhances efficacy of vascular targeted photodynamic therapy of mouse dorsal skin

Vascular-targeted photodynamic therapy(V-PDT)is an effective treatment for port wine stains(PWS).However,repeated treatment is usually needed to achieve optimal treatment outcomes,possibly due to the limited treatment light penetration depth in the PWS lesion.The optical clearing technique can increase light penetration in depth by reducing light scattering.This study aimed to investigate the V-PDT in combination with an optical clearing agent(OCA)for the therapeutic enhancement of V-PDT in the rodent skinfold window chamber model.Vascular responses were closely monitored with laser speckle contrast imaging(LSCI),optical coherence tomography angiography,and stereo microscope before,during,and after the treatment.We further quantitatively demonstrated the effects of V-PDT in combination with OCA on the blood flow and blood vessel size of skin microvasculature.The combination of OCA and V-PDT resulted in significant vascular damage,including vasoconstriction and the reduction of blood flow.Our results indicate the promising potential of OCA for enhancing V-PDT for treating vascular-related diseases,including PWS.

MACS-W:A modified optical clearing agent for imaging 3D cell cultures

Three-dimensional(3D)cell cultures have contributed to a variety of biological research fields by filling the gap between monolayers and animal models.The modern optical sectioning microscopic methods make it possible to probe the complexity of 3D cell cultures but are limited by the inherent opaqueness.While tissue optical clearing methods have emerged as powerful tools for investigating whole-mount tissues in 3D,they often have limitations,such as being too harsh for fragile 3D cell cultures,requiring complex handling protocols,or inducing tissue deformation with shrinkage or expansion.To address this issue,we proposed a modified optical clearing method for 3D cell cultures,called MACS-W,which is simple,highly efficient,and morphology-preserving.In our evaluation of MACS-W,we found that it exhibits excellent clearing capability in just 10 min,with minimal deformation,and helps drug evaluation on tumor spheroids.In summary,MACS-W is a fast,minimally-deformative and fluorescence compatible clearing method that has the potential to be widely used in the studies of 3D cell cultures.

Use of a tissue clearing technique combined with retrograde trans-synaptic viral tracing to evaluate changes in mouse retinorecipient brain regions following optic nerve crush

Successful establishment of reconnection between retinal ganglion cells and retinorecipient regions in the brain is critical to optic nerve regeneration.However,morphological assessments of retinorecipient regions are limited by the opacity of brain tissue.In this study,we used an innovative tissue cleaning technique combined with retrograde trans-synaptic viral tracing to observe changes in retinorecipient regions connected to retinal ganglion cells in mice after optic nerve injury.Specifically,we performed light-sheet imaging of whole brain tissue after a clearing process.We found that pseudorabies virus 724(PRV724)mostly infected retinal ganglion cells,and that we could use it to retrogradely trace the retinorecipient regions in whole tissue-cleared brains.Unexpectedly,PRV724-traced neurons were more widely distributed compared with data from previous studies.We found that optic nerve injury could selectively modify projections from retinal ganglion cells in the hypothalamic paraventricular nucleus,intergeniculate leaflet,ventral lateral geniculate nucleus,central amygdala,basolateral amygdala,Edinger-Westphal nucleus,and oculomotor nucleus,but not the superior vestibular nucleus,red nucleus,locus coeruleus,gigantocellular reticular nucleus,or facial nerve nucleus.Our findings demonstrate that the tissue clearing technique,combined with retrograde trans-synaptic viral tracing,can be used to objectively and comprehensively evaluate changes in mouse retinorecipient regions that receive projections from retinal ganglion cells after optic nerve injury.Thus,our approach may be useful for future estimations of optic nerve injury and regeneration.

Research Progress on Anti-tumor Mechanism of Clearing Heat-Toxin Intervention of Apoptosis-related Proteins

Apoptosis is a spontaneous programmed cell death process,which is closely related to the occurrence and development of tumors.Inducing apoptosis of tumor cells has become an important way of anti-tumor therapy.Studies have found that clearing heat-toxin Chinese medicine has a significant effect on inducing apoptosis,which may be the key mechanism of anti-tumor of Chinese medicine.By reviewing the theoretical origin of anti-tumor TCM of clearing heat-toxin Chinese herbs and the pharmacological research progress of intervening apoptosis-related proteins to promote apoptosis of tumor cells,this paper provides a basis for TCM to induce apoptosis of tumor cells to prevent and treat malignant tumors.

Automated Clearing System on Deposit Money Banks’Performance:Experience from the Nigerian Banks

This study majorly examined the effect of automated cheque clearing system on banks.Specifically,this study examined the effect of automated cheque clearing system on total deposits of deposit money banks in Nigeria.Data were extracted from the CBN Statistical Bulletin(2020).Using the ordinary least square method of analysis via E-views 10,the findings of the study revealed that deposit money banks’total deposits increased significantly owing to the decline in the value of cheques cleared,since the adoption of the automated cheque clearing system in Nigeria.Guided by the findings of this study,the researcher recommended that the regulatory authorities and banks should continue to liaise with each other on how to improve the efficiency of automated services so as to improve customer satisfaction and patronage.

A New Technique for Whole Clearing

[Objective] The research aimed to study a new whole clearing and preparation technology.[Method] After young seeds of Stephania sp.preserved in the alcohol solution,as well as the fresh pistil and nucellus of Sorghum bicolor were separated,they were soaked and cleared in the solution of 84 disinfectant.And the plant materials could be mounted by using three methods.[Result] The globular embryos and heart-shaped embryos at different stages of Stephania sp.as well as the pistil,nucellus,embryo sac,hypostase o...

Study on Microsatellite Distribution in Complete Genomes of Tobacco Vein Clearing Virus

MATLAB software and optimal complete subgraph algorithm were used to extract and reveal the microsatellite distribution features in the complete genomes of the tobacco vein clearing virus （NC-003 378.1） from the NCBI database.The results showed that the repetitions number and their location of the N-base group has been extracted and displayed.The largest repetitions of N-base group in the complete genomes of the tobacco vein clearing virus was decreased as the exponential function with the increasing of N.The method used in this study could be applied to the extraction and revealing of the microsatellite distribution features in the complete genomes of other viruses,thereby provided a basis for the research of the structure and the law of function,inheritance and variation by the using of the microsatellite distribution features.

ALTERATIONS IN AUTOFLUORESCENCE SIGNAL FROM RAT SKIN EX VIVO UNDER OPTICAL IMMERSION CLEARING

For the first time,the changes in autofluorescence spectra of ex vivo rat skin have been experimentally investigated using the combination of fluorescence spectroscopy and optical immersion clearing.The glucose,glycerol and propylene glycol solutions were used as clearing agents.The optical clearing was performed from the dermal side of skin imitating the in vivo injection of clearing agent under the dermal layers.In this contribution,the common properties of autofluorescence variation during optical immersion clearing were determined.The tendency of autofluorescence signal to decrease with reduction of scattering in tissue was noticed and discussed in detail.However,the differences in the shape of spectral curves under application of different clearing agents showed that optical clearing affects the autofluorescence properties of tissue differently depending on the type of clearing liquid.The results obtained are useful for the understanding of tissue optical clearing mechanisms and for improving techniques such as fluorescence spectroscopy.

QUANTITATIVE CONTROL OF OPTICAL CLEARING EFFECTS STUDIED WITH TISSUE-LIKE PHANTOM

Tissue optical clearing by use of optical clearing agents(OCAs)has been proven to have potential to reduce the highly scattering effect of biological tissues in optical techniques.However,the difference in tissue samples could lead to unreliable results,making it difficult to quantitatively control the dose of OCAs during the course of tissue optical clearing.In this work,in order to study the effects of optical clearing,we customized tissue-like phantoms with optical properties of some biological tissue.Diffuse reflectance and total transmittance of tissue-like phantoms with different OCAs(DMSO or glycerol)and porcine skin tissues were measured.Then optical property parameters were calculated by inverse adding-doubling(IAD)algorithm.Results showed that OCAs could lead to a reduction in scattering of tissue-like phantoms as it did to porcine skin tissue in vitro.Furthermore,a series of relational expressions could be fit to quantitatively describe the relationship between the doses of OCAs and the reduction of scattering effects.Therefore,proper tissue-like phantom could facilitate optical clearing to be used in quantitative control of tissue optical properties,and further promote the application potential of optical clearing to light-based noninvasive diagnostic and therapeutic techniques.

Monoclonal antibody-based serological detection of Citrus yellow vein clearing virus in citrus groves

Citrus yellow vein clearing virus （CYVCV） is considered as the causal agent of Citrus yellow vein clearing disease and belongs to the genus Mandarivirus in the family Alphaflexiviridae. Capsid protein （CP） of CYVCV Chongqing isolate （CYVCV- CQ） was produced using a prokaryotic expression system and used as the immunogen for monoclonal antibody （MAb） production. Four highly specific and sensitive murine MAbs and one polyclonal antibody were prepared in this study. Titers of the four MAbs in ascites fluids ranged from 10-6 to 10-7 as determined by indirect enzyme-linked immunosorbent assay （ELISA）. Three serological assays, including dot enzyme-linked immunosorbent assay （dot-ELISA）, tissue blot-ELISA, and double-antibody sandwich （DAS）-ELISA, were developed for quick and reliable detections of CYVCV in citrus samples. The developed dot-ELISA and DAS-ELISA methods could detect CYVCV in the infected citrus leaf crude extracts diluted at 1：2 560 and 1：10 240 （w/v, g mL^-1）, respectively. The detection result of 125 citrus leaf samples collected from citrus groves in Yunnan Province and Chongqing Municipality of China showed that approximately 36% samples were positive for CYVCV. This virus was, however, not＇detected in any sample collected from Zhejiang or Jiangxi Province, China.

Optical tissue clearing enables rapid,precise and comprehensive assessment of three-dimensional morphology in experimental nerve regeneration research

Morphological analyses are key outcome assessments for nerve regeneration studies but are historically limited to tissue sections.Novel optical tissue clearing techniques enabling three-dimensional imaging of entire organs at a subcellular resolution have revolutionized morphological studies of the brain.To extend their applicability to experimental nerve repair studies we adapted these techniques to nerves and their motor and sensory targets in rats.The solvent-based protocols rendered harvested peripheral nerves and their target organs transparent within 24 hours while preserving tissue architecture and fluorescence.The optical clearing was compatible with conventional laboratory techniques,including retrograde labeling studies,and computational image segmentation,providing fast and precise cell quantitation.Further,optically cleared organs enabled three-dimensional morphometry at an unprecedented scale including dermatome-wide innervation studies,tracing of intramuscular nerve branches or mapping of neurovascular networks.Given their wide-ranging applicability,rapid processing times,and low costs,tissue clearing techniques are likely to be a key technology for next-generation nerve repair studies.All procedures were approved by the Hospital for Sick Children’s Laboratory Animal Services Committee(49871/9)on November 9,2019.

Clinical Study on Therapy of Clearing Hallow Viscera in Treating Critical Patients with Gastro-enteric Function Disorder

Objective： To explore the clinical effect of therapy of clearing hallow viscera in treating critical patients with gastro-enteric function disorder （GEFD）. Methods： Retrospective analysis was carried out on 96 critical patients. They were 48 patients in the treated group treated with Dachengqi Decoction （ 大承气汤） and 48 patients in the control group treated with Western medicine for promoting gastric dynamic force. The recovery rate, recovery time of gastro-enteric function, incidence rate and fatality rate of multiple organ dysfunction syndrome （MODS）, as well as the level of plasma endotoxin （ET） before and after treatment between the two groups were compared. Results： Comparison between the two groups in gastro-enteric function recovery rate （81.3% vs 45.8 %）, functional disorder sustaining time in patients who got recovered （1.2± 0.3 daysvs4.0±1.1 days）, incidence rate （29. 17% vs 52.08%） and fatality rate （28.57% vs 56.00%） of MODS all showed significant difference （ P〈0.05 or P〈0.01 ）. The plasma level of ET after treatment in the treated group was significantly lower than that in the control group （P〈0.05）. Conclusion： Therapy of clearing hallow viscera has a good effect in treating critical patients with gastro-enteric function disorder, and could reduce the incidence and fatality of MODS.

Recent Advances on Citrus yellow vein clearing virus in Citrus

Citrus yellow vein clearing virus(CYVCV)is the causal agent of yellow vein clearing disease,a significant and devastating disease of most citrus species including lemon and sour orange.CYVCV,a single-strand positive-sense RNA virus containing six ORFs(Open Reading Frames),represents a new species in the genus Mandarivirus of the Alphaflexiviridae family.The virus can cause particularly serious damage,resulting in reduced tree vigor,lower yields,and decreased marketability of fruit production,and it has been found in India,Turkey,Pakistan,China and Iran.Here we described the geographical distribution of the virus,its transmission mode by grafting,mechanical inoculation,and insects,as well as currently available techniques for detection.In addition,we also discussed practical measures aimed at controlling the disease and provided theoretical guidance to prevent the acquisition and spread of the disease that is a significant step toward ensuring the health of the citrus industry.

Ex-vivo confocal Raman microspectroscopy of porcine skin with 633/785-NM laser excitation and optical clearing with glycerol/water/DMSO solution

Confocal Raman microspectroscopy(CRM)with 633-and 785-nm excitation wavelengths combined with optical clearing(OC)technique was used for ex-vivo study of porcine skin in the Raman fingerprint region.The optical clearing has been performed on the skin samples by applying a mixture of glycerol and distilled water and a mixture of glycerol,distilled water and chemical penetration enhancer dimethyl sulfoxide(DMSO)during 30 min and 60 min of treat-ment.It was shown that the combined use of the optical clearing technique and CRM at 633 nm allowed one to preserve the high probing depth,signal-to-noise ratio and spectral resolution simultaneously.Comparing the effect of different optical clearing agents on porcine skin showed that an optical clearing agent containing chemical penetration enhancer provides higher optical clearing efficiency.Also,an increase in treatment time allows to improve the optical clearing efficiency of both optical clearing agents.As a result of optical clearing,the detection of the amide-Ⅲ spectral region indicating well-distinguishable structural differences between the type-Ⅰ and type-Ⅳ collagens has been improved.

ULTRASOUND ENHANCED SKIN OPTICAL CLEARING:MICROSTRUCTURAL CHANGES

Our previous studies demonstrated the ultrasound-induced skin optical clearing enhancement with topical application of optical clearing agents on in vitro porcine skin and in vivo human skin.The objective of this study was to investigate the possible mechanisms of the enhanced skin optical clearing by ultrasound medications.Optical clearing effects of ex vivo guinea pig abdomen skin topically applied with 60% glycerol or the combination of 60% glycerol and ultrasound were studied by optical coherence tomography(OCT).Microstructure of skin surface was examined by scanning electron microscopy(SEM).Ultrasound with a frequency of 1MHz and a power of 0.75W over a 3-cm probe was simultaneously applied with glycerol solution for 15min.The combination of 60% glycerol and ultrasound results in a 19% increase in OCT 1/e light penetration depth after 30min,which is much better than 60% glycerol alone.SEM images demonstrated that changes in skin microstructure due to the tight order of the lipid bilayers in the stratum corneum disrupted and the separation of keratinocytes by the application of ultrasound contribute to the ultrasound-enhanced intact skin optical clearing effects.

Improving sampling depth of laser speckle imaging by topical optical clearing:A theoretical and in vivo study

The effect of optical cleaning method combined with laser speckle imaging(LSI)was discussed to improve the detection depth of LSI due to high scattering characteristics of skin,which limit its clinical application.A double-layer skin tissue model embedded with a single blood vessel was established,and the Monte Carlo method was used to simulate photon propagation under the action of light-permeating agent.808 nm semiconductor and 632.8 nm He–Ne lasers were selected to study the e®ect of optical clearing agents(OCAs)on photon deposition in tissues.Results show that the photon energy deposition density in the epidermis increases with the amount of tissue°uid replaced by OCA.Compared with glucose solution,polyethylene glycol 400(PEG 400)and glycerol can considerably increase the average penetration depth of photons in the skin tissue,thereby raising the sampling depth of the LSI.After the action of glycerol,PEG 400,and glucose,the average photon penetration depth is increased by 51.78%,51.06%,and 21.51%for 808nm,68.93%,67.94%,and 26.67%for 632.8 nm lasers,respectively.In vivo experiment by dorsal skin chamber proves that glycerol can cause a substantial decrease in blood°ow rate,whereas PEG 400 can signicantly improve the capability of light penetration without a®ecting blood velocity,which exhibits considerable potential in the monitoring of blood°ow in skin tissues.

IMPROVE OPTICAL CLEARING OF SKIN IN VITRO WITH PROPYLENE GLYCOL AS A PENETRATION ENHANCER

In order to enhance the optical clearing effect of topically applied optical clearing agents(OCAs),we evaluated the effect of propylene glycol(PG)as a chemical penetration enhancer(PE)on optical clearing of skin in vitro by observation and measurement of optical-transmittance and diffuse-reflectance spectra.Three OCAs,i.e.,glycerol,D-sorbitol and PEG400,and two other penetration enhancers,Azone and Thiazone,were used in this study.The results indicated that the decrease of reduced scattering coefficient caused by OCA/PG was larger than that by pure OCA,and the change by OCA/water was the least after the same treatment time.There were significant differences for the reduced scattering coefficient at 630 nm after 120 min application of agents between OCA and OCA/PG.The efficacy of optical clearing caused by OCA/PG depended on the OCA itself.When PEG400 was mixed with three different PEs,we found the optical clearing were different.The penetration enhancing ability of PG was much better compared to Azone,and suboptimal to Thiazone.Also,this study provides evidence for the use of PG as a PE in order to improve skin optical clearing.

Small RNA deep sequencing reveals full-length genome of Citrus yellow vein clearing virus in Chongqing,China

To identity the potential pathogen associated with the yellow vein clearing symptom on lemon trees, the profiles of virus-de- rived small interfering RNAs from citrus samples were obtained and analyzed by deep sequencing method in this study. Twenty-seven contigs almost cover the full length genome of Citrus yellow vein clearing virus （CYVCV） isolate YN were obtained using the small RNA deep sequencing technology. Analysis showed that this isolate CQ shared the highest nucleotide identity with isolate Y1 （JX040635） and YN （KP313242）, both of which belong to the genus Mandarivirus in the family Alphaflexiviridae. Mapping analysis of viral-derived siRNA （vsiRNA） profiles revealed an uneven distribution pattern of their generations along both positive and negative genome strands, and 22- and 21-nt vsiRNAs ranked the majority. BLAST against viroids and other viral databases confirmed that this sample was single-infected only by CYVCV, which indicated that CYVCV was the exact causal agent for the yellow clearing symptom occurring on lemon. This is the first CYVCV isolate detected in Chongqing and the second in China. This result could provide a molecular basis for the investigation of citrus viral diseases to protect citrus health in this region.

Distributed ledger technology for securities clearing and settlement: benefits, risks, and regulatory implications

This article outlines the benefits and risks of the distributed ledger technology(DLT)for the clearing and settlement of exchange-traded and OTC securities,followed by a description of the technology’s potential role for central counterparties and central securities depositories.Although the industry and scholars are attempting to solve the technological and operational issues that DLT systems still face,outstanding legal risks are such that the financial industry is asking for more regulatory guidance and intervention.This article wants to contribute to the public policy debate by presenting potential regulatory barriers that may have to be removed for DLT to be fully adopted.In addition,it identifies areas requiring an update of the legal framework in order to address certain prudential and conduct risks that this technology could introduce.