Flexible Graphene Paper Modified Using Pt&Pd Alloy Nanoparticles Decorated Nanoporous Gold Support for the Electrochemical Sensing of Small Molecular Biomarkers.

The exploration into nanomaterial-based nonenzymatic biosensors with superb performance in terms of good sensitivity and anti-interference ability in disease marker monitoring has always attained undoubted priority in sensing systems. In this work, we report the design and synthesis of a highly active nanocatalyst, i.e., palladium and platinum nanoparticles (Pt&Pd-NPs) decorated ultrathin nanoporous gold (NPG) film, which is modified on a homemade graphene paper (GP) to develop a high-performance freestanding and flexible nanohybrid electrode. Owing to the structural characteristics the robust GP electrode substrate, and high electrochemically catalytic activities and durability of the permeable NPG support and ultrafine and high-density Pt&Pd-NPs on it, the resultant Pt&Pd-NPs-NPG/GP electrode exhibits excellent sensing performance of low detection limitation, high sensitivity and anti-interference capability, good reproducibility and long-term stability for the detection of small molecular biomarkers hydrogen peroxide (H2O2) and glucose (Glu), and has been applied to the monitoring of H2O2 in different types of live cells and Glu in body fluids such as urine and fingertip blood, which is of great significance for the clinical diagnosis and prognosis in point-of-care testing.

A highly efficient blocking ELISA based on p72 monoclonal antibody for the detection of African swine fever virus antibodies and identification of its linear B cell epitope.

Due to the absence of effective vaccine and treatment, African swine fever virus (ASFV) control is entirely dependent on accurate and early diagnosis, along with culling of infected pigs. The B646L/p72 is the major capsid protein of ASFV and is an important target for developing a diagnostic assays and vaccines. Herein, we generated a monoclonal antibody (mAb) (designated as 2F11) against the trimeric p72 protein, and a blocking ELISA (bELISA) was established for the detection of both genotype I and II ASFV antibodies. To evaluate the performance of the diagnostic test, a total of 506 porcine serum samples were tested. The average value of percent of inhibition (PI) of 133 negative pig serum was 8.4 % with standard deviation (SD) 6.5 %. Accordingly, the cut-off value of the newly established method was set at 28 % (mean + 3SD). Similarly, a receiver operating characteristic (ROC) was applied to determine the cut off value and the p72-bELISA exhibited a sensitivity of 100 % and a specificity of 99.33 % when the detection threshold was set at 28 %. The bELISA was also able to specifically recognize anti-ASFV sera without cross-reacting with other positive serums for other major swine pathogens. Moreover, by designing a series of overlapped p72 truncated proteins, the linear B cell epitope recognized by 2F11 mAb was defined to be 283NSHNIQ288. Amino acid sequence comparison revealed that the amino acid sequence 283NSHNIQ288 is highly conserved between different ASFV isolates. Our findings indicate that the newly established mAb based blocking ELISA may have a great potential in improving the detection of ASFV antibodies and provides solid foundation for further studies.

Optical readout of charge carriers stored in a 2D memory cell of monolayer WSe2.

Owing to their superior charge retaining and transport characteristics, 2D transition metal dichalcogenides are investigated for practical applications in various memory-cell structures. Herein, we fabricated a quasi-one-terminal 2D memory cell by partially depositing a WSe2 monolayer on an Au electrode, which can be manipulated to achieve efficient charge injection upon the application or removal of external bias. Furthermore, the amount of charge carriers stored in the memory cell could be optically probed because of its close correlation with the fluorescence efficiency of WSe2, allowing us to achieve an electron retention time of ∼300 s at the cryogenic temperature of 4 K. Accordingly, the simplified device structure and the non-contact optical readout of the stored charge carriers present new research opportunities for 2D memory cells in terms of both fundamental mechanism studies and practical development for integrated nanophotonic devices.

The attenuated African swine fever vaccine HLJ/18-7GD provides protection against emerging prevalent genotype II variants in China.

Genetic changes have occurred in the genomes of prevalent African swine fever viruses (ASFVs) in the field in China, which may change their antigenic properties and result in immune escape. There is usually poor cross-protection between heterogonous isolates, and, therefore, it is important to test the cross-protection of the live attenuated ASFV vaccines against current prevalent heterogonous isolates. In this study, we evaluated the protective efficacy of the ASFV vaccine candidate HLJ/18-7GD against emerging isolates. HLJ/18-7GD provided protection against a highly virulent variant and a lower lethal isolate, both derived from genotype II Georgia07-like ASFV and isolated in 2020. HLJ/18-7GD vaccination prevented pigs from developing ASF-specific clinical signs and death, decreased viral shedding via the oral and rectal routes, and suppressed viral replication after challenges. However, HLJ/18-7GD vaccination did not provide solid cross-protection against genotype I NH/P68-like ASFV challenge in pigs. HLJ/18-7GD vaccination thus shows great promise as an alternative strategy for preventing and controlling genotype II ASFVs, but vaccines providing cross-protection against different ASFV genotypes may be needed in China.

Highly lethal genotype I and II recombinant African swine fever viruses detected in pigs.

African swine fever virus (ASFV) poses a great threat to the global pig industry and food security. Currently, 24 ASFV genotypes have been reported but it is unclear whether recombination of different genotype viruses occurs in nature. In this study, we detect three recombinants of genotype I and II ASFVs in pigs in China. These recombinants are genetically similar and classified as genotype I according to their B646L gene, yet 10 discrete fragments accounting for over 56% of their genomes are derived from genotype II virus. Animal studies with one of the recombinant viruses indicate high lethality and transmissibility in pigs, and deletion of the virulence-related genes MGF_505/360 and EP402R derived from virulent genotype II virus highly attenuates its virulence. The live attenuated vaccine derived from genotype II ASFV is not protective against challenge of the recombinant virus. These naturally occurring recombinants of genotype I and II ASFVs have the potential to pose a challenge to the global pig industry.

African Swine Fever Virus HLJ/18 CD2v Suppresses Type I IFN Production and IFN-Stimulated Genes Expression through Negatively Regulating cGMP-AMP Synthase-STING and IFN Signaling Pathways.

African swine fever is a fatal infectious disease caused by African swine fever virus (ASFV). The high mortality caused by this infectious disease is a significant challenge to the swine industry worldwide. ASFV virulence is related to its ability to antagonize IFN response, yet the mechanism of antagonism is not understood. Recently, a less virulent recombinant virus has emerged that has a EP402R gene deletion within the parental ASFV HLJ/18 (ASFV-ΔEP402R) strain. EP402R gene encodes CD2v. Hence we hypothesized that ASFV uses CD2v protein to evade type I IFN-mediated innate immune response. We found that ASFV-ΔEP402R infection induced higher type I IFN response and increased the expression of IFN-stimulated genes in porcine alveolar macrophages when compared with parental ASFV HLJ/18. Consistent with these results, CD2v overexpression inhibited type I IFN production and IFN-stimulated gene expression. Mechanistically, CD2v, by interacting with the transmembrane domain of stimulator of IFN genes (STING), prevented the transport of STING to the Golgi apparatus, and thereby inhibited the cGMP-AMP synthase-STING signaling pathway. Furthermore, ASFV CD2v disrupted IFNAR1-TYK2 and IFNAR2-JAK1 interactions, and thereby inhibited JAK-STAT activation by IFN-α. In vivo, specific pathogen-free pigs infected with the mutant ASFV-ΔEP402R strain survived better than animals infected with the parental ASFV HLJ/18 strain. Consistent with this finding, IFN-ß protein levels in the peripheral blood of ASFV-ΔEP402R-challenged pigs were significantly higher than in the blood of ASFV HLJ/18-challenged pigs. Taken together, our findings suggest a molecular mechanism in which CD2v inhibits cGMP-AMP synthase-STING and IFN signaling pathways to evade the innate immune response rendering ASFV infection fatal in pigs.

Adsorption coupling photocatalytic removal of gaseous n-hexane by phosphorus-doped g-C3N4/TiO2/Zn(OAc)2-ACF composites.

VOCs emission reduction in the petroleum and petrochemical industry is a hot and difficult topic at present. The single method may not be able to meet the actual treatment status. Therefore, the adsorption coupled photocatalytic degradation technology was used to remove VOCs. Phosphorus-doped carbon nitride (PCN) and PCN/TiO2 were prepared by hydrothermal synthesis and sol-gel method, and then PCN/TiO2/Zn(OAc)2-ACF composites were prepared by ultrasonic impregnation on zinc acetate modified activated carbon fibers (Zn(OAc)2-ACF). The removal efficiency of n-hexane by composite materials was explored in a self-made reactor, and the factors affecting removal efficiency, removal mechanism, and possible ways of degradation were investigated. The results showed that under the optimum reaction conditions (initial concentration of n-hexane 200 mg/m3, space velocity 1000 h-1, light intensity 24 W, mass fraction of doped PCN 6%, loading twice, calcination temperature 450 °C), PCN/TiO2/Zn(OAc)2-ACF composite has the highest removal efficiency of n-hexane (90.2%). The adsorption capacity of the composites after doping the P element was 215.3 mg/g, which did not enhance the adsorption performance compared with that before doping, but the removal rate of n-hexane was higher. This showed that doping P element was helpful to enhance the photocatalytic activity of the composites.

Development of an ELISA Method to Differentiate Animals Infected with Wild-Type African Swine Fever Viruses and Attenuated HLJ/18-7GD Vaccine Candidate.

African swine fever (ASF) is a highly contagious hemorrhagic disease of pigs, posing a significant threat to the world pig industry. Several researchers are investigating the possibilities for developing a safe and efficient vaccine against ASF. In this regard, significant progress has been made and some gene-deleted ASFVs are reported as potential live attenuated vaccines. A seven-gene-deleted live attenuated vaccine candidate HLJ/18-7GD (among which CD2v is included) has been developed in our laboratory and reported to be safe and protective, and it is expected to be commercialized in the near future. There is an urgent need for developing a diagnostic method that can clearly discriminate between wild-type-ASFV-infected and vaccinated animals (DIVA). In the present study, a dual indirect ELISA based on p54 and CD2v proteins was successfully established to specifically distinguish serum antibodies from pigs infected with wild-type ASFV or possessing vaccine immunization. To evaluate the performance of the assay, a total of 433 serum samples from four groups of pigs experimentally infected with the wild-type HLJ/18 ASFV, immunized with the HLJ/18-7GD vaccine candidate, infected with the new lower virulent variant, and specific-pathogen-free pigs were used. Our results showed that the positive rate of immunized serum was 96.54% (p54) and 2.83% (CD2v), and the positive rate of the infection by wild-type virus was 100% (p54) and 97.8% (CD2v). Similarly, the positive rate to infection by the new low-virulent ASFV variant in China was 100% (p54) and 0% (CD2v), indicating the technique was also able to distinguish antibodies from wild-type and the new low-virulent ASFV variant in China. Moreover, no cross-reaction was observed in immune sera from other swine pathogens, such as CSFV, PEDV, PRRSV, HP-PRRSV, PCV2, and PrV. Overall, the developed dual indirect ELISA exhibited high diagnostic sensitivity, specificity, and repeatability and will provide a new approach to differentiate serum antibodies between wild virulent and CD2v-unexpressed ASFV infection, which will play a great role in serological diagnosis and epidemiological monitoring of ASF in the future.

Thickness dependent properties of ultrathin perovskite nanosheets with Ruddlesden-Popper-like atomic stackings.

Ruddlesden-Popper perovskites possess a rich variety of multiple phases due to their mixed organic cations and variable layer numbers. However, the direct observation of these phases and their optical performance in ultrathin nanosheets, have rarely been reported. Here we demonstrate, through a one-pot CVD synthesis method to incorporate MA+ and NMA+ cations into PbI2 simultaneously, that the stackings of Ruddlesden-Popper phases with a distribution of a number of layers 〈n〉 can be produced within a single perovskite nanosheet. As featured by the micro-, time-resolved and temperature-dependent photoluminescence measurements, the optical properties are highly dependent on the nanosheet thickness.

Genotype I African swine fever viruses emerged in domestic pigs in China and caused chronic infection.

The Georgia-07-like genotype II African swine fever virus (ASFV) with high virulence has been prevalent in China since 2018. Here, we report that genotype I ASFVs have now also emerged in China. Two non-haemadsorbing genotype I ASFVs, HeN/ZZ-P1/21 and SD/DY-I/21, were isolated from pig farms in Henan and Shandong province, respectively. Phylogenetic analysis of the whole genome sequences suggested that both isolates share high similarity with NH/P68 and OURT88/3, two genotype I ASFVs isolated in Portugal in the last century. Animal challenge testing revealed that SD/DY-I/21 shows low virulence and efficient transmissibility in pigs, and causes mild onset of infection and chronic disease. SD/DY-I/21 was found to cause necrotic skin lesions and joint swelling. The emergence of genotype I ASFVs will present more problems and challenges for the control and prevention of African swine fever in China.

Synergistic effect of adsorption and photocatalysis for the degradation of toluene by TiO2 loaded on ACF modified by Zn(CH3COO)2.

Activated carbon fiber (ACF) was modified by Zn(NO3)2, ZnCl2, and Zn(CH3COO)2), respectively, and then, TiO2 was loaded on the modified ACFs. The adsorption and photocatalysis performance were explored through the removal of toluene, and TiO2/ACF-Ac modified by Zn(CH3COO)2) with the best toluene degradation performance was selected. The characterization results of a scanning electron microscope (SEM), X-ray diffraction spectra (XRD), and Fourier transform infrared spectrometer (FTIR) indicated that the samples were rough, and TiO2 was mainly loaded on the surface containing large amount of oxygen-containing functional groups in anatase phase. An ultraviolet-visible diffuse reflectance spectrophotometer (UV-vis DRS) revealed that the catalyst enhanced the light response range. The photoelectric chemical experiment results demonstrated that the modified ACFs remarkably improved the charge transmission and the separation efficiency of electrons and holes. The adsorption saturation time reached 40 h and toluene photodegradation rate was 70%. Four toluene degradation intermediate products were determined by GC-MS, and the removal mechanism of toluene by TiO2/ACF-Ac was discussed.

Emergence and prevalence of naturally occurring lower virulent African swine fever viruses in domestic pigs in China in 2020.

African swine fever virus (ASFV) has been circulating in China for more than two years, and it is not clear whether the biological properties of the virus have changed. Here, we report on our surveillance of ASFVs in seven provinces of China, from June to December, 2020. A total of 22 viruses were isolated and characterized as genotype II ASFVs, with mutations, deletions, insertions, or short-fragment replacement occurring in all isolates compared with Pig/HLJ/2018 (HLJ/18), the earliest isolate in China. Eleven isolates had four different types of natural mutations or deletion in the EP402R gene and displayed a non-hemadsorbing (non-HAD) phenotype. Four isolates were tested for virulence in pigs; two were found to be as highly lethal as HLJ/18. However, two non-HAD isolates showed lower virulence but were highly transmissible; infection with 106 TCID50 dose was partially lethal and caused acute or sub-acute disease, whereas 103 TCID50 dose caused non-lethal, sub-acute or chronic disease, and persistent infection. The emergence of lower virulent natural mutants brings greater difficulty to the early diagnosis of ASF and creates new challenges for ASFV control.

Characterization of Anti-p54 Monoclonal Antibodies and Their Potential Use for African Swine Fever Virus Diagnosis.

African swine fever (ASF) is a highly lethal hemorrhagic viral disease of domestic pigs caused by African swine fever virus (ASFV). Although a good advance has been made to understand the virus, a safe and effective vaccine against ASFV is still lacking and its eradication solely depends on its early and accurate diagnosis. Thus, improving the available diagnostic assays and adding some validated techniques are useful for a range of serological investigations. The aim of this study was to produce and characterize p54 monoclonal antibodies with an ultimate goal of developing a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for ASFV antibody detection. Five monoclonal antibodies against p54 protein expressed in Escherichia coli was generated and their characterizations were investigated. Furthermore, a competitive enzyme-linked immunosorbent assay (cELISA) based on a monoclonal antibody designated as 2A7 was developed. To evaluate the performance of the assay, a total of 365 pig serum samples (178 negative and 187 positive samples) were tested and a receiver-operating characteristic (ROC) analysis was applied to determine the cut-off value. Based on the ROC analysis, the area under the curve (AUC) was 0.982 (95% confidence interval: 96.9% to 99.4%), besides a sensitivity of 92.5% and a specificity of 98.9% was achieved when the percent inhibition of 20% was selected as a threshold. Moreover, the result showed an excellent agreement when compared to other commercially available blocking ELISA (kappa value = 0.912) and showed no reaction to other swine pathogens. Overall, the newly developed cELISA method offers a promising approach for a rapid and convenient ASFV serodiagnosis, which could be used as alternative to other serological assays for screening possible ASFV infection.

Recombinant bluetongue virus with hemagglutinin epitopes in VP2 has potential as a labeled vaccine.

Bluetongue (BT) is an arbovirus-borne disease of ruminants caused by bluetongue virus (BTV) that has the potential to have a serious economic impact. Currently available commercial vaccines include attenuated vaccines and inactivated vaccines, both of which have achieved great success in the prevention and control of BTV. However, these vaccines cannot distinguish between infected animals and immunized animals. To control outbreaks of BTV, the development of labeled vaccines is urgently needed. In this study, we used the plasmid-based reverse genetics system (RGS) of BTV to rescue four recombinant viruses in which HA (influenza hemagglutinin) tags were inserted at different sites of VP2. In vitro, the recombinant tagged viruses exhibited morphologies, plaque, and growth kinetics similar to the parental BTV-16, and expressed both VP2 and HA tag. Subsequently, the selected recombinant tagged viruses were prepared as inactivated vaccines to immunize IFNAR(-/-) mice and sheep, and serological detection results of anti-HA antibody provided discriminative detection. In summary, we used plasmid-based RGS to rescue BTV recombinant viruses with HA tags inserted into VP2, and detected several sites on VP2 that can accommodate HA tags. Some of the recombinant tagged viruses have potential to be developed into distinctive inactivated vaccines.

Development and optimization of a DNA-based reverse genetics systems for epizootic hemorrhagic disease virus.

Epizootic hemorrhagic disease virus (EHDV) is a member of the genus Orbivirus, family Reoviridae, and has a genome consisting of 10 linear double-stranded (ds) RNA segments. The current reverse genetics system (RGS) for engineering the EHDV genome relies on the use of in vitro-synthesized capped viral RNA transcripts. To obtain more-efficient and simpler RGSs for EHDV, we developed an entirely DNA (plasmid or PCR amplicon)-based RGS for viral rescue. This RGS enabled the rescue of infectious EHDV from BSR-T7 cells following co-transfection with seven helper viral protein expression plasmids and 10 cDNA rescue plasmids or PCR amplicons representing the EHDV genome. Furthermore, we optimized the DNA-based systems and confirmed that some of the helper expression plasmids were not essential for the recovery of infectious EHDV. Thus, DNA-based RGSs may offer a more efficient method of recombinant virus recovery and accelerate the study of the biological characteristics of EHDV and the development of novel vaccines.

Neutralization Mechanism of a Monoclonal Antibody Targeting a Porcine Circovirus Type 2 Cap Protein Conformational Epitope.

Porcine circovirus type 2 (PCV2) is an important pathogen in swine herds, and its infection of pigs has caused severe economic losses to the pig industry worldwide. The capsid protein of PCV2 is the only structural protein that is associated with PCV2 infection and immunity. Here, we report a neutralizing monoclonal antibody (MAb), MAb 3A5, that binds to intact PCV2 virions of the PCV2a, PCV2b, and PCV2d genotypes. MAb 3A5 neutralized PCV2 by blocking viral attachment to PK15 cells. To further explore the neutralization mechanism, we resolved the structure of the PCV2 virion in complex with MAb 3A5 Fab fragments by using cryo-electron microscopy single-particle analysis. The binding sites were located at the topmost edges around 5-fold icosahedral symmetry axes, with each footprint covering amino acids from two adjacent capsid proteins. Most of the epitope residues (15/18 residues) were conserved among 2,273 PCV2 strains. Mutations of some amino acids within the epitope had significant effects on the neutralizing activity of MAb 3A5. This study reveals the molecular and structural bases of this PCV2-neutralizing antibody and provides new and important information for vaccine design and therapeutic antibody development against PCV2 infections.IMPORTANCE PCV2 is associated with several clinical manifestations collectively known as PCV2-associated diseases (PCVADs). Neutralizing antibodies play a crucial role in the prevention of PCVADs. We demonstrated previously that a MAb, MAb 3A5, neutralizes the PCV2a, PCV2b, and PCV2d genotypes with different degrees of efficiency, but the underlying mechanism remains elusive. Here, we report the neutralization mechanism of this MAb and the structure of the PCV2 virion in complex with MAb 3A5 Fabs, showing a binding mode in which one Fab interacted with more than two loops from two adjacent capsid proteins. This binding mode has not been observed previously for PCV2-neutralizing antibodies. Our work provides new and important information for vaccine design and therapeutic antibody development against PCV2 infections.

Assessment for Less than 20-ppm Oil Leakage in Soil Using Terahertz Wave.

Although an accurate detection of trace oil leaks is of the utmost important for soil protection, the typically used techniques fail to provide rapid assessment of less than 20 parts per million (ppm) of oil in soil. Terahertz (THz) time-domain spectroscopy, an optical method with high sensitivity to polar organics, was used to characterize the content of crude oil in soils. A linear model was built between the concentration of crude oil and the THz attenuation coefficient, which predicted the limit of detection ranging from 4.11 to 16.2 ppm. Some organics, such as aromatic and aliphatic compounds, contribute to larger absorption in the THz range than minerals. Effective-medium theory was optimized to elucidate the crude oil content dependence of THz dielectric constants. Consequently, THz technology could be an effective method for detecting trace oil leakage in soil.

PCR-based reverse genetics strategy for bluetongue virus recovery.

BACKGROUND: Bluetongue virus (BTV), an emerging insect vector mediated pathogen affecting both wild ruminants and livestock, has a genome consisting of 10 linear double-stranded RNA genome segments. BTV has a severe economic impact on agriculture in many parts of the world. Current reverse genetics (RG) strategy to rescue BTV mainly rely on in vitro synthesis of RNA transcripts from cloned complimentary DNA (cDNA) corresponding to viral genome segments with the aid of helper plasmids. RNA synthesis is a laborious job which is further complicated with a need for expensive reagents and a meticulous operational procedure. Additionally, the target genes must be cloned into a specific vector to prepare templates for RNA transcription. RESULT: In this study, we have developed a PCR based BTV RG system with easy two-step transfection. Viable viruses were recovered following a first transfection with the seven helper plasmids and a second transfection with the 10 PCR products on the BSR cells. Further, recovered viruses were characterized with indirect immunofluorescence assays (IFA) and gene sequencing. And the proliferation properties of these viruses were also compared with wild type BTV. Interestingly, we have identified that viruses containing the segment 2 of the genome from reassortant BTV, grew slightly slower than the others. CONCLUSION: In this study, a convenient PCR based RG platform for BTV is established, and this strategy could be an effective alternative to the original available BTV rescue methods. Furthermore, this RG strategy is likely applicable for other Orbiviruses.

Generation, identification, and functional analysis of monoclonal antibodies against porcine epidemic diarrhea virus nucleocapsid.

The variant strains of porcine epidemic diarrhea virus (PEDV) severely threaten the pig industry worldwide and cause up to 100% mortality in suckling piglets. It is critically important and urgent to develop tools for detection of PEDV infection. In this study, we developed six monoclonal antibodies (mAbs) targeting N protein of PEDV and analyzed their applications on enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), western blot assay, and flow cytometry assay. The results demonstrated that all these six mAbs were IgG1 isotype and κ chain. Among these six mAbs, 3F12 recognizes a linear epitope (VAAVKDALKSLGI) while the other five mAbs recognize different conformational epitopes formed by a specific peptide fragment or the full length of N protein. The functional analysis showed that all these six mAbs were applicable to ELISA, western blot, IFA, and flow cytometry assay. In conclusion, we developed six mAbs against PEDV-N protein to facilitate the early detection of PEDV infection using ELISA, western blot, IFA, and flow cytometry.

CHST6 mutation screening and endoplasmatic reticulum stress in macular corneal dystrophy.

Macular corneal dystrophy (MCD) is an autosomal recessive disorder mainly caused by gene mutations of carbohydrate sulfotransferase (CHST6) leading to bilateral visual impairment. Because the mechanism underlying this degeneration remains poorly understood, we investigated molecular alterations and pathways that may be involved in MCD in this issue. Different mutation sites were screened by direct sequencing of the coding region of CHST6. In addition, we described morphological changes in MCD keratocytes by light microscopy and electron microscopy and determined the relationship between the development of this disease and the occurrence of apoptosis through flow cytometry, cell counting kit-8, colony formation assay and other experiments. Western blotting and quantitative real-time polymerase chain reaction were used to determine if endoplasmic reticulum (ER) stress was activated. We found 10 kinds of mutations among these families with 3 novel mutations included. The percentage of apoptotic keratocytes increased in MCD patients; furthermore, the expression of apoptosis related protein B-cell lymphoma-2 (Bcl-2) was down-regulated while Bcl-2 associated X protein was upregulated. Finally, ER stress was activated with the upregulation of glucose-regulated protein 78 and CCAAT-enhancer-binding protein homologous protein. Our clinical and in vitro results suggest that the CHST6 mutation associated with MCD is associated with apoptosis, and ER stress is probably involved in this apoptosis pathway.