Magnetic resonance imaging focused on the ferritin heavy chain 1 reporter gene detects neuronal differentiation in stem cells

The neuronal differentiation of mesenchymal stem cells offers a new strategy for the treatment of neurological disorders.Thus,there is a need to identify a noninvasive and sensitive in vivo imaging approach for real-time monitoring of transplanted stem cells.Our previous study confirmed that magnetic resonance imaging,with a focus on the ferritin heavy chain 1 reporter gene,could track the proliferation and differentiation of bone marrow mesenchymal stem cells that had been transduced with lentivirus carrying the ferritin heavy chain 1 reporter gene.However,we could not determine whether or when bone marrow mesenchymal stem cells had undergone neuronal differentiation based on changes in the magnetic resonance imaging signal.To solve this problem,we identified a neuron-specific enolase that can be differentially expressed before and after neuronal differentiation in stem cells.In this study,we successfully constructed a lentivirus carrying the neuron-specific enolase promoter and expressing the ferritin heavy chain 1 reporter gene;we used this lentivirus to transduce bone marrow mesenchymal stem cells.Cellular and animal studies showed that the neuron-specific enolase promoter effectively drove the expression of ferritin heavy chain 1 after neuronal differentiation of bone marrow mesenchymal stem cells;this led to intracellular accumulation of iron and corresponding changes in the magnetic resonance imaging signal.In summary,we established an innovative magnetic resonance imaging approach focused on the induction of reporter gene expression by a neuron-specific promoter.This imaging method can be used to noninvasively and sensitively detect neuronal differentiation in stem cells,which may be useful in stem cell-based therapies.

Induction of Cardiomyocyte Apoptosis by Anti-Cardiac Myosin Heavy Chain Antibodies in Patients with Acute Myocardial Infarction

Autoimmune is involved in the pathogenesis of ventricular remodeling in acute myocardial infarction (AMI).In the present study, we investigated the effect of anti-cardiac myosin heavy chain antibodies (AMHCA) from patients with AMI on rat cardiomyocyte apoptosis.Cardiomyocyte apoptosis was observed and measured by DNA end labeling and Annexin-Ⅴ/PI double-staining assay.The expression of apoptosis related p53 and Bcl-2 protein and the second messenger calcium were detected respectively by Western blotting, patch clamp and confocal calcium imaging.The results showed that AMHCA was able to induce cardiomyocyte apoptosis in a dose dependent manner.Apoptosis-accelerating nucleoprotein p53 was up-regulated, while apoptosis-inhibiting cytoplasmic protein Bcl-2 was down-regulated.In parallel, cytoplasmic calcium concentration was elevated.There was no effect on L-type calcium currents.It is concluded that AMHCA in patients with AMI as a novel triggering factor can induce cardiomyocyte apoptosis, which contributes to ventricular remodeling.

Isolation and Characterization of Recombinant Variable Domain of Heavy Chain Anti-idiotypic Antibodies Specific to Aflatoxin B_1

Some unique subclasses of Camelidae antibodies are devoid of the light chain, and the antigen binding site is comprised exclusively of the variable domain of the heavy chain （VHH）. The recombinant VHHs have a high potential as alternative reagents for the next generation of immunoassay. In particular, they might be very useful for molecular mimicry. The present study demonstrated an alpaca immunized with the F（ab＇）z fragment of anti-aflatoxin B1 mAb and developed an important anti-idiotypic （anti-ld） responses. Antigen-specific elution method was used for panning private anti-ld VHHs from the constructed alpaca VHH library. The selected VHHs were expressed, renatured, purified, and then identified by a competitive enzyme-linked immunosorbent assay （ELISA）. Our findings indicated that the VHH would be an alternative tool for haptens mimicry studies.

Effect of aerobic exercise on the contractile function of gastrocnemius myosin heavy chain

Objective To study the effect of 4-6 weeks’ treadmill training of male SD rats on the contractile function of their gastrocnemius myosin heavy chain (MHC). Methods Forty male SD rats were randomly divided into control group and training group. The treadmill training of the training group rats was incessantly performed for 4-6 weeks at an intensity of about 75% VO2max (18.5-24 m/min,gradient of 0°,each training session lasting 50 minutes,twice a day). The content of gastrocnemius MHC mRNA was tested by reverse transcription polymerase chain reaction (RT-PCR),and the changes of muscle fibre and its cross-section area (CSA) were measured using immunohistochemistry. Electric stimulation tests were used to determine the maximal tension of isometric contraction of the post-training gastrocnemius. Results ① After continuous treadmill training for 4-6 weeks,we found that the content of the total MHC,MHC Ⅰ,MHC Ⅱx,MHC Ⅱa mRNAs was 105%,105%,109% and 108% of that in the resting control group,respectively,and the MHC Ⅱb mRNA content did not change significantly. The percentage of MHC Ⅰ mRNA in the total MHC mRNA increased while that of MHC Ⅱ mRNA decreased after aerobic training. ② The slow type of fibre type Ⅰ was the main part of the MHC after training and the CSA of the muscle fibres increased simultaneously. ③ The maximal tension of isometric contraction by pulse stimulation of square wave in the training group increased significantly compared with that in the control group (P<0.01). Conclusion The findings indicate that aerobic exercise may promote an increase in the contractile function of MHC.

Influence of injection of Chinese botulinum toxin type A on the histomorphology and myosin heavy chain composition of rat gastrocnemius muscles

Background and objective:Botulinum toxin type A(BoNT/A)is a metalloprotease that blocks synaptic transmission via the cleavage of a synaptosomal-associated protein of 25 kDa(SNAP-25).It has gained widespread use as a treatment for cerebral palsy and skeletal muscle hypertrophy.In China,Chinese botulinum toxin type A(CBTX-A),a type of BoNT/A,is in widespread clinical use.However,the changes in the morphological and biochemical properties of treated muscles and in remote muscles from the CBTX-A injection site are relatively unknown.Therefore,we investigated the changes in histomorphology and myosin heavy chain(MyHC)isoform composition and distribution in rat gastrocnemius muscles after intramuscular injection of CBTX-A.Methods:The weakness of the injected muscles was assessed periodically to identify their functional deficiency.Muscle slices were stained with hematoxylin-eosin(HE)and adenosine triphosphatase(ATPase).MyHC isoform composition was analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis(SDS-PAGE)to uncover changes in morphological and biochemical properties.Results:Our findings demonstrate that following injection of CBTX-A 5 U into rat gastrocnemius muscles,shifts in MyHC isoform composition emerged on the third day after injection and peaked in the fourth week.The composition remained distinctly different from that of the control group after the twelfth week.More specifically,there was a decrease in the proportion of the type IIb isoform and an increase in the proportions of type IIx,type IIa,and type I isoforms.Conclusions:Data revealed that CBTX-A led to a shift in MyHC composition towards slower isoforms and that the MyHC composition remained far from normal six months after a single injection.However,no noticeable remote muscle weakness was induced.

Generation of cytotoxic T lymphocytes specific for B-cell acute lymphoblastic leukemia family-shared peptides derived from immunoglobulin heavy chain framework region

Background Immunoglobulin heavy chain variable region （IgHV） is a well-characterized tumor antigen for B-cell malignancies. It can function as a target for T cell-mediated immune response. Clinical trials of IgHV protein vaccines against lymphoma have demonstrated induction of tumor-specific cytotoxic T lymphocyte （CTL） responses. However, complementary determining regions-based individual vaccines have disadvantages for wide clinical application. Although a recent study demonstrated that immunogenic peptides are derived from framework regions （FR） shared among patients with B-cell lymphoma, how to choose the appropriate peptides for each patient is still unsolved. The aim of this study was to investigate whether immunoglobulin heavy chain FR-derived peptides shared in each IgHV family are potential CTL epitopes presented by B-cell acute lymphoblastic leukemia （B-ALL）. Such CTL epitopes might be beneficial to shifting vaccination strategies against B-ALL from individual specificity to family specificity.Methods Seven IgHV gene families were amplified respectively by PCR and sequenced directly from 71 childhood B-ALL cases. Bioinformatics was applied in analyzing characteristics of sequences available and predicting HLA-A^＊0201-restricted CTL epitopes for each IgHV family. An antigen-specific T cell expansion system was used to generate peptide-specific CTLs. The cytotoxicity of CTLs against B-ALL cells was assessed in the lactate dehydrogenase release assay. Results Complete IgHV rearrangements were identified in all of the 71 B-ALL cases. All of 40 sequences available showed ≥98% homology with the nearest germline IgHV genes, indicating IgHV genes in B-ALL of germline nature. Twelve nonapeptides of high HLA-A^＊0201-binding scores were obtained from 26 productive IgHV protein sequences. Ten （83%） of the peptides were located in FR1 and FR3 shared among the corresponding IgHV family. CTLs specific for the peptide QLVQSGAEV located in FR1 （3-11） shared among the IgHV1 family could be successfully generated from peripheral blood mononuclear cells of two HLA-A^＊0201＋ healthy donors in vitro and were capable of killing HLA-matched B-ALL cell clones belonging to the IgHV1 family. Conclusion Anti-B-ALL CTLs against immunoglobulin heavy chain FR-derived peptides have family-specific cytotoxicity.

Mutation of Arg723Gly in β-myosin heavy chain gene in five Chinese families with hypertrophic cardiomyopathy

Background Hypertrophic cardiomyopathy （HCM） is a form of cardiomyopathy with an autosomal dominant inherited disease, which is caused by mutations in at least one of the sarcomeric protein genes. Mutations in the beta-myosin heavy chain （β-MHC） are the most common cause of HCM. This study was to reveal the disease-causing gene mutations in Chinese population with HCM, and to analyze the correlation between the genotype and phenotype. Methods The exons 3 to 26 of MYH7 were amplified by PCR, and the PCR products were sequenced in five non-kin HCM patients. A 17-year-old patient was detected to be an Arg723Gly mutation carrier. Then his family was gene-screened, and the correlation between genotype and phenotype was analyzed. Results The mutation of Arg723Gly in a Chinese family with HCM was detected for the first time. With a C-G transversion in nucleotide 13 619 of the MYH7 gene, located at the essential light chain interacting region in S1, the replacement of arginine by glycine took place at amino acid residue 723. A two-dimensional echocardiogram showed moderate asymmetrical septal hypertrophy with left atria enlargement. There was no obstruction in the left ventricular outflow tract. In his family, a total of 13 individuals were diagnosed HCM and 5 of them were dead of congestive heart failure at a mean age of 66-year-old. Eight living members were all detected to carry the mutation, in which 3 developed progressive heart failure. Moreover, the heart function of the people evidently deteriorates when their age are older than 50. The mutation and the disease show co-separated. Conclusion The Arg723Gly mutation is a malignant type. In Chinese the mutation has the similar characters to the former report but has low degree malignant.

Clathrin heavy chain is essential for the development and reproduction of Locusta migratoria

Clathrin heavy chain(Chc)is a constituent of clathrin-coated vesicles and serves important functions in endocytosis and intracellular membrane trafficking but ap-pears to have physiological roles also at the organismal level.Most of what we know about Chc functions originates from studies performed in fungal or vertebrate cells.How-ever,the physiological functions of Chc in insects remain poorly understood.Here,we identified a Chc ortholog from a Locusta migratoria transcriptome database.RT-qPCR revealed that LmChc was constitutively expressed in fifth-instar nymphs.In this develop-mental stage,LmChc showed the highest expression in the ovary followed by hemolymph,testis,hindgut,midgut,and foregut.In isolated hemocytes,we detected the Chc protein in patches at the plasma membrane.To examine the role of LmChc in L.migratoria during development,RNA interference was performed by injecting dsRNA into the early fifth-instar nymphs.Silencing of LmChc caused a lethal phenotype with molting defect from nymph to adult.In addition,silencing of LmChc resulted in abnormal development of the ovaries,the size of which was significantly smaller than that in controls.Taken together,our results suggest that LmChc is a vital gene in L.migratoria that plays an important role in growth,development,and reproduction.LmChc may be used as an efficient RNAi target gene for developing dsRNA-based biological insecticides to manage insect pests.

Congophilic fibrils in the glomeruli with polyclonal immunoglobulin gamma staining-another cause for diagnostic overlap:A case report

BACKGROUND Glomerulopathy with fibrillary deposits is not uncommon in routine nephropathology practice,with amyloidosis and fibrillary glomerulonephritis being the two most frequently encountered entities.Renal amyloid heavy and light chain(AHL)is relatively uncommon and its biopsy diagnosis is usually limited to cases that show strong equivalent staining for a single immunoglobulin(Ig)heavy chain and a single light chain,further supported by mass spectrometry(MS)and serum studies for monoclonal protein.But polyclonal light chain staining can pose a challenge.CASE SUMMARY Herein we present a challenging case of renal AHL with polyclonal and polytypic Ig gamma(IgG)staining pattern by immunofluorescence.The patient is a 62-yearold Caucasian male who presented to an outside institution with a serum creatinine of up to 8.1 mg/dL and nephrotic range proteinuria.Despite the finding of a polyclonal and polytypic staining pattern on immunofluorescence,ultrastructural study of the renal biopsy demonstrated the presence of fibrils with a mean diameter of 10 nm.Congo red was positive while DNAJB9 was negative.MS suggested a diagnosis of amyloid AHL type with IgG and lambda,but kappa light chains were also present supporting the immunofluorescence staining results.Serum immunofixation studies demonstrated IgG lambda monoclonal spike.The patient was started on chemotherapy.The chronic renal injury however was quite advanced and he ended up needing dialysis shortly after.CONCLUSION Tissue diagnosis of AHL amyloid can be tricky.Thorough confirmation using other available diagnostic techniques is recommended in such cases.

A novel triple immunoenzyme staining enables simultaneous identification of all muscle fiber types on a single skeletal muscle cryosection from normal, denervated or reinnervated rats

Triple immunofluorescence staining has recently been developed to simultaneously identify all muscle fibers on a single cryosection which is helpful for clinical and basic research, but it has disadvantages such as fast photobleaching and unclear outlines of muscle fibers. Triple immunoenzyme staining（TIE） is likely to avoid these disadvantages. In this study, we aimed to establish a sensitive and specific TIE technique to identify fiber types in normal, denervated, and reinnervated rat muscles, and to develop a systematic sampling method for muscle fiber quantification. Tibialis anterior and soleus from normal, denervated, and reinnervated Lewis rat hind limbs were used. Five consecutive cryosections were cut from each muscle, including one for TIE and four for single immunoenzyme staining（SIE）. The TIE was performed using the polymerized reporter enzyme staining system for the first two antigens（A4.74 for My HC-IIA, BA-F8 for My HC-I） and alkaline phosphatase staining system for the third antigen（BF-F3 for My HC-IIB）, followed by corresponding detective systems and respective chromogens. The type of muscle fibers was quantified by systematic sampling at 12.5%, 25%, 33% and 50% of all muscle fibers, and was compared with that acquired from counting all the fibers（100%）. All muscle fiber phenotypes, including pure and hybrid, could be simultaneously identified on a single TIE cryosection with clear outlines. The fiber types on TIE slides matched well with their respective counterpart on the consecutive SIE slides with a 95% match rate. Systematic sampling of 12.5% fibers could represent the true fiber type distribution of the entire muscle section. Our results suggest that novel TIE can effectively visualize fiber types in normal, denervated or reinnervated rat muscles.

Current application of neurofilaments in amyotrophic lateral sclerosis and future perspectives

Motor neuron disease includes a heterogeneous group of relentless progressive neurological disorders defined and characterized by the degeneration of motor neurons.Amyotrophic lateral sclerosis is the most common and aggressive form of motor neuron disease with no effective treatment so far.Unfortunately,diagnostic and prognostic biomarkers are lacking in clinical practice.Neurofilaments are fundamental structural components of the axons and neurofilament light chain and phosphorylated neurofilament heavy chain can be measured in both cerebrospinal fluid and serum.Neurofilament light chain and phosphorylated neurofilament heavy chain levels are elevated in amyotrophic lateral sclerosis,reflecting the extensive damage of motor neurons and axons.Hence,neurofilaments are now increasingly recognized as the most promising candidate biomarker in amyotrophic lateral sclerosis.The potential usefulness of neurofilaments regards various aspects,including diagnosis,prognosis,patient stratification in clinical trials and evaluation of treatment response.In this review paper,we review the body of literature about neurofilaments measurement in amyotrophic lateral sclerosis.We also discuss the open issues concerning the use of neurofilaments clinical practice,as no overall guideline exists to date;finally,we address the most recent evidence and future perspectives.

Restricted nutrient intake does not alter serum-mediated measures of implant response in cell culture

Background： During nutritional stress, reduced intake may reduce the efficacy of anabolic implants. This study was conducted to evaluate basic cellular responses to a growth promotant implant at two intake levels. Methods： Sixteen crossbred steers （293 ± 19.3 kg） were used to evaluate the impact of anabolic implants in either an adequate or a restricted nutritional state. Steers were trained to individual Calan gates, and then randomly assigned to 1 of 4 treatments in a 2 × 2 factorial arrangement. Treatments consisted off presence or absence of an anabolic growth implant （Revalor-XS, 200 mg TBA and 40 mg estradiol; IMPLANT or CONTROL） and a moderate energy, pelleted, starting cattle diet fed at either 2.0 × or 1.0 × maintenance energy （NEM） requirements （HIGH or LOW）. Serum （d O, 14, and 28） was used for application to bovine muscle satellite cells. After treatment with the serum （20% of total media） from the trial cattle, the satellite cells were incubated for 72 h. Protein abundance of myosin heavy chain （MHC）, phosphorylated extracellular signal-related kinase （phospho-ERK）, and phosphorylated mammalian target of rapamycin （phospho-mTOR） were analyzed to determine the effects of implant, intake, and their interaction （applied via the serum）. Results： Intake had no effect on MHC （P = 0.85） but IMPLANT increased （P 〈 0.01） MHC abundance vs. CONTROL. Implant status, intake status, and the interaction had no effect on the abundance of phospho-ERK （P〉0.23）. Implanting increased phospho-mTOR （P 〈 0.01） but there was no effect （P 〉 0.51） of intake or intake x implant. Conclusions： The nearly complete lack of interaction between implant and nutritional status indicates that the signaling molecules measured herein respond to implants and nutritional status independently. Furthermore, results suggest that the muscle hypertrophic effects of anabolic implants may not be mediated by circulating IGF-1.

Rearranged Patterns of IgH and TcRγ Genes in Patients with Acute Lymphoblastic Leukemia

The rearrangement of immunoglobulin heavy chain gene(IgH) and T cell receptor γgene (ToRγ)was studied in 30 patients with acute lymphoblastic leukemia(ALL) by the polymerase chain reaction (PCR). 19 cases was found to have rearrangement of IgH gene,12 of TcRγ. Most of IgH rearrangement was characterized by one or two specific bands while some had more than two. Rearrangement of TcRγgene appeared as one specific band. A slight difference in number, size and lightness of bands was found among the patients. 4 different kinds of rearrangement were observed in the detection of IgH rearrangement in combination with TcRγgene. The rearranged patterns of IgH and TcRγgene as well as the clinical significance were discussed.

Selected Aspects of Camel Immune System and Immune Responses

The camel economy is of considerable importance for arid countries. In the last decade, studies about camel immune system and immune responses have recorded increasing interest. However, drawing a comprehensive picture of the camel immune system remains far from reached. A major part of this review is to cover the studies of the primary and secondary immune organs and the markers of the camel immune cells and certain lymphoid tissues. At the same time, immune responses to different diseases and the nature of effective immunity were included, with an emphasis on the most important zoonotic diseases in camels such as MERS CoV;brucellosis. New findings on the diversity mechanisms of camel immunoglobulin genes were addressed. However, detail of the mechanism of MHC-restricted cellular immunity and the mechanism of B lymphocyte activation in camels await further attention. Interestingly, the gross and the histological structure of the lymphoid tissues of the camel’s thymus, tonsils, and peyer’s patches have indicated significant differences from other animals in terms of structure and function. The most peculiar CD expression, such as LPAM-I, MAdCAM-1 and CX3CR1, in certain camel cells and tissues refers to possible extraordinary mechanisms of immune hemostasis in camel in comparison to other ruminants. The widely applied immunodiagnostic techniques to control camel diseases and to assist in improving the camel resistance were considered. Extensive studies of the camel immune system were greatly hampered by lack of specific reagents to camel markers and low funds in the field of camel immunology.

Bioactivity of silk fibroin peptides on vascular endothelial cells

To determine the contribution of non-repetitive domains to the bioactivity of the heavy chain in silk fibroin(SF)macromolecules,a gene motif f(1)encoding this fragment and its multimers(f(4)and f(8))were biosynthesized from Escherichia coli BL21.Based on the positive application potential of SF materials for the vascular tissue engineering,this study focused on examining the active response of these polypeptides to vascular endothelial cells.Biosynthetic polypeptides F(1),F(4),and F(8)were separately grafted onto the surfaces of bioinert polyethylene terephthalate(PET)films,resulting in remarkable improvements in the spread and proliferation of human umbilical vein endothelial cells(HUVECs).Using the same grafting dose,the activity of cells on polypeptide-modified PET films enhanced with the increase of the molecular weight of those grafted polypeptides from F(1)to F(8).Meanwhile,the growth of cells on the surface of the alkaline-treated PET film was improved,indicating that the hydrophilicity of the surface material had influence on the growth of HUVECs.Moreover,on surfaces with the same water contact angle,the spread and proliferation activity of cells on PET films were significantly lower than those on polypeptide-modified PET films.

A study of Hodgkin/Reed-Sternberg cells using single cell polymerase chain reaction

OBJECTIVE: To investigate the characteristics of Hodgkin/Reed-Stemberg (H/R-S) cells found in patients with various types of Hodgkin's disease (HD). METHODS: H/R-S cells were micropicked from frozen sections of tissues affected by HD. The DNA from these cells was amplified by polymerase chain reaction (PCR) using immunoglobulin heavy chain gene FR III a/JH primers and light chain gene family-specific primers. RESULTS: A total of 52/135 (35.8%) isolated cells showed the specific products in the reactions. IgH and V kappa 4 rearrangements were repeatedly found in many cells from a lymphocyte predominance type sample; repeated V kappa 4 and individual IgH/V kappa 2,4 rearrangements and individual IgH, V lambda 3/ V kappa 4 rearrangements were found in two different cases of the nodular sclerosis type; repeated IgH/ V lambda 3 and individual V lambda 2,4 rearrangements, repeated V kappa 2,4 rearrangements, repeated V kappa 4 and individual IgH/ V kappa 3 rearrangements, repeated IgH and individual V kappa 3/ V lambda 4 rearrangements were detected in 3 cases of the mixed cellularity type. Repeated and individual IgH rearrangements were found in other 2 cases. CONCLUSION: The H/R-S cells isolated from the lymphocyte predominance subtypes of HD have IgH and V lambda 4 gene rearrangements. This suggests that the lymphocyte predominance type is a proliferation of neoplastic B cells. The cells isolated from the mixed cellularity and nodular sclerosis types derive from B lineage cells at various stages of differentiation because of the presence of their IgH, kappa and/or lambda gene rearrangements. To our knowledge, this is the first time that the lambda gene rearrangement was detected in H/R-S cells.

Development of the expressed immunoglobulinμchain repertoire during maturation of mice B cells

In the bone marrow and spleen,the developing B cell populations undergo both negative and positive selections to shape their B cell receptor repertoire.To gain insight into the shift of the immunoglobulin heavy(IgH)chain repertoire during B cell development,we undertook large scale Igμchain repertoire analysis of pre-B,immature B and spleen B cell populations.We found that the majority of VH gene segments,VH families,JH and D gene segments,were observed to have significantly different usage frequencies when three B cell populations were compared,but the usage profile of the VH,D,and JH genes between different B cell populations showed high correlations.In both productive and nonproductive rearrangements,the length of CDRH3 shortened significantly on average when B cells entered the periphery.However,the CDRH3 length distribution of nonproductive rearrangements did not follow a Gaussian distribution,but decreased successively in the order 3n–2,3n–1 and 3n,suggesting a direct correlation between mRNA stability and CDRH3 length patterns of nonproductive rearrangements.Further analysis of the individual components comprising CDRH3 of productive rearrangements indicated that the decrease in CDRH3 length was largely due to the reduction of N addition at the 5′and 3′junctions.Moreover,with development,the amino acid content of CDRH3 progressed toward fewer positively charged and nonpolar residues but more polar residues.All these data indicated that the expressed Igμchain repertoire,especially the repertoire of CDRH3,was fine-tuned when B cells passed through several checkpoints of selection during the process of maturation.

Generation of B Cell-Deficient Pigs by Highly Efficient CRISPR/Cas9-Mediated Gene Targeting

Generating B cell-deficient mutant is the first step to produce human antibody repertoires in large animal models. In this study, we applied the clustered regularly interspaced short palindromic repeat （CRISPR）/CRISPR-associated （Cas） system to target the JH region of the pig IgM heavy chain gene which is crucial for B cell development and differentiation. Transfection of IgM-targeting Cas9 plasmid in primary porcine fetal fibroblasts （PFFs） enabled inducing gene knock out （KO） in up to 53.3% of colonies analyzed, a quarter of which harbored biallelic modification, which was much higher than that of the traditional homologous recombination （HR）. With the aid of somatic cell nuclear transfer （SCNT） technology, three piglets with the biallelic lgM heavy chain gene mutation were produced. The piglets showed no antibody-producing B cells which indicated that the biallelic mutation of the lgM heavy chain gene effectively knocked out the function of the IgM and resulted in a B cell-deficient phenotype. Our study suggests that the CRISPR/Cas9 system combined with SCNT technology is an efficient genome-editing approach in pigs.

Clinical significance and pathogenic role of anti-cardiac myosin autoantibody in dilated cardiomyopathy

In order to explore the possible roles played by the autoimmune mechanism in the progression of myocarditis into dilated cardiomyopathy (DCM) using an animal model, we investigated whether autoimmune myocarditis might develop into DCM Methods Experimental Balb/C mice (n=20) were immunized with cardiac myosin with Freun d’s complete adjuvant at days 0, 7 and 30 The control Balb/C mice (n=10) were immunized with Freund’s complete adjuvant in the same mannere Serum and my ocardium samples were collected after the first immunization at days 15, 21 and 120 The anti-myosin antibody was examined by enzyme-linked immunosorbent assay and immunoblotting Results Pathological findings demonstrated that there was myocardial necrosis or inflammatory infiltration during acute stages and fibrosis mainly in the late phase of experimental group, but the myocardial lesions were not found in the control group Autoimmunity could induce myocarditis and DCM in the absence of viral infection High titer anti-myosin IgG antibodies were found in the experimental group, but not in the control group Furthermore, the anti-myosin heavy chain (200 KD) antibody was positive in 21 of 48 patients with DCM and viral myocarditis, but only 4 of 20 patients with coronary heart disease, including 1 case and 3 c ases that reacted with heavy and light chains (27 5 KD), respectively The antibodies were not detected in healthy donors Conclusion Cardiac myosin might be an autoantigen that provokes autoimmunity and leads to the transformation of myocarditis into DCM Detection of anti-myosin heavy chain antibody might contribute to diagnosis for DCM and viral myocarditis

Immunogenetic factors driving formation of ultralong VH CDR3 in Bos taurus antibodies

The antibody repertoire of Bos taurus is characterized by a subset of variable heavy(VH)chain regions with ultralong third complementarity determining regions(CDR3)which,compared to other species,can provide a potent response to challenging antigens like HIV env.These unusual CDR3 can range to over seventy highly diverse amino acids in length and form uniqueβ-ribbon‘stalk’and disulfide bonded‘knob’structures,far from the typical antigen binding site.The genetic components and processes for forming these unusual cattle antibody VH CDR3 are not well understood.Here we analyze sequences of Bos taurus antibody VH domains and find that the subset with ultralong CDR3 exclusively uses a single variable gene,IGHV1-7(VHBUL)rearranged to the longest diversity gene,IGHD8-2.An eight nucleotide duplication at the 3′end of IGHV1-7 encodes a longer V-region producing an extended Fβ-strand that contributes to the stalk in a rearranged CDR3.A low amino acid variability was observed in CDR1 and CDR2,suggesting that antigen binding for this subset most likely only depends on the CDR3.Importantly a novel,potentially AID mediated,deletional diversification mechanism of the B.taurus VH ultralong CDR3 knob was discovered,in which interior codons of the IGHD8-2 region are removed while maintaining integral structural components of the knob and descending strand of the stalk in place.These deletions serve to further diversify cysteine positions,and thus disulfide bonded loops.Hence,both germline and somatic genetic factors and processes appear to be involved in diversification of this structurally unusual cattle VH ultralong CDR3 repertoire.