OBJECTIVETo construct point mutation plasmids expressing HCV NS3/4A with different secondary structures at amino-terminal, and express the constructs in Huh 7 cells.

METHODSUsing pSG5/M-H05-5/4A as the template (A1-1) and primers designed according to the typing criteria, 4 single point mutation plasmids, namely pSG5/M-H05-5(A1-2)/4A(A1-2) (Y56F), pSG5/M-H05-5(B1-1)/4A(B1-1) (L80Q), pSG5/M-H05-5(B2-1)/4A(B2-1) (V51A), and pSG5/M-H05-5(B2-2)/4A(B2-2) (S61A), were constructed. With A1-2, B2-1, and B2-2 as the templates, the leucine to glutamine mutation at position 80 (L80Q) was induced to construct another 3 double point mutation plasmids pSG5/M-H05-5(B1-2)/4A(B1-2), pSG5/M-H05-5(A2-1)/4A(A2-1), and pSG5/M-H05-5(A2-2)/4A(A2-2), respectively. DNA sequencing was performed for confirmation of the mutations. Huh 7 cells were transfected with the constructs using FuGene 6 transfection reagents. Indirect immunofluorescence staining and Western blotting were used to detect the expression of the constructs.

RESULTSIndirect immunofluorescence assay revealed 4 subcellular localization patterns of NS3 protein, including dot-like staining, diffuse staining, doughnut-like staining, and rod-shape staining. Western blotting also demonstrated successful expression of the constructs and weak in cis and in trans NS3 serine protease activities of subtypes A2-1 and B2-1 in comparison with other subtypes.

CONCLUSIONThe point mutation plasmids expressing HCV NS3/4A with different secondary structures at amino-terminal are constructed successfully, which provides the basis for further study of different subtypes of HCV.

Amino Acid Sequence ; Cell Line ; Gene Expression ; Genetic Engineering ; methods ; Hepacivirus ; enzymology ; Immunohistochemistry ; Intracellular Space ; metabolism ; Molecular Sequence Data ; Plasmids ; genetics ; Point Mutation ; Protein Structure, Secondary ; Protein Transport ; Viral Nonstructural Proteins ; chemistry ; genetics ; metabolism

OBJECTIVETo assess the clinicopathological features of atypical cystic duct (ACD) as a precancerous lesion of the breast.

METHODSWhole mammary gland serial sections were performed on 200 cases of breast cancer without pre-operative biopsy (prior operation, fine needle aspiration or needle biopsy were routinely performed in each case). The clinicopathological findings and immunohistochemical features of ACD were investigated.

RESULTSForty-four (22%) of the 200 breast cancer patients had ACD breast lesions. The frequency of patients with ACD increased in premenopausal women (P=0.001). A number of ACD lesions displayed a histological transition to adjacent ductal carcinoma in-situ. In 16 of 44 (36%) patients with ACD, carcinoma cells stained positive for p53. In 12 of these 16 cases (75%), ACD cells also stained positive for p53 protein (P=0.001). Myoepithelial cells of ACD appeared attenuated and stained strongly for alpha-smooth muscle actin. There was no correlation between the ACD-present group and the ACD-absent group in tumor size, nodal metastasis, and immunostaining patterns of estrogen receptor (ER), progesterone receptor (PR), p53, c-erbB-2 and Ki-67 labeling index of cancerous tissues. All 44 ACD lesions showed a negative staining of c-erbB-2, regardless of the staining result in their corresponding carcinomas. The mean Ki-67 labeling index of ACD lesions was low.

CONCLUSIONSACD is frequently associated with breast cancer. It may represent a precancerous mammary lesion, supported by the frequent histological continuum between ACD and malignancy, and simultaneous p53 over-expression present in both ACD and its corresponding breast carcinoma.

Biomarkers, Tumor ; analysis ; Breast Neoplasms ; chemistry ; pathology ; surgery ; Carcinoma in Situ ; chemistry ; pathology ; surgery ; Carcinoma, Ductal, Breast ; chemistry ; pathology ; surgery ; Female ; Humans ; Precancerous Conditions ; chemistry ; pathology ; surgery ; Receptors, Estrogen ; analysis ; Receptors, Progesterone ; analysis

Alport syndrome (AS) is a progressive inherited kidney disease characterized by hearing loss and ocular abnormalities.There are three forms of AS depending on inheritance mode: X-linked Alport syndrome (XLAS), autosomal recessive AS (ARAS), and autosomal dominant AS (ADAS). XLAS is caused by pathogenic variants in COL4A5, which encodes type IV collagen α5 chain, while ADAS and ARAS are caused by variants in COL4A3 or COL4A4, which encode type IV collagen α3 or α4 chain, respectively. In male XLAS or ARAS cases, end-stage kidney disease (ESKD) develops around a median age of 20 to 30 years old, while female XLAS or ADAS cases develop ESKD around a median age of 60 to 70 years old. The diagnosis of AS is dependent on either genetic or pathological findings. However, determining the pathogenicity of the variants detected by gene tests can be difficult. Recently, we applied the following molecular investigation tools to determine pathogenicity: 1) in silico and in vitro trimer formation assay of α345 chains to assess triple helix formation ability, 2) kidney organoids constructed from patients’ induced pluripotent stem cells to identify α5 chain expression on the glomerular basement membrane, and 3) in vitro splicing assay to detect aberrant splicing to determine the pathogenicity of variants. In this review article, we discuss the genetic background and novel assays for determining the pathogenicity of variants. We also discuss the current treatment approaches and introduce exon skipping therapy as one potential treatment option.

Alport syndrome (AS) is a progressive inherited kidney disease characterized by hearing loss and ocular abnormalities.There are three forms of AS depending on inheritance mode: X-linked Alport syndrome (XLAS), autosomal recessive AS (ARAS), and autosomal dominant AS (ADAS). XLAS is caused by pathogenic variants in COL4A5, which encodes type IV collagen α5 chain, while ADAS and ARAS are caused by variants in COL4A3 or COL4A4, which encode type IV collagen α3 or α4 chain, respectively. In male XLAS or ARAS cases, end-stage kidney disease (ESKD) develops around a median age of 20 to 30 years old, while female XLAS or ADAS cases develop ESKD around a median age of 60 to 70 years old. The diagnosis of AS is dependent on either genetic or pathological findings. However, determining the pathogenicity of the variants detected by gene tests can be difficult. Recently, we applied the following molecular investigation tools to determine pathogenicity: 1) in silico and in vitro trimer formation assay of α345 chains to assess triple helix formation ability, 2) kidney organoids constructed from patients’ induced pluripotent stem cells to identify α5 chain expression on the glomerular basement membrane, and 3) in vitro splicing assay to detect aberrant splicing to determine the pathogenicity of variants. In this review article, we discuss the genetic background and novel assays for determining the pathogenicity of variants. We also discuss the current treatment approaches and introduce exon skipping therapy as one potential treatment option.