Ultrashort peptides have higher stability, tissue penetrability, biocompatibility, and less immunogenicity, and are widely applied in biology and medicine. GHK (glycyl-l-histidyl-l-lysine) and GQPR (glycyl-l-glutamyl-l-prolyl-l-arginine) can stimulate collagen renewal and inhibit collagen degradation. GHK and GQPR have been used in cosmetic anti-wrinkle skincare and make-up products. The most common approach for ultrashort peptide production is the solid-phase synthesis, which is eco-unfriendly due to heavy usage of organic chemical reagents during the manufacturing process. Here we report a new approach to the production of ultrashort peptides. Recombinant expression of ultrashort peptides is usually unfeasible because of the short amino acid sequences. A vector pET28a-Trxm harboring the thioredoxin gene was first constructed for subsequent fusion expression. The tandem repeats of GHK and GQPR genes were used as the templates for rolling circle amplification (RCA). The RCA reaction was tuned to incorporate noncanonical nucleotides 5-methylcytosine to obtain long DNA fragments. Gene sequences with various lengths were generated through double digestion of Acc65 Ⅰ and Apa Ⅰ. The resulting digestion products were gel recovered by size (from 500 bp to 1 500 bp) and cloned into pET28a-Trxm to obtain the recombinant vector pET28a-Trxm-(TRSP)_n. The pET28a-Trxm-(TRSP)_n was introduced into E. coli BL21(DE3) to generate a library of Trxm-(TRSP)_n sequences with a controlled distribution of lengths. Through double digestion and sequencing, positive clones with tandem repeats n=1, 2, 3, 4, 6, 7, 8, 9 were obtained. Protein expression results showed protein bands with corresponding molecular weight, and the protein expression level decreased as the tandem repeats increased. The expression level of Trxm-(TRSP)₁ achieved 50% of the total protein, while the expression level of Trxm-(TRSP)₂ was 30% of the total protein. The crude extracts from cell pellets were further treated with enterokinase cleavage, and the supernatants containing (TRSP)₁ were collected after ultrafiltration and then subjected to trypsin cleavage. HPLC analysis indicated that the ultrashort peptides GHK and GQPR were successfully obtained through two-step cleavage. This study may facilitate the commercial production of ultrashort peptides.
Escherichia coli/metabolism* ; Peptides/chemistry* ; Amino Acid Sequence ; Gene Library ; Tandem Repeat Sequences

OBJECTIVE: To study the effects of FLT3-ITD length on 32D cell proliferation, apoptosis and sensitivity to FLT3 inhibitor, so as to provide references for stepwise therapy of FLT3-ITD mutated acute myeloid leukemia patients. METHODS: Three different FLT3-ITD mutants with same or adjacent insert sites were selected and constructed in an eukaryotic expression vector. FLT3-ITD mutants stably expressed 32D cell strains were selected with the help of lentivirus system and IL3 free cell culture medium. The proliferation and apoptosis of 32D cell strains after AC220 treatment were detected. RESULTS: FLT3-ITD mutants (ITD1, ITD2 and ITD3) stably expressed 32D cell strains were constructed successfully. In the absence of IL3 factor, the proliferation number of ITD1, ITD2 and ITD3 cell strains were mounted up to 2.3 folds, 3.7 folds, and 4.3 folds after 48 hours, respectively. Under the exposure of FLT3 inhibitor AC220, the IC CONCLUSION FLT3-ITD mutant expressed cell strains with longer ITD show higher capacity of proliferation and higher tolerance to AC220 treatment.
Apoptosis ; Cell Proliferation ; Humans ; Leukemia, Myeloid, Acute/genetics* ; Mutation ; Protein Kinase Inhibitors ; Tandem Repeat Sequences ; fms-Like Tyrosine Kinase 3/genetics*

tandem repeat (STR), variable number tandem repeat (VNTR) tests, or informative single nucleotide polymorphism (SNP) analysis can be used to confirm the origin of leukemic cells from donor cells. Here, we report a case of DCL in a female patient after allogeneic peripheral stem cell transplantation from a male donor for the treatment of acute promyelocytic leukemia (APL) with PML-RARA. DCL developed 6 years after stem cell transplantation and leukemic cells of donor origin were confirmed by the presence of Y chromosome on the X/Y FISH analysis of bone marrow aspirate specimen. This is the first case of DCL reported in an APL patient in Korea.]]>
Bone Marrow ; Diagnosis ; Female ; Fluorescence ; Humans ; In Situ Hybridization ; Korea ; Leukemia ; Leukemia, Promyelocytic, Acute ; Male ; Microsatellite Repeats ; Peripheral Blood Stem Cell Transplantation ; Polymorphism, Single Nucleotide ; Stem Cell Transplantation ; Tandem Repeat Sequences ; Tissue Donors ; Y Chromosome

Anthrax, caused by Bacillus anthracis, is a non-contagious infectious disease that affects a wide range of animal species (primarily ruminants) including humans. Due to the often-fatal outcome in humans, quick administration of definitely effective antimicrobials is crucial either as prophylaxis or as a clinical case therapy. In this study, 110 B. anthracis strains, temporally, geographically, and genetically different, isolated during anthrax outbreaks in Italy from 1984 to 2017, were screened using a broth microdilution method to determine their susceptibility to 16 clinically relevant antimicrobial agents. The strains were isolated from various matrices (human, animal, and environmental samples) and were representative of thirty distinct genotypes previously identified by 15-loci multiple-locus variable-number of tandem repeats analysis. The antimicrobials tested were gentamicin, ceftriaxone, streptomycin, penicillin G, clindamycin, chloramphenicol, vancomycin, linezolid, cefotaxime, tetracycline, erythromycin, rifampin, amoxicillin, ciprofloxacin, doxycycline, and trimethoprim. All isolates were susceptible to most of the tested antimicrobials, with the exception of trimethoprim for which all of them showed high minimal inhibitory concentration values. An intermediate level of susceptibility was recorded for ceftriaxone and cefotaxime. Although the Centers for Disease Control and Prevention recommend the use of doxycycline, ciprofloxacin, penicillin G, and amoxicillin for treatment of human cases and for post-exposure prophylaxis to anthrax spores, this study shows a high degree of in vitro susceptibility of B. anthracis to many other antimicrobials, suggesting the possibility of an alternative choice for prophylaxis and therapy.
Amoxicillin ; Animals ; Anthrax ; Anti-Infective Agents ; Bacillus anthracis ; Bacillus ; Cefotaxime ; Ceftriaxone ; Centers for Disease Control and Prevention (U.S.) ; Chloramphenicol ; Ciprofloxacin ; Clindamycin ; Communicable Diseases ; Disease Outbreaks ; Doxycycline ; Erythromycin ; Genotype ; Gentamicins ; Humans ; In Vitro Techniques ; Italy ; Linezolid ; Methods ; Microbial Sensitivity Tests ; Penicillin G ; Post-Exposure Prophylaxis ; Rifampin ; Spores ; Streptomycin ; Tandem Repeat Sequences ; Tetracycline ; Trimethoprim ; Vancomycin

OBJECTIVES: Psychological resilience is the ability to cope with stress. The genetic background behind psychological resilience is not much known. The serotonin transporter and dopamine transporter are implicated in stress related psychology and emotional processing. The aim of this study is to investigate a possible genetic role of functional polymorphisms of serotonin and dopamine transporters for psychological resilience. METHODS: A total of 951 healthy adult subjects were included. Psychological resilience was measured using Connor-Davidson Resilience Scale (CD-RISC). Genotyping was performed for serotonin transporter gene (SERT) promoter variable number tandem repeat (VNTR) and dopamine transporter gene (DAT1) 3'-untranslated region (UTR) VNTR. Genetic association analysis was conducted between genotypes and the CD-RISC score. RESULTS: No genetic association was observed for SERT promoter VNTR or DAT1 3'-UTR VNTR with CD-RISC score. No genetic interaction between SERT promoter VNTR and DAT1 3'-UTR VNTR with CD-RISC score was detected. CONCLUSIONS: Either serotonin or dopamine transporter did not seem to play a significant role for psychological resilience in this sample.
Adult ; Dopamine Plasma Membrane Transport Proteins ; Dopamine* ; Genetic Background ; Genotype ; Humans ; Psychology ; Resilience, Psychological* ; Serotonin Plasma Membrane Transport Proteins ; Serotonin* ; Tandem Repeat Sequences

OBJECTIVES: Chronic otitis media (COM) is followed by irreversible tissue damage and destruction of the middle ear structures, with the possibility of complications under the maintenance of inflammation. Inflammatory mediators such as cytokines play a crucial role in the initial stage of inflammation. The aim of this study was to evaluate the association of the polymorphisms in two innate immunity/inflammation cascade genes from interleukin-1 (IL-1) gene cluster with COM with regard to cholesteatoma. METHODS: In the cross-sectional case-control study, DNA samples were collected from 189 patients with COM and 119 controls from a population of Serbia. The +3953 C/T (rs1143634), TaqI polymorphism in interleukin-1 beta (IL-1β) gene and 86 bp variable number tandem repeat (VNTR, rs2234663) polymorphism in the IL-1 receptor antagonist (IL-1RA) gene were analyzed by polymerase chain reaction. RESULTS: The IL-1β TaqI polymorphism was not significantly different in patients compared with the control group. The significant difference between patients and controls was observed for both, genotype and allele frequencies of IL-1RA VNTR polymorphism (chi-square P < 0.01). We found that carriers of IL-1RA allele 2 (odds ratio, 0.47; 95% confidence interval, 0.29 to 0.76; P=0.004) have a favorable association with COM, using multivariate logistic analysis that included both polymorphisms, age and sex. The IL-1RA allele frequency distribution was significantly different with regard to cholesteatoma. CONCLUSION: The carriers of allele 2 of VNTR IL-1RA polymorphism had a decreased odds ratio for COM, which is in agreement with findings in other inflammatory disease and its previous association with higher IL-1RA levels. Possible down-regulation of IL-1 mediated proinflammatory signaling pathways via IL-1RA in COM as well as results of our study should be further investigated and replicated.
Alleles* ; Case-Control Studies ; Cholesteatoma ; Cytokines ; DNA ; Down-Regulation ; Ear, Middle ; Gene Frequency ; Genotype ; Humans ; Inflammation ; Interleukin 1 Receptor Antagonist Protein* ; Interleukin-1 ; Interleukin-1beta ; Multigene Family ; Odds Ratio ; Otitis Media* ; Otitis* ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Serbia ; Tandem Repeat Sequences

OBJECTIVETo investigate the clinical characteristics of acute myeloid leukemia(AML) patients with FLT3-ITD(Fms-like tyrosine kinase3, intenal tandem duplication) mutation and their response to treatment.

METHODSRetrospective analysis of 128 newly diagnosed AML (except type M3) patients was performed between January 2014 and July 2017. Patients were divided into FLT3-ITD mutated group and non-mutated group. Mutation detection was carried out by using polymerase chain reaction(PCR) and gene sequencing analysis. Standard 3 + 7 or CAG regimen were taken as the first induction chemotherapy, 4 cases received sorafenib, overall survival (OS) was calculated by Kaplan-Meier.

RESULTSNinety-seven patients can be evaluable for clinical data available; 4 patients were FLT3-TKD mutated, which accounted for 4.1%; 19 patients were FLT3-ITD mutated, which accounted for 19.59%(19/97). Median white blood cell count (WBC), percentage of peripheral blasts and LDH value were significantly higher in FLT3-ITD group than those in non-mutated group [64.65(1.07-587.92)×10/L vs 39.68 (0.45-203.81) ×10/L](P<0.01), [69.62(16-99)% vs 36.35(0-92) %](P<0.01 ) and [LDH 526(124-2 729)U/L vs 265(20-1977)U/L](P<0.05), respectively. The frequency of coexisting NPM1 mutation was higher in FLT3-ITD group than that in non-mutated group [36.8(7/19)% vs 6.8 (5/74) %](P<0.01). The CR+PR was lower in FLT3-ITD group than that in non-mutated group [31.6(6/19)% vs 64.9 (48/74)%](P<0.05). OS in FLT3-ITD group was significantly shorter than that in non-mutated group (5 vs 18 months)(P<0.05). There is no significant difference in OS between FLT3-ITD concomitant with and without NPM1 mutation groups(5 vs 5 months)(P>0.05). The median OS was 13 months for the FLT3-ITD patients taking sorafenib.

CONCLUSIONThe FLT3-ITD is a common mutation in AML, FLT3-ITD mutated AML is more likely concomitant with NPM1 mutation with higher number of WBC, percentage of peripheral blasts and LDH value, thus CR is low after the 1st treatment and survival is poor.

Humans ; Leukemia, Myeloid, Acute ; Mutation ; Prognosis ; Retrospective Studies ; Tandem Repeat Sequences ; fms-Like Tyrosine Kinase 3

Bovine tuberculosis is a chronic contagious disease responsible for major agricultural economic losses. Abattoir monitoring and trace-back systems are an appropriate method to control bovine tuberculosis, particularly in beef cattle. In the present study, a trace-back system was applied to bovine tuberculosis cases in Korean native Hanwoo beef cattle. Bovine tuberculosis was detected in three index beef cattle during abattoir monitoring in Jeonbuk Province, Korea, and the original herds were traced back from each index cow. All cattle in each original herd were subjected to tuberculin skin test. The positive rates in the tuberculin skin test were 64.6% (62 of 96), 4.8% (2 of 42), and 8.1% (3 of 37) at farms A, B, and C, respectively. On post-mortem examination of 56 tuberculin-positive cattle, 62% had granulomatous lesions, and Mycobacterium bovis was cultured from 40 (71.4%) of the cattle. Molecular typing by spoligotyping and the mycobacterial interspersed repetitive unit-variable-number tandem repeat assay revealed the genotype of the M. bovis strains from the index cattle were same as the M. bovis genotype in each original herd. The results suggest that tracing back from index cattle to the original herd is an effective method to control bovine tuberculosis in beef cattle.
Abattoirs ; Agriculture ; Animals ; Autopsy ; Cattle ; Disease Outbreaks ; Genotype ; Jeollabuk-do ; Korea ; Methods ; Molecular Typing ; Mycobacterium bovis ; Red Meat ; Skin Tests ; Tandem Repeat Sequences ; Tuberculin ; Tuberculosis, Bovine

The aim of this study was to describe the genetic diversity of Mycobacterium avium subsp. paratuberculosis (MAP) obtained from individual cows in Korea. Twelve MAP-positive fecal DNA samples and 19 MAP isolates were obtained from 10 cattle herds located in 5 provinces in Korea. In addition, 5 MAP isolates obtained from the Czech Republic and Slovakia and 3 isolates from Australia were genotyped for comparison with the domestic isolates. The most prevalent strains in Korea were of the “bison-type” genotype (23 of 31 fecal DNA/isolates) and were distributed nationwide. The remaining MAP isolates (8) and all of the foreign isolates were identified as “cattle-type”. The bison-type strains which were discriminated only as INMV 68 in variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR) typing. Multilocus short sequence repeat (MLSSR) typing differentiated the bison-type strains into 3 different subtypes. The cattle-type strains were divided into 3 subtypes by MIRU-VNTR and 8 subtypes by MLSSR. The allelic diversities in the MIRU-VNTR and MLSSR results were calculated as 0.567 and 0.866, respectively. These results suggest that MIRU-VNTR typing cannot provide a sufficient description of the epidemiological situation of MAP. Therefore, an alternative method, such as MLSSR, is needed for typing of MAP strains to elucidate the molecular epidemiology of MAP infections. Overall, this study is the first epidemiological survey report in Korea using both MIRU-VNTR and MLSSR typing methods, and it has provided basic data necessary to elucidate the characteristics of MAP infections in Korea.
Animals ; Australia ; Cattle ; Czech Republic ; DNA ; Genetic Variation ; Genotype ; Korea ; Methods ; Molecular Epidemiology ; Mycobacterium avium subsp. paratuberculosis ; Mycobacterium avium ; Mycobacterium ; Paratuberculosis ; Slovakia ; Tandem Repeat Sequences

BACKGROUNDDuring the past decades, the incidence of invasive aspergillosis (IA) caused by Aspergillus fumigatus has increased dramatically. The aims of this study were to investigate the susceptibility of clinical isolates of A. fumigatus to triazole and the underlying cyp51A mutations in triazole-resistant A. fumigatus.

METHODSA total of 126 A. fumigatus clinical isolates from 126 patients with proven or probable IA were obtained from four large tertiary hospitals in Nanjing, China, between August 2012 and July 2015. The determination of minimal inhibitory concentrations (MICs) for itraconazole, voriconazole, and posaconazole was performed by broth microdilution according to the European Committee on Antimicrobial Susceptibility Testing reference method.

RESULTSA total of 4 A. fumigatus isolates (3.17%) were confirmed to be itraconazole resistant, with MICs of ≥8 mg/L, and one isolate (0.8%) was confirmed to be voriconazole resistant and posaconazole resistant, with MICs of 4 mg/L and 0.5 mg/L, respectively. We found that two of the 4 isolates of triazole-resistant A. fumigatus had the L98H amino acid substitution in combination with a 34-base pair tandem repeat in the promoter region, one isolate had an M220I mutation, and another itraconazole-resistant isolate did not have a substitution in the cyp51A gene.

CONCLUSIONSThis study shows that triazole-resistant A. fumigatus clinical isolates are present in Nanjing, China, which is a new challenge to the clinical management of IA.

Antifungal Agents ; pharmacology ; Aspergillus fumigatus ; drug effects ; genetics ; China ; Drug Resistance, Fungal ; Itraconazole ; pharmacology ; Microbial Sensitivity Tests ; Promoter Regions, Genetic ; genetics ; Tandem Repeat Sequences ; genetics ; Triazoles ; pharmacology ; Voriconazole ; pharmacology