Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
Tandemly arrayed genes (TAGs) or gene clusters are prevalent in higher eukaryotic genomes. For example, approximately 17% of genes are organized in tandem in the model plant Arabidopsis thaliana. The genetic redundancy created by TAGs presents a challenge for reverse genetics. As molecular scissors, engineered zinc finger nucleases (ZFNs) make DNA double-strand breaks in a sequence-specific manner. ZFNs thus provide a means to delete TAGs by creating two double-strand breaks in the gene cluster. Using engineered ZFNs, we successfully targeted seven genes from three TAGs on two Arabidopsis chromosomes, including the well-known RPP4 gene cluster, which contains eight resistance (R) genes. The resulting gene cluster deletions ranged from a few kb to 55 kb with frequencies approximating 1% in somatic cells. We also obtained large chromosomal deletions of ~9 Mb at approximately one tenth the frequency, and gene cluster inversions and duplications also were achieved. This study demonstrates the ability to use sequence-specific nucleases in plants to make targeted chromosome rearrangements and create novel chimeric genes for reverse genetics and biotechnology.
zinc finger nuclease (ZFN); tandemly arrayed genes (TAGs); deletion; inversion; Arabidopsis
Human serum albumin (HSA) is widely utilized for medical purposes and biochemical research. Transgenic rice has proved to be an attractive bioreactor for mass production of recombinant HSA (rHSA). However, transgene spread is a major environmental and food safety concern for transgenic rice expressing proteins of medical value. This study aimed to develop a selectively terminable transgenic rice line expressing HSA in rice seeds, and a simple process for recovery and purification of rHSA for economical manufacture. An HSA expression cassette was inserted into a T-DNA vector encoding an RNA interference (RNAi) cassette suppressing the CYP81A6 gene. This gene detoxifies the herbicide bentazon and is linked to the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) cassette which confers glyphosate tolerance. ANX Sepharose Fast Flow (ANX FF) anion exchange chromatography coupled with Butyl Sepharose High Performance (Butyl HP) hydrophobic interaction chromatography was used to purify rHSA. A transgenic rice line, HSA-84, was obtained with stable expression of rHSA of up to 0.72% of the total dry weight of the dehusked rice seeds. This line also demonstrated high sensitivity to bentazon, and thus could be killed selectively by a spray of bentazon. A two-step chromatography purification scheme was established to purify the rHSA from rice seeds to a purity of 99% with a recovery of 62.4%. Results from mass spectrometry and N-terminus sequencing suggested that the purified rHSA was identical to natural plasma-derived HSA. This study provides an alternative strategy for large-scale production of HSA with a built-in transgene safety control mechanism.
Recombinant human serum albumin (rHSA); Selectively terminable transgenic rice; Purification
The objective of this study was to investigate the tempo-spatial distribution of paddy rice in Northeast China using moderate resolution imaging spectroradiometer (MODIS) data. We developed an algorithm for detection and estimation of the transplanting and flooding periods of paddy rice with a combination of enhanced vegetation index (EVI) and land surface water index with a central wavelength at 2 130 nm (LSWI2130). In two intensive sites in Northeast China, fine resolution satellite imagery was used to validate the performance of the algorithm at pixel and 3×3 pixel window levels, respectively. The commission and omission errors in both of the intensive sites were approximately less than 20%. Based on the algorithm, annual distribution of paddy rice in Northeast China from 2001 to 2009 was mapped and analyzed. The results demonstrated that the MODIS-derived area was highly correlated with published agricultural statistical data with a coefficient of determination (R
2) value of 0.847. It also revealed a sharp decline in 2003, especially in the Sanjiang Plain located in the northeast of Heilongjiang Province, due to the oversupply and price decline of rice in 2002. These results suggest that the approaches are available for accurate and reliable monitoring of rice cultivated areas and variation on a large scale.
Paddy rice; Moderate resolution imaging spectroradiometer (MODIS); Northeast China; Enhanced vegetation index; Land surface water index
AIM: To investigate whether transforming growth factor-β1 (TGF-β1) signaling pathway is involved in the pathogenesis of primary biliary cirrhosis (PBC).
METHODS: A murine model of PBC was developed by injection of polyinosinic polycytidylic acids (poly I: C) in C57BL/6 mice, and the liver expressions of TGF β1, TGF-β receptor I (TβRI), TGF-β receptor II (TβRII), p-Smad2/3, monoclonal α-smooth muscle actin antibody (α-SMA) and α1 (I) collagen in the mouse model and control mice were evaluated by immunohistochemistry, immunoblotting and real-time polymerase chain reaction (RT-PCR). Lymphocyte subsets in liver were analyzed using flow cytometry.
RESULTS: The mouse model had several key phenotypic features of human PBC, including elevated levels of alkaline phosphatase, antimitochondrial antibodies, portal bile ducts inflammation, and progressive collagen deposition. Compared with control mice, protein and mRNA levels of TGF β1, TβRI, TβRII, p-Smad2/3, α-SMA and α1 (I) collagen in liver (1.7 ± 0.4 vs 8.9 ± 1.8, 0.8 ± 0.2 vs 5.1 ± 1.5, 0.6 ± 0.01 vs 5.1 ± 0.1, 0.6 ± 0.3 vs 2.0 ± 0.3, 0.9 ± 0.4 vs 3.4 ± 0.6, 0.8 ± 0.4 vs 1.7 ± 0.3, 1.1 ± 1.2 vs 11.8 ± 0.6, P < 0.05), and the total number and percentage of CD4+ CD25+ FOXP3+ and CD8+ lymphocytes (0.01 ± 0.001 vs 0.004 ± 0.00, 0.12 ± 0.04 vs 0.52 ± 0.23, P < 0.01) were higher in the mouse model.
CONCLUSION: TGFβ1 might play a dual role in the development of PBC: it suppresses inflammatory response but operates to enhance fibrogenesis. The aberrant activity of TGF-β1 signaling contributes to the development of PBC.
Primary biliary cirrhosis; Transforming growth factor-β1; Regulatory T cell; Liver
The basolateral amygdala (BLA) plays a crucial role in emotional learning irrespective of valence1–5. While the BLA projection to the nucleus accumbens (NAc) is hypothesized to modulate cue-triggered motivated behaviors4, 6, 7,, our understanding of the interaction between these two brain regions has been limited by the inability to manipulate neural circuit elements of this pathway selectively during behavior. To circumvent this limitation, we used in vivo optogenetic stimulation or inhibition of glutamatergic fibers from the BLA to the NAc, coupled with intracranial pharmacology and ex vivo electrophysiology. We show that optical stimulation of the BLA-to-NAc pathway in mice reinforces behavioral responding to earn additional optical stimulations of these synaptic inputs. Optical stimulation of BLA-to-NAc glutamatergic fibers required intra-NAc dopamine D1-type, but not D2-type, receptor signaling. Brief optical inhibition of BLA-to-NAc fibers reduced cue-evoked intake of sucrose, demonstrating an important role of this specific pathway in controlling naturally occurring reward-related behavior. Moreover, while optical stimulation of medial prefrontal cortex (mPFC) to NAc glutamatergic fibers also elicited reliable excitatory synaptic responses, optical self-stimulation behavior was not observed by activation of this pathway. These data suggest that while the BLA is important for processing both positive and negative affect, the BLA-to-NAc glutamatergic pathway in conjunction with dopamine signaling in the NAc promotes motivated behavioral responding.
Gallstone ileus (GI) is characterized by occlusion of the intestinal lumen as a result of one or more gallstones. GI is a rare complication of gallstones that occurs in 1%-4% of all cases of bowel obstruction. The mortality associated with GI ranges between 12% and 27%. Classical findings on plain abdominal radiography include: (1) pneumobilia; (2) intestinal obstruction; (3) an aberrantly located gallstone; and (4) change of location of a previously observed stone. The optimal management of acute GI is controversial and can be: (1) enterotomy with stone extraction alone; (2) enterotomy, stone extraction, cholecystectomy and fistula closure; (3) bowel resection alone; and (4) bowel resection with fistula closure. We describe a case to highlight some of the pertinent issues involved in GI management, and propose a scheme to minimize recurrent disease and postoperative complications. We conclude that GI is a rare condition affecting mainly the older population with a female predominance. The advent of computed tomography and magnetic resonance imaging has made it easier to diagnose GI. Enterotomy with stone extraction alone remains the most common surgical method because of its low incidence of complications.
Gallstone ileus; Fistula closure; Intestinal obstruction; Bowel obstruction; Enterolithotomy
The association between polymorphisms on 5p12 and breast cancer (BC) has been widely evaluated since it was first identified through genome-wide association approach; however, the studies have yielded contradictory results. We sought to investigate this inconsistency by performing a comprehensive meta-analysis on two wildly studied polymorphisms (rs10941679 and rs4415084) on 5p12.
Databases including Pubmed, EMBASE, Web of Science, EBSCO, and Cochrane Library databases were searched to find relevant studies. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of association. The random-effects model was applied, addressing heterogeneity and publication bias.
A total of 19 articles involving 100,083 cases and 163,894 controls were included. An overall random-effects per-allele OR of 1.09 (95% CI: 1.06–1.12; P = 4.5×10−8) and 1.09 (95% CI: 1.05–1.12; P = 4.2×10−7) was found for the rs10941679 and rs4415084 polymorphism respectively. Significant results were found in Asians and Caucasians when stratified by ethnicity; whereas no significant associations were found among Africans/African-Americans. Similar results were also observed using dominant or recessive genetic models. In addition, we find both rs4415084 and rs10941679 conferred significantly greater risks of ER-positive breast cancer than of ER-negative tumors.
Our findings demonstrated that rs10941679-G allele and rs4415084-T allele might be risk-conferring factors for the development of breast cancer, especially in Caucasians and East-Asians.
Hawthorn (Crataegus spp.) is an important pome with a long history as a fruit, an ornamental, and a source of medicine. Fruits of hawthorn are marked by hard stony endocarps, but a hawthorn germplasm with soft and thin endocarp was found in Liaoning province of China. To elucidate the molecular mechanism underlying the soft endocarp of hawthorn, we conducted a de novo assembly of the fruit transcriptome of Crataegus pinnatifida and compared gene expression profiles between the soft-endocarp and the hard-endocarp hawthorn varieties. De novo assembly yielded 52,673 putative unigenes, 20.4% of which are longer than 1,000 bp. Among the high-quality unique sequences, 35,979 (68.3%) had at least one significant match to an existing gene model. A total of 1,218 genes, represented 2.31% total putative unigenes, were differentially expressed between the soft-endocarp hawthorn and the hard-endocarp hawthorn. Among these differentially expressed genes, a number of lignin biosynthetic pathway genes were down-regulated while almost all the flavonoid biosynthetic pathway genes were strongly up-regulated, concomitant with the formation of soft endocarp. In addition, we have identified some MYB and NAC transcription factors that could potentially control lignin and flavonoid biosynthesis. The altered expression levels of the genes encoding lignin biosynthetic enzymes, MYB and NAC transcription factors were confirmed by quantitative RT-PCR. This is the first transcriptome analysis of Crataegus genus. The high quality ESTs generated in this study will aid future gene cloning from hawthorn. Our study provides important insights into the molecular mechanisms underlying soft endocarp formation in hawthorn.
Curcuminoids are well known for their capabilities to combat risk factors that are associated with ageing and cellular senescence. Recent reports have demonstrated that curcuminoids can extend the lifespan of model organisms. However, the underlying mechanisms by which these polyphenic compounds exert these beneficial effects remain unknown. In this study, t-BHP-induced premature senescence model in human fibroblasts was chosen to explore the protective effects of a curcuminoid, bisdemethoxycurcumin (BDMC), on cellular senescence. The results demonstrated that BDMC attenuated oxidative stress-induced senescence-like features which include the induction of an enlarged cellular appearance, higher frequency of senescence-associated β-galactosidase staining activity, appearance of senescence-associated heterochromatic foci in nuclei, decrease in proliferation capability, and alteration in related molecules such as p16 and retinoblastoma protein. Notably, we found that BDMC treatment activated Sirt1/AMPK signaling pathway. Moreover, downregulating Sirt1 by the pharmacological inhibitor nicotianamine or small interfering RNA blocked BDMC-mediated protection against t-BHP-mediated decrease in proliferation. These results suggested that BDMC prevented t-BHP-induced cellular senescence, and BDMC-induced Sirt1 may be a mechanism mediating its beneficial effects.
Preconditioning has been considered promising for the treatment of ischemic flaps. In this study, the therapeutic effect of postconditioning was compared with that of preconditioning during ischemia/reperfusion (I/R) injury, and a role of inducible nitric oxide synthase (iNOS) in postconditioning treatment was also explored.
Sixty rats were randomly divided into four groups with 15 rats in each group. Ischemic injury was induced in a rat’s gracilis muscle flap model. Preconditioning and postconditioning were performed respectively on the flaps in the pre-con group and the post-con group. No treatment was given to the flaps in the control group, and flaps without I/R injury were used as a sham control. Muscle viability ratio, histology, and gene expression of iNOS were examined at different time intervals (3, 12, and 18 h).
A significantly higher survival ratio was observed in both the pre-con group (78.98 ± 3.39, 62.74 ± 3.7, and 54.42 ± 4.45 %) and the post-con group (77.42 ± 4.14, 59.74 ± 6.67, and 49.52 ± 4.13 %) than the control group (45.22 ± 3.69, 42.44 ± 3.76, and 33.2 ± 3.29 %) at 3, 12, and 18 h postoperatively (P < 0.05). There was no statistical difference between the pre-con group and the post-con group (P > 0.05). Histological examination showed delayed and attenuated tissue damage in both the pre-con group and the post-con group when compared to that of the control group. A higher expression of iNOS was observed in both the pre-con group and the post-con group than the control group and the sham group (P < 0.05).
Significant improvement of flap survival could be achieved by both preconditioning and postconditioning treatments; however, better protection could be provided by preconditioning. The higher expression of iNOS may play an important role in the therapeutic effect of postconditioning during I/R injury.
Ischemic postconditioning; Ischemic preconditioning; Ischemia/reperfusion (I/R) injury; Inducible nitric oxide synthase
The targeting of nucleases to specific DNA sequences facilitates genome editing. Recent work demonstrated that the CRISPR-associated (Cas) nuclease Cas9 can be targeted to sequences in vitro simply by modifying a short7 CRISPR RNA (crRNA) guide. Here we use this CRISPR-Cas system to introduce marker-free mutations in Streptococcus pneumoniae and Escherichia coli. The approach involves re-programming Cas9 by using a crRNA complementary to a target chromosomal locus and introducing a template DNA harboring a desired mutation and an altered crRNA recognition site for recombination with the target locus. We exhaustively analyze Cas9 target requirements to define the range of targetable sequences and show strategies for editing sites that do not meet these requirements. Alone or together with recombineering, CRISPR assisted editing induces recombination at the targeted locus and kills non-edited cells leading to a recovery of close to a 100% of edited cells. Multiple crRNA can be used to modify several loci simultaneously. Our results show that CRISPR-mediated genome editing only requires programming of the crRNA and template sequences and thus constitutes a useful tool for genetic engineering.
Pilon fractures are challenging to manage because of the complexity of the injury pattern and the risk of significant complications. The soft tissue injury and handling of the soft tissue envelope are crucial in pilon fracture outcomes. The purpose of this study was to evaluate the early rate of complications using the strategy of “soft tissue control” for operative treatment of complex pilon fractures.
Materials and Methods:
36 complex pilon fractures were treated with the “soft tissue control” strategy. Patients followed the standard staged protocol, anterolateral approach to the distal tibia, the “no-touch” technique and incisional negative pressure wound therapy for pilon fractures. Patients were examined clinically at 2-3 weeks and then 8 weeks for complications associated with the surgical technique.
All fractures were AO/OTA (Orthopaedic Trauma Association) type C fractures (61% C3, 22% C2 and 16% C1). Only one patient developed superficial infection and resolved with antibiotics and local wound care. None developed deep infection.
The strategy of soft tissue control for treatment of pilon fractures resulted in relatively low incidence of early wound complications in patients with complex pilon fractures.
Negative pressure wound therapy; pilon fractures; soft tissue control; staged protocol
Multiple osteochondromas (MO) is an inherited skeletal disorder, and the molecular mechanism of MO remains elusive. Exome sequencing has high chromosomal coverage and accuracy, and has recently been successfully used to identify pathogenic gene mutations. In this study, exome sequencing followed by Sanger sequencing validation was first used to screen gene mutations in two representative MO patients from a Chinese family. After filtering the data from the 1000 Genome Project and the dbSNP database (build 132), the detected candidate gene mutations were further validated via Sanger sequencing of four other members of the same MO family and 200 unrelated healthy subjects. Immunohistochemisty and multiple sequence alignment were performed to evaluate the importance of the identified causal mutation. A novel frameshift mutation, c.1457insG at codon 486 of exon 6 of EXT1 gene, was identified, which truncated the glycosyltransferase domain of EXT1 gene. Multiple sequence alignment showed that codon 486 of EXT1 gene was highly conserved across various vertebrates. Immunohistochemisty demonstrated that the chondrocytes with functional EXT1 in MO were less than those in extragenetic solitary chondromas. The novel c.1457insG deleterious mutation of EXT1 gene reported in this study expands the causal mutation spectrum of MO, and may be helpful for prenatal genetic screening and early diagnosis of MO.
AIM: To establish the frequency and clinical features of connective tissue diseases (CTDs) in a cohort of Chinese patients with primary biliary cirrhosis (PBC).
METHODS: Three-hundred and twenty-two Chinese PBC patients were screened for the presence of CTD, and the systemic involvement was assessed. The differences in clinical features and laboratory findings between PBC patients with and without CTD were documented. The diversity of incidence of CTDs in PBC of different countries and areas was discussed. For the comparison of normally distributed data, Student’s t test was used, while non-parametric test (Wilcoxon test) for the non-normally distributed data and 2 × 2 χ2 or Fisher’s exact tests for the ratio.
RESULTS: One-hundred and fifty (46.6%) PBC patients had one or more CTDs. The most common CTD was Sjögren’s syndrome (SS, 121 cases, 36.2%). There were nine cases of systemic sclerosis (SSc, 2.8%), 12 of systemic lupus erythematosus (SLE, 3.7%), nine of rheumatoid arthritis (RA, 2.8%), and 10 of polymyositis (PM, 3.1%) in this cohort. Compared to patients with PBC only, the PBC + SS patients were more likely to have fever and elevated erythrocyte sedimentation rate (ESR), higher serum immunoglobulin G (IgG) levels and more frequent rheumatoid factor (RF) and interstitial lung disease (ILD) incidences; PBC + SSc patients had higher frequency of ILD; PBC + SLE patients had lower white blood cell (WBC) count, hemoglobin (Hb), platelet count, γ-glutamyl transpeptidase and immunoglobulin M levels, but higher frequency of renal involvement; PBC + RA patients had lower Hb, higher serum IgG, alkaline phosphatase, faster ESR and a higher ratio of RF positivity; PBC + PM patients had higher WBC count and a tendency towards myocardial involvement.
CONCLUSION: Besides the common liver manifestation of PBC, systemic involvement and overlaps with other CTDs are not infrequent in Chinese patients. When overlapping with other CTDs, PBC patients manifested some special clinical and laboratory features which may have effect on the prognosis.
Cirrhosis; Biliary; Connective tissue disease; Sjögren’s syndrome; Systemic sclerosis; Raynaud phenomenon
Designer DNA-binding proteins based on transcriptional
effectors (TALEs) and zinc finger proteins (ZFPs) are easily tailored
to recognize specific DNA sequences in a modular manner. They can
be engineered to generate tools for targeted genome perturbation.
Here, we review recent advances in these versatile technologies with
a focus on designer nucleases for highly precise, efficient, and scarless
gene modification. By generating double stranded breaks and stimulating
cellular DNA repair pathways, TALE and ZF nucleases have the ability
to modify the endogenous genome. We also discuss current applications
of designer DNA-binding proteins in synthetic biology and disease
modeling, novel effector domains for genetic and epigenetic regulation,
and finally perspectives on using customizable DNA-binding proteins
for interrogating neural function.
TAL effector; TALEN; zinc finger; gene targeting; designer nuclease; genome engineering
An alteration in the mitochondrial DNA (mtDNA) copy number has been detected in numerous human cancers. However, certain changes in the mtDNA copy number that occur during the initiation and progression of gastric cancer remain undetected. In the present study, using quantitative PCR analysis, the quantitative changes in mtDNA were observed during the initiation and progression of gastric cancer. Furthermore, the possible correlation between the changes in mtDNA and the clinicopathological stage were also investigated. However, the mechanism by which the change in mtDNA copy number occurs remains to be elucidated. Epigenetic changes are believed to play a significant role in regulating the mtDNA content. In order to determine whether there is a potential correlation between DNA methylation and mtDNA regulation, in vitro demethylation experiments were performed. Tumor tissues and corresponding non-cancerous tissues were surgically resected from 76 gastric cancer patients between 2010 and 2011. The results revealed that the average relative mtDNA copy numbers were 94.71±28.11 in the cancer tissues and 111.68±21.84 in the corresponding non-cancerous tissues (P<0.01). The quantitative changes in mtDNA demonstrated a significant decrease in gastric cancer, particularly in ill-defined stage III and IV cases, but had no association with gender. The mtDNA copy numbers demonstrated a marked increase (P<0.05) following demethylation treatment. The present results indicate that the mtDNA copy number plays a significant role during the progression of colorectal cancer, particularly during the late clinicopathological stages, and that the change in the mtDNA copy number may correlate with DNA methylation.
gastric cancer; mitochondrial DNA; copy number; clinicopathological stage
The purpose of the study was to understand the function and expression of ATF2 by JNK and p38 signal pathways in the chondrocytes apoptosis of articular cartilage of the Kashin-Beck disease (KBD).
The changes of ATF2, JNK and p38 mRNAs and proteins were investigated between cartilage and chondrocyte as well as KBD and normal. JNK and p38 inhibitors were used as treatments to prevent apoptosis in chondrocytes from KBD patients.
It was found that the protein levels of p-p38, p-JNK, ATF2 and p-ATF2 increased in KBD human cartilage which is in line with the higher mRNA levels of p38, JNK and ATF2 as compared both with normal cartilage and KBD chondrocytes. In addition, p-ATF2 was only detected in KBD cartilage. Furthermore, JNK inhibitor was more effective than p38 inhibitor in preventing chondrocyte apoptosis at equal concentrations of 10 μM.
These findings indicated the expression of p-ATF2 by JNK and p38 signal pathways involved in the chondrocyte apoptosis in cartilage with KBD.
JNK and p38 pathways; ATF2; Apoptosis; Chondrocytes; Cartilage; Kashin-Beck disease
The present study aimed to investigate the molecular basis of lung cancer development using a microarray to identify the differentially-expressed genes associated with the various tumor-node-metastasis (TNM) stages of lung adenocarcinoma. This subtype of lung cancer has increased in incidence within recent years in China. A 35K oligo gene array covering ~25,100 genes was used to screen the differentially-expressed genes among 90 lung adenocarcinoma samples of various TNM stages. To verify the data from the gene arrays, three genes [human zinc finger-containing, Miz1, PIAS-like protein on chromosome 7 (Zimp7), GINS complex subunit 2 (GINS2) and NSAID activated gene 1 (NAG-1)] were validated using quantitative (q)PCR in an alternative set of samples to the gene array. A total of 640 genes were identified that were differentially-expressed in lung adenocarcinoma compared with the surrounding normal lung tissues. From these 640 candidate genes, 10 were observed to be differentially-expressed among TNM stages I, II and IIIA, of which, the Zimp7, GINS2 and NAG-1 genes were reported for the first time to be expressed at high levels in lung adenocarcinoma. The results of the qPCR for the three genes were consistent with those from the gene array. In total, 10 candidate genes were identified to be associated with the various TNM stages of lung adenocarcinoma in the population studied, which may provide new insights into the molecular basis underlying the development of lung adenocarcinoma and offer new targets for the diagnosis, therapy and prognosis.
lung adenocarcinoma; tumor-node-metastasis stage; gene expression profile
We sought to evaluate the efficacy and effects of low-dose tacrolimus (FK506) to recipients with living donor liver transplantation (LDLT). A total of 66 patients who underwent LDLT between 2001 and 2007 were enrolled in this study. According to different doses of tacrolimus, the recipients were randomly divided into two groups: the low-dose tacrolimus group (group A) and the normal-dose tacrolimus group (group B). The blood concentration of tacrolimus and its side effects including infection, hyperglycemia, hypertension, high blood creatinine and jaundice were monitored once a week at the perioperative period, and once a month thereafter. Besides, the survival rates of the recipients were analyzed at the 1- and 3-year time point after operation. Among these patients, no significant acute rejection was detected after LDLT. The incidences of infection, hyperglycemia, liver dysfunction and renal impairment in group A were markedly lower than those in group B. However, no significant differences were detected in the incidence of hypertension between the two groups. Moreover, the recipients in each group had a similar survival rate according to the results of 1- and 3-year follow-up. The incidence of side effects that associated with tacrolimus positively correlated with tacrolimus blood concentration. In conclusion, long-term and low-dose administration of tacrolimus is a safe and effective treatment for LDLT recipients.
living donor liver transplantation; tacrolimus; low dose; side effect; survival rate
HIV-infected individuals currently cannot be completely cured because existing antiviral therapy regimens do not address HIV provirus DNA, flanked by long terminal repeats (LTRs), already integrated into host genome. Here, we present a possible alternative therapeutic approach to specifically and directly mediate deletion of the integrated full-length HIV provirus from infected and latently infected human T cell genomes by using specially designed zinc-finger nucleases (ZFNs) to target a sequence within the LTR that is well conserved across all clades. We designed and screened one pair of ZFN to target the highly conserved HIV-1 5′-LTR and 3′-LTR DNA sequences, named ZFN-LTR. We found that ZFN-LTR can specifically target and cleave the full-length HIV-1 proviral DNA in several infected and latently infected cell types and also HIV-1 infected human primary cells in vitro. We observed that the frequency of excision was 45.9% in infected human cell lines after treatment with ZFN-LTR, without significant host-cell genotoxicity. Taken together, our data demonstrate that a single ZFN-LTR pair can specifically and effectively cleave integrated full-length HIV-1 proviral DNA and mediate antiretroviral activity in infected and latently infected cells, suggesting that this strategy could offer a novel approach to eradicate the HIV-1 virus from the infected host in the future.
We examined the association between household solid fuel exposure and lung function in a densely populated district in urban Shanghai, China.
Spirometry was performed in 12,506 subjects, aged 18 and over, residing the Putuo District in Shanghai, China, in a cross-sectional survey. Exposure to solid fuel use at home was assessed by administered questionnaire, estimating duration and total amount of solid fuel use at home during the lifetime.
After adjusting for confounders, the subjects with exposure to household solid fuel had a 1.3% [95% confidence interval (CI) 0.57 to 2.02] decrease in forced expiratory volume in 1 sec (FEV1) percent predicted and 3.5% (95% CI 2.74 to 4.18) decrease in forced vital capacity (FVC) percent predicted, respectively. Trends towards decreased pulmonary function measures were seen for longer duration and greater amount of household fuel use at home, in the highest compared with lowest tertile (P values for trend < 0.001). We observed decrease in FEV1 and FVC percent predicted across increase in tertile of BMI in association with in-home solid fuel exposure.
This study suggests that in-home solid fuel exposure is associated with reduced lung function in an urban population.
solid fuel; lung function; body mass index; Chinese
Transcription activator-like effectors (TALEs) are a class of naturally occurring DNA binding proteins found in the plant pathogen Xanthomonas sp. The DNA binding domain of each TALE consists of tandem 34-amino acid repeat modules that can be rearranged according to a simple cipher to target new DNA sequences. Customized TALEs can be used for a wide variety of genome engineering applications, including transcriptional modulation and genome editing. Here we describe a toolbox for rapid construction of custom TALE transcription factors (TALE-TFs) and nucleases (TALENs) using a hierarchical ligation procedure. This toolbox facilitates affordable and rapid construction of custom TALE-TFs and TALENs within one week and can be easily scaled up to construct TALEs for multiple targets in parallel. We also provide details for testing the activity in mammalian cells of custom TALE-TFs and TALENs using, respectively, qRT-PCR and Surveyor nuclease. The TALE toolbox described here will enable a broad range of biological applications.
TALE; TAL effector; TALE-TF; TALE nuclease; TALEN; zinc finger; ZFN; transcription factor; gene activation; genome engineering; meganuclease; gene knockout; synthetic biology; homologous gene targeting
The mammalian brain poses a formidable challenge to the study and treatment of neuropsychiatric diseases – owing to the complex interaction of genetic, epigenetic, and circuit-level mechanisms underlying pathogenesis. Technologies that facilitate functional dissection of distinct brain circuits are necessary for systematic identification of disease origin and therapy. Recent developments in the optogenetics technology have begun to address this challenge by enabling precise perturbation of distinct cell types based on molecular signatures, functional projections, and intracellular biochemical signaling pathways. With high temporal precision and reversible neuromodulation, optogenetics promises to improve existing disease models and advance our understanding of psychiatric conditions. In this review, we will describe the current state of molecular optogenetic tools and future directions of development.
optogenetics; channelrhodopsin; neuropsychiatric disease; neuromodulation; gene therapy; brain machine interface