Carrier DNA Research Articles

P-439 Analysis of Products of Conception (POC) using Whole Genome Next Generation Sequencing (NGS) in pregnancy loss after euploid or mosaic embryo transfer

Abstract Study question What is the outcome of genetic assessment for fetal chromosomal aberrations in miscarriages after euploid/mosaic embryo transfers (ET)? Summary answer POC testing should be recommended in patients with E/M-ET to screen for chromosomal aberrations occurring post-implantation or beyond the detection limit of PGT-A testing What is known already Spontaneous abortion is the most common complication of pregnancy, affecting approximately 15% of clinically recognized pregnancies. Although the etiology is diverse, fetal chromosomal aberrations account for 50-70% of first trimester miscarriages. Assessment of the fetal chromosomal composition is recommended by ACOG guidelines and aids in counselling and management of patients’ reproductive options. The value of this assessment in patients with miscarriage after euploid/mosaic ET has not evaluated. At the same time there is generally a lack of follow-up studies to examine the accuracy of PGT-A testing and investigate the rates of post-implantation errors causing miscarriage. Study design, size, duration This retrospective study had REB approval. This study was performed at the CReATe Fertility Centre and its Reproductive Genetics Laboratory in Toronto, Canada, between January 2019-December 2023.We evaluated the molecular karyotype of POC samples from first trimester <12GW miscarriages after ART: IVF-PGT-group euploid (E) and mosaic (M) embryo transfers (ET) and IVF-group; IUI and natural conception-NC groups; while controlling for maternal cell contamination (MCC) by high-resolution whole genome NGS. Participants/materials, setting, methods 549 POC samples from early miscarriages after fertility treatment were obtained by suction-dilatation-and curettage. Selection of fetal tissue/chorionic villi (4 representative samples for each case) was performed under a dissecting microscope. Maternal and paternal DNA was obtained to test for MCC. MCC was determined by testing maternal/gestational carrier and fetal DNA (fDNA) with highly informative STRs (AmpFLSTR Identifiler kit). Whole genome low-pass NGS (0.1xcoverage) was performed using the Illumina platform and analyzed with NxClinical Software. Main results and the role of chance Fetal tissue was obtained from 90.7% (498/549) of tested samples and the rest had MCC (9.3%, 51/549): IVF-PGT-A(n = 183), IVF(n = 92), IUI(n = 46) and NC(n = 173). The overall mean maternal age was 37±3.2years. The average gestational age at D&C was 8w4d (range 5w-12w6d). Overall, NGS analysis of fDNA showed 44.6% male and 55.3% female fetuses, 57.6% (287/498) were euploid, 39.5% (197/498) were aneuploid and 1.8% (9/498) were mosaic. In IVF-PGT-A group there were 169 POC from euploid ET and 14 from mosaic ET. 98.2% (n = 166/169) of POCs from IVF-PGT-A euploid-ETs were confirmed as euploid, and 3 had confined placental mosaicism (CPM) (trisomy-11, trisomy-4, monosomy-X). POC from mosaic-ET (n = 14), showed CPM in 3/14(21.4%): TE mosaicism was confirmed in 1/14(7.1%), 2/14(14.3%) had CPM differing from the PGT-A result, 11/14(78.6%) were euploid. POC euploidy rate in IVF (no PGT-A) group was 59.8%(55/92), in IUI 34.8%(16/46) and in NC 24.3%(42/173). In NC group CPM was detected in 1.7%(3/173). The frequency of aberrations detected in POC(n = 197) were: T22, 16.5%; T16,12.9%; T15, 11.3%; T20, 5.2%; T8, 3.6%; T7, T9 and T13, 3.1%; T10 and T14, 2.6%; T18 and T21, 2.1%; T12, 1.5%; 1% of each T2, T4 and T11; monosomy-X,6.7% and 11.3% triploid (69,XXY or 69,XXX). Limitations, reasons for caution MCC is relatively high in POC samples from early pregnancies (9.3%) and controlling for it is warranted. Although 4 representative samples of placental tissue were tested, the true extent and existence of CPM may not be captured correctly. Wider implications of the findings NGS analysis of POC from first trimester miscarriages after fertility treatment would improve diagnostic accuracy, and could aid in patient counselling and management. We observed high PGT-A accuracy (100% for gender) and 98.2% for fetal ploidy with CPM rate of 1.8% (which is lower than observed after CVS (4%)). Trial registration number not applicable

Short-homology-mediated PCR-based method for gene introduction in the fission yeast Schizosaccharomyces pombe.

Schizosaccharomyces pombe is a commonly utilized model organism for studying various aspects of eukaryotic cell physiology. One reason for its widespread use as an experimental system is the ease of genetic manipulations, leveraging the natural homology-targeted repair mechanism to accurately modify the genome. We conducted a study to assess the feasibility and efficiency of directly introducing exogenous genes into the fission yeast S. pombe using Polymerase Chain Reaction (PCR) with short-homology flanking sequences. Specifically, we amplified the NatMX6 gene (which provides resistance to nourseothricin) using PCR with oligonucleotides that had short flanking regions of 20bp, 40bp, 60bp and 80bp to the target gene. By using this purified PCR product, we successfully introduced the NatMX6 gene at position 171 385 on chromosome III in S. pombe. We have made a simple modification to the transformation procedure, resulting in a significant increase in transformation efficiency by at least 5-fold. The success rate of gene integration at the target position varied between 20% and 50% depending on the length of the flanking regions. Additionally, we discovered that the addition of dimethyl sulfoxide and boiled carrier DNA increased the number of transformants by ~60- and 3-fold, respectively. Furthermore, we found that the removal of the pku70+ gene improved the transformation efficiency to ~5% and reduced the formation of small background colonies. Overall, our results demonstrate that with this modified method, even very short stretches of homologous regions (as short as 20bp) can be used to effectively target genes at a high frequency in S. pombe. This finding greatly facilitates the introduction of exogenous genes in this organism.

Equine Piroplasmosis in Asymptomatic Horses of Western Iran: Comparison of Microscopic Examination and Multiplex PCR.

Piroplasmosis is responsible for anemia, fever, loss of physical activity and even death in equines. In epidemiological studies, accurate diagnostic tests are essential for detecting asymptomatic carriers. This study aimed to investigate the prevalence of infection in asymptomatic horses from Lorestan province, western Iran by developing a multiplex PCR. Blood samples were examined by microscopy and multiplex PCR targeting the SSU rRNA gene of Theileria equi and Babesia caballi. Out of the total of 165 horses, 19 (11.51%) and 31 (18.78%) cases were positive for piroplasms by microscopy and PCR, respectively. The detection rates of both genera were significantly higher in multiplex PCR compared to microscopy (p < 0.0001). Compared with multiplex PCR, the sensitivities of microscopy for the detection of Babesia were only 28.5%. The prevalence of T. equi infection was significantly higher in summer (p = 0.035). The prevalence of B. caballi was significantly higher in males (p = 0.038). Findings indicate that the multiplex PCR described here is a sensitive technique for the detection of piroplasm DNA in carriers. Furthermore, asymptomatic carriers must be considered as an important source of infection for equids living in this region.

NASCarD (Nanopore Adaptive Sampling with Carrier DNA): A Rapid, PCR-Free Method for SARS-CoV-2 Whole-Genome Sequencing in Clinical Samples.

Whole-genome sequencing (WGS) represents the main technology for SARS-CoV-2 lineage characterization in diagnostic laboratories worldwide. The rapid, near-full-length sequencing of the viral genome is commonly enabled by high-throughput sequencing of PCR amplicons derived from cDNA molecules. Here, we present a new approach called NASCarD (Nanopore Adaptive Sampling with Carrier DNA), which allows a low amount of nucleic acids to be sequenced while selectively enriching for sequences of interest, hence limiting the production of non-target sequences. Using COVID-19 positive samples available during the omicron wave, we demonstrate how the method may lead to >99% genome completeness of the SARS-CoV-2 genome sequences within 7 h of sequencing at a competitive cost. The new approach may have applications beyond SARS-CoV-2 sequencing for other DNA or RNA pathogens in clinical samples.

Salmon sperm DNA increases sample recovery from cotton swabs

Forensic samples with low DNA template amounts are difficult to analyze and interpret. There is a large body of research demonstrating that adding carrier nucleic acid to storage tubes, solid phase extractions, or filtering devices can improve yields of target DNA. However, the addition of carrier nucleic acid to sampling substrates, like cotton swabs, has not yet been attempted. In this proof-of-concept study, carrier nucleic acids in the form of either Poly (A) RNA or salmon sperm DNA were spotted onto cotton swabs, followed by human genomic DNA, to determine if introducing the carrier prior to sample collection would increase recovery from the swabs post-extraction. Extracts were also evaluated to determine whether adding the carrier nucleic acids to human DNA would interfere with downstream forensic DNA analysis processes such as real-time PCR quantitation, PCR amplification of STR loci, or capillary electrophoresis. The RNA carrier did not improve human sample recovery from cotton swabs. The extraction efficiency of human DNA from cotton swabs was increased when the DNA carrier was applied to the swabs prior to sample deposition, and the scale of the increase depended on the amount of carrier DNA used. When applying the salmon sperm DNA carrier to cotton swabs, with each increase from no carrier to 0.001–1–10 µg, human DNA recovery went from ∼29 % to ∼50 % to ∼75 % to ∼100 %. Additionally, no inhibitory effects from the carrier DNA were observed post-extraction with quantitation or in the DNA profile after amplification. Therefore, salmon sperm DNA carrier will increase human DNA yield from cotton swabs without negative effects on downstream forensic DNA profiling methods, with the optimal carrier amount being 10 µg.

High-Efficiency Transformation and Expression of Genomic Libraries in Yeast.

Saccharomyces cerevisiae is a powerful system for the expression of genome-wide or combinatorial libraries for diverse types of screening. However, expressing large libraries in yeast requires high-efficiency transformation and controlled expression. Transformation of yeast using electroporation methods is more efficient than chemical methods; however, protocols described for electroporation require large amounts of linearized plasmid DNA and often yield approximately 106 cfu/µg of plasmid DNA. We optimized the electroporation of yeast cells for the expression of whole-genome libraries to yield up to 108 cfu/µg plasmid DNA. The protocol generates sufficient transformants for 10-100× coverage of diverse genome libraries with small amounts of genomic libraries (0.1 µg of DNA per reaction) and provides guidance on calculations to estimate library size coverage and transformation efficiency. It describes the preparation of electrocompetent yeast cells with lithium acetate and dithiothreitol conditioning step and the transformation of cells by electroporation with carrier DNA. We validated the protocol using three yeast surface display libraries and demonstrated using nanopore sequencing that libraries' size and diversity are preserved. Moreover, expression analysis confirmed library functionality and the method's efficacy. Hence, this protocol yields a sufficient representation of the genome of interest for downstream screening purposes while limiting the amount of the genomic library required.

Detection of HTLV-1 proviral DNA in cell-free DNA: Potential for non-invasive monitoring of Adult T cell leukaemia/lymphoma using liquid biopsy?

Fragmented genomic DNA is constitutively released from dying cells into interstitial fluid in healthy tissue. In cancer, this so-called 'cell-free' DNA (cfDNA) released from dying malignant cells encodes cancer-associated mutations. Thus, minimally invasive sampling of cfDNA in blood plasma can be used to diagnose, characterise and longitudinally monitor solid tumours at remote sites in the body. ~5% of carriers of Human T cell leukaemia virus type 1 (HTLV-1) develop Adult T cell leukaemia/lymphoma (ATL), and a similar percentage develop an inflammatory CNS disease, HTLV-1 associated myelopathy (HAM). In both ATL and HAM, high frequencies of HTLV-1 infected cells are present in the affected tissue: each carrying an integrated DNA copy of the provirus. We hypothesised that turnover of infected cells results in the release of HTLV-1 proviruses in cfDNA, and that analysis of cfDNA from infected cells in HTLV-1 carriers might contain clinically useful information pertaining to inaccessible sites in the body- e.g. for early detection of primary or relapsing localised lymphoma type ATL. To evaluate the feasibility of this approach, we tested for HTLV-1 proviruses in blood plasma cfDNA. CfDNA (from blood plasma) and genomic DNA (gDNA, from peripheral blood mononuclear cells, PBMC) was isolated from blood from 6 uninfected controls, 24 asymptomatic carriers (AC), 21 patients with HAM and 25 patients with ATL. Proviral (HTLV-1 Tax) and human genomic DNA (the beta globin gene, HBB) targets were quantified by qPCR using primer pairs optimised for fragmented DNA. Pure, high quality cfDNA was successfully extracted from blood plasma of all study participants. When compared with uninfected controls, HTLV-1 carriers had higher concentrations of cfDNA circulating in their blood plasma. Patients with ATL who were not in remission had the highest levels of blood plasma cfDNA in any group studied. HTLV-1 proviral DNA was detected in 60/70 samples obtained from HTLV-1 carriers. The proviral load (percentage of cells carrying proviruses) was approximately tenfold lower in plasma cfDNA than in PBMC genomic DNA, and there was a strong correlation between the proviral load in cfDNA and PBMC genomic DNA in HTLV-1 carriers that did not have ATL. cfDNA samples in which proviruses were undetectable also had very low proviral load in PBMC genomic DNA. Finally, detection of proviruses in cfDNA of patients with ATL was predictive of clinical status: patients with evolving disease had higher than expected total amount of proviruses detectable in plasma cfDNA. We demonstrated that (1) HTLV-1 infection is associated with increased levels of blood plasma cfDNA, (2) proviral DNA is released into blood plasma cfDNA in HTLV-1 carriers and (3) proviral burden in cfDNA correlates with clinical status, raising the possibility of developing assays of cfDNA for clinical use in HTLV-1 carriers.

Assessing the specificity of the Rosette agent DNA amplification: An optimized protocol for the detection of standard DNA among studies.

Assessing the specificity of the Rosette agent DNA amplification: An optimized protocol for the detection of standard DNA among studies.

Non-homologous End Joining-Mediated Insertional Mutagenesis Reveals a Novel Target for Enhancing Fatty Alcohols Production in Yarrowia lipolytica.

Non-homologous end joining (NHEJ)-mediated integration is effective in generating random mutagenesis to identify beneficial gene targets in the whole genome, which can significantly promote the performance of the strains. Here, a novel target leading to higher protein synthesis was identified by NHEJ-mediated integration that seriously improved fatty alcohols biosynthesis in Yarrowia lipolytica. One batch of strains transformed with fatty acyl-CoA reductase gene (FAR) showed significant differences (up to 70.53-fold) in fatty alcohol production. Whole-genome sequencing of the high-yield strain demonstrated that a new target YALI0_A00913g (“A1 gene”) was disrupted by NHEJ-mediated integration of partial carrier DNA, and reverse engineering of the A1 gene disruption (YlΔA1-FAR) recovered the fatty alcohol overproduction phenotype. Transcriptome analysis of YlΔA1-FAR strain revealed A1 disruption led to strengthened protein synthesis process that was confirmed by sfGFP gene expression, which may account for enhanced cell viability and improved biosynthesis of fatty alcohols. This study identified a novel target that facilitated synthesis capacity and provided new insights into unlocking biosynthetic potential for future genetic engineering in Y. lipolytica.

Protoplast transformation of Kluyveromyces marxianus

The dairy yeast Kluyveromyces marxianus is a promising cell factory for producing bioethanol and heterologous proteins, as well as a robust synthetic biology platform host, due to its safe status and beneficial traits, including fast growth and thermotolerance. However, the lack of high-efficiency transformation methods hampers the fundamental research and industrial application of this yeast. Protoplast transformation is one of the most commonly used fungal transformation methods, but it yet remains unexplored in K. marxianus. Here, we established the protoplast transformation method of K. marxianus for the first time. A series of parameters on the transformation efficiency were optimized: cells were collected in the late-log phase and treated with zymolyase for protoplasting; the transformation was performed at 0°C with carrier DNA, CaCl2 , and PEG; after transformation, protoplasts were recovered in a solid regeneration medium containing 3-4% agar and 0.8m sorbitol. By using the optimized method, plasmids of 10, 24, and 58kb were successfully transformed into K. marxianus. The highest efficiency reached 1.8×104 transformants per μg DNA, which is 18-fold higher than the lithium acetate method. This protoplast transformation method will promote the genetic engineering of K. marxianus that requires high-efficiency transformation or the introduction of large DNA fragments.

In silico study of PEI-PEG-squalene-dsDNA polyplex formation: the delicate role of the PEG length in the binding of PEI to DNA.

Biocompatible hydrophilic polyethylene glycol (PEG) is widely used in biomedical applications, such as drug or gene delivery, tissue engineering or as an antifouling component in biomedical devices. Experimental studies have shown that the size of PEG can weaken polycation-polyanion interactions, like those between branched polyethyleneimine (b-PEI) and DNA in gene carriers, but details of its cause and underlying interactions on the atomic scale are still not clear. To better understand the interaction mechanisms in the formation of polyplexes between b-PEI-PEG based carriers and DNA, we have used a combination of in silico tools and experiments on three multicomponent systems differing in PEG MW. Using the PEI-PEG-squalene-dsDNA systems of the same size, both in the all-atom MD simulations and in experimental in-gel electrophoresis measurements, we found that the binding between DNA and the vectors is highly influenced by the size of PEG, with the binding efficiency increasing with a shorter PEG length. The mechanism of how PEG interferes with the binding between PEI and DNA is explained using a two-step MD simulation protocol that showed that the DNA-vector interactions are influenced by the PEG length due to the hydrogen bond formation between PEI and PEG. Although computationally demanding we find it important to study molecular systems of the same size both in silico and in a laboratory and to simulate the behaviour of the carrier prior to the addition of bioactive molecules to understand the molecular mechanisms involved in the formation of the polyplex.

573: A non-invasive prenatal test for Alpha Thalassemia Hb Barts and Hb Constant Spring

Hb Barts and Hb Constant Spring are the two most frequent severe forms of alpha thalassemia. The UNITYTM Carrier screen is a Lab Developed Test that performs reflex alpha thalassemia NIPT when the mother is determined to be an alpha-thalassemia carrier. This study analyzes the ability of the UNITYTM NIPT to exclude inheritance of Hb Barts and detect maternal inheritance of Hb Constant Spring. The UNITYTM Alpha Thalassemia NIPT was performed on simulated cell-free DNA samples. The assay is a multiplex PCR reaction that employs multiple technologies to evaluate the fetal genotype. Polymorphic alleles contained within the SEA deletion region are assayed to exclude inheritance of double deletions and therefore Hb Barts. Quantitative counting templates are used to accurately measure the number of DNA molecules present in the reaction. Relative mutation dosage is performed to determine the maternal and paternal inheritance of the Constant Spring allele. Gap-PCR is used to detect paternal inheritance of an SEA deletion. A likelihood ratio is used to combine these measurements and determine the Hb Constant Spring status of the fetus. Simulated cell-free DNA from pregnant mothers was prepared by shearing and combining genomic DNA samples. Hb Barts exclusion was tested using SEA carrier DNA mixed with healthy or Hb Barts DNA. Hb Barts was excluded from all healthy DNA samples and was not excluded from all affected samples (n=96). Maternal inheritance of the Constant Spring variant was tested on Constant Spring carrier DNA mixed with healthy SEA carrier DNA. All calls were correct at 5%, 10% and 20% fetal fractions, two 5% samples resulted in no-calls (n=12). These results analytically demonstrate the ability of the UNITYTM alpha thalassemia NIPT to identify healthy fetuses in carrier mothers. The UNITYTM alpha thalassemia carrier screen with reflex NIPT will enable detection of both maternal carrier status and fetal disease risk from a single blood tube. This removes the need for paternal DNA and decreases the turn around time to identify high-risk fetuses.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Long-term adaptive evolution of Shewanella oneidensis MR-1 for establishment of high concentration Cr(VI) tolerance

Acclimation is the main method to enhance the productivity of microorganisms in environmental biotechnology, but it remains uncertain how microorganisms acquire resistance to high concentrations of pollutants during long-term acclimation. Shewanella oneidensis MR-1 was acclimated for 120 days with increasing hexavalent chromium (Cr(VI)) concentrations from 10 to 190 mg/L. The bacterium was able to survive from the highly toxic Cr(VI) environment due to its enhanced capability to reduce Cr(VI) and the increased cell membrane surface. We sequenced 19 complete genomes from 7 populations of MR-1, including the ancestral strain, the evolved strains in Cr(VI) environment on days 40, 80 and 120 and their corresponding controls. A total of 27, 49 and 90 single nucleotide polymorphisms were found in the Cr(VI)-evolved populations on days 40, 80 and 120, respectively. Nonsynonymous substitutions were clustered according to gene functions, and the gene mutations related to integral components of the membrane, including efflux pumps and transporters, were the key determinants of chromate resistance. In addition, MR-1 strengthened the detoxification of Cr(VI) through gene variations involved in adenosine triphosphate binding, electron carrier activity, signal transduction and DNA repair. Our results provide an in-depth analysis of how Cr(VI) resistance of S. oneidensis MR-1 is improved by acclimation, as well as a genetic understanding of the impact of long-term exposure of microorganisms to pollution.

Comparative metatranscriptome analysis revealed broad response of microbial communities in two soil types, agriculture versus organic soil

BackgroundStudying expression of genes by direct sequencing and analysis of metatranscriptomes at a particular time and space can disclose structural and functional insights about microbial communities. The present study reports comparative analysis of metatranscriptome from two distinct soil ecosystems referred as M1 (agriculture soil) and O1 (organic soil). ResultsAnalysis of sequencing reads revealed Proteobacteria as major dominant phyla in both soil types. The order of the top 3 abundant phyla in M1 sample was Proteobacteria >Ascomycota >Firmicutes, whereas in sample O1, the order was Proteobacteria >Cyanobacteria >Actinobacteria. Analysis of differentially expressed genes demonstrated high expression of transcripts related to copper-binding proteins, proteins involved in electron carrier activity, DNA integration, endonuclease activity, MFS transportation, and other uncharacterized proteins in M1 compared to O1. Of the particular interests, several transcripts related to nitrification, ammonification, stress response, and alternate carbon fixation pathways were highly expressed in M1. In-depth analysis of the sequencing data revealed that transcripts of archaeal origin had high expression in M1 compared to O1 indicating the active role of Archaea in metal- and pesticide-contaminated environment. In addition, transcripts encoding 4-hydroxyphenylpyruvate dioxygenase, glyoxalase/bleomycin resistance protein/dioxygenase, metapyrocatechase, and ring hydroxylating dioxygenases of aromatic hydrocarbon degradation pathways had high expression in M1. Altogether, this study provided important insights about the transcripts and pathways upregulating in the presence of pesticides and herbicides. ConclusionAltogether, this study claims a high expression of microbial transcripts in two ecosystems with a wide range of functions. It further provided clue about several molecular markers which could be a strong indicator of metal and pesticide contamination in soils. Interestingly, our study revealed that Archaea are playing a significant role in nitrification process as compared to bacteria in metal- and pesticide-contaminated soil. In particular, high expression of transcripts related to aromatic hydrocarbon degradation in M1 soil indicates their important role in biodegradation of pollutants, and therefore, further investigation is needed.

Monitoring long-term DNA storage via absolute copy number quantification by ddPCR

Long-term storage of DNA is a routine practice in biomedical research, diagnostics and drug discovery. Periodic monitoring is important for early detection of changes in DNA quality and quantity. Existing methods include agarose gel, ultraviolet (UV) absorbance, fluorometric reading and qPCR. However, these methods are either limited by sensitivity or depend on DNA standards, which face the same storage challenges. In this paper, we tested the state-of-the-art droplet digital PCR (ddPCR) technology that can quantify the absolute DNA copy number with no need of a standard curve. We found that ddPCR was very accurate in determining the level of a plasmid DNA standard and was sensitive to DNA loss due to degradation or adsorption. With the ddPCR technology, we found a gradual process of DNA adsorption to several types of low binding tubes, which was unnoticed before. Although modest, adsorption significantly affected recovery of highly diluted DNA (<0.2 μg/mL), which could be rescued by addition of carrier DNA. In conclusion, this paper not only demonstrated that ddPCR is an ideal method for monitoring DNA storage, but also provided an effective approach to improving recovery of highly diluted DNA, which may have broad implications in assay development, diagnostics and forensic sciences.

An Overview of the Randomized Placebo-Controlled Trials of Chinese Herbal Medicine Formula Granules.

Objective To summarize the characteristics and the outcomes of the Randomized Placebo-Controlled Trials of Chinese Herbal Medicine Granules manufactured by China Resources Sanjiu Pharmaceutical Co., Ltd. Methods Databases including China National Knowledge Infrastructure, VIP, Wanfang, PubMed, Cochrane Library, and clinicaltrials.gov were searched in March 2018 for relevant randomized controlled trials (RCTs). Two reviewers independently screened for and selected studies, extracted data, and checked data extraction. Methodological quality was evaluated using the Cochrane Risk of Bias tool. For the outcome, the characteristics of the study, the cure rate, the effectiveness rate, and advert events were described with a method of bibliometrics. Also, we performed meta-analysis only if there were ≥2 studies treated by the same intervention and evaluated by the same outcome. Results A total of 40 placebo-controlled RCTs treated for 17 diseases were included in our analysis involving 4,632 patients. 16 of 19 studies treated by CHM granules only showed positive result in patients with HBV, HCV, fever, depression, nonalcoholic fatty liver disease, AIDS, and asthma while negative result was shown in patients with migraine. 17 of 21 studies treated by combination therapy against conventional therapy showed positive result in patients with HBV, herpes simplex keratitis, COPD, liver cirrhotic ascites, Parkinson's disease, and diabetic peripheral neuropathy while negative result was shown in patients with myasthenia gravis, angina pectoris, and depression. The pooled result cannot demonstrate that the notifying kidney formula granules had the superior effect with placebo on the clearance of serum HBV DNA and HBeAg in HBV carriers with a RR (and the 95% CI) of 2.97 [0.74,11.91] and 1.99 [0.93,4.29], respectively. But, the CHM granules can reduce within-group HBV DNA levels by more than 2 lgIU/ml; the RR (and 95% CI) was 4.64 [2.89,7.45]. Qizhu granules had a significant effect on clearance of HCV RNA with a RR (and 95% CI) of 6.26 [2.16,18.16]. And, the heat-clearing and detoxifying formula granules were superior to placebo in resolution of cold symptom among patients with fever with a RR and 95% CI of 2.58 [1.40,4.74]. Based on the conventional therapy, the pooled result demonstrated that the Regulating liver formula granules were superior to placebo on the clearance of serum HBeAg in chronic hepatitis B patients with a RR (and the 95% CI) of 1.73 [1.30,2.31]. The EeChen decoction granules were superior to placebo in COPD patients with a RR (and the 95% CI) of 1.13 [1.06,1.22]. 28 of the 40 studies reported adverse events. There were 51 adverse events in CHM formula granules group or combination group (n=2,483) and 26 in control group (n=2,122) totally. Most of the adverse symptoms spontaneously resolved after completing the courses of treatment and the other adverse symptoms improved after symptomatic treatment. Conclusion 16 of 19 studies treated by CHM granules only showed positive result in 7 diseases and negative result in 1 disease. 17 of 21 studies treated by combination therapy against conventional therapy showed positive result in 6 diseases and negative result in 3 diseases. However, both the absolute and relative effectiveness of CHM formula granules compared with placebo need to be considered clinically.

Large Interruptions of GAA Repeat Expansion Mutations in Friedreich Ataxia Are Very Rare.

Friedreich ataxia is a multi-system autosomal recessive inherited disorder primarily caused by homozygous GAA repeat expansion mutations within intron 1 of the frataxin gene. The resulting deficiency of frataxin protein leads to progressive mitochondrial dysfunction, oxidative stress, and cell death, with the main affected sites being the large sensory neurons of the dorsal root ganglia and the dentate nucleus of the cerebellum. The GAA repeat expansions may be pure (GAA)n in sequence or may be interrupted with regions of non-GAA sequence. To our knowledge, there has been no large-scale study of FRDA patient DNA samples to determine the frequency of large interruptions in GAA repeat expansions. Therefore, we have investigated a panel of 245 Friedreich ataxia patient and carrier DNA samples using GAA repeat PCR amplification and MboII restriction enzyme digestion. We demonstrate that the vast majority (97.8%) of Friedreich ataxia GAA repeat expansion samples do not contain significant sequence changes that would result in abnormal MboII digestion profiles, indicating that they are primarily pure GAA repeats. These results show for the first time that large interruptions in the GAA repeats are very rare.

Electric Single-Molecule Hybridization Detector for Short DNA Fragments.

By combining DNA nanotechnology and high-bandwidth single-molecule detection in nanopipets, we demonstrate an electric, label-free hybridization sensor for short DNA sequences (<100 nucleotides). Such short fragments are known to occur as circulating cell-free DNA in various bodily fluids, such as blood plasma and saliva, and have been identified as disease markers for cancer and infectious diseases. To this end, we use as a model system an 88-mer target from the RV1910c gene in Mycobacterium tuberculosis, which is associated with antibiotic (isoniazid) resistance in TB. Upon binding to short probes attached to long carrier DNA, we show that resistive-pulse sensing in nanopipets is capable of identifying rather subtle structural differences, such as the hybridization state of the probes, in a statistically robust manner. With significant potential toward multiplexing and high-throughput analysis, our study points toward a new, single-molecule DNA-assay technology that is fast, easy to use, and compatible with point-of-care environments.

High-efficiency transformation of Yarrowia lipolytica using electroporation.

Yarrowia lipolytica is an industrial host organism with incredible potential for metabolic engineering. However, the genetic tools and capacities in this host lag behind those of conventional counterparts. In this study, we sought to increase the transformation efficiency of Y. lipolytica by creating a simple protocol using electroporation. Efficiency was increased by optimizing wash buffers, pre-culture growth time, OD600 of competent cells, voltage, competent cell volume, DNA concentration, and recovery time. The outcome of these optimizations led to a simple protocol with maximum linear fragment transformation efficiency of 1.6×104 transformants per μg DNA and 2.8×104 transformants per μg DNA for episomal plasmid transformation. The protocol presented here is superior to other Y. lipolytica transformation protocols as it requires no lengthy pretreatment and no required carrier DNA to achieve efficiencies on par with, or exceeding, previously reported methods.

Transfer of plasmid into the pentose-fermenting yeast Pachysolen tannophilus

The pentose-fermenting yeast Pachysolen tannophilus can convert glucose and xylose in lignocellulosic hydrolysates to ethanol. However, it performs poorly in industrially relevant lignocellulosic hydrolysates containing mixed sugars and inhibitors. Efforts have been directed at improving the performance of this yeast to enable efficient lignocellulosic biomass conversion. While some successes have been reported using random mutagenesis and/or hybridization-based approaches, further genetic improvement of this yeast is hampered by the lack of efficient gene transfer methods as well as limited genetic information to guide further construction of robust strains of P. tannophilus. In this study, we aimed to address this short-coming by establishing the optimal conditions needed for efficient gene transfer into P. tannophilus. We ascertained that plasmids can be transferred into P. tannophilus through trans-kingdom conjugation or lithium acetate (LiAc) transformation. The efficiency of plasmid YEp13 (2-micron, LEU2) transferred into a P. tannophilus leucine auxotroph (Leu−) reached as high as 1.93 × 10−2 transconjugants per input recipient and 3.25 × 104 transformants per μg plasmid DNA through trans-kingdom conjugation and transformation, respectively. In trans-kingdom conjugation, the number of recipient P. tannophilus cells played an important role, while the ratio of donor (Escherichia coli) to recipient cells was less important. For efficient transformation in P. tannophilus, the use of PEG 3350 was essential, as no transformants were obtained in its absence. The transformation efficiency increased with the addition of single-stranded carrier DNA and incubation at 30 °C for >60 min. Plasmids with different replication origins or 2-micron plasmids with different CUG codon-optimized antibiotic resistance markers were unable to transform P. tannophilus under our experimental conditions. The results are of interest in the genetic manipulation and improvement of P. tannophilus.