Multiple machine-learning tools identifying prognostic biomarkers for acute Myeloid Leukemia.

BACKGROUND: Acute Myeloid Leukemia (AML) generally has a relatively low survival rate after treatment. There is an urgent need to find new biomarkers that may improve the survival prognosis of patients. Machine-learning tools are more and more widely used in the screening of biomarkers. METHODS: Least Absolute Shrinkage and Selection Operator (LASSO), Support Vector Machine-Recursive Feature Elimination (SVM-RFE), Random Forest (RF), eXtreme Gradient Boosting (XGBoost), lrFuncs, IdaProfile, caretFuncs, and nbFuncs models were used to screen key genes closely associated with AML. Then, based on the Cancer Genome Atlas (TCGA), pan-cancer analysis was performed to determine the correlation between important genes and AML or other cancers. Finally, the diagnostic value of important genes for AML was verified in different data sets. RESULTS: The survival analysis results of the training set showed 26 genes with survival differences. After the intersection of the results of each machine learning method, DNM1, MEIS1, and SUSD3 were selected as key genes for subsequent analysis. The results of the pan-cancer analysis showed that MEIS1 and DNM1 were significantly highly expressed in AML; MEIS1 and SUSD3 are potential risk factors for the prognosis of AML, and DNM1 is a potential protective factor. Three key genes were significantly associated with AML immune subtypes and multiple immune checkpoints in AML. The results of the verification analysis show that DNM1, MEIS1, and SUSD3 have potential diagnostic value for AML. CONCLUSION: Multiple machine learning methods identified DNM1, MEIS1, and SUSD3 can be regarded as prognostic biomarkers for AML.

Pan-cancer analysis of the PDE4DIP gene with potential prognostic and immunotherapeutic values in multiple cancers including acute myeloid leukemia.

Phosphodiesterase 4D interacting protein (PDE4DIP) interacts with cAMP-specific phosphodiesterase 4D and its abnormal expression promotes the development of hematological malignancies, breast cancer, and pineal cell carcinoma. However, there is currently no systematic pan-cancer analysis of the association between PDE4DIP and various cancers. Thus, this study aimed to elucidate the potential functions of PDE4DIP in various cancers. Based on the multiple public databases and online websites, we conducted comprehensive analyses for PDE4DIP in various cancers, including differential expression, prognosis, genetic variation, DNA methylation, and immunity. We thoroughly analyzed the specific role of PDE4DIP in acute myeloid leukemia (LAML). The results indicated that there were differences in PDE4DIP expression in cancers, and in kidney chromophobe, LAML, pheochromocytoma and paraganglioma, thymoma, and uveal melanoma, PDE4DIP had potential prognostic value. PDE4DIP expression was also correlated with genetic variation, DNA methylation, immune cell infiltration, and immune-related genes in cancers. Functional enrichment analysis showed that PDE4DIP was mainly related to immune-related pathways in cancers, and in LAML, PDE4DIP was mainly related to immunoglobulin complexes, T-cell receptor complexes, and immune response regulatory signaling pathways. Our study systematically revealed for the first time the potential prognostic and immunotherapeutic value of PDE4DIP in various cancers, including LAML.

Association of CYP19A1 rs28757157 polymorphism with lung cancer risk in the Chinese Han population.

BACKGROUND: Lung cancer is the leading cause of cancer death globally. Recent studies have revealed that CYP19A1 gene plays a crucial role in cancer initiation and development. The aim of this study was to assess the association of CYP19A1 genetic polymorphisms with the risk of lung cancer in the Chinese Han population. METHODS: This study randomly recruited 489 lung cancer patients and 467 healthy controls. The genotypes of four single nucleotide polymorphisms (SNPs) of the CYP19A1 gene were identified by the Agena MassARRY technique. Genetic model analysis was used to assess the association between genetic variations and lung cancer risk. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to evaluate the effect of four selected SNPs on lung cancer risk. RESULTS: CYP19A1 rs28757157 might contribute to an increased risk of lung cancer (p = 0.025, OR = 1.30, 95% CI 1.03-1.64). In stratified analysis, rs28757157 was associated with an increased cancer risk in the population aged under 60 years, females, smokers, and drinkers. Besides, rs3751592 and rs59429575 were also identified as risk biomarkers in the population under 60 years and drinkers. Meanwhile, a relationship between an enhanced risk of squamous cell carcinoma and rs28757157 was found, while the rs3751592 CC genotype was identified as a risk factor for lung adenocarcinoma development. CONCLUSIONS: This study has identified revealed that the three SNPs (rs28757157, rs3751592, and rs59429575) of CYP19A1 are associated with lung cancer in the Chinese Han population. These findings will provide theoretical support for further functional studies of CYP19A1 in lung cancer.

Analysis of Very Important Pharmacogenomics Variants in the Chinese Lahu Population.

BACKGROUND: Genetic polymorphism, obviously, has a potential clinical role in determining differences in drug efficacy; however, there are no reports about the pharmacogenomic information of the Lahu population. Therefore, our research aimed to screen the genotypic frequencies of the very important pharmacogenomics (VIP) mutations and determined the differences between Lahu and the other 11 populations. METHODS: Agena MassARRAY (AgenaMassARRAY) single nucleotide polymorphism (SNP) genotyping technique was used to detect 81 VIP mutations of pharmacogenomics genes in Lahu, and their genotypic frequencies were compared with the other major 11 populations. Chi-square tests were used to identify different loci among these populations. Finally, the genetic structure and pairwise Fst values of Lahu and the other 11 populations were analyzed. RESULTS: We found that the distribution of allele frequencies within different pharmacogenes in Lahu showed significantly different with other populations. Additionally, the pairwise F-statistics (Fst) values and genetic structure revealed the variants in the Lahu population as well were mostly related to the Han Chinese in Beijing, China (CHB) and the Japanese population in Tokyo, Japan (JPT) genetically. CONCLUSION: This study will provide a theoretical basis for safe drug use and help to establish the appropriate individualized treatment strategies in the Lahu population.

Dual plastic film and straw mulching boosts wheat productivity and soil quality under the El Nino in semiarid Kenya.

The extreme climate events such as El Nino seriously threaten crop production and agro-ecological sustainability because of the aggravated environmental stresses worldwide, particularly in sub-Saharan Africa. To address this issue, we investigated the effects of dual plastic film and straw mulching in ridge-furrow (RF) system on wheat productivity, soil carbon and nitrogen stocks in a semiarid area in Kenya from 2015 to 2017. The experimental site represents a typical semiarid continental monsoon climate, and soil type is chromic vertisols. Field experiment with randomized block design consisted of six RF treatments as follows: 1) dual black plastic film and straw mulching (RFbS), 2) dual transparent plastic film and straw mulching (RFtS), 3) sole black plastic film mulching (RFb), 4) sole transparent plastic mulching RF (RFt), 5) sole straw mulching (RFS) and 6) no mulching (CK). The results indicated that seasonal dynamics of rainfall and air temperature fit in with the weather type of El Nino over four growing seasons. RFbS, RFtS, RFb and RFt significantly increased soil water storage (SWS), topsoil temperature, aboveground biomass, grain yield and water use efficiency across four growing seasons (p < 0.05) as compared with CK. Among all the treatments, RFbS and RFtS achieved the greatest SWS, AgB, grain yield and WUE, owing to improved soil hydro-thermal status in both treatments. Critically, RFbS and RFtS significantly improved soil organic carbon and total nitrogen, soil bulk density and the C:N ratio following four growing seasons, comparing with other treatments (p < 0.05). Besides, RFbS and RFtS gave the highest economic returns among all treatments. For the first time, we found that dual plastic film and straw mulching could serve as a sustainable land management to boost wheat productivity and improve soil quality under El Nino in semiarid areas of SSA.

Genetic polymorphisms of pharmacogenomic VIP variants in the Dai population from Yunnan province.

BACKGROUND: Pharmacogenomics plays a crucial role in individualized therapy, but the variant information of pharmacogenomics in the Dai population is limited. We therefore aimed to screen very important pharmacogenetic (VIP) in the Dai population and compared differences between Dai and other 25 populations. METHODS: In this study, we genotyped 73 VIP variants from the PharmGKB and compared genotype distribution of variants in Dai with other 25 populations by χ2 test. To assess the genetic relationship among 26 populations, we performed the structure analysis. In addition, pair-wise F-statistics (Fst) was calculated to measure the population differentiation. RESULTS: We found 12, 10, 13, 17, 11, 39, 46, 46, 45, 43, 49, 46, 46, 46, 49, 45, 41, 42, 48, 53, 45, 50, 50, 51, 47, and 50 significantly different variants in Dai compared with other 25 populations. Genetic structure analysis showed Dai had close relationships with CDX (Chinese Dai in Xishuangbanna), CHB (Han Chinese in Beijing), JPT (Japanese in Tokyo), and KHV (Kinh in Ho Chi Minh City, Vietnam). Moreover, Dai is the most similar to KHV according to Fst analysis. CONCLUSIONS: Our study complement the pharmacogenomics information of Dai population from Yunnan province and provide a theoretical basis for personalized medicine.

Global prevalence of human pegivirus-1 in healthy volunteer blood donors: a systematic review and meta-analysis.

BACKGROUND AND OBJECTIVES: The local prevalence of HPgV-1 has been reported from different countries worldwide, but the global prevalence of HPgV-1 remains unknown. The aim of this systematic review and meta-analysis was to gather data from the literature to estimate the prevalence of HPgV-1 in healthy volunteer blood donors in the world. MATERIALS AND METHODS: We searched PubMed, EMBASE, Scopus and Google Scholar databases for records up to January 2019 and included studies reporting HPgV-1 virus prevalence amongst healthy volunteer blood donors based on the detection of HPgV-1 RNA. RESULTS: In all, we included 79 studies for the systematic review and 63 for the meta-analysis. Based on the random effect meta-analysis of 35 468 volunteer blood donors, we found the global prevalence of HPgV-1 to be 3·1% (95% CI, 2·4-4·1). The pooled prevalences of HPgV-1 were 1·7% (95% CI, 1·1-2·6) in North America, 9·1% (95% CI, 6·4-12·7) in South America, 2·3% (95% CI, 2%, 2·8) in Europe and 2·4% (95% CI, 1·4-4) in Asia. Subgroup analyses based on age, gender or risk factors were not possible. CONCLUSION: Approximately 3 in 100 blood donations worldwide are positive for HPgV-1 increasing the risk of infection from transfusion of their components to subsequent recipients. Further research on virus pathogenicity is required before recommending routine screening of HPgV-1 for healthy volunteer blood donors.

Genetic polymorphisms analysis of pharmacogenomic VIP variants in Bai ethnic group from China.

BACKGROUND: The pharmacogenomics study has been widely used for the study of very important pharmacogenetic (VIP) variants among different ethnic groups. However, there is little known about the pharmacogenomics information regarding Bai family. Our study aimed to screen the polymorphism of the VIP gene in Bai nationality. METHODS: We genotyped 81 VIP variants (selected from the PharmGKB database) in the Bai population and then compared them to the other 11 major HapMap populations by chi-square test, structure and F-statistics (Fst) analysis. RESULTS: Our results indicated that rs20417 (PTGS2), rs4148323 (UGT1A), and rs1131596 (SLC19A1) were most different in Bai compared with most of the 11 populations from the HapMap data set. Furthermore, population structure and F-statistics (Fst) analysis also demonstrated that the Bai population has the closest genetic relationship with Han Chinese in Beijing, China (CHB), followed by Japanese in Tokyo, Japan (JPT), and the farthest population from the Yoruba in Ibadan, Nigeria (YRI). CONCLUSIONS: Our study not only presented the genotype frequency difference between the selected population of the Bai population and the other 11 populations, but also showed that the Bai population is most similar to the CHB populations, followed by JPT. These findings would contribute to the development of individualized medicine for the Bai population.

Genetic polymorphisms in IL-7 and IL-7R are correlated with lung cancer risk in the Chinese Han population.

PURPOSE: IL-7/IL-7R axis participates in the initiation and progression of lung cancer (LC). This study aimed to explore the potential influence of IL-7/IL-7R polymorphisms on LC risk. PATIENTS AND METHODS: In total, 1,010 participants (507 LC patients and 503 healthy controls) were enrolled. Five single-nucleotide polymorphisms (SNPs) in IL-7R and one SNP in IL-7 were genotyped in included samples with Agena MassARRAY system. OR and 95% CIs were computed by logistic regression analysis after adjusting for age and gender. Stratified analyses with demographic and clinical characteristics were also performed. Finally, linkage disequilibrium (LD) analysis was conducted with the PLINK version 1.07 software . RESULTS: IL-7R rs10053847 variant was related to a decreased LC risk under the allele gene (OR =0.78, P=0.043) and additive model (OR =0.77, P=0.042). The results of stratified analysis indicated that this SNP was associated with a lower LC risk among nonsmokers (AA/GG: OR =0.09, P=0.033; AA/AG+GG: OR =0.10 P=0.037) or nondrinkers (AA/GG: OR =0.07, P=0.047; AA/AG+GG: OR =0.18 P=0.049). Moreover, carriers of IL-7R rs10213865-C allele had an increased lung adenocarcinoma risk (CA/AA: OR =1.60, P=0.011; CC+CA/AA: OR =1.62, P=0.007; CA/CA/AA: OR =1.50, P=0.007). Additionally, AGAA haplotype (rs10213865, rs969129, rs118137916 and rs10053847) increased LC risk (OR =1.30, P=0.041). CONCLUSION: IL-7R rs10053847 was correlated with a decreased LC risk, while IL-7R rs10213865 was correlated with an elevated lung adenocarcinoma risk, implying these two SNPs might play essential roles in LC risk evaluation.

Genetic analysis of pharmacogenomic VIP variants in the Blang population from Yunnan Province of China.

BACKGROUND: Genetic polymorphisms in numerous pharmacogenetics studies were regarded as the essential factors involved in the response to or metabolism of drugs. These genetic variants called very important pharmacogenetic (VIP) variants played a role in drugs metabolism, which have been summarized in the PharmGKB database. In this study, we genotyped 80 VIP variants from the PharmGKB in 100 members of Blang volunteers from Yunnan province. METHODS: Based on the PharmGKB database, we genotyped 80 VIP variants loci located in 47 genes. We used χ2 tests to evaluate the significant loci between Blang and the other populations, including ASW, CEU, CHB, CHD, GIH, JPT, LWK, MEX, MKK, TSI, and YRI. The global variation distribution of the significant variants was observed from the ALlele FREquency Database. And then, we used F-statistics (Fst), genetic structure, and phylogenetic tree analyses to ascertain the genetic affinity among 12 populations. RESULTS: Comparing the Blang with the other 11 populations from the HapMap Project, the statistical results revealed that rs3814055 (NC_000003.12:g.119781188C>T) of nuclear receptor subfamily 1 group I member 2 (NR1I2, OMIM# 603,065) was the most significant variant, followed by rs1540339 (NC_000012.12:g.47863543C>T) of vitamin D receptor (VDR, OMIM#601,769). Furthermore, we found that genotype frequency of rs3814055 in the Blang was closer to the populations distributed in Miao. And genetic structure and F-statistics indicated that the Blangs had a relatively closer affinity with CHD, CHB, and JPT populations. In addition, the Han nationality in Shaanxi was closer to it. CONCLUSIONS: Our results will complement the pharmacogenomics information of the Blang ethnic group and provide a theoretical basis for safer drug administration for Blang.

Population genetic difference of pharmacogenomic VIP gene variants in the Lisu population from Yunnan Province.

Individual differences in drug clinical response are related to pharmacogenomics. The genetic variation of drug-metabolizing enzymes, drug receptors, and their downstream protein genes is the main factor causing individual differences in drug response. The genetic backgrounds among different ethnic groups are quite different. In this study, we aimed to detect the distribution difference of genotype frequency in very important pharmacogenetic (VIP) gene variants in the Lisu.Using the chi-squared test, we compared the genotype frequencies of the VIP variants in 105 Lisu people with those in 26 populations from the 1000 Genome project separately. Bonferroni's multiple adjustment was also conducted (P < .05/(26*49)). Moreover, Arlequin v3.5 and Structure v2.3.4 software were used to analyze the genetic distance and genetic structure.There were 9, 9, 11, 12, 11, 11, 9, 17, 13, 13, 16, 5, 3, 5, 3, 4, 17, 14, 16, 17, 16, 10, 13, 12, 10, and 9 single nucleotide polymorphisms that differed in frequency distribution, when Lisu people compared with the 26 populations separately. Only CYP2E1 rs2070676 was different in the Lisu population compared with the 26 groups from the 1000 Genome project. PTGS2 rs5275 and CYP2D6 rs1065852 were different in the Lisu population compared with most of the populations. Additionally, genetic backgrounds of Lisu and Han Chinese in Beijing were closest according to the lowest F-statistics value and resemblance in genetic structures.Our results complete the information of the Lisu population in pharmacogenomics database.

Therapy of Dredging the Bowels Enhanced the Neuroprotective Effect of Nourishing Kidney Herbs on Hippocampal Cholinergic System in Alzheimer's Disease Model Rat Induced by Aß 1-42.

BACKGROUND: Therapy of nourishing kidney has been used for treating memory deficits of Alzheimer's disease (AD) for thousands of years based on traditional Chinese medicine. However, we found the therapy of dredging the bowels could alleviate both memory deficits and mental symptoms of AD in clinic. OBJECTIVE: To determine whether the therapy of dredging the bowels could enhance the neuroprotective effect of nourishing kidney herbs for treating AD rats, and to explore the underlying mechanism of the combination of nourishing kidney and dredging the bowels (NKDB) herbs. METHODS: 60 rats were randomly divided into sham-operated group (SOG), model group (MG), nourishing kidney group (NKG), dredging the bowels group (DBG), nourishing kidney and dredging the bowels group (NKDBG), and donepezil hydrochloride group (DHG). The model establishment was performed by injecting Aß 1-42 into the hippocampal CA1 region. Animals received aqueous solution of Chinese herbal medicine or western medicine while SOG received only distilled water. Ability of learning and memory were assessed by Morris water maze. Acetylcholinesterase(AChE) and choline acetyltransferase (ChAT) activity and positive cells in the hippocampus were detected by the biochemical and immunofluorescent assay. RESULTS: All rats were in the same baseline. While after model establishment, ability of learning and memory of MG, NKG, DBG, NKDBG, and DHG were significantly impaired compared with SOG. Whereas after treatment, ability of learning and memory of NKG, DBG, NKDBG, and DHG were significantly improved compared with MG. Additionally, AChE activity of NKG, DBG, and NKDBG was significantly decreased, meanwhile ChAT activity showed an increased tendency. The number of AChE-positive cells and ChAT-positive cells of both NKDBG and DHG were significantly decreased and increased respectively, superior to those when compared with NKG and DBG. What's more, there was no significant difference between NKDBG and DHG. CONCLUSION: Therapy of dredging the bowels could enhance the neuroprotective effect of nourishing kidney herbs by reversing morphological damage of hippocampal cholinergic system. Furthermore, treatment with NKDB herbs could be effectively against AD, providing a practical therapeutic strategy in clinic.

MicroRNA-135a Inhibits Nasopharyngeal Carcinoma Cell Proliferation Through Targeting Interleukin-17.

BACKGROUND/AIMS: The objective of this study was to investigate the potential role of IL-17 in the development of nasopharyngeal carcinoma (NPC) and to screen microRNAs (miRNAs) that potentially target IL-17 in NPC cells. METHODS: Blood was collected from NPC patients and normal subjects, and plasma IL-17 concentration was quantified by enzyme-linked immunosorbent assay. An immortalized normal human nasopharyngeal epithelial cell line, NP69, was treated with or without human IL-17 (15 ng/mL) for various times, and expression of IL-1ß, IL-6, IL-12, and TNF-α mRNA was assessed by real-time reverse transcription PCR. The candidate miRNAs that potentially target IL-17 were predicted by a bioinformatics strategy. The selected miR-135a mimic was transfected into primary NPC cells, and cell proliferation was assessed by MTT assay. RESULTS: The concentration of plasma IL-17 was significantly higher in the NPC patients (92.5 ± 7.3 pg/mL) than in the control subjects (56.8 ± 2.9 pg/mL). In response to IL-17 treatment, the mRNA expression of IL-1ß and IL-6 was significantly upregulated and reached a peak at 12 h, followed by a slight decrease at 24 h, while the mRNA expression of IL-12 and TNF-α was significantly upregulated at 12 h and remained high even at 48 h after exposure to IL-17. Moreover, miR-135a specifically targets IL-17 and was dramatically downregulated in NPC cells compared with NP69 cells. Transfection of exogenous miR-135a mimic resulted in significant suppression of IL-17 secretion and subsequent inhibition of NPC cell proliferation. CONCLUSIONS: Blood IL-17 was significantly higher in NPC patients compared with normal subjects. Expression of miR-135a in the cancer cells isolated from nasopharyngeal tumors was significantly lower than that in NP69 cells, and suppression of IL-17 by miR-135a mimic resulted in significant inhibition of NPC cell proliferation. These findings suggested that downregulation of miR-135a may contribute to the development of NPC via the mechanism of IL-17 stimulation of proinflammatory cytokine expression.

Genetic polymorphisms in ALDH2 are associated with drug addiction in a Chinese Han population.

We investigated the association between single nucleotide polymorphisms (SNPs) in ALDH2, which has been associated with alcohol dependence and several types of diseases, and the risk of drug addiction in a Chinese Han population. In a case-control study that included 692 cases and 700 healthy controls, eight SNPs in ALDH2 were selected and genotyped using the Sequenom MassARRAY platform. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using unconditional logistic regression after adjusting for age and gender. We determined that rs671 is significantly associated with a 1.551-fold increased drug addiction risk (95% CI = 1.263-1.903; p < 0.001). In the genetic model analysis, we found that rs671 is associated with an increased risk of drug addiction under additive, dominant and recessive models (p < 0.001), while rs886205, rs441 and rs4646778 displayed a decreased drug addiction risk under additive and recessive model, respectively (p < 0.05). SNP rs671 remained significant after Bonferroni correction (p<0.00125). Additionally, we observed that haplotype "GTCAC" was associated with increased drug addiction risk (OR = 1.668; 95% CI, 1.328-2.094, p < 0.001); in contrast, "ATCGC" was a protective haplotype for drug addiction risk (OR = 0.444; 95% CI, 0.281-0.704, p < 0.001). Our findings showed that ALDH2 polymorphisms are significantly associated with the risk of drug addiction in the Chinese Han population.

The Semiflexible Polymer Translocation into Laterally Unbounded Region between Two Parallel Flat Membranes.

Using the dynamic Monte Carlo method, we investigate dynamics of semiflexible polymer translocation through a nanopore into laterally unbounded region between two parallel flat membranes with separation R in presence of an electric field inside the pore. The average translocation time τ initially decreases rapidly with increase of R in the range of R < 10 and then almost keeps constant for R ≥ 10, and the decline range increases with increase of dimensionless bending stiffness κ. We mainly study the effect of chain length N, κ and electric field strength E on the translocation process for R = 5. The translocation dynamics is significantly altered in comparison to an unconfined environment. We find τ ~ Nα, where the exponent α increases with increase of E for small κ. α initially increases slowly with increase of E and then keeps constant for moderate κ. α decreases with increase of E for large κ. However, α decreases with increase of κ under various E. In addition, we find τ ~ κß. ß decreases with increase of N under various E. These behaviors are interpreted in terms of the probability distribution of translocation time and the waiting time of an individual monomer segment passing through the pore during translocation.

Fine encapsulation of dual-particle electronic ink by incorporating block copolymer for electrophoretic display application.

The design of the oppositely charged ink particles based on titanium dioxide and carbon black for the monochrome electrophoretic display (EPD) was reported. The white ink particles with acidic surface and black ink particles with basic surface were synthesized and sterically stabilized by long alkyl chains, which were charged oppositely by mixing with basic surfactant (OLOA 1200) and acidic surfactant (Span 80), respectively. The electrophoretic mobility and the Zeta potential were -3.87×10(-10)m(2)V(-1)s(-1) and -25.1 mV for the white ink particles, 3.79×10(-10)m(2)V(-1)s(-1) and 24.6 mV for the black ink particles. In addition, the block copolymer, poly(lauryl methacrylate)-b-poly(2-(dimethylamino)ethyl methacrylate) (PLMA-b-PDMAEMA) synthesized by atom transfer radical polymerization (ATRP), was first incorporated in the modification of the pigments for the fine encapsulation. Then, a stable dual-particle electronic ink with contrast ratio of 120:1 was prepared and encapsulated with the gelatin (GE)/sodium carboxymethylcellulose (NaCMC)/sodium dodecyl sulfate (SDS) microcapsules by complex coacervation method. Finally, the matrix character display prototype driven at a low voltage exhibited excellent performance, the contrast ratio of which was 8:1 at 9 V DC.