Letter to the Editor

Regarding Paper “Stratified Fisher's exact test and its sample size calculation” by Sin‐Ho Jung Biometrical Journal (2014) 56 (1): 129–140 Article: http://dx.doi.org/10.1002/bimj.201300048     

Germline recombination in a novel Cre transgenic line,Prl3b1‐Cre mouse

Spermatogenesis is a complex and highly regulated process by which spermatogonial stem cells differentiate into spermatozoa. To better understand the molecular mechanisms of the process, the Cre/loxP system has been widely utilized for conditional gene knockout in mice. In this study, we generated a transgenic mouse line that expresses Cre recombinase under the control of the 2.5 kbp of the Prolactin family 3, subfamily b, member 1 (Prl3b1) gene promoter (Prl3b1‐cre). Prl3b1 was initially reported to code for placental lactogen 2 (PL‐2) protein in placenta along with increased expression toward the end of pregnancy. PL‐2 was found to be expressed in germ cells in the testis, especially in spermatocytes. To analyze the specificity and efficiency of Cre recombinase activity in Prl3b1‐cre mice, the mice were mated with reporter R26GRR mice, which express GFP ubiquitously before and tdsRed exclusively after Cre recombination. The systemic examination of Prl3b1‐cre;R26GRR mice revealed that tdsRed‐positive cells were detected only in the testis and epididymis. Fluorescence imaging of Prl3b1‐cre;R26GRR testes suggested that Cre‐mediated recombination took place in the germ cells with approximately 74% efficiency determined by in vitro fertilization. In conclusion, our results suggest that the Prl3b1‐cre mice line provides a unique resource to understand testicular germ‐cell development. genesis 54:389–397, 2016. © 2016 Wiley Periodicals, Inc.     

Enantiomeric 4‐Acylamino‐6‐alkyloxy‐2 Alkylthiopyrimidines As Potential A3 Adenosine Receptor Antagonists: HPLC Chiral Resolution and Absolute Configuration Assignment by a Full Set of Chiroptical Spectroscopy

The chiral separation of enantiomeric couples of three potential A₃ adenosine receptor antagonists: (R/S)‐N‐(6‐(1‐phenylethoxy)‐2‐(propylthio)pyrimidin‐4‐yl)acetamide ( 1 ), (R/S)‐N‐(2‐(1‐phenylethylthio)‐6‐propoxypyrimidin‐4‐yl)acetamide ( 2 ), and (R/S)‐N‐(2‐(benzylthio)‐6‐sec‐butoxypyrimidin‐4‐yl)acetamide ( 3 ) was achieved by high‐performance liquid chromatography (HPLC). Three types of chiroptical spectroscopies, namely, optical rotatory dispersion (ORD), electronic circular dichroism (ECD), and vibrational circular dichroism (VCD), were applied to enantiomeric compounds. Through comparison with Density Functional Theory (DFT) calculations, encompassing extensive conformational analysis, full assignment of the absolute configuration (AC) for the three sets of compounds was obtained. Chirality 28:434–440, 2016. © 2016 Wiley Periodicals, Inc.     

Generation of an estrogen receptor beta‐iCre knock‐in mouse

A novel knock‐in mouse that expresses codon‐improved Cre recombinase (iCre) under regulation of the estrogen receptor beta (Esr2) promoter was developed for conditional deletion of genes and for the spatial and/or temporal localization of Esr2 expression. ESR2 is one of two classical nuclear estrogen receptors and displays a spatiotemporal expression pattern and functions that are different from the other estrogen receptor, ESR1. A cassette was constructed that contained iCre, a polyadenylation sequence, and a neomycin selection marker. This construct was used to insert iCre in front of the endogenous start codon of the Esr2 gene of a C57BL/6J embryonic stem cell line via homologous recombination. Resulting Esr2‐iCre mice were bred with ROSA26‐lacZ and Ai9‐RFP reporter mice to visualize cells of functional iCre expression. Strong expression was observed in the ovary, the pituitary, the interstitium of the testes, the head and tail but not body of the epididymis, skeletal muscle, the coagulation gland (anterior prostate), the lung, and the preputial gland. Additional diffuse or patchy expression was observed in the cerebrum, the hypothalamus, the heart, the adrenal gland, the colon, the bladder, and the pads of the paws. Overall, Esr2‐iCre mice will serve as a novel line for conditionally ablating genes in Esr2‐expressing tissues, identifying novel Esr2‐expressing cells, and differentiating the functions of ESR2 and ESR1. genesis 54:38–52, 2016. © 2016 Wiley Periodicals, Inc.     

Identification of N‐octylnortadalafil and its Stereoisomers in Dietary Supplements with Chiral Liquid Chromatography–Circular Dichroism

A direct chiral liquid chromatography–circular dichroism (LC‐CD) method was developed for the simple and rapid identification of N‐octylnortadalafil [(6R, 12aR)‐6‐(1,3‐benzodioxol‐5‐yl)‐2‐octyl‐2,3,6,7,12,12a‐hexahydropyrazino[1’,2’:1,6]pyrido[3,4‐b]indole‐1,4‐dione; RR‐OTDF] and its stereoisomers in dietary supplements. Samples were extracted with methanol. Compounds were then separated by chiral LC‐CD using Chiralcel OD‐RH (4.6 × 1 50 mm, 5 µm) with 5 mM ammonium formate (pH 3)/0.1% formic acid in acetonitrile (95:5, v/v) mixture solution (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). The isocratic elution used was mobile phase A / mobile phase B (3:7, v/v) at a flow rate of 1 ml/min. The column temperature was held at 30°C. RR‐OTDF and its stereoisomers were separated within 20 min with the resolution factors being over 2.0. Using this method, RR‐OTDF and (6R, 12aS)‐6‐(1,3‐benzodioxol‐5‐yl)‐2‐octyl‐2,3,6,7,12,12a‐hexahydropyrazino[1’,2’:1,6]pyrido[3,4‐b]indole‐1,4‐dione were detected in a dietary supplement. Chirality 28:204–208, 2016. © 2016 Wiley Periodicals, Inc.     

Identification of active constraints in dynamic flux balance analysis

This study deals with the calibration of dynamic metabolic flux models that are formulated as the maximization of an objective subject to constraints. Two approaches were applied for identifying the constraints from data. In the first approach a minimal active number of limiting constraints is found based on data that are assumed to be bounded within sets whereas, in the second approach, the limiting constraints are found based on parametric sensitivity analysis. The ability of these approaches to finding the active limiting constraints was verified through their application to two case studies: an in‐silico (simulated) data‐based study describing the growth of E. coli and an experimental data‐based study for Bordetella pertussis (B. pertussis). © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:26–36, 2017     

Simple Isocratic HPLC Method for Determination of Enantiomeric Impurity in Besifloxacin Hydrochloride

Besifloxacin is a unique chiral broad‐spectrum flouroquinolone used in the treatment of bacterial conjunctivitis. R‐form of besifloxacin hydrochloride shows higher antibacterial activity as compared to the S‐isomer. Therefore, it is necessary to establish chiral purity. To establish chiral purity a high‐performance liquid chromatography (HPLC) method for determination of R‐besifloxacin and S‐besifloxacin (BES impurity A) was developed and validated for in‐process quality control and stability studies. The analytical performance parameters such as linearity, precision, accuracy, specificity, limit of detection (LOD), and lower limit of quantification (LOQ) were determined according to International Council for Harmonization ICH Q2(R1) guidelines. HPLC separation was achieved on Chiralpak AD‐H (250 x 4.6 mm, 5 μm) column using n‐heptane: ethanol: ethylenediamine: acetic acid (800:200:0.5:0.5) (v/v/v/v) as the mobile phase in an isocratic elution. The eluents were monitored by UV/Visible detector at 290 nm. The resolution between S‐isomer and besifloxacin hydrochloride was more than 2.0. Based on a signal‐to‐noise ratio of 3 and 10 the LOD of besifloxacin was 0.30 μg/mL, while the LOQ was 0.90 μg/mL. The calibration curves were linear in the range of 0.9–7.5 μg/mL. Precision of the method was established within the acceptable range. The method was suitable for the quality control enantiomeric impurity in besifloxacin hydrochloride. Chirality 28:628–632, 2016. © 2016 Wiley Periodicals, Inc.     

Pdgfrb‐Cre targets lymphatic endothelial cells of both venous and non‐venous origins

The Pdgfrb‐Cre line has been used as a tool to specifically target pericytes and vascular smooth muscle cells. Recent studies showed additional targeting of cardiac and mesenteric lymphatic endothelial cells (LECs) by the Pdgfrb‐Cre transgene. In the heart, this was suggested to provide evidence for a previously unknown nonvenous source of LECs originating from yolk sac (YS) hemogenic endothelium (HemEC). Here we show that Pdgfrb‐Cre does not, however, target YS HemEC or YS‐derived erythro‐myeloid progenitors (EMPs). Instead, a high proportion of ECs in embryonic blood vessels of multiple organs, as well as venous‐derived LECs were targeted. Assessment of temporal Cre activity using the R26‐mTmG double reporter suggested recent occurrence of Pdgfrb‐Cre recombination in both blood and lymphatic ECs. It thus cannot be excluded that Pdgfrb‐Cre mediated targeting of LECs is due to de novo expression of the Pdgfrb‐Cre transgene or their previously established venous endothelial origin. Importantly, Pdgfrb‐Cre targeting of LECs does not provide evidence for YS HemEC origin of the lymphatic vasculature. Our results highlight the need for careful interpretation of lineage tracing using constitutive Cre lines that cannot discriminate active from historical expression. The early vascular targeting by the Pdgfrb‐Cre also warrants consideration for its use in studies of mural cells. genesis 54:350–358, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc.     

A possible aid in targeted insertion of large DNA elements by CRISPR/Cas in mouse zygotes

The CRISPR/Cas system has rapidly emerged recently as a new tool for genome engineering, and is expected to allow for controlled manipulation of specific genomic elements in a variety of species. A number of recent studies have reported the use of CRISPR/Cas for gene disruption (knockout) or targeted insertion of foreign DNA elements (knock‐in). Despite the ease of simple gene knockout and small insertions or nucleotide substitutions in mouse zygotes by the CRISPR/Cas system, targeted insertion of large DNA elements remains an apparent challenge. Here the generation of knock‐in mice with successful targeted insertion of large donor DNA elements ranged from 3.0 to 7.1 kb at the ROSA26 locus using the CRISPR/Cas system was achieved. Multiple independent knock‐in founder mice were obtained by injection of hCas9 mRNA/sgRNA/donor vector mixtures into the cytoplasm of C57BL/6N zygotes when the injected zygotes were treated with an inhibitor of actin polymerization, cytochalasin. Successful germ line transmission of three of these knock‐in alleles was also confirmed. The results suggested that treatment of zygotes with actin polymerization inhibitors following microinjection could be a viable method to facilitate targeted insertion of large DNA elements by the CRISPR/Cas system, enabling targeted knock‐in readily attainable in zygotes. genesis 54:65–77, 2016. © 2016 Wiley Periodicals, Inc.     

Validation of a Chiral Liquid Chromatographic Method for the Degradation Behavior of Flumequine Enantiomers in Mariculture Pond Water

In this work, flumequine (FLU) enantiomers were separated using a Chiralpak OD‐H column, with n‐hexane‐ethanol (20:80, v/v) as the mobile phase at a flow rate of 0.6 mL/min. Solid phase extraction (SPE) was used for cleanup and enrichment. The limit of detection, limit of quantitation, linearity, precision, and intra/interday variation of the chiral high‐performance liquid chromatography (HPLC) method were determined. The developed method was then applied to investigate the degradation behavior of FLU enantiomers in mariculture pond water samples. The results showed that the degradation of FLU enantiomers under natural, sterile, or dark conditions was not enantioselective. Chirality 28:649–655, 2016. © 2016 Wiley Periodicals, Inc.     

Synthesis and Application of C2 and C3 Symmetric (R)‐Phenylglycinol‐Derived Chiral Stationary Phases

A C3 symmetric (R)‐phenylglycinol N‐1,3,5‐benzenetricarboxylic acid‐derived chiral stationary phase (CSP) and three C2 symmetric (R)‐phenylglycinol CSPs were newly synthesized using o‐, m‐, and p‐phthaloyl dichlorides. © 2016 Wiley Periodicals, Inc. These CSPs were used to compare the resolution of 25 chiral samples using a previously reported 3,5‐dinitrobenzoyl (R)‐phenylglycinol‐derived CSP. Even though all CSPs have the same chiral moiety, the C3 symmetric CSP showed the best resolution. Chirality 28:186–191, 2016.© 2016 Wiley Periodicals, Inc.     

Enantiomeric Neolignans and a Sesquiterpene from Solanum erianthum and Their Absolute Configuration Assignment

One pair of new C‐8–C‐3′/C‐7–O–C‐4′ linked neolignan enantiomers ( 1a / 1b ) and one new guaiane sesquiterpene ( 2 ) first featuring the 1(2),9(10)‐conjugated double bond were isolated from the stems of Solanum erianthum (Solanceae). Their structures were characterized on the basis of extensive spectroscopic analyses, especially from their 2D nuclear magnetic resonance (NMR) spectra. The absolute configurations of 1a / 1b were rigorously elucidated by electronic circular dichroism (ECD) experiments combined with the reversed helicity rule for the 2,3‐dihydrobenzo[b]furan chromophore, and compound 2 is the first report on the sterochemical assignment of a guaiane sesquiterpene by using the allylic axial chirality rule for the conjugated diene chromophore in combination with the calculated ECD spectrum. Chirality 28:259–263, 2016. © 2016 Wiley Periodicals, Inc.     

Anticonvulsant Activity of Enantiomeric N‐trans‐Cinnamoyl Derivatives of 2‐Aminopropan‐1‐ols and 2‐Aminobutan‐1‐ols

Epilepsy, one of the most frequent neurological disorders, is still insufficiently treated in about 30% of patients. As a consequence, identification of novel anticonvulsant agents is an important issue in medicinal chemistry. In the present article we report synthesis, physicochemical, and pharmacological evaluation of N‐trans‐cinnamoyl derivatives of R and S‐2‐aminopropan‐1‐ol, as well as R and S‐2‐aminobutan‐1‐ol. The structures were confirmed by spectroscopy and for derivatives of 2‐aminopropan‐1‐ols the configuration was evaluated by means of crystallography. The investigated compounds were tested in rodent models of seizures: maximal electroshock (MES) and subcutaneous pentetrazol test (scPTZ), and also in a rodent model of epileptogenesis: pilocarpine‐induced status prevention. Additionally, derivatives of 2‐aminopropan‐1‐ols were tested in benzodiazepine‐resistant electrographic status epilepticus rat model as well as in vitro for inhibition of isoenzymes of cytochrome P450. All of the tested compounds showed promising anticonvulsant activity in MES. For R(–)‐(2E)‐N‐(1‐hydroxypropan‐2‐yl)‐3‐phenylprop‐2‐enamide pharmacological parameters were found as follows: ED₅₀ = 76.7 (68.2–81.3) mg/kg (MES, mice i.p., time = 0.5 h), ED₅₀ = 127.2 (102.1–157.9) mg/kg (scPTZ, mice i.p., time = 0.25 h), TD₅₀ = 208.3 (151.4–230.6) mg/kg (rotarod, mice i.p., time = 0.25 h). Evaluation in pilocarpine status prevention proved that all of the reported compounds reduced spontaneous seizure activity and act as antiepileptogenic agents. Both enantiomers of 2‐aminopropan‐1‐ols did not influence cytochrome P450 isoenzymes activity in vitro and are likely not to interact with CYP substrates in vivo. Chirality 28:482–488, 2016. © 2016 Wiley Periodicals, Inc.     

Cooperative Effects Between Arginine and Glutamic Acid in the Amino Acid‐Catalyzed Aldol Reaction

Catalysis of the aldol reaction between cyclohexanone and 4‐nitrobenzaldehyde by mixtures of L‐Arg and of L‐Glu in wet dimethyl sulfoxide (DMSO) takes place with higher enantioselectivity (up to a 7‐fold enhancement in the anti‐aldol for the 1:1 mixture) than that observed when either L‐Glu or L‐Arg alone are used as the catalysts. These results can be explained by the formation of a catalytically active hydrogen‐bonded complex between both amino acids, and demonstrate the possibility of positive cooperative effects in catalysis by two different α‐amino acids. Chirality 28:599–605, 2016. © 2016 Wiley Periodicals, Inc.     

Enantioselective Degradation and Chiral Stability of Metalaxyl‐M in Tomato Fruits

Metalaxyl is an important chiral acetanilide fungicide, and the activity almost entirely originates from the R‐enantiomer. Racemic metalaxyl has been gradually replaced by the enantiopure R‐enantiomer (metalaxyl‐M). In this study a chiral residue analysis method for metalaxyl and the metabolite metalaxyl acid was set up based on high‐performance liquid chromatography tandem mass spectroscopy (HPLC‐MS/MS). The enantioselective degradation and chiral stability of metalaxyl‐M in tomato fruits in two geographically distinct regions of China (Heilongjiang and Hunan Province) were evaluated and the enantioselectivity of metalaxyl acid was also investigated. Tomato plants grew under field conditions with a one‐time spray application of metalaxyl‐M wettable powder. It was found that R‐metalaxyl was not chirally stable and the inactive S‐metalaxyl was detected in tomato fruits. At day 40, S‐metalaxyl derived from R‐metalaxyl accounted for 32% and 26% of the total amount of metalaxyl, respectively. The metabolites R‐metalaxyl acid and S‐metalaxyl acid were both observed in tomato, and the ratio of S‐metalaxyl acid to the sum of S‐ and R‐metalaxyl acid was 36% and 28% at day 40, respectively. For both metalaxyl and metalaxyl acid, the half‐life of the S‐enantiomer was longer than the R‐enantiomer. The results indicated that the enantiomeric conversion should be considered in the bioactivity evaluation and environmental pollution assessment. Chirality 28:382–386, 2016. © 2016 Wiley Periodicals, Inc.     

Chiral HPLC Separation and Modeling of Four Stereomers of DL‐Leucine‐DL‐Tryptophan Dipeptide on Amylose Chiral Column

Chiral high‐performance liquid chromatography (HPLC) separation and modeling of four stereomers of DL‐leucine‐tryptophan DL‐dipeptide on AmyCoat‐RP column are described. The mobile phase applied was ammonium acetate (10 mM)‐methanol‐acetonitrile (50:5:45, v/v). The flow rate of the mobile phases was 0.8 mL/min with UV detection at 230 nm. The values of retention factors for LL‐, DD‐, DL‐, and LD‐ stereomers were 2.25, 3.60, 5.00, and 6.50, respectively. The values of separation and resolution factors were 1.60, 1.39, and 1.30 and 7.76, 8.05, and 7.19. The limits of detection and quantitation were ranging from 1.0–2.3 and 5.6–14.0 μg/mL. The simulation studies established the elution orders and the mechanism of chiral recognition. It was seen that π–π connections and hydrogen bondings were the main forces for enantiomeric resolution. The reported chiral HPLC method may be applied for the enantiomeric separation of DL‐leucine‐DL‐tryptophan in unknown matrices. Chirality 28:642–648, 2016. © 2016 Wiley Periodicals, Inc.     

A multifunctional reporter mouse line for Cre‐ and FLP‐dependent lineage analysis

The Cre/lox and FLP/FRT recombination systems have been used extensively for both conditional knockout and cell lineage analysis in mice. Here we report a new multifunctional Cre/FLP dual reporter allele (R26^NZG) that exhibits strong and apparently ubiquitous marker expression in embryos and adults. The reporter construct, which is driven by the CAG promoter, was knocked into the ROSA26 locus providing an open chromatin domain for consistent expression and avoiding site‐of‐integration effects often observed with transgenic reporters. R26^NZG directs Cre‐dependent nuclear‐localized β‐galactosidase (β‐gal) expression, and can be converted into a Cre‐dependent EGFP reporter (R26^NG) by germline excision of the FRT‐flanked nlslacZ cassette. Alternatively, germline excision of the floxed PGKNEO cassette in R26^NZG generates an FLP‐dependent EGFP reporter (R26^ZG) that expresses β‐gal in FLP‐nonexpressing cells. Finally, by the simultaneous use of both Cre and FLP deleters, R26^NZG allows lineage relationships to be interrogated with greater refinement than is possible with single recombinase reporter systems. genesis 47:107–114, 2009. © 2009 Wiley‐Liss, Inc.     

Isolation,Stereochemical Study,and Antioxidant Activity of Benzofuranone Derivatives from a Mangrove‐derived Fungus Eurotium rubrum MA‐150

Enantiomers of a 2‐benzofuran‐1(3H)‐one derivative [(–)‐ 1 and (+)‐ 1 ] and four known analogs ( 2 , 3 , 4 , 5 ) were isolated and identified from the culture extract of Eurotium rubrum MA–150, a fungus obtained from the mangrove‐derived rizospheric soil. Their structures were established by detailed interpretation of nuclear magnetic resonance (NMR) data and the structure of (±)‐ 1 was confirmed by single‐crystal X‐ray diffraction analysis. The absolute configuration of the enantiomers (–)‐ 1 and (+)‐ 1 was determined by means of online high‐performance liquid chromatography – electronic circular dichroism (HPLC‐ECD) measurements and time‐dependent Density Functional Theory – electronic circular dichroism (TDDFT‐ECD) calculations. Compounds (±)‐ 1 as well as 2 and 3 exhibited potent DPPH radical scavenging activities with IC₅₀ values of 1.23, 2.26, and 3.99 μg/mL, respectively. Chirality 28:581–584, 2016. © 2016 Wiley Periodicals, Inc.     

Chemoenzymatic Route for the Synthesis of (S)‐Moprolol,a Potential β‐Blocker

A biocatalytic route for the synthesis of a potential β‐blocker, (S)‐moprolol is reported here. Enantiopure synthesis of moprolol is mainly dependent on the chiral intermediate, 3‐(2‐methoxyphenoxy)‐propane‐1,2‐diol. Various commercial lipases were screened for the enantioselective resolution of (RS)‐3‐(2‐methoxyphenoxy)propane‐1,2‐diol to produce the desired enantiomer. Among them, Aspergillus niger lipase (ANL) was selected on the basis of both stereo‐ and regioselectivity. The optimized values of various reaction parameters were determined such as enzyme (15 mg/mL), substrate concentration (10 mM), organic solvent (toluene), reaction temperature (30 °C), and time (18 h).The optimized conditions led to achieving >49% yield with high enantiomeric excess of (S)‐3‐(2‐methoxyphenoxy)propane‐1,2‐diol. The lipase‐mediated catalysis showed regioselective acylation with dual stereoselectivity. Further, the enantiopure intermediate was used for the synthesis of (S)‐moprolol, which afforded the desired β‐blocker. Chirality 28:313–318, 2016. © 2016 Wiley Periodicals, Inc.