Conversion of a normal maize hybrid into a waxy version using in vivo CRISPR/Cas9 targeted mutation activity

Waxy maize is a specialty maize that produces mainly amylopectin starch with special food or industrial values. The objective of this study was to overcome the limitations of wx mutant allele acquisition and breeding efficiency by conversion of parental lines from normal to waxy maize. The intended mutation activity was achieved by in vivo CRISPR/Cas9 machinery involving desired-target mutation of the Wx locus in the ZC01 background,abbreviated as ZC01-DTM^(wx). Triple selection was applied to segregants to obtain high genome background recovery with transgene-free wx mutations. The targeted mutation was identified, yielding six types of mutations among progeny crossed with ZC01-DTM^(wx).The amylopectin contents of the endosperm starch in mutant lines and hybrids averaged94.9%, while those of the wild-type controls were significantly(P < 0.01) lower, with an average of 76.9%. Double selection in transgene-free lines was applied using the Bar strip test and Cas9 PCR screening. The genome background recovery ratios of the lines were determined using genome-wide SNP data. That of lines used as male parents was as high as98.19% and that of lines used as female parents was as high as 86.78%. Conversion hybrids and both parental lines showed agronomic performance similar to that of their wild-type counterparts. This study provides a practical example of the efficient extension of CRISPR/Cas9 targeted mutation to industrial hybrids for transformation of a recalcitrant species.

Therapeutic Activity of Partially Purified Fractions of Emblica officinalis （Syn, Phyllanthus embfica） Dried Fruits against Trypanosoma evansi

Emblica officinalis （E. oJficinalis） dried fruits were evaluated for its antitrypanosomal activity and cytotoxic effects. Vero cell line maintained in DMEM （Dubecco＇s Modified Eagle Medium） and incubated with Trypanosoma evansi for more than 12 h. MPE was added to the Vero cell culture medium at different concentrations （250-1,000 μg/mL） with trypanosomes concentration （1 × 106 trypanosomes/mL in each ELISA plate well） and incubated at appropriate conditions for 72 h. In-vitro cytotoxieity of MPE of E. officinalis was determined on Vero cells at concentrations （（1.56-100 ~tg/mL）. Acute toxicity and in-vivo infectivity tests were done in mice. Obtained MPE ofE. officinalis underwent process of purification via column chromatography, preparative chromatography and HPLC （higher performance liquid chromatography） with bioassay at different strata on Alsever＇s medium. In-vivo assay for trypanocidal activity, MPE and PPFs （partially purified fractions） of E. officinalis with two sets of mice, each mouse was inoculated with 1 × 104/mL oftrypanosomes and treated （48 h post inoculation） at concentrations （12.5, 25, 50, 100 and 200 mg/kg body weight） were administered at dose rate of 100 [tL per mouse via intraperitoneal route （in treating parassitemic mice） to different groups of mice, 6 mice per concentration. HPLC of partially purified fractions ofE. officinalis was carried out with mobile phase ofacetonitdle： water （40：60） in gradient mode. In vitro, MPE induced immobilization and killing of the parasites in concentration-time dependent manner. Significant reduction of trypanosomes counts from concentration of 250μg/mL and complete killing of trypanosomes at 5th hour of observation, which was statistically equivalent to 4th hour of Diminazine Aceturate （Berenil）, standard reference drug used. HPLC of the partially purified fractions revealed two major prominent peaks at retention time of 1-4 min. In vivo, both MPE and PPFs of test material did prolong lives of mice by 6-9 days but could not cure them. At concentration of 2,000 kg/kg body weight of MPE in acute test, all mice survived. For in-vivo infectivity test, mice injected with immobilized trypanosomes developed parasitemia and died while, the other group survived. MPE, PPFs and Diminazine Aceturate were toxic to Vero cells at all concentrations exception of 1.56, 1.56-3.13 and 1.56-6.25 μg/mL, respectively. From this report, PPFs ofE. officinalis dried fruits demonstrated potential pathway for a new development oftrypanocide in near future if additional investigations are put in place.

In vivo antioxidant activity of rabbiteye blueberry(Vaccinium ashei cv.'Brightwell')anthocyanin extracts

Blueberries are rich in phenolic compounds including anthocyanins which are closely related to biological health functions.The purpose of this study was to investigate the antioxidant activity of blueberry anthocyanins extracted from'Brightwell'rabbiteye blueberries in mice.After one week of adaptation,C57BL/6J healthy male mice were divided into different groups that were administered with 100,400,or 800 mg/kg blueberry anthocyanin extract(BAE),and sacrificed at different time points(0.1,0.5,1,2,4,8,or 12 h).The plasma,eyeball,intestine,liver,and adipose tissues were collected to compare their antioxidant activity,including total antioxidant capacity(T-AOC),superoxide dismutase(SOD)activity and glutathione-peroxidase(GSH-PX/GPX)content,and the oxidative stress marker malondialdehyde(MDA)level.The results showed that blueberry anthocyanins had positive concentration-dependent antioxidant activity in vivo.The greater the concentration of BAE,the higher the T-AOC value,but the lower the MDA level.The enzyme activity of SOD,the content of GSH-PX,and messenger RNA(mRNA)levels of Cu,Zn-SOD,Mn-SOD,and GPX all confirmed that BAE played an antioxidant role after digestion in mice by improving their antioxidant defense.The in vivo antioxidant activity of BAE indicated that blueberry anthocyanins could be developed into functional foods or nutraceuticals with the aim of preventing or treating oxidative stress-related diseases.

Chemical and Biological Characterization from Condafia microphylla Fruits, a Native Species of Patagonia Argentina

Condalia microphylla Cav. （Rhamnaceae）, popularly known as ＂piquillin＂, is widely distributed in Patagonia. The drupes are consumed as fresh fruits by Argentine communities. The aim of this work was to quantify the nutritional value of C. microphylla fruit and the phenolic compounds present and to determine the functional antioxidant properties in vitro and in vivo. The nutritional value was determined according to the Association of Official Analytical Chemists （AOAC） methodology, and phenolic compounds were quantified by diode-array detection （HPLC-DAD）. Antioxidant activity in vitro and in vivo was analyzed through the use of the radical species 2,2-diphenyl-l-picrylhydrazyl （DPPH） and zebrafish model, respectively. Quercetin-3-O-rutinoside （rutin） was the single principal phenolic compound. The extracts contained in vitro antioxidant activities and total phenolic contents （TPCs） between 1,143 ~ 112 lag and 4,633 ~ 174 lag gallic-acid equivalents （GAEs） per 100 g dry weight （DW）, though no relationship was found between the latter parameter and the antioxidant activity of the extracts. When zebrafish larvae were exposed to oxidative stress （2.4% v/v H2O2）, a concentration as low as 1.44 lag of GAEs/mL of piquillin-derived polyphenols inhibited lipid oxidation by up to 40%. Thus, in view of these advantageous functional food properties and the opportunity to exploit this Patagonian natural resource, piquillin consumption should be promoted worldwide.

Study on the Effect of Aqueous Extract of Houttuynia cordata on Serum TNF-αand IL-6 Content in Mice Infected with Five Common Pathogenic Bacteria

[Objectives]To observe the effect of aqueous extract of Houttuynia cordata on the content of serum TNF-αand IL-6 in mice with abdominal infection caused by Staphylococcus aureus,Escherichia coli,Pseudomonas aeruginosa,Proteus vulgaris and Klebsiella pneumoniae,so as to investigate the antibacterial effect of aqueous extract of Houttuynia cordata in vivo.[Methods]A total of 72 KM mice were randomly divided into six groups:model group,blank group,cefdinir dispersible tablet group(0.25 g/kg),high,medium and low dose groups of aqueous extract of Houttuynia cordata(40,20,10 g/kg crude drug).There were 12 mice in each group,half male and half female,continuously given intragastric administration for three days,once a day.The blank group and the model group were intragastrically infused with normal saline,and the other groups were given corresponding drugs with a volume of 0.02 mL/g.After 30 min of administration on the third day,the mice were injected intraperitoneally with 0.5 mL of bacterial suspension to make bacterial infection.The mice in the blank group were intraperitoneally injected with the same amount of normal saline,followed by intragastric administration 6 h after intraperitoneal injection and intragastric administration again after 24 h.The mice were observed for three days,and the changes of body weight before and after infection were recorded.On the fourth day of infection,eyeball blood was taken,serum was separated,and the concentration of inflammatory factors TNF-αand IL-6 in serum was determined.[Results]Compared with the model group,the high,medium and low dose groups of H.cordata aqueous extract had protective effects on mice with abdominal infection caused by S.aureus,E.coli,P.aeruginosa,P.vulgaris and K.pneumoniae,and decreased the content of TNF-αand IL-6 in serum.[Conclusions]The aqueous extract of H.cordata had a certain antibacterial effect on acute abdominal infection caused by S.aureus,E.coli,P.aeruginosa,P.vulgaris and K.pneumoniae,and its mechanism was related to TNF-αand IL-6.

Antioxidant Activities of Crude Phlorotannins from Sargassum hemiphyllum

Brown algae are well known as a source of biologically active compounds, especially those having antioxidant activities, such as phlorotannins. In this study we examined the antioxidant activities of crude phlorotannins extracts（CPEs） obtained from Sargassum hemiphyllum（SH） and fractionated according to the molecular weights. When CPEs were administrated at a dose of 30 mg/kg to Kunming mice pre-treated with carbon tetrachloride（CCl4）, the levels of oxidative stress indicators in the liver, kidney and brain were significantly reduced in vivo. All the components of various molecular weight fractions of CPEs exhibited greater scavenging capacities in clearing hydroxyl free radical and superoxide anion than the positive controls gallic acid, vitamin C and vitamin E. Particularly, the components greater than 30 k D obtained from ethyl acetate phase showed the highest antioxidant capacities. These results indicated that SH is a potential source for extracting phlorotannins, the algal antioxidant compounds.

Anthelmintic, antimicrobial, antioxidant and cytotoxic activity of Caltha palustris var. alba Kashmir, India

The methanolic extract obtained from the root portion of Caltha palustris var. alba was evaluated for its anthelmintic efficacy against gastrointestinal nematodes of sheep under both in vitro and in vivo conditions using worm motility inhibition(WMI) assay and fecal egg count reduction(FECR) assay, respectively. The extract was subjected to antimicrobial activity using agar-well diffusion method against different bacterial strains. In addition the extract was evaluated for cytotoxic and antioxidant activity against cultured THP-1(Leukemia), A-549(Lung), HCT-15(Colon), Cervix(HeLa) and PC-3(Prostrate) cell lines by SRB and DPPH radical scavenging assays. The extract used resulted in mean %WMI of 94.44%, as observed when the worms were put in lukewarm buffer for 30 min after exposure to different treatments. The mean mortality index of the sample was 0.95. The lethal concentration(LC50) was 0.11 mg·mL-1. Cell lines were exposed to concentration of 100 μg·mL-1 of extract for 48 h, which reduced the viability of these cell lines. The same plant extract also showed 55.58% DPPH radical scavenging activity.

Homo-PROTAC mediated suicide of MDM2 to treat non-small cell lung cancer

The dose-related adverse effects of MDM2-P5 3 inhibitors have caused significant concern in the development of clinical safe anticancer agents.Herein we report an unprecedented homo-PROTAC strategy for more effective disruption of MDM2-P53 interaction.The design concept is inspired by the capacity of sub-stoichiometric catalytic PROTACs enabling to degrade an unwanted protein and the dual functions of MDM2 as an E3 ubiquitin ligase and a binding protein with tumor suppressor P53.The new homo-PROTACs are designed to induce self-degradation of MDM2.The results of the investigation have shown that PROTAC 11 a efficiently dimerizes MDM2 with highly competitive binding activity and induces proteasome-dependent self-degradation of MDM2 in A549 non-small cell lung cancer cells.Furthermore,markedly,enantiomer 11 a-1 exhibits potent in vivo antitumor activity in A549 xenograft nude mouse model,which is the first example of homo-PROTAC with in vivo therapeutic potency.This study demonstrates the potential of the homo-PROTAC as an alternative chemical tool for tumorigenic MDM2 knockdown,which could be developed into a safe therapy for cancer treatment.