Generation of double knockout cattle via CRISPR-Cas9 ribonucleoprotein(RNP)electroporation

Background Genome editing has been considered as powerful tool in agricultural fields.However,genome editing progress in cattle has not been fast as in other mammal species,for some disadvantages including long gestational periods,single pregnancy,and high raising cost.Furthermore,technically demanding methods such as microinjection and somatic cell nuclear transfer(SCNT)are needed for gene editing in cattle.In this point of view,electroporation in embryos has been risen as an alternative.Results First,editing efficiency of our electroporation methods were tested for embryos.Presence of mutation on embryo was confirmed by T7E1 assay.With first combination,mutation rates for MSTN and PRNP were 57.6%±13.7%and 54.6%±13.5%,respectively.In case of MSTN/BLG,mutation rates were 83.9%±23.6%for MSTN,84.5%±18.0%for BLG.Afterwards,the double-KO embryos were transferred to surrogates and mutation rate was identified in resultant calves by targeted deep sequencing.Thirteen recipients were transferred for MSTN/PRNP,4 calves were delivered,and one calf underwent an induction for double KO.Ten surrogates were given double-KO embryos for MSTN/BLG,and four of the six calves that were born had mutations in both genes.Conclusions These data demonstrated that production of genome edited cattle via electroporation of RNP could be effectively applied.Finally,MSTN and PRNP from beef cattle and MSTN and BLG from dairy cattle have been born and they will be valuable resources for future precision breeding.

Development of genome engineering technologies in cattle: from random to specific

The production of transgenic farm animals(e.g., cattle) via genome engineering for the gain or loss of gene functions is an important undertaking. In the initial stages of genome engineering, DNA micro-injection into one-cell stage embryos(zygotes) followed by embryo transfer into a recipient was performed because of the ease of the procedure.However, as this approach resulted in severe mosaicism and has a low efficiency, it is not typically employed in the cattle as priority, unlike in mice. To overcome the above issue with micro-injection in cattle, somatic cell nuclear transfer(SCNT) was introduced and successfully used to produce cloned livestock. The application of SCNT for the production of transgenic livestock represents a significant advancement, but its development speed is relatively slow because of abnormal reprogramming and low gene targeting efficiency. Recent genome editing technologies(e.g.,ZFN, TALEN, and CRISPR-Cas9) have been rapidly adapted for applications in cattle and great results have been achieved in several fields such as disease models and bioreactors. In the future, genome engineering technologies wil accelerate our understanding of genetic traits in bovine and wil be readily adapted for bio-medical applications in cattle.

Additive potential of ginger starch on antifungal potency of honey against Candida albicans

Objective:To evaluate the additive action of ginger starch on the antifungal activity of honey against Candida albicans(C.albicans).Methods:C.albicans was used to determine the minimum inhibitory concentration(MIC)of four varieties of Algerian honey.Lower concentrations of honey than the MIC were incubated with a set of concentrations of starch and then added to media to detennine the minimum additive inhibitory concentration(MAIC).Results:The MIC for the four varieties of honey without starch against C.albicans ranged between 38%and 42%(v/v).When starch was incubated with honey and then added to media,a MIC drop was noticed with each variety.MAIC of the four varieties ranged between 32%honey(v/v)with 4%starch and 36%honey(v/v) with 2%starch.Conclusions:The use of ginger starch allows honey benefit and will constitute an alternative way against the resistance to antifungal agents.

Antifungal activity of four honeys of different types from Algeria against pathogenic yeast:Candida albicans and Rhodotorula sp.

Objective:To evaluate the antifungal activity of four honeys of different types from Algeria against pathogenic yeast i.e.Candida albicans(C.albicans) and Rhoddtorula sp.Methods:Four Algeria honeys of different botanical origin were analvzed to test anlilungal effect against C.albicans,and Rhodotorula sp.Different concentrations(undiluted,10%,30%,50%and 70%w/v) of honey were studied in vitro for their antifugal aclivity using C.albicans and Rhodotorula sp.as fungal strains.Results:The range of the c liameter of zone of inhibition of various concentrations ol tested honeys was(7-23 mm) for Rhodotorula sp.,while C.albicans showed clearly resistance towards all concentrations used.The MICs of tested honey concentrations against C.albicans and Rhodotorula sp.were(70.09-93.48)%and(4.90-99.70)%v/v,respectively.Conclusions:This study demonstrales that,in vitro,these natural products have clearly an antifungal activity against Rhodotorula sp.and C.albicans.

Role of inflammasome regulation on immune modulators

Inflammatory responses are essential in eliminating harmful substrates from damaged tissue and inducing recovery.Several cytokines participate in and facilitate this response. Certain cytokines such as interleukin(IL)-1β and IL-18 are initially produced in precursor form in response to toll-like receptor(TLR) ligands and undergo maturation by inflammasomes, which are cytosolic multi-protein complexes containing nucleotide-binding oligomerization domain(NOD)-containing protein 2-like receptors(NLRs). Immune modulators targeting inflammasomes have been investigated to control inflammatory diseases such as metabolic syndrome. However, most immune modulators possessing anti-inflammasome properties attenuate production of other cytokines, which are essential for host defense. In this review, we analyzed the effect of anti-inflammasome agents on the production of cytokines which are not regulated by inflammasome and involving in initial immune responses. As a result, the infiammasome inhibitors are put into three categories: non-effector, stimulator, or inhibitor of cytokine production. Even the stimulator of cytokine production ameliorated symptoms resulting from inflammasome activation in mouse models. Thus, we suggest ideal immune modulators targeting inflammasomes in order to enhance cytokine production while inhibiting cytokine maturation.

Antibacterial activity of various honey types of Algeria against Staphylococcus aureus and Streptococcus pyogenes

Objective:To assess the in vitro antibacterial activity of honey from different geographical location on Gram negative organismes.Methods:Different concentrations(Undiluted honey,10%, 30%,50%and 70%wt/vol) of honey were studied in vitro using Staphylococcus aureus(S.aureus) and Streptococcus pyogenes(S.pyogenes),briefly,two-fold dilutions of honey solutions were tested to determine the minimum inhibitory concentration(MIC) against each type of microorganism,followed by more assays within a narrower dilution range to obtain more precise MIC values.MICs were determined by both visual inspection and spectrophotometric assay at 620 nm.These honey samples were compared with standard antibiotics like ampicillin,penicillin G,amoxicillin,gentamycin,tobramycin,erythromycin and chloramphenicol was determined by the disc diffusion method.Results:The diameter of zone of the inhibition(ZDI) of honey has various concentrations tested for the isolates ranged 0-46 mm for S.aureus,0-44 mm for S.pyogenes.While the MIC(%) ranged 12%-95%,25%-73%respectively.Conclusions:Algeria honey,in-vitro,possess antibacterial activity.

Intra-articular transplantation of porcine adipose-derived stem cells for the treatment of canine osteoarthritis: A pilot study

AIM: To test whether intra-articular injection of porcine adipose-derived stem cells(ADSCs) can treat canine osteoarthritis(OA).METHODS: To enroll in this study dogs must have stifle joint OA that had lasted ≥ 3 mo and been treated with OA medication without significant improvement. Three dogs fulfilled these criteria and were thus subjects for ADSCs treatment. ADSCs were isolated from abdominal adipose tissue of a 2-mo-old female Yorkshire pig. Their stem cell marker expression was examined by immunofluorescence staining. For treatment, 5 million ADSCs were injected into the diseased joint of each dog. In the next 48 h, the patient was observed for signs of inflammatory and allergic reactions. Thepatient was then discharged to the owner and, at 2, 6, and 12 wk, followed up with orthopedic assessment, owner questionnaire, X-ray imaging, and force-plate gait analysis.RESULTS: Porcine ADSCs expressed mesenchymal stem cell markers CD90 and CD105. Injection of porcine ADSCs into canine stifle joints did not cause any inflammatory or allergic reactions. Orthopedic evaluation found improvements in two dogs, particularly at the longest time point. Owners' evaluation found increased capacity and decreased pain in all three dogs' activities such as walking and running. Radiographic evaluation did not find statistically significant differences before and after treatment. Force-plate analysis found significant improvements in all three dogs after treatment.CONCLUSION: Xenotransplantation of ADSCs for the treatment of OA is feasible. Further studies are needed to validate this novel treatment modality, which can then be implemented for the routine treatment of OA in veterinary medicine.

Changes in energetic profile of pregnant ewes in relation with the composition of the fetal fluids

Objective: To evaluate the energetic profile of fetal fluids and to make comparisons of the concentrations of the constituents present with those in the maternal plasma.Methods: A study was conducted in 102 gravid sheep uteri. The four stages of gestation as Stage I(0–60 days), Stage II(61–90 days), Stage III(91–120 days) and Stage IV(121–145 days) were identified based on the crown anus length of the embryo/fetus. The amniotic and allantoic fluids collected from the gravid uteri of each group were subjected to biochemical analysis of glucose, cholesterol and triglyceride.Results: The levels of glucose and triglyceride in maternal plasma were lower(P < 0.05)on late pregnancy as well as in amniotic and allantoic fluids. No significant variation(P > 0.05) of plasma cholesterol levels was detected between the sampling periods.Contrariwise, cholesterol concentrations of fetal fluids were higher in Stages III and IV of pregnancy when compared with the Stages I and II.Conclusions: The influence of pregnancy on the biochemical composition of fetal fluids was statistically significant.

An ethnopharmacological survey of medicinal plants used in the traditional treatment of human infertility in eastern Algeria

Objective:To summarize medicinal plants used in the treatment of couple fertility disorders.Methods:An ethnopharmacological survey was conducted with 30 traditional healers from some localities of Skikda region in eastern Algeria.A standard questionnaire was used in the interview which focused mainly:the treated fertility disorders,the used medicinal plants species,parts used,and preparation methods.The relative frequency of citation and percentages of infertility troubles were calculated and analyzed.Results:The most important treated disorders in men were dysfunction of libido(44.22%),low sperm concentration and motility(25.33%),low semen volume(17.67%)and prostate disorders(5.66%).Women were treated mainly for menstruation disorders(35.32%),breast problems(29.53%)and sexual asthenia(25.82%).The survey identified 28 plant species belonging to 15 families,used by different ethnic groups,particularly women(58.00%),to address different fertility disorders.Three plant families were largely used viz Lamiaceae,Asteraceae,and Apiaceae.In term of relative frequency citation,ten dominated plant species were:Zingiber officinalis L.(0.96),Nigella sativa L.(0.83),Lepidium sativum L.(0.80),Capsicum annuum L.(0.60),Cuminum cyminum L.(0.56),Origanum vulgare L.(0.55),Allium sativum L.(0.50),Petroselinum sativum L.(0.43),Salvia officinalis L.(0.42),and Foeniculum vulgare L.(0.40).A number of investigated plants were scientifically confirmed by phytochemical and pharmacological studies to have one or more significant effects on couple fertility.However,much controversy was found in literature concerning the efficacy of some cited plants.Conclusions:The study highlights the important use of medicinal plants in management of couple infertility in eastern Algeria.More experimental studies are recommended to confirm or to refute these traditional uses and to ascertain the safety of these medicinal plants to consumers.

Tungsten-based nanocatalysts with different structures for visible light responsive photocatalytic degradation of bisphenol A

Environmental pollution,such as water contamination,is a critical issue that must be absolutely addressed.Here,three different morphologies of tungsten-based photocatalysts(WO_(3)nanorods,WO_(3)/WS_(2)nanobricks,WO_(3)/WS_(2)nanorods)are made using a simple hydrothermal method by changing the solvents(H_(2)O,DMF,aqueous HCl solution).The as-prepared nanocatalysts have excellent thermal stability,large porosity,and high hydrophilicity.The results show all materials have good photocatalytic activity in aqueous media,with WO_(3)/WS_(2)nanorods(NRs)having the best activity in the photodegradation of bisphenol A(BPA)under visible-light irradiation.This may originate from increased migration of charge carriers and effective prevention of electron–hole recombination in WO_(3)/WS_(2)NRs,whereby this photocatalyst is able to generate more reactive·OH and·O_(2)^(–)species,leading to greater photocatalytic activity.About 99.6% of BPA is photodegraded within 60 min when using 1.5 g/L WO_(3)/WS_(2)NRs and 5.0 mg/L BPA at pH 7.0.Additionally,the optimal conditions(pH,catalyst dosage,initial BPA concentration)for WO_(3)/WS_(2)NRs are also elaborately investigated.These rod-like heterostructures are expressed as potential catalysts with excellent photostability,efficient reusability,and highly active effectivity in different types of water.In particular,the removal efficiency of BPA by WO_(3)/WS_(2)NRs reduces by only 1.5% after five recycling runs and even reaches 89.1%in contaminated lake water.This study provides promising insights for the nearly complete removal of BPA from wastewater or different water resources,which is advantageous to various applications in environmental remediation.

Hepatocyte growth factor gene therapy prevents radiation-induced liver damage

AIM: To transfer human HGF gene into the liver of rats by direct electroporation as a means to prevent radiationinduced liver damage.METHODS: Rat whole liver irradiation model was accomplished by intra-operative approach. HGF plasmid was injected into liver and transferred by electroporation using a pulse generator. Control rats (n = 8) received electrogene therapy (EGT) vehicle plasmid and another 8rats received HGF-EGT 100 μg 48 h before WLIR.Expression of HGF in liver was examined by RT-PCR and ELISA methods. Apoptosis was determined by TUNEL assay. Histopathology was evaluated 10 wk after whole liver irradiation.RESULTS: Marked decrease of apoptotic cells and downregulation of transforming growth factor-beta 1 (TGF-β1)mRNA were observed in the HGF-EGT group 2 d after liver irradiation compared to control animals. Less evidence of radiation-induced liver damage was observed morphologically in liver specimen 10 wk after liver irradiation and longer median survival time was observed from HGF-EGT group (14 wk) compared to control rats (5 wk). (P = 0.031).CONCLUSION: For the first time it has been demonstrated that HGF-EGT would prevent liver from radiation-induced liver damage by preventing apoptosis and down-regulation of TGF-β1.

Glucose metabolism and neurogenesis in the gerbil hippocampus after transient forebrain ischemia

Recent evidence exists that glucose transporter 3（GLUT3） plays an important role in the energy metabolism in the brain.Most previous studies have been conducted using focal or hypoxic ischemia models and have focused on changes in GLUT3 expression based on protein and m RNA levels rather than tissue levels.In the present study,we observed change in GLUT3 immunoreactivity in the adult gerbil hippocampus at various time points after 5 minutes of transient forebrain ischemia.In the sham-operated group,GLUT3 immunoreactivity in the hippocampal CA1 region was weak,in the pyramidal cells of the CA1 region increased in a time-dependent fashion 24 hours after ischemia,and in the hippocampal CA1 region decreased significantly between 2 and 5 days after ischemia,with high level of GLUT3 immunoreactivity observed in the CA1 region 10 days after ischemia.In a double immunofluorescence study using GLUT3 and glial-fibrillary acidic protein（GFAP）,we observed strong GLUT3 immunoreactivity in the astrocytes.GLUT3 immunoreactivity increased after ischemia and peaked 7 days in the dentate gyrus after ischemia/reperfusion.In a double immunofluorescence study using GLUT3 and doublecortin（DCX）,we observed low level of GLUT3 immunoreactivity in the differentiated neuroblasts of the subgranular zone of the dentate gyrus after ischemia.GLUT3 immunoreactivity in the sham-operated group was mainly detected in the subgranular zone of the dentate gyrus.These results suggest that the increase in GLUT3 immunoreactivity may be a compensatory mechanism to modulate glucose level in the hippocampal CA1 region and to promote adult neurogenesis in the dentate gyrus.

Beta-nerve growth factor gene therapy alleviates pyridoxine-induced neuropathic damage by increasing doublecortin and tyrosine kinase A in the dorsal root ganglion

Beta-nerve growth factor(β-NGF)is known to be a major leading cause of neuronal plasticity.To identify the possible action mechanisms ofβ-NGF gene therapy for sciatic nerve recovery,experimental dogs were randomly divided into control,pyridoxine,and pyridoxine+β-NGF groups.We observed chronological changes of morphology in the dorsal root ganglia in response to pyridoxine toxicity based on cresyl violet staining.The number of large neurons positive for cresyl violet was dramatically decreased after pyridoxine intoxication for 7 days in the dorsal root ganglia and the neuron number was gradually increased after pyridoxine withdrawal.In addition,we also investigated the effects ofβ-NGF gene therapy on neuronal plasticity in pyridoxine-induced neuropathic dogs.To accomplish this,tyrosine kinase receptor A(TrkA),βIII-tubulin and doublecortin(DCX)immunohistochemical staining was performed at 3 days after the last pyridoxine treatment.TrkA-immunoreactive neurons were dramatically decreased in the pyridoxine group compared to the control group,but strong TrkA immunoreactivity was observed in the small-sized dorsal root ganglia in this group.TrkA immunoreactivity in the dorsal root ganglia was similar betweenβ-NGF and control groups.The numbers ofβⅢ-tubulin-and DCX-immunoreactive cells decreased significantly in the pyridoxine group compared to the control group.However,the reduction ofβⅢ-tubulin-and DCX-immunoreactive cells in the dorsal root ganglia in theβ-NGF group was significantly ameliorated than that in the pyridoxine group.These results indicate thatβ-NGF gene therapy is a powerful treatment of pyridoxine-induced neuropathic damage by increasing the TrkA and DCX levels in the dorsal root ganglia.The experimental protocol was approved by the Institutional Animal Care and Use Committee(IACUC)of Seoul National University,South Korea(approval No.SNU-060623-1,SNU-091009-1)on June 23,2006 and October 9,2009,respectively.

CTX-M producing Escherichia coli isolated from cattle feces in Bogor slaughterhouse,Indonesia

Objective: To determine the occurrence of CTX-M producing Escherichia coli(E. coli)from cattle feces in Bogor slaughterhouse, Indonesia.Methods: A total of 220 cattle feces samples were collected from Bogor slaughterhouse from March to April 2015. Presence of extended-spectrum beta-lactamase(ESBL) producing E. coli was detected by disc diffusion test based on the recommendation from Clinical and Laboratory Standards Institute(2014). Bacterial strains which were confirmed as producing ESBLs were further analyzed for the presence of bla genes of the ESBL by PCR.Results: The results showed that CTX-M producing E. coli isolates were detected in 19 samples from 220 samples(8.6%). The b-lactamase genes detected were CTX-M-1(n = 10) and CTX-M-9(n = 9). All of the CTX-M producing E. coli isolates showed multidrug resistance phenotypes to at least four antibiotics. The highest incidence of antibiotics resistance was showed to ampicillin(100.0%), cefotaxime(100.0%), and cefpodoxime(100.0%), followed by streptomycin(84.3%), trimethoprim-sulfamethoxazole(73.7%), erythromycin(52.6%), kanamycin(26.3%), doxycycline(10.5%), and ceftazidime(0.0%).Conclusions: Detection of CTX-M-producing E. coli in cattle feces raises important questions as they can represent a potential risk factor to public health.

Milk losses due to bovine tropical theileriosis(Theileria annulata infection) in Algeria

The authors studied the impact of tropical theileriosis onset on milk yield decrease in 10 local bred cows in Skikda(Northern Algeria) during 2015 summer season.The milk yield decrease estimated weekly during two months was 2.76 L/day/cow corresponding to31.92% of the total milk yield.This decrease corresponds to 110.5 Algerian Dinars(1.02US$)/day/diseased cow.The relative variation of milk yield showed a dramatic decrease from 82.72% to 0.76% at Day 21 then became constant.Further studies are needed to improve these estimations of financial losses due to bovine tropical theileriosis in Algeria.

Experimental infection with Yersinia pseudotuberculosis in European brown hare(Lepus europaeus,Pallas)

Objective:To investigate clinicopathological,bacteriological and pathological aspects of an experimental infection with Yersinia pseudotuberculosis(Y.pseudotuberculosis)in hares in order to verify the efficacy of serology for the in vivo diagnosis.Moreover,the pathogenicity of two Y.pseudotuberculosis strains was investigated in order to detect potential differences.Methods:Twelve European brown hares(Lepus europaeus,Pallas)were experimentally infected per os and via conjunctival mucosae with Y.pseudotuberculosis:six subjects were infected with a strain isolated from a naturally infected hare(YpH)and six subjects with a strain isolated from a naturally infected rabbit(YpR).Two hares were used as negative controls.All animals were subjected to clinical,bacteriological and serological examinations during 9 weeks following the infection and,at the end of the control period,subjects still alive were euthanized and submitted to a complete post mortem examination.Results:All faecal samples collected during the control period were positive for bacteriological examinations and to a PCR for the inv gene of Y.pseudotuberculosis,while only one Yp H-infected hare showed a positive haemocultures.From the 2nd to the 9th week post infection(pi),serological analysis revealed specific antibodies with titers ranging from 1:10 to 1:160 in all YpH-infected and two YpR-infected subjects.All the Yp H-infected and two Yp R-infected hares scored positive for Y.pseudotuberculosis by means of bacteriological investigations.Grossly,suppurative multifocal lesions were detected in liver,spleen,kidney and sub-mandibular lymph nodes in both YpH-and YpR-infected hares and confirmed with histopathology.Pulmonary lesions were observed only in Yp H-infected subjects.Immunohistochemistry confirmed the presence of bacterial antigen in all infected animals.Conclusion:Results of this study revealed that YpH strain is more pathogenic for hares than the YpR strain;moreover the serological test performed in this study could be used for the diagnosis of pseudotuberculosis in hares,whereas post mortem diagnosis should be confirmed by means of bacteriological examination,PCR,histopathology and immunohistochemistry.

Preliminary study on synergistic combinations of raw honey with gentamicin against Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa of veterinary origin

Objective:To search for further synergistic combinations of gentamicin and raw honey that might have potential in treating wounds.Methods:The antibacterial activity and synergistic interaction of raw honey and gentamicin was assessed by using agar well diffusion method.Two Gram-negative(Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 2154)bacteria were selected for antibacterial activity assay.The cultures of bacteria were maintained in their appropriate agar slants at 4°C throughout the study and used as stock cultures.Results:Raw honey and gentamicin interacted synergistically to inhibit Escherichia coli and Pseudomonas aeruginosa.Conclusions:These results suggest that combinations of raw honey and gentamicin have therapeutic benefits in prophylaxis of infections caused by multidrug-resistant Gram-negative bacilli.

Anti-hypercholesterolemic effect of Pistacia lentiscus fatty oil in egg yolk-fed rabbits: a comparative study with simvastatin

AIM: The current study was undertaken to assess anti-hyperlipidemic activity of Pistacia lentiscus fatty oil(PLFO) in rabbits following a hyperlipidemic diet. METHOD: Twenty healthy female(WNZ) rabbits were divided into four groups of five animals each:(a) normal control(NC group) receiving standard diet,(b) hyperlipidemic control(EY) group receiving standard diet and gavaged daily with egg yolk(10 mL),(c) hyperlipidemic + PLFO(EY + PLFO) group receiving as the EY group and treated daily with PLFO(2 mL/kg BW,(d) hyperlipidemic + simvastatin(EY + SVS) group receiving as the EY group and treated once daily with 2.5 mg/kg BW of simvastatin. At the end of the six-week experimental period, the lipidemic profiles of the different groups were investigated. RESULTS: In the EY group, the egg yolk resulted in a significant increase of total cholesterol(TC), triglycerides(TG), HDL-C, LDL-C, and the LDL-C/HDL-C ratio. Both the EY + PLFO and EY + SVS groups, when compared to the EY group, showed a significant decrease of TC, TG, LDL-C, and the LDL-C/HDL-C ratio. However, with respect to HDL-C the differences were not significant. The TGs were significantly lower(P < 0.001) in the simvastatin-treated group when compared to rabbits treated in the PLFO group. CONCLUSION: The study concludes that P. lentiscus fatty oil(PLFO) possesses anti-hyperlipidemic properties at least in reducing total cholesterol, LDL-cholesterol and triglycerides.

Comparison of efficacy of unheated and heat-treated Sahara honey on wound healing in rabbits

Objective:To investigate the efficacy of unheated and heat-treated Sahara honey on wound healing in rabbits on the basis of macroscopic observation changes.Methods:Eight female rabbits were used.Using aseptic surgical technique,a 3 cm incision was made on the back of each rabbit and two rabbits with injuries in each group were treated daily with a topical application of unheated and heated honey,sulfadiazine and sterile saline,respectively.Results:The unheated honey demonstrated the highest activity on the wound compared to reference ointment silver sulfadiazine,heat-treated honey and sterile saline respectively.Further the present investigation proves that unheated honey is possessing superior wound healing activity than that of heat-treated honey.Conclusions:The result of this study confirms that unheated honey had the best wound healing effect even better than heat-treated honey.