Mycoplasma pulmonis in mice, and SeV, rat parvo virus (RPV), Kilham’s rat virus (KRV) and sialodacryoadenitis virus (SDAV) in rats, by sandwich ELISA. It was observed that MHV was the most prevalent agent followed by Mycoplasma pulmonis and MVM in mice, and SDAV followed by RPV were prevalent in rats. On the other hand, none of the samples were positive for ECTV in mice, or SeV or KRV in rats. Multiple infections were common in both mice and rats. The incidence of MHV and Mycoplasma pulmonis was higher in facilities maintained by public organizations than in vivaria of private organizations, although the difference was not statistically different. On the other hand the prevalence of rodent pathogens was significantly higher in the northern part of India than in the South. These studies form the groundwork for detailed sero-prevalence studies which should further lay the foundations for country-specific guidelines for health monitoring of laboratory animals.Health monitoring is an integral part of laboratory animal quality standards. However, current or past prevalence data as well as regulatory requirements dictate the frequency, type and the expanse of health monitoring. In an effort to understand the prevalence of rodent pathogens in India, a preliminary study was carried out by sero-epidemiology. Sera samples obtained from 26 public and private animal facilities were analyzed for the presence of antibodies against minute virus of mice (MVM), ectromelia virus (ECTV), lymphocytic choriomeningitis virus (LCMV), mouse hepatitis virus (MHV), Sendai virus (SeV), and  The suitability of an animal species for a specific experiment is dictated by the physiology of the animal . PathogeThe identification of agents prevalent at individual or population level can be achieved by sero-epidemiology or other studies . FollowiIndia has emerged as a significant player in the global biotech arena, with large-scale outsourcing of work by several pharmaceutical drug discovery firms. Consequently, state-of-the-art facilities established by many contract research organizations and pharmaceutical industries have opted for accreditation of their vivaria by India’s National Good Laboratory Practice Compliance Monitoring Authority as well as the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), USA, in order that study reports are acceptable globally. However, these have been mostly need-based and driven by regulatory requirements, and not standard practices. Of the more than 1400 animal facilities registered with the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), which regulates animal experimentation in India, very few meet international standards and many do not have health monitoring programmes due to prohibitive costs. In addition, no systematic data is available on the prevalence of laboratory rodent pathogens in India. The present study was undertaken to determine the prevalence of various rodent pathogens in laboratory animals in some parts of India where majority of the animal facilities are located.Serum samples (500 μL) were sought from five mice and five rats maintained at 26 vivaria in public and private organizations located in different parts of India. These samples were either collected by a veterinarian or by the trained personnel under the supervision of a veterinarian. Majority of the animal facilities obtained Institutional Animal Ethics Committee (IAEC) approval specific to the health monitoring programme in their respective institutes/organizations, and the rest collected serum samples from the control group of animals from the on-going IAEC approved animal experiments. The sera were shipped on freezer packs and stored at -80°C until further analysis.Mycoplasma pulmonis in mice, and SeV, rat parvovirus (RPV), Kilham’s rat virus (KRV) and sialodacryoadenitis virus (SDAV) in rats, by sandwich ELISA [The serum samples were analyzed for the presence or absence of antibodies against for minute virus of mice (MVM), ectromelia virus (ECTV), lymphocytic choriomeningitis virus (LCMV), mouse hepatitis virus (MHV), Sendai virus (SeV), and ch ELISA , using ct-test. GraphPad Prism (Version 5) was used for the preparation of graphs. EPI-Infosoftware  and digitized maps of India were used for preparing distribution of pathogens.The data between samples from government and private organizations as well as between those from North and South India were analysed for statistical significance by the paired One hundred and thirty serum samples each from mice and rats were analyzed for six pathogens of mice and four pathogens of rats by ELISA. For the examined set of pathogens, 53.84% of the mice were found to be infected with one or more pathogens whereas the rate of infection in rats was substantially lower (20%).Mycoplasma pulmonis (22.30%) and MVM (13.84%). On the other hand, the incidence of SeV and LCMV were very low , and none of the samples were positive for ECTV , and this was followed by for ECTV . In ratsfor ECTV .Fifty four per cent of the sera tested positive for one or the other agent, and multiple infections were common, with 14.75% of the sera being positive for three or more agents. In mice, MHV appeared to co-exist with other pathogens (55.74%) rather than singly (39.34%). On the other hand, MVM was never found singly . WhereasMycoplasma pulmonis in mice was more in North India  compared to South India , and the only positive sample for LCMV was from North India. In rats, the incidence of SDAV and RPV was higher in North India  compared to South India   . OverallMycoplasma pulmonis was high in Uttar Pradesh, Delhi and Kerala  compared to public (9.09%) organizations Click here for additional data file.S2 TableNote: OD values >0.30 were considered as positive as per the instructions by the ELISA kit manufacturer.(DOCX)Click here for additional data file.


Genome-wide RNA interference (RNAi) screening is an emerging and powerful technique for genetic screens, which can be divided into arrayed RNAi screen and pooled RNAi screen/selection based on different screening strategies. To date, several genome-wide RNAi screens have been successfully performed to identify host factors essential for influenza virus replication. However, the host factors identified by different research groups are not always consistent. Taking influenza virus screens as an example, we found that a number of screening parameters may directly or indirectly influence the primary hits identified by the screens. This review highlights the differences among the published genome-wide screening approaches and offers recommendations for performing a good pooled shRNA screen/selection. RNA interference (RNAi) is a revolutionary technique for studying the biological functions of a particular gene by silencing its gene expression that can be applied in mammalian systems. The manipulation of gene expression of any particular gene by RNAi provides insight into the genetic networks related to the target gene. Thus far, the main approaches for RNAi screening can be divided into two screening formats, arrayed screen and pooled selection/screen. For arrayed screen, each RNAi reagent is assigned to a unique well in a microplate, and a variety of cell-based assays are performed in the microplate format. After the assay, cells with significant changes can be identified and the related RNAi reagents can be traced by using a database. For pooled selection/screen, all of the RNAi reagents are pooled together and added randomly to cells. There are two strategies for pooled selection: positive selection, which only detects surviving cells and does not require an untreated control, and negative selection, which includes an untreated control for comparison to allow the detection of RNAi reagents that make the cells resistant or sensitive to the selective reagent. The significant RNAi reagents are subsequently deconvoluted by using Next Generation Sequencing (NGS) or barcode microarray.Influenza virus causes annual epidemics and recurring pandemics which potentially threaten public health and the global economy. Influenza A viruses (IAVs) are enveloped RNA viruses with single-stranded, negative-sense viral RNAs encoding 11 viral proteins [The first genome-wide RNAi screen for the identification of host factors required for influenza virus replication was performed in a Drosophila cell line by Hao et al.  in 2008,In 2009, Brass et al.  performeKönig et al.  and KarlRather than performing the arrayed RNAi screens (dsRNA or siRNA) as mentioned above, we established a genome-wide pooled RNAi screen  based on a positive selection strategy to identify host factors required for influenza virus replication . Under pSimultaneously, Tran et al.  also useA total of 1,362 host factors were identified as potential cellular mediators in relation to influenza virus replication by the seven aforementioned RNAi screens. More than 90% of the hits  were identified by only one of the RNAi screens. 113 overlapping host genes were identified in two independent RNAi screens, 14 overlapping genes  were identified in three RNAi screens, and 6 overlapping genes  were identified in four individual screens  characteristics of influenza virus  and host cell line ; (ii) features of the RNAi library screened, particularly the knockdown efficiency of the RNAi resources, and the quality of the RNAi library; (iii) screening method/time points for analysis; and (iv) hit-selection criteria and evaluation of proper controls, e.g., at least two siRNAs or shRNAs targeting to the same gene, and z-score of RNAi screen. Screens that used similar screening resources or approaches showed a higher degree of concordance. For example, Brass et al. and Shapira et al. used the same source of siRNA library (Dharmacon) and influenza virus strain (A/Puerto Rico/8/34) for arrayed siRNA screens, and in their lists of primary gene hits there were 16 overlapping host factors. Similarly, König et al. and Karlas et al. used the same siRNA library (Qiagen) and influenza virus strain (A/WSN/33) for arrayed siRNA screens and identified 25 overlapping host factors  reveals the host factor(s) essential for supporting influenza virus replication.Although several virus replication-related host factors have been successfully identified by the genome-wide arrayed RNAi screens, the high-cost of the siRNA library and the requirement for a robotic liquid handling system for high throughput screening are two major drawbacks of this approach. In addition, the duration of gene silencing by siRNA transfection usually lasts for less than 1 week, which limits the usage of siRNA-based screen if long-term knockdown is needed. The lentivirus-based arrayed RNAi screen or pooled RNAi screen, on the other hand, can overcome the short-term effect of siRNA to discover host factors crucial for influenza virus replication. However, the much higher cost (compared to an siRNA library) and the requirement for a robotic liquid handling system for genome-wide arrayed shRNA screen are again major concerns. On the other hand, the cost of the pooled RNAi reagent is much lower and screen/selection can be conducted in most laboratories.We performed a genome-wide pooled RNAi screen and identified host factors using a survival-dependent screening strategy . InfluenThe optimal concentration of siRNA used for gene silencing varies (1–30 nM) from study to study, and is highly dependent on the cell type and its target. Transfection of siRNA by using improper concentrations of siRNAs may result in unwanted off-target effects or incomplete gene silencing during arrayed siRNA screens, which may produce misleading results. It may be difficult to determine an optimal siRNA concentration for performing an arrayed siRNA screen. On the contrary, pooled shRNA screens take advantage of lentiviral transduction at low MOI  to deliver single copy of shRNA into cells. This low MOI remarkably reduces the probability of off-target effects. However, heterogeneity of functional siRNAs may be generated by improper processing of a shRNA hairpin, thus causing off-target effects. To minimize the off-target effects in both cases, targeted genes knocked down by at least 2 unique siRNAs (or shRNAs), which target different regions within the same gene transcript are recommended as the selection criteria, since the probability of off-target effects triggered by 2 independent siRNAs (or shRNAs) to the same target gene is extremely low. Unfortunately, the knockdown efficiency of individual siRNA in a given arrayed smart pool RNAi library is usually not known although the manufacturer guarantees that at least one of the provided siRNAs has good knockdown efficiency of 70% or more. Such knockdown information is less informative for researchers who intend to minimize the off-target effects by using two good individual siRNAs. By contrast, more than 40% of the shRNAs from the TRC RNAi library have been validated and the knockdown information of these shRNAs would provide great advantage for analysis of primary hits.Genome-wide pooled shRNA screens are technically complicated, and numerous factors directly or indirectly affect the efficiency and accuracy of the screening results. Several experimental details have to be taken into consideration when performing a large-scale pooled RNAi screen. First, individual shRNA (shRNA-expressing lentivirus) should be enlarged to more than 250 representatives to guarantee that the effective shRNA(s) is not lost in subsequent experimental procedures and to compensate for the low expression level resulting from random integration sites of the shRNA expression cassette . Second,Third, when performing a pooled RNAi screen, the use of polybrene (hexadimethrine bromide) during lentivirus transduction should be avoided. Polybrene is a common polycation that increases the infectivity of lentivirus 3–5 fold (our unpublished data). The cationic polymer enhances virus adsorption and transduction by neutralizing the charge between viral envelope and cellular membrane . Moreovehttp://rnai.genmed.sinica.edu.tw/file/protocol/PooledScreen_SequencingProtocol.pdf.Unlike the arrayed RNAi screen, the potential hits (the content of shRNAs) selected/enriched by a pooled RNAi screen must be further identified by other methods, such as barcode microarray or next generation sequencing (NGS). The barcode microarray-based method employs PCR amplification of shRNA template sequence pools, labeling of fluorophore, and hybridization to complementary DNA microarray chip(s) from an experimental group as well as a reference group. After hybridization, the fluorescent signal intensity reflects the abundance of cells expressing a certain shRNA under test conditions as compared to the reference. However, the dissimilar amplification resulting from self-annealing of hairpin sequences of shRNA may cause detection bias during microarray analysis. The external barcode tags help to optimize hybridization conditions for each probe and avoid technique bias of PCR amplification caused by the secondary structures of the shRNA template sequence. Artifacts caused by cross-hybridization, also known as unspecific probe-target interaction, are the major concern of barcode microarray hybridization in identification of target shRNA from genome-wide pooled RNAi screen. NGS or namely deep sequencing technology provides a new approach for the analysis of the hits selected by pooled RNAi screens. In brief, the experimental procedure of NGS consists of: (i) isolation of genomic DNA; (ii) PCR amplification of shRNA; (iii) restriction enzyme digestion and gel purification of the PCR products; and (iv) ligation of PCR products to an adaptor for subsequent NGS analysis. In general, maintaining the fidelity of shRNA population during preparation of PCR fragments for NGS is important for subsequent hit determination. Our recommendations for preparing shRNA-containing PCR fragments for NGS are as follows: (i) Prepare appropriate amounts of genomic DNA without loss of shRNA complexity. Low amounts of genomic DNA may not cover the whole population of shRNA in a typically genome-wide-scale negative selection experiment; (ii) Avoid creating conditions that generate sequencing bias during PCR amplification, such as over-amplification and two-rounds of PCR. Exponential PCR cycles without generating heteroduplex that are derived from annealing of two shRNA sequences occurring at the later stage of PCR are recommended; (iii) Prepare PCR fragments with half-arm of shRNA cassette for NGS, if possible, because full-length shRNA may impede sequencing; (iv) Templates should contain a 5' end phosphate and a 3' end hydroxyl group for efficiently adding adaptor for NGS; (v) Purify restricted PCR fragments by gel electrophoresis for deep sequencing. A protocol for genome-wide pooled RNAi screen can be found at: By detection of the amounts of the shRNA sequence, the NGS approach may hold greater promise for accuracy than that of the barcode microarray-based method for deconvolution of genome-wide pooled RNAi screening output. There are at least four advantages of using the NGS method in place of the barcode microarray hybridization. First, NGS technology is a cost-effective approach because it measures the presence of large quantities of distinct shRNA sequences in a short time. Secondly, NGS greatly improves the sensitivity and efficacy of detection by offering a digital readout of even very small amounts of shRNA species. Third, the NGS method provides a greater detection range and better resolution of measurements, which enables clear discrimination of true hits from background noise. Finally, NGS is a more flexible approach for the identification of hits from a genome-wide RNAi screen, which can be easily incorporated into any high-complexity shRNA library without generating new information of barcode and linkage to individual shRNA sequence.Genome-wide RNAi screen is a useful tool for studying the gene functions by manipulation of gene expression. Both arrayed RNAi screen and pooled RNAi screen have been successfully conducted to identify host factors crucial for influenza virus replication. Two previous influenza screening reviews focusing on arrayed RNAi screens have uncovered host factors and cellular networks involved in the influenza virus replication ,21. Here


In this paper, we observed a discrepancy of penile hemodynamics dependent on location by using near infrared spectroscopy (NIRS) sensor, and showcase NIRS as a potentially suitable sensor in supplementing the diagnosis and treatment of erectile dysfunction.To observe the effect that location has on penile hemodynamics, the NIRS sensor was placed on the top and the side of genital organ, and oxy- (HbO), deoxy-(RHb), and total (HbT) hemoglobin concentration changes were acquired. Our results from 6 healthy subjects show that hemodynamic changes vary depending on where the probe was placed. To observe a statistical difference between the signals, a Wilcoxon signed-rank test was performed.p < 0.05) between concentration changes of RHb and HbT depending on the probes’ location. Moreover, the sensor placed on the top of the organ shows a rise of HbO and HbT concentration while RHb concentration decreased. However, hemodynamics from the side of the organ showed that RHb concentration increased along with HbO.The result shows a significant difference (The outcomes demonstrates an ability of NIRS to be sensitive enough to detect the different hemodynamic changes in various locations of a healthy male genital organ during visual sexual stimulation. The results also show the importance of sensor location on the genital organ for the resulting hemodynamic changes. We can foresee our results as a way for clinicians to obtain more accurate hemodynamic measurements from the penis, and also show the likelihood for NIRS enhanced diagnosis tool of male erectile dysfunction over the current standards. Male erectile dysfunction (ED) is a disease that affects a large group of men and can have a severe negative effect on the subject’s life. ED has been found to affect 5-20% of the world’s male population . ED is dThere are many ways to assess the vascular functionality during penile erection including: selective pudendal angiography ,5, dupleNear Infrared Spectroscopy (NIRS) has shown to be a suitable sensor to detect the hemodynamic changes in the body under certain conditions. NIRS has been applied to detect hemodynamic changes in subjects with diseases ranging from epilepsy, Alzheimer’s, and including erectile dysfunction -19. The The aim of our study is to show that NIRS is a convenient, portable, and most importantly, a sensitive enough system to detect HbO and RHb changes during an erection of the genital organ. We also demonstrate that penile hemodynamics varies depending on the probe location on the genital organ. The dependency of penile hemodynamics according to NIRS probe location can be used to improve the diagnosis, treatment and overall understanding of male erectile dysfunction.Our in house-built continuous wave NIRS probe consisted of one light emitting diodes (LED) (Epitex Inc. L735/850-40D32) and one monolithic photodiodes (Texas Instruments Inc. OPT101). The light emitting diode and photodiode was fixed onto the probe with a separation of 1 cm by means of a polydimethylsiloxane (PDMS) probe holder. The LED sequenced between the two wavelengths of 735 and 850 nm in order to obtain relative concentration changes of HbO and RHb. The sampling rate of the system was 2 Hz.L is an optical path length between the light source and detector.The detailed description of calculation of HbO, RHb, and HbT can be found in our previous report . In the L is not exactly equal to the source-detector separation, d, but rather approximated as L = dDPF  [In a scattering medium,  factor) . If the The raw data have been passed through Butterworth lowpass filter with cutoff frequency at 0.3 Hz, to eliminate physiological artifacts such as: heartbeat, respiration etc.Our study consisted of three different placements of the probes, for three different experiments: a single probe placed on the side of the genital organ, a single probe placed on the top of the genital organ, and two identical probes placed on the top and side of the genital organ Figure . The subThis study has been reviewed and approved by the Gwangju Institute of Science and Technology’s institutional review board (IRB 20140319-HR-10-02-02). The first two experiments were run for 6 healthy male subjects, age ranging from 28 ± 3 years old, with no previous history of ED. The informed written consent was obtained from each subject before the experiments. Each subject was placed in a dark experiment room with a chair and monitor. Before the experiment began, the administrator instructed the subject how to attach the probe. After that, the experiment was administered remotely by an experiment administrator in another room. A single probe was attached on the subject’s left-middle of the genital organ for the first experiment and a single probe was attached on the top and middle of the genital organ for the second experiment.  of the genital organ for one trial and a probe placed on top of the genital organ for another trial. The trials were independent of each other with appropriate rest in between and different visual sexual stimulations as to create the same sensation in the subject. The collection and post processing of the data occurred in entirely by using Matlab software.Figure Figure In addition, the third experiment results show not only the different behavior of RHb but also the difference of response time between HbO and RHb occuring after the visual sexual stimulation. The probe placed on the side of genital organ showed that HbO change occurs before the change of RHb while the reversed response time between HbO and RHb was observed from the probe located on the top of the genital organ. Figure c and d Tp < 0.05) while HbO concentration change did not show the statistical difference (p = 0.07).The changes of HbO, RHb and HbT concentration during the visual sexual stimulation are averaged from all three experiments and are compared between the top and the side of the genital organ. Figure  HbO fromThereby, the results from the three experiments demonstrate that the distinction of physiological process occurs at different location during penile erection. The penile hemodynamics from the side of genital organ is similar to the hemodynamics from venous occlusion of the arm, which shows an increase of both HbO and RHb. This can be explained by understanding the process of erection as follows. Visual sexual stimulation causes a sexual arousal in brain which in turn releases the nitric oxide from nerve endings near blood vessels within the corpora cavernosa and spongiosum. This induces the dilation of penile arteries, and the resulting rapid increase of blood volume fills the corporal tissue. The increased blood volume in erectile chambers compresses the thin-walled penile venules which prevents the venous drainage of blood to maintain the erection. Figure  ThereforUnlike the hemodynamics observed at the side of genital organ, the penile hemodynamics from the top of genital organ showed a decrease of RHb while HbO increased during visual sexual stimulation. The probe on the top of genital organ collects less signals from corporal cavernosa which is shown as a smaller increase of HbO than that from the side of genital organ. The decrease of RHb tells us that the expansion of corporal tissue caused less compression of dorsal vein due to its bigger diameter than the penile venules located on the side of genital organ so that there is less accumulation of venous blood but still with an increase of arterial blood on the top region of genital organ. All the results proved that NIRS is sensitive to monitor the penile hemodynamics during erection and multi-location measurements may tell us the details of erectile dysfunction and weaker erections.This preliminary study has shown strong indication that NIRS can be a very important device for the understanding of erectile dysfunction due to its ability to detect different hemodynamic changes in various locations of a healthy male genital organ during visual sexual stimulation. Our data set shows that different locations of the genital organ may show a substantial increase in HbO, RHb and HbT, or a rise of HbO and HbT, but a decrease in RHb during visual sexual stimulation. Especially in the case of the studies of the genital organ and erectile dysfunction, it is vastly important that the measurement system should be comfortable for the subject, while at the same time, provide sufficient and high quality data for researchers and clinicians alike. We hope that the unique insight gained by applying NIRS can be useful in the diagnosis of male erectile dysfunction and weak erection.For our future studies, we hope to expand upon our findings in this paper to understand what other physiological changes the body undergoes during sexual stimulation. Since a healthy penile erection is a result of many complex psychological and physiological reactions, and the impairments in any of these reactions may cause ED, it is important to develop a system to observe these reactions. NIRS presents a realistic solution to conveniently observe the psychological and physiological changes that the body undergoes during sexual stimulation by using a single system. In turn, we would like to apply our system to the cases of erectile dysfunction patient to see if NIRS can observe different hemodynamic changes in ED cases than from the healthy subjects we have shown in this study. As it can be seen, NIRS gives a promising outlook for a biosensor that can greatly enhance the diagnosis and treatment of erectile dysfunction and weak erection.


Spodoptera lituramultiple nucleopolyhedrovirus before and after passage through the gut of the predatory stink bug,Eocanthecona furcellataWolff (Hemiptera: Pentatomidae) was compared through field bioassay studies. Three sets ofE. furcellatawere used for bioassays and these were allowed to feed on a single meal of five third instar Oriental leaf worm,Spodoptera litura(Fabricius) (Lepidoptera: Noctuidae), that were infected with polyhedra before passage, after passage, and healthy (control) larvae 1 day prior to the trial. The predators were subsequently released on cabbage plants that were infested with 100 healthyS. lituralarvae. The median lethal dose (LD50) and survival time (ST50) values before and after passage through the gut were not significantly different. Additional mortality due to virus infection increased 13– 17% before and after treatments but within these treatments the mortality did not vary significantly. It was concluded thatE. furcellatadisseminated the virus through their feces into the ecosystem and infectivity of the SpltMNPV was not altered after passage through the gut of the predator.Infectivity of polyhedra ofAbbreviations: NPV, nucleopolyhedrovirus;SpltMNPV,Spodoptera lituramultiple nucleopolyhedrovirus;PIBs, polyhedral inclusion Eocanthecona furcellataWolff (Hemiptera: Pentatomidae) is regarded as potential predator of pest insects at both the nymphal and adult stages.E. furcellatais a generalist predator; it may consume any soft-bodied arthropods it encounters in the ecosystem.Baculoviruses are a group of double-stranded DNA viruses with a narrow host range. They are well known due to their potential as agents of biological control of pests in agriculture and forestry. The predatory bugPredator-mediated baculovirus dispersal has been demonstrated to be an important mechanism for virus dissemination in diverse crop and forest habitats . This dSpodoptera lituramultiple nucleopolyhedrovirus (SpltMNPV) before and after passage through the gut ofE. furcellataand investigated the role of predator-mediated virus dissemination in enhancing pest suppression in the field.Although the consumption of infected larvae and subsequent release of viable virus by predatory bugs are likely to occur in nature, the ecological implication of this phenomenon is poorly understood. The importance of predaceous arthropods in the dissemination of insect viruses depends on three factors: the acceptance of virus-infected insects as food, the effect of passage through the predator gut on virus infectivity, and the interactive behavior of predator and prey in relation to virus acquisition. However, effective integration of baculoviruses into existing pest management systems depends on their compatibility with other components of the systems. A full understanding of the spread of baculovirus by predators is therefore necessary for the successful application of biopesticides and also to predict the environmental fate of natural and genetically modified microorganisms . For inSpodoptera litura(Fabricius) (Lepidoptera: Noctuidae) insects used in this study were from the offspring of 34thgeneration female moths obtained from the Indian Agriculture Research Institute, New Delhi. These insects had been continuously maintained at 28 ± 2°C and 60% RH with a 16:8 L:D photoperiod on artificial diet as described byThethgeneration female bugs that emerged from field-collected eggs laid onPartheniumleaves , followed by filtration through muslin cloth and subsequent pelleting through continuous sucrose (40 to 66% w/w) gradient centrifugation for 1 hr at 50,000g. After several washes in TE , the polyhedra were resuspended in a small volume of distilled water and stored in aliquots at -20°C. The virus was quantified using a haemocytometer and phase contrast microscope at ×800 magnification.The SpltMNPV used in this study was originally isolated from naturally-infected (in the field), India . The viE. furcellata,two sets of SpltMNPV were used in the study: one set consisting of SpltMNPV before passage and the other after passage through the gut ofE. furcellata. To obtain the SpltMNPV after passage,E. furcellatawas allowed to feed on moribund infected fourth-instarS. lituraand placed in individual petri dishes (80 mm × 15 mm). Feces were collected up to 48 hours after feeding on infected prey, as no significant loss of infectivity of polyhedra occurred during passage through the gut within 24–48 hours. For feces collection, predators were used only once throughout the experiment. Feces were titrated in 100 µL of sterile water and each suspension was assayed for viable virus by feeding the virus to each of fifty second instar host larvae using the diet plug system (www.nbaii.res.in). One larva was then placed in each compartment. A total of 100 larvae were used for each concentration. Larvae, having eaten the entire diet disc within 24 hours, were transferred to fresh uncontaminated diet. Mortality was recorded daily until pupation. Dead larvae were checked for SpltMNPV infection. The experiment was replicated five times. A preliminary experiment comparing the estimation of polyhedra in a series of dilutions by the above method and by haemocytometer count indicated a highly significant regression coefficient (P< 0.001).To observe the infectivity of SpltMNPV before and after passage through the gut of system . CirculE. furcellatato disperse the virus, three sets of predators that were fed with polyhedra before passage, after passage through the gut, and healthy hosts (control) were released in the field on individual cabbage plants that did not show signs ofS. liturainfestation. The plants were randomly selected and allocated to three different treatments. Each experimental cabbage plant was at an average distance of 170 cm (or seven cabbage plants) away from any other experimental plant, such that each plant represented a single independent replicate. One day prior to the trial, pre-starved adult predators (three days old) were fed a single meal of five healthy or SpltMNPV-infected (before passage and after passage) third instars ofS. litura(48 hr postinfection) of uniform size and equal weight (65-70 mg). The infected larvae that were offered to predators were treated with a similar dose of 1 × 103PIBs/insect using the diet plug system as described above. Only those predators that consumed all the larvae within 24 hours were used in the experiment. Treatments involved placing one adult and 100 healthy third instarS. liturafrom the laboratory culture on the top third portion of the cabbage plant, which was then enclosed by a nylon mesh bag 35 cm tall and 30 cm wide, gently tied at the base to minimize insect movement. Each treatment was replicated five times. After six days, the bags were opened, the predators were removed, and remainingS. lituralarvae were recovered, taken to the laboratory, and individually transferred to semi-synthetic diet and checked daily for virus-induced mortality for 10 days. All virus deaths were confirmed by microscopic examination through Giemsa staining.To compare the ability of50± 95% CL) and median survival times (ST50); corresponding confidence interval for each dose was calculated using log rank test under Kaplan-Meier analysis. Data on cumulative mortality percentage ofS. lituralarvae caused by SpltMNPV before and after passage through the gut ofE. furcellatawere subjected to a “pairwise”t-test. The data on recovery and viral mortality ofS. liturain the field were subjected to one-way ANOVA.The data were subjected to Probit analysis (SPSS-16) for the calculation of median lethal dose = 50.823,P= 0.001) and after passage = 56.6684,P= 0.001) of SpltMNPV through the gut ofE. furcellata. A gradual increase in the cumulative mortality was observed as the dose increased from 187.5 to 3,000 PIBs/larva in both cases, with a significant positive correlation  between dose and mortality. However, when the cumulative mortality percentage ofS. lituralarvae caused by SpltMNPV before and after passage through the gut ofE. furcellatawas compared among similar doses, no significant difference was observed (50) estimates of 817 and 1,144 PIBs/larva before and after passage of SpltMNPV through the gut of the predator, with confidence limits of 532–1107 and 904– 1418 respectively, but as the confidence limits overlapped, the differences in infectivity were not significant.The effect of virus dose on the cumulative mortality was significant both for before passage  were 155.4, 163.6, 173.8, 201.4, and 209.6 hours before passage, respectively, and 162.6, 171.2, 177.0, 214.4, and 231.2 hours, respectively, after passage of SpltMNPV through the gut ofE. furcellata= 4.12,P= 0.001). However, when the viral mortality was compared on plants that hosted predators that were fed with two kinds of polyhedra , the observed differences were not significant  on plants where virus-fed predators were released when compared to plants that hosted healthy predators that did not show viral infection in recovered larvae  showed thatHarpalus rufipescould excrete viable virus for up to 15 days after feeding on an infected prey. However,The results suggest that PIBs of SpltMNPV were still infectious and the infectivity of the virus was not altered after passage through the gut of the predator. The observed values for LDr feces . This ir feces . HoweveE. furcellata).However, when the viral mortality was compared among these two sets of predators, the observed differences were nonsignificant. As no significant variation in larval recovery up to six days was observed, we assume that there was nearly equal predation by virus-fed and non-virus-fed bugs. Mortality in the field, although not high, showed that predators released infective virus in a form that could be acquired by susceptible lepidopteran hosts. Previous experiments involving the release of virus-fed predators (Carabidae) into the field that were subsequently infested with host larvae resulted in lower levels of infection (< 5%) of larvae . This is because in comparison to carabids, pentatomids are quite mobile, exhibiting frequent contact between predator and prey with a mean searching time of 3.5 minutes. This higher infectivity was due to higher mobility of the predator as compared to host larvae, which may increase the speed of inoculum dispersal . In field conditions, virus disseminated in the feces of insect predators is sufficient to initiate epizootics in larval populations. This has been reported in predators such asSycanus leucomesusWalk.  (Podisus maculiventrisSay (Nabis roseipennis(Reuter)  on those plants where the predators were fed with the two kinds of polyhedra  ,PodisurisSay , andNaReuter) . In our


P < 0.05). More important is that although the antibody titers of all infected ducks were similar from 9 to 17 dpi, reduced clearance of virus was observed in the youngest groups comparing with the other two groups, indicating that immune system maturity was more important than the presence of neutralizing antibody. In summary, this study demonstrates that viral pathogenesis is strongest in 1-week-old ducks and the age-related immune response plays an important role in the pathogenesis of DTMUV in ducks.The effect of host age on the outcome of duck tembusu virus (DTMUV) infection was studied in ducks. Three groups of Cherry Valley ducks at 1, 3, and 7 weeks of age were intramuscularly infected with DTMUV to systematically observe the clinical symptoms, pathological changes, tissue viral loads, and immune responses. Severe clinical symptoms and neurological dysfunction were observed in 1-week-old ducks as early as 2 days post infection (dpi) and some died at 5–7 dpi. Three weeks-old ducks showed similar but milder symptoms and no deaths. However, 7-weeks-old ducks showed only transient loss of appetite. Gross lesions gradually reduced in severity as ducks matured. One-week-old ducks showed endocardial hemorrhage, splenomegaly, swelling in the lymph follicles of the ileum, liver, and kidney swelling with degeneration, and meningeal hyperemia. Three-weeks-old ducks showed only mild pathological lesions. No visible lesions were observed in 7-weeks-old ducks. However, pathological histology analysis demonstrated all infected ducks displayed viral encephalitis. DTMUV could be detected in the brains of 1-week-old ducks as early as 1 dpi and virus titers of most organs in 1-week-old ducks were significantly higher than that of 3- and 7-weeks-old ducks at 3–5 dpi. The patterns of IFN-γ, IL-2, and serum neutralizing antibodies were similar, and there were significant difference between the youngest ducks and the older ducks at early infection stage ( Duck tembusu virus (DTMUV) can result in an acute, contagious infection characterized by decline in egg production of laying ducks . The infAlthough DTMUV was first isolated from egg ducks, differently aged ducks could be infected with the virus, especially ducks less than 3 weeks-old . We note6.1 median tissue culture infective dose (TCID50)/mL by infection of duck embryo fibroblasts and calculation of the titers by the Reed and Muench method . The ducks were housed in SPF chicken isolators that were ventilated under negative pressure. Feed and water were provided ad libitum. Samples were collected from ducks before inoculation to ensure that the ducks were serologically and virologically DTMUV-negative as determined by a blocking ELISA and RT-PCR , respect∘C. Parts of the tissues  were fixed with 10% neutral buffered formalin solution for histopathological examination, the rest were stored at -80∘C for RNA extraction. The remaining ducks at the end of this study were euthanized by the intravenous administration of sodium pentobarbital (100 mg/kg body weight).Eighty-one-days-old ducks were randomly divided into two groups of 50, and housed in the isolators separately. The ducks in group 1 were used for an infection experiment at 1 week, the other one was control group. Forty ducks in group 1 were intramuscular injection with 0.4 ml of virus stocks, control group were inoculated with 0.4 ml sterile phosphate-buffered saline (PBS) in the same manner. Ducks were observed continuously for 21 days, clinical symptoms and results of the autopsy were recorded. On 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 day post-inoculation (dpi), three ducks from each group were euthanized and necropsied. Serums were collected and stored at -20One-day-old ducks were raised in isolators until 3- and 7-weeks-old, respectively. The experimental design was the same as that of 1-week-old ducks.TM RT Regent Kit with gDNA Eraser Kit . The primers for viral E gene were designed as previously reported  following the manufacturer’s instructions. The concentration of each sample was measured using an ultraviolet spectrophotometer . First cDNA was synthesized from 1 μg total RNA using PrimeScript reported . For conreported . QuantitThe serums were detected by using the ELISA Kit of duck IL-2 and IFN-γ  following the manufacturer’s instruction. The serum neutralizing antibodies were determined by a blocking ELISA .P < 0.05.All data were expressed as means ± SD and processed by GraphPad Prism 5.0 . One-way ANOVA with Duncan’s multiple range test was used for evaluating data using SAS 9.1 software . Statistical significance was set at No clinical symptoms or necrotic lesions were observed throughout the experiment in control ducks. There were about twenty infected ducks showed loss of appetite and depression in the youngest challenged group after 2 dpi. At 3–4 dpi, almost 80% of ducks were reluctant to move, and showed white water-like diarrhea. At 5 dpi, six infected ducks died and three more were also dead at 7 dpi, moreover, about five infected ducks developed neurological signs, characterized by dystaxia and paralysis. At 9 dpi, the symptoms of the infected ducks gradually disappeared. In 3-weeks-old ducks inoculated with DTMUV, inappetence and decreased activity were observed from about eighteen ducks at 3 dpi. At 6 dpi, five ducks showed mild neurological signs, characterized by walking instability. At 7 dpi, feed intake in this group gradually recovered. Furthermore, no mortality was observed during the experiment. In 7-weeks-old ducks, about 20% infected ducks showed transient loss of appetite and depression at 3 dpi, but soon recovered at 6 dpi. No ducks displayed neurological symptoms or succumbed to infection.Figures 1A,B) and they also demonstrated a swollen liver with hemorrhage dot , splenomegaly with mottled surface , meningeal hyperemia , and mucosal swelling of the lymphoid follicles of the ileum . Taken together, gross lesions were similar between the 1- and 3-weeks-old ducks, but the latter displayed milder lesions with a lower lesion detection rate. No visible lesions were found in control ducks and 7-weeks-old ducks infected with DTMUV.At necropsy, bleeding of endocardium and epicardium of 1-week-old infected ducks was a significant lesion . Perivascular inflammatory infiltrates, glial nodules, and neuronophagia were observed at 5 dpi in the dead ducks , while at this time, the 3-weeks-old ducks showed widened gaps around small blood vessels. At 7 dpi, viral encephalitis became more severe in the youngest group, while the 3- and 7-weeks-old ducks demonstrated obvious but milder encephalitic lesions . At 9 dpi, the viral encephalitis in the ducks of all three ages gradually decreased.Histological analysis showed that the differently aged ducks inoculated with DTMUV had different histopathological changes. In 1-week-old ducks, cerebral vascular hyperemia and endothelial cell degeneration and swelling were found in the brain at 3 dpi . However, the older inoculated groups showed no significant lesions. At 5 dpi, degeneration and necrosis with scattered bleeding were observed in the youngest group of ducks , 3 weeks-old ducks showed granular degeneration and mild lymphocytic infiltration , but only cellular degeneration could be observed in the oldest ducks . Myocardium hemorrhage and a small amount of inflammatory infiltration were observed in 1-week-old ducks, but ducks in the other two groups merely showed granular degeneration at 7–9 dpi. No significant histological changes were observed in any infected ducks at 11 dpi.In the heart, 1-week-old ducks showed granular degeneration of myocardial fibers and lymphocytic infiltration at 3 dpi , while ducks in the other groups displayed moderate vacuolated degeneration, and heterophilic granulocyte and lymphocyte infiltration . At 9–11 dpi, degeneration of the liver decreased but the inflammatory infiltration increased in all infected ducks.In the liver, 1-week-old ducks displayed vacuolated degeneration of liver cells at 3 dpi, followed by granular degeneration in 3-weeks-old ducks, but 7-weeks-old ducks had no obvious lesions. At 5–7 dpi, examinations of the youngest ducks revealed severe vacuolated degeneration, necrosis, scattered bleeding and heterophilic granulocyte infiltration . This was also observed in the 3-weeks-old ducks, with decreased lymphocytes , but the eldest group of ducks demonstrated heterophilic granulocyte infiltration . At 7 dpi, all groups inoculated with DTMUV showed plentiful heterophilic granulocyte infiltration.In the spleen, obvious lymphocytic necrosis was observed in 1-week-old ducks infected with DTMUV at 3 dpi but not in the other experimental groups. The number of lymphocytes in the white pulp decreased significantly with bleeding and heterophilic granulocyte infiltration in the youngest group of ducks at 5 dpi , while the older groups only showed blood capillary congestion of pulmonary alveoli at 5–9 dpi.In the lung, blood capillary congestion of pulmonary alveoli was observed in 1-week-old ducks at 1–3 dpi. The youngest infected ducks displayed interstitial pneumonia with excessive inflammatory cell infiltration , while the older inoculated ducks showed only lymphoid hyperplasia and mild necrosis.Finally, inflammatory cell infiltration and mild necrosis of the lymphocytes were observed in the lymphoid follicles in the mucosal lamina propria of the ileum at 3 dpi in all three age groups. However, at 5–7 dpi, significant necrosis of lymphocytes was observed in 1-week-old ducks  in 1-week-old ducks at 1 dpi. Of particular note was that the concentration of virus reached 103.3 copies in the brain, yet no virus was detected in the brains of the older ducks, which indicating that DTMUV could more easily pass through the blood–brain barrier in younger ducks. Moreover, the highest virus titers were observed in spleens from all three experimental groups at 1 dpi. The virus titers in the lung from the youngest ducks were very significantly higher than those of 3- or 7-weeks-old infected ducks . At 3 dpi, maximum virus titers were achieved in the investigated tissues, except the spleen, and the virus could also be detected in the brains of 3- and 7-weeks-old ducks at this time point . Comparing between groups, the virus titers obtained from the youngest ducks were highest, and virus titers in heart and lung were 101.5–102.3 higher than those of the other two groups, with highly significant difference . In addition, virus titers in the brains from youngest ducks were 101.5 times more than in the other two groups, which was statistically significant . The viral titers of almost tissues began to decrease at 5 dpi, and it was noteworthy that DTMUV was not detectable in the liver and lung of 7-weeks-old infected ducks . However, the virus titers of heart, kidney, and brain in 1-week-old ducks were at high levels, and there was a statistically significant compared with the other two groups . Virus titers in the ducks succumbing to infection were higher than that of the ducks euthanised on the designated days (data not shown). At 7 dpi, virus titers of all the infected ducks continually declined, and it was undetectable in the heart, liver, lung, and brain of 7-weeks-old ducks as well as the liver and lung of 3-weeks-old ducks . At 9 dpi, all tissues from 1-weeks-old ducks showed detectable virus, but the virus titers of various tissues were lower than that obtained at 3 dpi in this age group, and titers were mostly maintained at 102–103 copies . Virus titers were undetectable in the liver, spleen, lung, and brain of 3-weeks-old ducks, and similarly, no virus was found in 7-weeks-old ducks . Generally speaking, virus titers obtained from the youngest age group of ducks were higher than those of the older during the tested time.The results of qRT-PCR to detect DTMUV copy numbers in tissues were shown in Figure 4A, IFN-γ levels increased rapidly in 1-week-old ducks at 1 dpi, and had a highly statistically significant difference compared with that of other two groups (P < 0.01). At 3 dpi, although IFN-γ levels of the two older groups increased rapidly, the levels of IFN-γ continued to rise and reached a peak value in the youngest group, and there was a significant difference between 1- and 3-weeks-old ducks (P < 0.05). However, IFN-γ levels of 1-week-old ducks began to decline at 5 dpi, while that of 3-weeks-old ducks reached the maximum, and that of the oldest group continued to increase and had a significant difference compared with the youngest group (P < 0.05). At 7 dpi, the expression of IFN-γ reached the peak in the 7-weeks-old ducks, which resulted in a highly significant difference between the youngest and oldest groups (P < 0.01). So did the levels of IFN-γ at 9 and 11 dpi . Subsequently, the levels of IFN-γ in all groups were similar. However, there was a significant difference between the older groups and 1-week-old ducks at 21 dpi (P < 0.05).As shown in Figure 4B, IL-2 levels of the three age groups were increased at 3 dpi and that of 1-week-old ducks reached a peak but there was no statistically significant difference. Consistent with the IFN-γ data, IL-2 levels of 1-week-old ducks decreased at 5 dpi, but that of the older age groups continued to increase and significant difference was exist between 1 and 3-weeks-old ducks (P < 0.05). At 7 dpi, IL-2 in the oldest ducks reached its maximum and had a very significantly difference compared with that of 1-week-old ducks (P < 0.01). Like the levels of IFN-γ, IL-2 levels also had a very significant difference among all groups at 9 dpi (P < 0.01). Of note was the significant difference at 19 dpi between the oldest and youngest groups (P < 0.05).As shown in Figure 5, positive serum could be detected in 7-weeks-old ducks as early as 3 dpi. A small amount of neutralizing antibody could be observed in 1-week-old ducks at 5 dpi, but the antibody titers of both older age groups had developed to high levels and were significantly higher than that of the youngest ducks (P < 0.05). Although the antibody titers of younger ducks continued to rise until reaching a peak value at 17 dpi, those of 3- and 7-weeks-old ducks remained at a high level at 19 and 21 dpi, and more important was that there was a significant difference between the youngest and oldest group (P < 0.05). Control ducks showed no seroconversion, as determined by blocking ELISA, throughout the experiment.As can be seen from Age-related outcomes of infection in ducks have been observed with West Nile virus, Japanese encephalitis virus, Hepatitis A/B virus and highly pathogenic avian influenza virus . As expeFigure 3). These results suggested that DTMUV had a special affinity for the central nervous system, and could cross the blood–brain barrier , which would result in immunosuppression and facilitate bacterial infections. Moreover, mucosal swelling of the lymphoid follicles of the ileum and the lymphocyte necrosis suggested DTMUV might also cause damage to the gastrointestinal tissues and mucosal immunity.The gross anatomical analysis of 1-week-old ducks infected with DTMUV, especially those dying from infection, showed significant symptoms, characterized by endocardium hemorrhage, hepatomegaly, and splenomegaly, swelling of the lymphoid follicles of the ileum and meningeal hyperemia. The results of necropsy in 3-weeks-old ducks were mild, mainly restricted to endocardium bleeding, hepatomegaly, splenomegaly, and meningeal hyperemia. Furthermore, there were no significant lesions in 7-weeks-old ducks. West Nile virus could cause obvious neurological signs in host . In the  barrier . FurtherP < 0.01, Figure 3B), which indicates that DTMUV could replicate more rapidly in 1-week-old ducks. At 5 dpi, virus titers in tissues from inoculated ducks declined quickly, particularly the 7-weeks-old ducks, virus became undetectable in the liver and lung, this phenomenon was more obvious in 3- and 7-weeks-old ducks from 7 to 9 dpi, this result showed that the older ducks could clear the virus more quickly.The pathogenicity of virus in ducks correlated with the viral titers in tissues. Virus was detectable in all tissues except brains of 3- and 7-weeks-old ducks at 1 dpi, indicating that DTMUV could rapidly spread after it entered the body and it was a pantropic virus. Amongst the investigated tissues, virus titers in spleens from all three age groups were highest at 1 dpi, suggesting the spleen might be a target organ of DTMUV, consistent with its microscopic lesion and the findings of other researchers . HoweverP < 0.01, Figure 4), which showed the immune response to DTMUV in 1-week-old ducks was lower than that of the older ducks. In addition, the levels of IL-2 and IFN-γ of the youngest ducks were significant lower than those of the older ducks at 19 and 21 dpi, respectively , which indicate 1-week-old ducks had shorter durations of immune response to virus. Neutralizing antibodies have been shown to contribute to the clearance of the virus , and significant difference was observed between 3- and 7-weeks-old ducks . The phenomenon may result from the destruction of the immune organs and (or) the immaturity of the immune system. Although the antibody titers of all three groups were similar from 9 to 17 dpi, reduced clearance of virus were observed in 1-week-old ducks comparing with the other two groups , which suggesting that immune system maturity was more important than the presence of antibody, despite the fact that neutralizing antibodies of 7-weeks-old ducks only existed for 3 days , but virus titers of multiple tissues were not detected at 5 dpi . In contrast, it recently reported that the titers of neutralizing antibodies of 5-weeks-old and 5-days-old infected ducks were detected at 7 and 10 dpi, respectively . The kinetics of neutralizing antibodies in infected ducks were consistent with that of IFN-γ or IL-2, which indicted that immune response to DTMUV in 1-week-old ducks was weaker than the older ducks, and this may be caused by immaturity of the immune system in 1-week-old ducks. On the whole, this study revealed the healthy 1-week-old ducks were more susceptible to DTMUV than the older ducks, and further demonstrated that the age-related immune response played a key role in resistance to DTMUV.Taken together, differently aged ducks had a significantly different susceptibility to DTMUV based on the clinical symptoms, necropsy analysis, histological changes and virus titers in tissues, and resistance increased as ducks matured. Here, we detected the levels of IFN-γ, IL-2, and neutralizing antibody in serum. The levels of IFN-γ and IL-2 rapidly increased but were maintained only for a short time, and highly significant difference was exist between the 1- and 7-weeks-old ducks at 7 dpi (he virus . The patectively . The reaectively . Howeverectively . Due to The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ph1 locus, which controls chromosome pairing and affects replication in wheat, was also studied by flow cytometry. Here we showed that the Ph1 locus plays an important role on the length of meiotic DNA replication in wheat, particularly affecting the rate of replication during early meiosis in wheat.Meiosis is a specialised cell division that involves chromosome replication, two rounds of chromosome segregation and results in the formation of the gametes. Meiotic DNA replication generally precedes chromosome pairing, recombination and synapsis in sexually developing eukaryotes. In this work, replication has been studied during premeiosis and early meiosis in wheat using flow cytometry, which has allowed the quantification of the amount of DNA in wheat anther in each phase of the cell cycle during premeiosis and each stage of early meiosis. Flow cytometry has been revealed as a suitable and user-friendly tool to detect and quantify DNA replication during early meiosis in wheat. Chromosome replication was detected in wheat during premeiosis and early meiosis until the stage of pachytene, when chromosomes are associated in pairs to further recombine and correctly segregate in the gametes. In addition, the effect of the  Meiosis is a specialised type of cell division common to sexually developing eukaryotes that generates four haploid gametes from a single diploid cell. The evolutionary trends of cell cycle including DNA replication, growth control and cell division are mechanistically well conserved among eukaryotes Duplication of the genome during S phase of the cell cycle is a highly organised process, usually followed in germinal cells by chromosome pairing of homologous  chromosomes, recombination and synapsis Ph1 locus Triticum aestivum L.) is a staple food for most of the world population, and understanding its genetics and genome organisation is of great value for genetics and plant breeders. The Ph1 locus controls homologous chromosome pairing in wheat Ph1-like gene in wheat shares some homology to Cdk2 in mammals, which regulates the progression of replication through controlling chromatin decondensation during S phase Ph1-like gene regulates premeiotic replication, chromatin condensation, transcription of the earliest meiotic gene (Asy1), homologue pairing/synapsis, resolution of incorrect pairing at pachytene and recombination Ph1 locus may affect replication through either an increment in the activation of origins and hence the rate of replication of the dispersed chromatin or, a delay in the initiation of heterochromatin replication in the absence of the Ph1 locus Pre-meiotic replication has been found to be connected to later events occurring in meiosis such as recombination and reductional chromosome segregation Flow cytometry has become a useful method for studying the characteristics of eukaryotic cells, with applications in crop and horticultural science Ph1 locus on the timing and on the rate of replication during early meiosis in wheat is also discussed.In this work we aimed to further our knowledge of pre-meiotic and meiotic replication in wheat, focusing in the early meiosis stages using flow cytometry. To achieve this, we established a quick and user-friendly flow cytometry-based method to investigate replication during meiosis in wheat through the quantification of the amount of DNA in each meiotic stage. Flow cytometry has been revealed as a rapid and robust method to quantify the amount of DNA during the five sub-stages  of early meiosis (prophase I) in wheat, and allowed a correlation between the amount of DNA and the level of replication at each stage during early meiosis in bread wheat. In addition, the effect of the Triticum aestivum L., 2n = 2x = 42) cv. Chinese Spring (CS) in the presence and in the absence of the Ph1 locus were kindly provided by Dr. Steve Reader from The John Innes Centre . DNA from wheat lines either in the presence or in the absence of the Ph1 locus were extracted from young frozen leaf tissue using the CTAB method ph1 mutants were checked for the ph1 deletion using the ABC920 SCAR marker as described previously Seeds of bread wheat (Seeds were germinated in the dark at 25°C on moistened filter paper in petri dishes for 2 days and then transferred into pots and grown in the greenhouse at 26°C during the day and 22°C at night with a photoperiod of long days (16 h of daylight).in situ hybridisation. Young leaves from both wheat lines were used as somatic control in flow cytometry experiments.Spikes were collected from plants entering meiosis, and fixed in 100% ethanol: acetic acid  for at least one week. Florets from fixed wheat spikes were checked under a phase-contrast microscope  for correct assignment of the meiotic stage. Each floret has three synchronous anthers, thus one anther per floret was squashed in 45% acetic acid in water and assigned to each meiotic stage by observation under a PrimoStar light microscope . The two remaining anthers were fixed in 100% ethanol: acetic acid 3∶1 (v/v) and used for flow cytometric analysis and in situ hybridisation. The telomeric sequence was amplified by PCR using the (5′-TTTAGGG-3′) and (5′- CCCTAAA-3′) primers in the absence of template DNA in situ hybridisation protocol was performed according to Fixed anthers were squashed in 45% acetic acid in water for Ph1 locus, were carried out on different days. In addition, 3 replicate measurements of each sample were taken for each biological replicate. At least 5000 nuclei were counted in each sample either in the presence or in the absence of the Ph1 locus and the coefficient of variation (CV) for each sample was always under 8.0%. The histograms were analysed using the Cylchred Software from Cardiff University developed by Terry Hoy, which is a cell cycle analysis software based on previously developed algorithms Wheat anthers at each meiotic stage were ground with a pestle in 400 µl nuclear extraction buffer of the Partec CyStain UV Precise T kit  for 3 min at room temperature following the instructions of the supplier. The suspension was filtered through a 30 µm nylon mesh filter to discard cell debris. Finally, each sample was stained with 4′, 6-diamidino-2-phenylindole (DAPI) for 1 min to measure the amount of nuclear DNA using a CyFlow Ploidy Analyser  equipped with an UV LED. Three independent experiments, consisting of the measurement of the DNA content of samples in leptotene, zygotene, pachytene, diplotene, diakinesis and metaphase I either in the presence or in the absence of the Statistical analyses were performed using STATISTIX 9.0 software . The analysis of variance (ANOVA) was based on randomised blocks. Means were separated using the Least Significant Difference (LSD) test with a probability level of 0.05.Ph1 locus. Each sample was then separated in three aliquots of 50 anthers each with the aim of having three independent replicates of each stage of meiosis, either in the presence or in the absence of the Ph1 locus for three independent experiments. In addition, three different flow cytometric measurements were taken from each sample in each experiment to account for equipment deviations.To study the progression of replication during early meiosis in wheat, one anther per floret was carefully checked to determine the meiotic stage using a light microscopy. Since all the anthers in the same flower are synchronised, the two remaining anthers were stored in 100% ethanol: acetic acid  at 4°C. The identification, selection and isolation of anthers was carried out until a total of 150 anthers were accumulated in each meiosis stage of prophase I  and metaphase I, either in the presence or in the absence of the Ph1 locus, was distributed in a histogram with two peaks corresponding to G0/G1 phases  and G2/M phases , respectively , to have a basal reference of unreplicated cells, replicated cells and somatic replication of each wheat line and, in addition, to monitor instrument or staining variations  was always at least five times lower than the minimum replication value obtained in early meiosis    and 3.in situ hybridisation was carried out to allow the visualisation of chromosome dynamics by labelling centromeres and telomeres  (+1.2) to pachytene (18.5+0.8). Then, replication remained constant from pachytene to diakinesis, but higher (15.5+1.2) than the replication value obtained for the somatic control (3.1+0.2). These results suggested that residual synthesis of DNA occurred in wheat after pachytene, when chromosomes are already associated in pairs.Interestingly enough, the number of cells under active replication in wheat anthers in premeiosis was much higher and significantly different than in the somatic control (30.7ctively) . In factctively) , Table 2+4.0 and 11.3+0.4 respectively), which correlates with replication in premeiosis in wheat anther. At the onset of meiosis the number of replicated cells increased from premeiosis (14.1+4.0) up to pachytene (21.0+1.0), being 1.9 times higher in pachytene than in the somatic control and confirming that replication actively occurs during early meiosis in wheat   was higher and statistically different in premeiosis than in the somatic tissue  .Cells going under active replication (S phase) were also clearly detected in early meiosis in wheat in the absence of the ytometry . In factctively) . Replicactively) . The levctively) , suggest+0.6 and 10.0+0.6, respectively). Moreover, the number of replicated cells did also increase during leptotene up to zygotene, where the level of replicated cells detected was almost double the number of replicated cells in premeiosis  during either premeiosis or any stage of prophase I . These differences may be due to the differences in mature anther size in relation to the presence and absence of the Ph1 locus, given that anthers in the ph1 mutant are slightly smaller than anthers in the presence of the Ph1 locus.The effect of the h1 locus . No diffophase I , Table 4Ph1 locus. Our results showed that at the onset of meiosis replication occurred similarly in both wheat lines with no statistical differences in the amount of DNA either in the presence or in the absence of the Ph1 locus (Ph1+ and Ph1-)  meanwhile in the absence of the Ph1 locus replication had already decreased and reached the basal level  , Table 4at lines ; Table 4Ph1 locus in any meiotic stage but in zygotene  in the absence of the Ph1 locus. Moreover, the gradient of the line of the cells number in G0/G1, G2/M and S phases was calculated during early meiosis until pachytene, as no significant differences were found between both lines after this meiotic stage at any time, either in the presence or in the absence of the Ph1 locus  in the absence of the Ph1 locus than in its presence, showing that the replication rate is higher in the absence of the Ph1 locus. As expected, the gradient of the line for the G2/M phase was also higher (1.76 times) in the absence of the Ph1 than in its presence, indicating that the rate of the increment in the number of replicated cells is higher in the absence of the Ph1 locus. All these results suggest that replication timing is affected by the presence of the Ph1 locus, in particular the rate of replication during early meiosis in wheat. The replication rate during meiosis is lower in the presence of the Ph1 locus. Consequently replication during meiosis in wheat lasts longer in the presence of the Ph1 locus.These data also suggest that there is no significant differences for the G2/M value between wheat lines in the presence and in the absence of the zygotene , Table 4h1 locus . Results3H] thymidine, 5-bromo-2′-deoxyuridine (BrdU) or 5-ethynyl-2′-deoxyuridine (EdU), which is highly sensitive Lactuca sativa L., Solanum melongena L., and Lycopersicon esculentatum Mill., among other species Ph1 locus, which controls chromosome pairing Ph1 locus, similarly to the observations of Ph1 locus. Thus replication last longer (until pachytene) during early meiosis in the presence of the Ph1 locus, or in other words, replication finished earlier (zygotene) when the Ph1 locus was absent. In fact, our analysis of the slope of the lines at early meiosis indicates that the rate of replication during meiosis is higher in the absence of the Ph1 locus. Due to the fact that the Ph1 locus is similar to Cdk2 Ph1 locus, in that the activation of origins of replication might be increased and consequently the rate of replication.The cell cycle is a much studied process due to its importance in plant growth and development. The significance of replication during the cell cycle is critical to ensure proper chromosome association, recombination and segregation in meiosis, which is directly related to viability of gametes and therefore to fertility. This paper presents a simple and robust method for the determination of the synthesis of DNA during early meiosis by means of flow cytometric measurements in nuclei released from fixed wheat anthers. The synthesis of DNA has been studied using different deoxynucleosides, such as [Ph1 locus, respectively, when chromosomes are associated in pairs. Replication at this stage of meiosis corresponds not only to heterochromatin regions which replicate later in meiosis Residual replication was also detected in wheat anthers at later meiosis stages, after pachytene and zygotene in wheat in the presence and in the absence of the Ph1 locus to facilitate correct chromosome associations, and consequently replication takes longer. This could also suggest that only homologous chromosomes are replicating at the same, which could be associated with similar homologous chromosome conformation at early meiosis in wheat, as it has previously described Ph1 locus, explained as an increment in the activation of origins and hence the rate of replication of disperse chromatin in wheat-rye hybrids in the absence of the Ph1 locus The structure of chromatin has been shown determinant for the initiation of replication Ph1 locus is affecting the rate of replication during early meiosis in wheat, being lower in the presence of the Ph1 locus and consequently, replication during early meiosis lasts longer and finishes later than in the absence of the Ph1 locus. The biological significance of replication at early meiosis and the effect of the Ph1 locus in replication suggest a solid connection between DNA replication and chromosome associations at the onset of meiosis in a polyploid like wheat. Further studies are needed to build upon these results for unravelling the underlying molecular mechanisms.In summary, flow cytometry has been revealed as a suitable tool to detect and quantify DNA replication during early meiosis in wheat. Replication was detected in wheat during premeiosis and early meiosis until the stage of pachytene, when chromosomes are associated in pairs to further recombine and correctly segregate in the gametes. Moreover, flow cytometric results suggested that the


Plantago ovata F.) husk mucilage- (IHM-) alginate mucoadhesive beads containing glibenclamide by ionotropic gelation technique. The effects of sodium alginate (SA) to IHM and cross-linker (CaCl2) concentration on the drug encapsulation efficiency , as well as cumulative drug release after 10 hours , were optimized using 32 factorial design based on response surface methodology. The observed responses were coincided well with the predicted values by the experimental design. The optimized mucoadhesive beads exhibited 94.43 ± 4.80% w/w of DEE and good mucoadhesivity with the biological membrane in wash-off test and sustained drug release profile over 10 hours. The beads were also characterized by SEM and FTIR analyses. The in vitro drug release from these beads was followed by controlled release (zero-order) pattern with super case-II transport mechanism. The optimized glibenclamide-loaded IHM-alginate mucoadhesive beads showed significant antidiabetic effect in alloxan-induced diabetic rats over prolonged period after oral administration.The current study deals with the development and optimization of ispaghula ( The mucoadhesive polymer containing oral drug delivery systems has the capacity to prolong residence time of drugs at the absorption site and facilitate intimate contact with underlying absorptive surface to enhance bioavailability . PolymerLaminaria hyperborean, Ascophullum nodosum, and Macrocystis pyrifera [β-D-mannuronic acid and α-L-guluronic acid [2+ to form calcium alginate due to an ionic interaction between carboxylic acid groups of alginate chain and Ca2+ [In recent years, a growing interest has been identified in the development of natural polymer-based drug delivery systems due to their biodegradability, biocompatibility, aqueous solubility, swelling ability, easy availability, and cost-effectiveness . Amongstpyrifera . It is cnic acid . Sodium and Ca2+ . Sodium and Ca2+ , 10. Theand Ca2+ , 11, 12.and Ca2+ .Plantago ovata F.) husk is an indigenous product of south Asia and is widely used herbal product both in traditional and modern medicines [Ispaghula  , 19. Its2 (two factors and three levels) factorial design and response surface methodology was employed to investigate the effects of two independent process variables (factors), that is, SA: IHM and cross-linker (CaCl2) concentration on the properties of glibenclamide-loaded ionotropically gelled IHM-alginate beads such as drug encapsulation and drug release.In the development of any pharmaceutical formulation, an important issue is to design a formulation with optimized quality in a short time period and minimum number of trials , 21. TraGlibenclamide , SA , ispaghula husk , and calcium chloride  were used for this investigation. All other chemicals and reagents used were of analytical grade.Plantago ovata F.) husk was extracted according to the previously reported method [Plantago ovata F. husk was added. Heating was resumed and the process of husk dissolution was watched. After total change of color, the flask was finally removed from heat and the solution was filtered through a clean muslin cloth while still hot. In order to separate residual traces of mucilage, the seeds were washed twice with 5 mL of hot water and the solution obtained each time was filtered. The combined filtrate, containing the dissolved mucilage, was mixed with 60 mL of 95% ethyl alcohol, stirred, and allowed to stand for 5 hours. Finally, the supernatant liquid was decanted and the precipitate in the beaker was dried in an oven at 50°C. The dried IHM cake was grounded with a mortar and passed through a sieve (0.15 mm mesh size). The isolated IHM powders were packed in a plastic bag and kept airtight desiccators until further use.The mucilage from ispaghula  as cross-linker. Briefly, SA and IHM aqueous dispersions were prepared separately using distilled water. These dispersions were well mixed with stirring for 15 min at 1000 rpm using a magnetic stirrer . Afterwards, glibenclamide was added to the dispersion mixture. The ratio of drug to polymer was maintained 1 : 2 in all formulations. The final mixtures were homogenized for 15 min at 1000 rpm using a homogenizer  and ultrasonicated for 5 minutes for debubbling. The resulting dispersion was then added via a 21-gauge needle. The added droplets were retained in the CaCl2 solution for 15 min to complete the curing reaction and to produce rigid beads. The wet beads were collected by decantation and washed two times with distilled water and dried at 37°C for 24 h. The dried glibenclamide-loaded IHM-alginate beads were stored in a desiccator until used.The glibenclamide-loaded IHM-alginate beads were prepared by ionotropic-gelation technique using calcium chloride  and concentration of CaCl2 (X2) as the prime selected independent variables, which were varied at three levels, low (–1), medium (0), and high (+1). The drug encapsulation efficiency  and 10 hours  were used as dependent variables (responses). Design-Expert Version 8.0.6.1 software  was used for the generation and evaluation of the statistical experimental design. The matrix of the design including investigated responses, that is, DEE and R10 h, is shown in 2 factorial design [Y is the response, b0 is the intercept, and b1, b2, b3, b4, b5 are regression coefficients. X1 and X2 are individual effects; X12 and X22 are quadratic effects; X1X2 is the interaction effect. One-way ANOVA was applied to estimate the significance (P < 0.05) of generated models. Individual response parameters were evaluated using the F-test. The response surface methodology was applied to analyze the effect of independent factors (SA: IHM and CaCl2 concentration) on the measured responses (DEE and R10 h). A 3l design :(1)Y=b0 Accurately weighed, 100 mg of beads were taken and were crushed using pestle and mortar. The crushed powders of drug containing beads were placed in 500 mL of phosphate buffer, pH 7.4, and kept for 24 hours with occasionally shaking at 37 ± 0.5°C. After the stipulated time, the mixture was stirred at 500 rpm for 15 min on a magnetic stirrer. The polymer debris formed after disintegration of bead was removed filtering through Whatman filter paper (No. 40). The drug content in the filtrate was determined using a UV-VIS spectrophotometer  at 228.5 nm. The DEE of beads was calculated using this following formula: Particle size of 100 dried beads from each batch was measured by optical microscopic method for average particle size using an optical microscope (Olympus). The ocular micrometer was previously calibrated by stage micrometer. Samples were gold coated by mounted on a brass stub using double-sided adhesive tape and under vacuum in an ion sputter with a thin layer of gold (3 ~ 5 nm) for 75 seconds and at 20 kV to make them electrically conductive, and their morphology was examined by scanning electron microscope .−1 at a resolution of 4 cm−1 with scan speed of 1 cm/second.Samples were reduced to powder and analyzed as KBr pellets by using a Fourier transform-infrared (FTIR) spectroscope . The pellet was placed in the sample holder. Spectral scanning was taken in the wavelength region between 4000 and 400 cm The release of glibenclamide from various IHM-alginate beads was tested using a dissolution apparatus USP . The baskets were covered with 100-mesh nylon cloth to prevent the escape of the beads. The dissolution rates were measured at 37 ± 1°C under 50 rpm speed. Glibenclamide-loaded IHM-alginate beads equivalent to 30 mg glibenclamide were taken in 900 mL of dissolution medium . An amount of 5 mL of aliquots was collected at regular time intervals, and the same amount of fresh dissolution medium was replaced into the dissolution vessel to maintain sink condition throughout the experiment. The collected aliquots were filtered and suitably diluted to determine absorbance using a UV-VIS spectrophotometer  at 228.5 nm against appropriate blank.in vitro release behaviour of glibenclamide from formulated glibenclamide-loaded IHM-alginate beads, it is necessary to fit into a suitable mathematical model. The in vitro drug release data were evaluated kinetically in different mathematical models [(i)Zero-Order Model: Q = kt + Q0, where Q represents the drug released amount in time t, and Q0 is the start value of Q; k is the rate constant;(ii)First-Order Model: Q = Q0ekt, where Q represents the drug released amount in time t, and Q0 is the start value of Q; k is the rate constant;(iii)Higuchi Model: Q = kt0.5, where Q represents the drug released amount in time t, and k is the rate constant;(iv)Korsmeyer-Peppas Model: Q = ktn, where Q represents the drug released amount in time t, k is the rate constant, and n is the diffusional exponent, indicative of drug release mechanism.In order to predict and correlate the l models , 6: (i)in vitro drug release behaviour analysis of these formulations to distinguish between competing release mechanisms [n is ≤0.43, it is Fickian release. The n value between 0.43 and 0.85 is defined as non-Fickian release. When n ≥ 0.85, it is case-II transport.The Korsmeyer-Peppas model was also employed in the chanisms , 6: Fickex vivo wash-off method [The mucoadhesivity of optimized glibenclamide-loaded IHM-alginate beads was evaluated by f method , 12. Fre In vivo studies were performed in alloxan-induced diabetic albino rats [ad libitum. All experiments were performed between 8 AM to 12 PM to minimize circadian influences. The experimental protocol was subjected to the scrutiny of the Institutional Animal Ethical Committee and was cleared before starting. The animals were handled as per guidelines of committee for the purpose of control and supervision on experimental animals (CPCSEA). All efforts were made to minimize both the suffering and number of animals used. ino rats  of eithe The rats were made diabetic by intraperitoneal administration of freshly prepared alloxan solution at a dose of 150 mg/kg dissolved in 2 mM citrate buffer (pH 3.0). After one week of alloxan administration, alloxanized rats with fasting blood glucose of 300 mg/dL or more were considered diabetic and were employed in the study for 12 hours. The alloxan-induced diabetic rats were divided randomly into 2 groups of 6 rats each and treated as follows. Group A was administered with pure glibenclamide in suspension form, and Group B was administered with optimized formulation of glibenclamide-loaded IHM-alginate beads, both at a dose equivalent to 5 mg glibenclamide/kg body weight using oral feeding needle. Blood samples were withdrawn (0.1 mL) from tail tip of each rat at regular time intervals under mild ether anesthesia and were analyzed for blood glucose by oxidase-peroxidase method using Accu-Chek Sensor Comfort  test strips.in vivo data were tested for significant differences (P < 0.05) by paired samples t-test. All other data was analyzed with simple statistics. The simple statistical analysis and paired samples t-test were conducted using MedCalc software, version 11.6.1.0.Statistical optimization was performed using Design-Expert Version 8.0.6.1 software . The Plantago ovata F.) husk, and the average yield of IHM was found 39.86% w/w. IHM was isolated from ispaghula  and glibenclamide were dropped into the solutions containing calcium ions, gelled glibenclamide-loaded IHM-alginate beads were formed instantaneously due to electrostatic interaction between negatively charged alginate ions and positively charged calcium ions present in the cross-linking solutions. For the 3R10 h) was presented in R10 h, measured for all trial formulations, were fitted in the 32 factorial design to get model equations. The Design-Expert Version 8.0.6.1 software provided quadratic model equations involving individual main factors and interaction factors for all response parameters. The results of the ANOVA indicated that these models were significant for all response parameters . The model equation relating DEE  (%) as response became DEE  (%) = 87.24−  5.22R10 h(%) as response became R10 h  (%) = 83.94+  2.37X1+  0.01X2+  0.34X1X2−  0.13X12 − 0.23X22  . The model equation relating P > 0.05) from previously mentioned model equations [X1 − 2.49X2 − 0.06X1X2 + 0.41X12+  0.35X22 and R10 h(%) = 83.94 + 2.37X1 + 0.01X2 + 0.34X1X2 − 0.23X22.Model simplification was carried out by eliminating nonsignificant terms  were further elucidated by response surface methodology. Response surface methodology is a widely proficient approach in the development and optimization of drug delivery devices [s) [X1) and increasing of CaCl2 concentration (X2). However, an increment in R10 h values with the increasing of SA: IHM (X1) and lowering of CaCl2 concentration (X2) is indicated by the three-dimensional response surface plot relating R10 h  . The thrices [s) . The thrg R10 h . All theg R10 h  and 8 shX1 ≤ 1.50 and 9.50 ≤ X2 ≤ 11.50, whereas the desirable ranges of responses were restricted to 95.00 ≤ DEE ≤ 100.00% and 60.00 ≤ R10 h ≤ 65.00%. The optimal values of responses were obtained by numerical analysis using the Design-Expert Version 8.0.6.1 software based on the criterion of desirability. The desirability plot indicating desirable regression ranges for optimal process variable settings was presented in 2 factorial design, optimized glibenclamide-loaded IHM-alginate beads were prepared using one of the optimal process variable settings proposed by the design (prediction R2 = 1). The selected optimal process variable setting used for the formulation of optimized formulation was X1 = 1.35 and X2 = 10.99. The optimized beads containing glibenclamide (F-O) were evaluated for DEE  (%) and R10 h(%). R10 h of 65.78 ± 3.44% with small error values , indicating that mathematical models obtained from the 32 factorial design were fitted well. Numerical optimization technique using the desirability approach was employed to develop optimized formulations with desired response . The desirable ranges of the independable variables (factors) were restricted to 1.00 ≤ 2. Again, the DEE of these beads was increased with increasing CaCl2 concentration in cross-linking solutions, due to the high degree of cross-linking by the concentrated calcium ions. The glibenclamide-loaded IHM-alginate beads prepared using lower CaCl2 concentration might have larger pores due to insufficient cross-linking, and drug leaching may occur through the pores that may result in lower drug encapsulation [The DEE  (%) of all these glibenclamide-loaded IHM-alginate beads was within the range between 68.03 ± 1.77 and 94.43 ± 4.80% w/w  for first 2 hours and then in phosphate buffer  for next 8 hours. All these beads showed prolonged glibenclamide release over 10 hours  was within the range of 65.78 ± 3.44% to 92.07 ± 4.05%, and this was found to be higher with the decreasing SA to IHM ratio in the polymer-blend and increasing CaCl2 concentration in cross-linking solution. In case of comparatively higher IHM containing beads, the more hydrophilic property of IHM could bond better with water to form viscous gel-structure. This might blockade the pores on the surface of beads and sustain drug release profile. Again, the glibenclamide release from IHM-alginate beads prepared using higher CaCl2 concentration was comparatively sustained than the beads formulated with that of lower concentration. The higher concentration of CaCl2 (cross-linker) could produce high degree of cross-linking and thereby slower the drug release from highly cross-linked glibenclamide-loaded IHM-alginate beads. The 10 hours . Glibencin vitro drug release data from various glibenclamide-loaded IHM-alginate beads were evaluated kinetically using various mathematical models like zero-order, first-order, Higuchi, and Korsmeyer-Peppas models. The accuracy and prediction ability of these models was determined using regression analysis. The result of the curve fitting (R2) into various mathematical models is given in R2 of glibenclamide-loaded IHM-alginate beads was compared, it was found to follow the zero-order model (R2 = 0.992 to 0.997) over a period of 10 hours. This was also observed to be closest to Korsmeyer-Peppas model (R2 = 0.985 to 0.994). The best fit of zero-order model indicated that the glibenclamide release from these IHM-alginate beads followed controlled-release pattern. The values of diffusional exponent (n) determined from Korsmeyer-Peppas model ranged from 1.025 to 1.115, indicating the drug release from these glibenclamide-loaded IHM-alginate beads following the super case-II transport mechanism controlled by swelling and relaxation of polymeric-blend (SA-IHM) matrix. This could be attributed due to polymer dissolution and polymeric chain enlargement or relaxation. The ex vivo wash-off behavior of optimized glibenclamide-loaded IHM-alginate beads (F-O) using goat intestinal mucosa was found faster in intestinal pH (7.4) than that in gastric pH (1.2). In gastric pH, the percentage of beads adhered onto the goat intestinal mucosal tissue varied from 64.88 ± 5.06% over 10 hours, whereas this was 30.47 ± 3.86% in intestinal pH  are presented in Figures P < 0.05) were found between the blood glucose levels after administration of pure glibenclamide and optimized glibenclamide-loaded IHM-alginate mucoadhesive beads (F-O) at each time point measured. However, the reductions in glucose level were increased gradually with the increment of time in case of Group B  and were sustained over 10 hours. A 25% reduction in glucose level is considered a significant hypoglycemic effect [In alloxan-induced diabetic rats, the comparative c effect . TherefoIn this investigation, glibenclamide-loaded IHM-alginate mucoadhesive beads were successfully developed and optimized. These developed optimized mucoadhesive beads demonstrated high drug encapsulation, good mucoadhesivity with the biological membrane, sustained drug release profile at a controlled rate, and significant antidiabetic activity in alloxan-induced diabetic rats over prolonged period after oral administration. Therefore, these glibenclamide-loaded IHM-alginate mucoadhesive beads were found suitable for prolonged systemic absorption of glibenclamide through sustained drug release and mucoadhesive properties after oral administration maintaining tight blood glucose level and improved patient compliance in the management of non-insulin-dependent diabetes mellitus. Moreover, the technique for the preparation of these beads was found simple, economical, and consistent. This type of beads can also be exploited for drug delivery of other drugs to improve their bioavailability and therapeutic efficacy.