mteb/CovidQA huggingface dataset:
Cells of the Colon. Initially, the cytotoxicity of Cu(BrHAP) 2 was tested on HT-29 and CCD 841 cell lines. The IC 50 values of the Schiff base compound were determined based on the result collected from three independent MTT experiments. As indicated in Table 1 , Cu(BrHAP) 2 elicited a significant cytotoxicity and cell inhibitory effect after 24, 48, and 72 h of treatment on HT-29 cell. 2 -Induced LDH Release. Lactate dehydrogenase (LDH) release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of the Cu(BrHAP) 2 compound, as determined by the LDH release assay, was quantified on HT-29 cells treated with various concentrations of the Schiff base compound for 48 h. Cu(BrHAP) 2 induced a significant elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 g/mL concentrations compared to the control cells ( Figure 2 ).
Microscopy and AO/PI Double Staining. Morphological changes in HT-29 cells treated with Cu(BrHAP) 2 compound were observed under a fluorescent microscope at 24, 48, and 72 h. The cells were scored under a fluorescent microscope to analyze viable cells, early apoptosis, and late apoptosis. Early apoptosis, defined as intervening AO within the fragmented DNA, was observed under bright green fluorescence. At the same time, control cells were visualized with a green intact nuclear structure. After 24 and 48 h of treatment with Cu(BrHAP) 2 , moderate apoptosis was observed in the form of blebbing and nuclear chromatin condensation. Furthermore, in the late stage of apoptosis, changes, such as the presence of a reddish-orange color due to binding of PI to denatured DNA, were observed after 72 h of treatment ( Figure 3) . The results showed that the Cu(BrHAP) 2 compound induced morphological features of apoptosis in a time-dependent manner. Figure 4 , demonstrated that there is no cell cycle
arrest in the S/M phases. The lack of cell cycle arrest in the S/M phases suggested possible cell cycle arrest in the G 1 /G 2 phases. To determine the exact arrested phase, treated HT-29 cells were analyzed for cell cycle progression using flow cytometry. As expected, there was no significant arrest in the S/M phases. Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment ( Figure 5 ).
Assay. Antioxidant capacity was measured by ORAC assay, which is the only assay that involves the use of peroxyl radical as a prooxidant and quantifies activity via the area under the curve (AUC) technique. In our experiment, quercetin was used as a positive control. The result demonstrated that Cu(BrHAP) 2 exhibited low to moderate antioxidant activity compared to quercetin ( Table 2) .

Formation. HT-29 cells were treated with different concentrations of Cu(BrHAP) 2 for 24 h and stained with DHE dye to determine the influence of the Schiff base compound on ROS production. The fluorescence intensities of DHE oxidization by ROS were quantified using a fluorescence microplate reader. As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration.
To investigate the induction of apoptosis by Cu(BrHAP) 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation. As shown in Figure 7 , Hoechst 33342 staining demonstrated that nuclear condensation, which is directly related to apoptotic chromatin changes, emerged in some cells after treatment with Cu(BrHAP) 2 . Meanwhile, the permeability of treated cells was also elevated. Mitochondria are the main source for the production of ROS and adenosine triphosphate (ATP) and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu(BrHAP) 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells.
Activation. The elevation in ROS production associated with a collapse in MMP may lead to the activation of the caspase cascade. To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu(BrHAP) 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu(BrHAP) 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway.
is a transcription factor that has a critical role in cytokine gene expression. NF-B activation and translocation to the nucleus to enable DNA-binding activity and facilitate target gene expression are mediated by inflammatory cytokines such as tumor necrosis factor-(TNF-). The Cu(BrHAP) 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus (Figure 9 ).
Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression [21] . One of the defense procedures in multicellular organisms is the destruction of undesirable cell development, which is defined as programmed cell death. Apoptosis is the most noticed programmed cell death mechanism and is characterized by distinct morphological changes such as membrane permeability, cell shrinkage, disruption of the mitochondrial membrane, and chromatin condensation [22, 23]. The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence [24] , and agents that can induce apoptosis are known to have potential anticancer effects [25, 26] . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention. Numerous chemopreventive drugs have been determined to regulate key events or molecules in apoptosis-inducing signal
transduction pathways [27] . In the present study, the Cu(BrHAP) 2 Schiff base compound was evaluated for its ability to inhibit the growth of HT-29 cells using an MTT assay. HT-29 cells have recently been characterized as a suitable model for colon cancer studies [28] [29] [30] . human colon cancer cells in a time-and dose-dependent manner. Meanwhile, the nontumorigenic colon cell line (CCD 841) showed no cytotoxicity after treatment with the compound. The cytotoxic effect of the Cu(II) compound was also confirmed by measuring the level of LDH release from treated cells. Considerably elevated LDH release showed that the cytotoxicity of the Cu(BrHAP) 2 compound possibly occurred via the loss of membrane integrity, whether through activation of apoptosis or the necrosis pathway [31] . The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and AO/PI double staining following treatment of HT-29 cells with the compound included some signs of apoptosis,
namely, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation [32, 33] . We found that the number of cells with early apoptosis features was higher at earlier stages of treatment. However, when treatment time increased to 72 h, late apoptosis or necrosis characterizations were dominant among treated HT-29 cells. Concurrent detection of late apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis may have progressed into necrosis due to the prolonged incubation with the Schiff base compound.
To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu(II) complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone H3 staining along with flow cytometry [34] [35] [36] . BrdU dye can attach to the synthesized DNA of replicating cells during the S phase of the cell cycle, while Phospho-Histone H3 dye stains cells in different mitotic stages. The cell cycle results from the BrdU and Phospho-Histone H3 double staining assay indicated that there were no significant changes in the number of cells in the S/M phases after the exposure of HT-29 cells to the Schiff base compound. This result suggests the possibility that the cells were arrested in the G 1 or G 2 phase of the cell cycle. Thus, the flow cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the results demonstrated significant cell cycle arrest at G 1 after 24 and 48 h of treatment, suggesting proliferative suppression via
induction of apoptosis [37, 38] .
Perturbation of mitochondrial membrane potential is one of the earliest intracellular events that occur following the induction of apoptosis [39] . As the main source of cellular ROS and adenosine triphosphate (ATP), mitochondria are the key regulators of mechanisms controlling the survival or death of cells. After confirming that the Cu(BrHAP) 2 Schiff base compound did not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the induction of ROS production in treated cells was analyzed. According to our study, after exposing the Cu(II) compound to HT-29 cells and analyzing the levels of ROS, it was demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a compound concentration of 6.25 g/mL.
In metal-induced apoptosis, the mitochondria have the crucial role in mediating apoptosis through metal-induced ROS [40] . The intrinsic or mitochondrial-dependent signaling pathway involves different factors of nonreceptor-mediated stimuli that induce intracellular signals. These signals, mainly through the p53 protein, act on the mitochondrialinitiated events. Excessive ROS production is a negative signal that can result in the failure of suppression of antiapoptotic factors, thereby triggering apoptosis. Therefore, we used mitochondrial membrane potential (MMP) fluorescent probes to examine the effect of elevated ROS production on the function of mitochondria in treated HT-29 cells. As shown in Figure 7 , changes in MMP after treatment with the Cu(BrHAP) 2 Schiff base compound leading to the membrane depolarization of the mitochondria were demonstrated by Rhodamine 123 release to the cytoplasm from the mitochondria matrix. The result implies that the induction of apoptosis by
Cu(II) Schiff base complexes may be associated with the mitochondrial pathway [26, 41, 42] . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome . The release of cytochrome into the cytosol and reduction of its levels in the mitochondria have been shown to occur as a result of changes in MMP [30] . As the result illustrated, the synthetic Schiff base compound also led to an increase in the level of cytochrome in the cytosol compared to the control.
The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets [43] . In the extrinsic pathway, apoptosis is mediated by death receptors. As an example, FAS ligand interacts with the FAS receptor, leading to the activation of caspase-8 [44] . Caspase-8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 [45, 46] . In our study, the Cu(BrHAP) 2 schiff base compound induced significant elevation in the caspases 3/7 and 9 activities compared to the control. Meanwhile, there was no activation of caspase-8, suggesting that the apoptosis induced in HT-29 cells was mediated via the intrinsic mitochondrial pathway but not the extrinsic, death receptor-linked
caspase-8 pathway.
The supporting evidence of LDH release, ROS production, MMP suppression, elevation in the level of cytochrome , and activation of caspases 3/7 and 9 demonstrated the promising anticancer activity of the Cu(BrHAP) 2 Schiff base compound against the HT-29 colon cancer cell line via the intrinsic mitochondrial pathway.
Multivalent HA DNA Vaccination Protects against Highly Pathogenic H5N1 Avian Influenza Infection in Chickens and Mice

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2657001/

SHA: ef872b80cf38917f64c42bfa52a57beb4399897a

Authors: Rao, Srinivas; Kong, Wing-Pui; Wei, Chih-Jen; Yang, Zhi-Yong; Nason, Martha; Styles, Darrel; DeTolla, Louis J.; Sorrell, Erin M.; Song, Haichen; Wan, Hongquan; Ramirez-Nieto, Gloria C.; Perez, Daniel; Nabel, Gary J.
Date: 2008-06-18
DOI: 10.1371/journal.pone.0002432
License: cc0
Abstract: BACKGROUND: Sustained outbreaks of highly pathogenic avian influenza (HPAI) H5N1 in avian species increase the risk of reassortment and adaptation to humans. The ability to contain its spread in chickens would reduce this threat and help maintain the capacity for egg-based vaccine production. While vaccines offer the potential to control avian disease, a major concern of current vaccines is their potency and inability to protect against evolving avian influenza viruses. METHODOLOGY / PRINCIPAL FINDINGS: The ability of DNA vaccines encoding hemagglutinin (HA) proteins from different HPAI H5N1 serotypes was evaluated for its ability to elicit neutralizing antibodies and to protect against homologous and heterologous HPAI H5N1 strain challenge in mice and chickens after DNA immunization by needle and syringe or with a pressure injection device. These vaccines elicited antibodies that neutralized multiple strains of HPAI H5N1 when given in combinations containing up to 10 HAs.
The response was dose-dependent, and breadth was determined by the choice of the influenza virus HA in the vaccine. Monovalent and trivalent HA vaccines were tested first in mice and conferred protection against lethal H5N1 A/Vietnam/1203/2004 challenge 68 weeks after vaccination. In chickens, protection was observed against heterologous strains of HPAI H5N1 after vaccination with a trivalent H5 serotype DNA vaccine with doses as low as 5 µg DNA given twice either by intramuscular needle injection or with a needle-free device. CONCLUSIONS/SIGNIFICANCE: DNA vaccines offer a generic approach to influenza virus immunization applicable to multiple animal species. In addition, the ability to substitute plasmids encoding different strains enables rapid adaptation of the vaccine to newly evolving field isolates.
Text: The highly pathogenic H5N1 influenza virus causes lethal multi-organ disease in poultry, resulting in significant economic losses and a public health concern in many parts of the world. The greatest threats posed by this virus are its ability to cause mortality in humans, its potential to compromise food supplies, and its possible economic impacts. Viral maintenance in poultry potentiates the risk of human-to-human transmission and the emergence of a pandemic strain through reassortment. An effective, safe poultry vaccine that elicits broadly protective immune responses to evolving flu strains would provide a countermeasure to reduce the likelihood of transmission of this virus from domestic birds to humans and simultaneously would protect commercial poultry operations and subsistence farmers.
DNA vaccines have been shown to elicit robust immune responses in various animal species, from mice to nonhuman primates [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] . In human trials, these vaccines elicit cellular and humoral immune responses against various infectious agents, including influenza, SARS, SIV and HIV. In addition to their ability to elicit antibody responses, they also stimulate antigenspecific and sustained T cell responses [1] [2] [3] 6, 12, 13] . DNA vaccination has been used experimentally against various infectious agents in a variety of mammals, including cattle (against infectious bovine rhinotracheitis/bovine diarrhea virus, leptospirosis and mycobacteriosis) [14, 15] , pigs (against classical swine fever virus and mycoplasmosis) [16] , and horses (against West Nile virus and rabies) [17] . In addition, DNA vaccines have been tested against avian plasmodium infection in penguins [18] and against influenza and infectious bursal disease in chickens [7, 8, 19] ,
duck hepatitis B virus in ducks [6] , and avian metapneumovirus and Chlamydia psittaci in turkeys [20, 21] (reviewed in ref. [22] ). While they have been used in chickens to generate antisera to specific influenza viruses and confer protection against the low pathogenicity H5N2 strain [23] , there is only one previous report of a monovalent DNA vaccine effective against H5N1 (and that only against a matched H5N1 isolate) [24] ; no protection with multivalent DNA vaccines against heterologous strains has been reported.
Development and characterization of a DNA vaccine modality for use in poultry offers a potential countermeasure against HPAI H5N1 avian influenza outbreaks. The virus can infect humans, typically from animal sources, including commercial and wild avian species, livestock, and possibly other non-domesticated animal species [25] [26] [27] . While there is marked diversity in the host range of type A influenza viruses, many experts have speculated that a pandemic strain of type A influenza could evolve in avian species or avian influenza viruses could contribute virulent genes to a pandemic strain through reassortment [28, 29] . Thus, there is reason to consider vaccination of poultry that would stimulate potent and broad protective immune responses [7, 30, 31] . In undertaking such efforts, it is important that there be a differentiation of infected from vaccinated animals [32] so that animals can be protected and permit monitoring of new infections using proven and sensitive
In this study, we used an automated high capacity needle-free injection device, Agro-JetH (Medical International Technology, Inc., Denver, CO) to explore the feasibility of DNA vaccination of poultry. After optimization of injection conditions, alternative multivalent DNA vaccine regimens were analyzed and compared for magnitude and breadth of neutralizing antibodies, as well as protective efficacy after challenge in mouse and chicken models of HPAI H5N1 infection. The findings suggest that it is possible to develop a multivalent DNA vaccine for poultry that can protect against multiple HPAI H5N1 strains and that could keep pace with the continued evolution of avian influenza viruses.
Immunogenicity and neutralizing antibody specificity of alternative HA DNA vaccines in mice To evaluate the efficacy of multivalent DNA vaccines, initial studies were performed in mice. Expression vectors encoding HAs from ten phylogenetically diverse strains of influenza viruses [33] were generated by synthesis of cDNAs (see Materials and Methods) in plasmid expression vectors, pCMV/R or pCMV/R 8kB, which mediates high level expression and immunogenicity in vivo [34, 35, 36] . Animals were immunized with each expression vector intramuscularly (IM) at three week intervals, and the antisera were evaluated on day 14 after the third immunization for their ability to neutralize HPAI H5N1 pseudotyped lentiviral vectors as previously described [35, 36] . We have previously shown that lentiviral assay inhibition (LAI) yields similar results to microneutralization and HAI analyses with higher sensitivity in mice [35, 36]
To determine whether immunization with multiple HAs simultaneously could expand the breadth of the neutralizing antibody response without significant loss of magnitude, a combination of 10 HA DNA vaccine immunogens was administered IM at proportionally lower concentration (1.5 mg per immunogen) into groups of 10 mice (see Materials and Methods). Remarkably, despite a log lower DNA concentration of each component, significant neutralizing antibody titers were generated to each of the 10 immunogens, with .80% neutralization against 6 out of 12 H5 HA pseudoviruses at dilutions of up to 1:400 ( Fig. 2A) .
To evaluate whether similar breadth of immunity could be generated with fewer immunogens, two different combinations of 5 immunogens were selected, based on the phylogenetic diversity of HA among the avian influenza viruses [33] and the crossreactivity of the neutralizing antibody responses of select individual immunogens (Fig. 1 ). As expected, there were substantial differences in the breadth of neutralization between these two sets of 5 immunogen multivalent vaccines (Fig. 2 , B vs. C). In one set, while neutralization of homologous strains was comparable to the monovalent and the 10 immunogen multivalent immune response, fewer cross-reactive antibodies were detected, directed most prominently against A/Iraq Protection of DNA-vaccinated mice against challenge with heterologous H5N1 A/Vietnam/1203/2004 influenza virus Mice immunized as described above were challenged with a heterologous H5N1 virus 68 weeks after the final DNA vaccination. Animals were then challenged with 10 LD 50
of the highly pathogenic A/Vietnam/1203/2004 virus intranasally, and morbidity and mortality were monitored for 21 days after the viral challenge. The control animals, injected with the plasmid expression vector with no insert, died within 10 days of infection. Complete survival was observed in the groups immunized with the 10 component and set 2 of the 5 component multivalent DNA vaccines (Fig. 3) . Immunization with HA derived from the A/ Indonesia/05/2005 strain or set 1 of the 5 component multivalent DNA vaccine showed a survival rate approaching 90%. In contrast, animals injected with HA plasmid DNA derived from A/ Anhui/1/2005, which has diverged more from A/Vietnam/ 1203/2004, showed a lower percent survival (70%) after lethal viral challenge. Survival differences between groups were assessed using a log-rank test and the Gehan-Wilcoxon test on the survival curves for pairs of groups. A test was deemed significant if the pvalue was ,0.01. Mice injected IM with different HAs, A/
Indonesia/5/05, A/Anhui/1/05, 10HA, 5 HA (Set 1), or 5 HA (Set 2) showed a significant difference compared to control (all p values,0.001). Among the HA-immunized groups, there was no significant difference between any two groups (p.0.08 for all comparisons). For example, no significant difference was observed between the A/Anhui/1/05 group, which had the least survival among the HA immunized groups (7 out of 10), and other HA groups: A/Indonesia/5/05 (p = 0.377), 10 HA (p = 0.082), 5 HA (Set 1) (p = 0.101), or 5 HA (Set 2) (p = .411). Therefore, we cannot exclude the possibility that the 3 deaths in the A/Anhui/1/05 group may have been due to random chance.
Since it is desirable to confer protective immunity in poultry and HA DNA vaccination was effective in mice, we next examined the breadth and potency of single or multiple HA plasmid immunization in chickens. The ability of chickens to generate specific antibodies was assessed with three strains that showed broad cross protection in mouse studies (A/Vietnam/1203/2004, A/Anhui/ 1/2005 and A/Indonesia/05/2005), administered individually or in combination, by different injection methods. In addition to needle injection, a needle-free repetitive injection device, Agro-JetH (Medical International Technology, Inc., Denver, CO), was analyzed. This device disperses the 0.1 to 5 ml injection doses into the dermal, subcutaneous, or intramuscular tissue depending upon the pressure adjustments, powered by a CO 2 gas pressure plunger [39] . The injection conditions were determined by histologic analysis of tissues that received injections of India ink; a pressure of 48 psi was chosen since it
enabled consistent delivery into intradermal and subcutaneous tissues (Fig. S1 ).