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Broad protection with an inactivated vaccine against primary-isolated lethal enterovirus 71 infection in newborn mice
Junliang Chang1,
Jingliang Li1,
Xin Liu1,
Guanchen Liu1,
Jiaxin Yang1,
Wei Wei1,2,
Wenyan Zhang1 &
Xiao-Fang Yu1,2
Circulating enterovirus 71 (EV-A71)-associated hand, foot, and mouth disease is on the rise in the Asian-Pacific region. Although animal models have been developed using mouse-adapted EV-A71 strains, mouse models using primary EV-A71 isolates are scarce. Lethal animal models with circulating EV-A71 infection would contribute to studies of pathogenesis as well as vaccine development and evaluation.
In this study, we established a lethal mouse model using primary EV-A71 isolates from patients infected with serotypes that are currently circulating in humans. We also characterized the dose-dependent virulence and pathologic changes of circulating EV-A71 in this mouse model. Most importantly, we have established this mouse model as a suitable system for EV-A71 vaccine evaluation. An inactivated EV-A71 vaccine candidate offered complete protection from death induced by various circulating EV-A71 viruses to neonatal mice that were born to immunized female mice. The sera of the immunized dams and their pups showed higher neutralization titers against multiple circulating EV-A71 viruses.
Thus, our newly established animal model using primary EV-A71 isolates is helpful for future studies on viral pathogenesis and vaccine and drug development.
Human enterovirus 71 (EV-A71) is a non-enveloped, single-stranded positive-sense RNA virus that belongs to the Enterovirus species A genogroup in the Picornaviridae family. It began circulating in the Netherlands as early as 1963 and was first described in the USA in 1969 [1, 2]. EV-A71 and Coxsackievirus A16 (CV-A16) are the two major etiological agents that cause hand, foot, and mouth disease (HFMD); periodic large epidemics have occurred in recent decades, and it has become a severe public health problem [3–9].
Previous studies have shown that EV-A71 usually causes HFMD with severe neurological complications, including aseptic meningitis, brainstem encephalitis, poliomyelitis, encephalomyelitis, and even death [10–20]. In 1997, a large outbreak of HFMD caused by highly neurovirulent EV-A71 emerged in Malaysia and led to 41 deaths among young children [21]. In 1998, a large outbreak of enterovirus infection occurred in Taiwan that resulted in 405 severe cases in children and 78 deaths. Of the 78 children who died, 71 (91 %) were under 5 years of age [22]. In 2011, the largest recorded outbreak of EV-A71-associated HFMD occurred in mainland China, comprising >1.7 million cases and including 27,000 patients who exhibited severe neurological complications and 905 deaths [23].
EV-A71 has one serotype and can be classified into three genotypes (A, B, and C) and many subtypes (A, B0, B1-B5, and C1-C5). In Taiwan, the major subtypes of EV-A71 were C2 in 1998, B4 in the 2002 epidemic, C4 in the 2004-2005 epidemic, C5 in the 2006-2007 epidemic, B5 in the 2008-2009 epidemic, C4 in the 2010 epidemic, and B5 in the 2011-2012 epidemic [24, 25]. The predominant EV-A71 genotypes detected in Singapore were B3 in 1997-1999, B4 in 2000-2003, C1 in 2002, and B5 in 2006-2008. In mainland China in 1998-2011, all the strains were clustered in the C4 subgenotype of EV-A71.
Most research has been focused on developing vaccines against EV-A71 [26–35]. Given the successful experience in the development of inactivated whole viruses for poliovirus, influenza virus, and rabies virus, inactivated EV-A71 whole-virus vaccines have been produced by five manufacturers in mainland China, Taiwan, and Singapore. These vaccines have completed Phase III (mainland China) and Phase I (Taiwan and Singapore), respectively [32]. In mainland China, Beijing Vigoo Biological Co., Ltd (Vigoo), Sinovac Biotec Co., Ltd (Sinovac), and the Chinese Academy of Medical Science (CAMS) have used EV-A71 subgenotype C4 as a virus seed because it is the prevalent genotype in mainland China; however, Vigoo and Sinovac chose distinct strains, FY and H07, respectively, which were all isolated from Anhui province in South China [36, 37]. Thus far, no vaccine has effectively prevented EV-A71 infection in HFMD patients is available.
Previously, lethal mouse model in EV-A71 infection has been a pivotal evaluation role in the development of EV-A71 vaccines [27, 29, 33, 35]. However, EV-A71 viral isolates from HFMD patients in northeastern China [38] have not been previously studied in a mouse model or for vaccine development. Our group has isolated and identified several circulating EV-A71 strains from hospitalized HFMD children in northeastern China who had either severe or mild disease. We determined that these strains are complex recombinant viruses involving multiple type A human enterovirus (HEV) [38]. In the present study, we examined and compared the virulence, pathological changes, and progression induced by the circulating EV-A71 viruses, including Changchun (CC, Northeast China) and Fuyang (FY, South China) strains, in a neonatal mouse model. These strains showed different virulence, and a series of lethal strains could be used as a tool for vaccine evaluation. Furthermore, the EV-A71 vaccine candidate CC063 strain with the highest virulence also presented a broadly cross-neutralizing capacity and protection to neonatal mice from lethal-dose infect with various EV-A71 viruses. At the same time, the sera of the immunized dams and their pups showed higher neutralization titers against various EV-A71 viruses. The lethal challenge and protection in mouse model from circulating primary EV-A71 strains and the select vaccine candidates can be very helpful for vaccine development and evaluation.
Cells and viruses
African green monkey kidney epithelial cell line, Vero cells (Cat no. CCL-81; ATCC) were cultured in modified Eagle's medium (MEM) (Gibco, Invitrogen, USA) containing 10 % fetal bovine serum (FBS) (Gibco, Invitrogen, USA) and 3 % L-glutamine at 37 °C with 5 % CO2. The CC strains of EV-A71 viruses were isolated from throat swabs of HFMD patients in Changchun, China in 2010. Viruses were continuously subcultured to the tenth passage in order to ensure stable titers and genetic features. FY0805 (GenBank accession No. HQ882182.1) and SHZH98 (GenBank accession No. AF302996.1) viruses were gifts from Dr. Qi Jin (Institute of Pathogen Biology, Chinese Academy of Medical Science). BrCr (GenBank accession No. U00871.1) viruses were received from Dr. PY. Mao (302 Military Hospital of China). CV-A16-CC024 is preserved in our laboratory and was isolated from a HFMD patient in Changchun, China (GenBank accession No. KF055238). Viruses were harvested when the cytopathogenic effect (CPE) reached 90 %, and the viral titers were determined in Vero cells by the microplate CPE method and calculated by the Reed–Muench method [39].
Neonatal mouse infection model
One-day-old specific-pathogen-free (SPF) ICR neonatal mice (Experimental Animal Center, Jilin University) were used to establish the animal model of viral infection. All welfare and experimental procedures were carried out strictly in accordance with the guide for the care and use of laboratory animals and the related ethical regulations of the First Hospital of Jilin University. All efforts were made to minimize animal's suffering.
The neonatal mice were divided into different groups, randomly and each group contains three litters (n = 8 ~ 10 per litter), and inoculated intracerebrally with EV-A71 viruses or MEM (10 μl/mouse), respectively. The survival rates and mean clinical symptoms were monitored daily for 21 days post-infection. The mean clinical symptoms were scored as follows: 0, healthy; 1, lethargy or weakness; 2, wasting; 3, limb shake; 4, paralysis in hind limb; 5, moribund or dead. The control mice were healthy throughout the experiments. The median lethal dose (LD50) was calculated as described by the Reed–Muench method [39].
Histopathological and immunohistochemical analysis (IHC)
Three mice with grade 1 to 5 of clinical disease from each of three experimental groups, EV-A71CC063 (106.5 CCID50/ml), BrCr (106.5 CCID50/ml), EV-A71CC072 (106.5 CCID50/ml), and three mice of grade 0 to 1 from the control group were subjected to histopathological and IHC analysis at 5 days post-infection. After the mice were anesthetized, tissues and organs, including lung, intestine, liver, kidney, hind limb muscle, spleen, heart, spinal muscle and brain, were harvested and immersion-fixed with 10 % formaldehyde solution for 5 days. Then, all the samples were dehydrated via an ethanol gradient, clarified through dimethylbenzene, and embedded in paraffin, and 4-μm sections were obtained for hematoxylin and eosin (HE) staining. For IHC examination, 4-μm sections of tissue samples were dewaxed and hydrated through an ethanol gradient. Antigens were then restored by microwaving for 15 min at 95 °C in citrate buffer. Used hydrogen peroxide (2.5 %) treatment to inhibit endogenous peroxidase activity of the samples. EV-A71 viral antigens were detected by anti-EV-A71 polyclonal antibody of rabbit (developed in our laboratory) and streptavidin-peroxidase anti-rabbit IgG kit (Fuzhou Maixin Biotechnology Development Co., Ltd, Fuzhou, China).
Viral loads in newborn infected mouse tissues
Injected intracerebrally with EV-A71CC063 (106.5CCID50/ml) or control medium, 12 viral challenged mice and 3 negative control mice were then used to detecting viral loads.
All samples including blood, lung, intestine, liver, kidney, hind limb muscle, spleen, heart, spinal muscle and brain were harvested from the experimental group (n = 3 per time point) on days 2, 4, and 6 post-infection and from the control group (n = 3) with no challenge on day 1. All the collected tissue samples were weighed individually and stored at -80 °C for further viral detection. The collected tissue samples were disrupted and homogenized in sterile phosphate buffer with the freeze-thaw/grinding method, then centrifuged. The clarified supernatants were collected, and viral loads in the tissue supernatant and blood were determined by real-time fluorescence quantitative reverse transcriptase PCR (qRT-PCR) and the results were expressed in log10 copies/ml of blood or log10 copies/mg of tissue. The 95 % confidence interval of the negative control values determined in various organs and tissues were regarded as the reference value for methodological sensitivity (0 copies/mg or ml).
Inactivation, purification, and immunogenicity of EV-A71 virus candidate vaccine
The Vero cells infected by the seven strains of EV-A71 candidate virus were collected and centrifuged at 4500 × g for 30 min; the collected supernatants were inactived with formalin at 4 °C for 72 h. The effect of viral inactivation was tested by CPE, The inactived viruses were concentrated by ultrafiltration, following purified on Sepharose 4 Fast Flow gel. The protein concentration of the final virus elution was measured with a BCA kit (Thermo Scientific, Inc.), and the viral purity was determined by silver stain plus reagent (Bio-Rad, Inc.). The purified, inactivated EV-A71 derived viral proteins and negative control antigens prepared in similar fashion were mixed with alum and 0.5 ml (10 μg/ml) of the alum adjuvant vaccine was used for each immunization via intraperitoneal injection (I.p.).
Seven groups (n = 8 per group) of female adult ICR mice were immunized i.p. with a different EV-A71 vaccine candidate, CC063, CC072, CC077, CC080, CC085, SHZH98, or FY0805 two times at 2-week intervals. Serum was collected on the fourth week after immunization, and neutralization titers against the various EV-A71 viruses were measured by TCID50 assay in Vero cells.
Serum neutralization test
Serum neutralization titers (NTAb) were determined by the TCID50 reduction assay in Vero cells. Isopycnic mixing (50 μl/well, respectively) of the serially diluted sera was carried out with the working concentration (100 TCID50/ml) variously circulating EV-A71 strain stocks; the mixtures were incubated at 37 °C for 2 h in 96-well plates. Subsequently, the Vero cells (2 × 105/ml) were seeded onto 96-well plates (100 μl/well) for infection at 35 °C with 5 % CO2 and cultured for 7 days. The CPE of the Vero cells was observed by light microscopy. The highest serum dilution that could inhibit CPE in >50 % of the wells were determined as serum neutralization titers.
Protective efficacy of vaccine-induced maternal antibody for neonatal mice
Eight-week-old female ICR mice (n = 3, each group) were injected i.p. with inactivated EV-A71CC063 vaccine or negative control phosphate-buffered saline (PBS) on weeks 0 and 2, respectively. The inactivated vaccine contained 10 μg of EV-A71CC063 virus. The first injection was 1 h after mate. After delivery, two experiments were carried out to confirm the protective efficacy of the humoral immune response of the EV-A71 vaccine candidate. First, three immunized maternal mice and their pups in the vaccine and negative control groups were euthanized, sera were collected for neutralizing antibodies assays against various EV-A71 strains. Second, four groups of pups (n = 3 litters per group, and n = 8 ~ 10 per litter) were intracerebrally challenged with different lethal doses of EV-A71 strains on day 1. Challenged mice were monitored for 21 days.
The clinical scores, viral loads, and antibody titers were analyzed with nonparametric one-way ANOVA tests. The survival rates were determined by log-rank test. All statistical analysis were expressed as means ± the standard error of the mean (SEM). P < 0.05 was considered significant.
Establishment of a neonatal mice model using primate EV-A71 viruses that produce strain-specific morbidity and mortality
Although it has been determined that the dominant strains of EV-A71 have maintained a C4 genotype since 1998 in mainland China, there have been no reports of the development and use of a lethal animal model employing primary EV-A71 isolates. In 2010, multiple EV-A71 viruses (EV-A71CC063, EV-A71CC077, EV-A71CC072, EV-A71CC080, and EV-A71CC085) were isolated from infected HFMD patients in Northeast China by our group and determined to be recombinant forms of EV-A71 viruses [38]. To determine whether these EV-A71 viruses are lethal to neonatal mice and could be developed as a tool for vaccine evaluation, we intracerebrally injected multiple circulating EV-A71 viruses, i.e., CC063, CC077, CC072, CC080, CC085, FY0805, and SHZH98, as well as prototype EV-A71 BrCr (each at 106.5 CCID50/ml), into one-day-old mice (Fig. 1). The clinical scores and survival rates of infected mice were monitored for 21 days. The results showed that the mice that were infected with CC063, CC077, CC080, and FY0805 became sick on day 4 post-infection and present a gradual aggravation tropism, with a clinical score of grade 5 on days 9, 9, 7, 9, respectively; and CC063, CC080, FY0805 infected mice all were dead by days 9, 12, 9, respectively, CC077 group had 78 % survival rate (Fig. 1). However, CC072, CC085, SHZH98, and BrCr did not cause mortality of the neonatal mice; the mice only showed some mild lethargy and inactivity in the early days of infection (mean, 2 ~ 7d) (Fig. 1). As expected, the mice of negative control group had a grade 0 of clinical score and 100 % survival rate.
Distinct pathogenicity of circulating CC063, CC077, CC072, CC080, CC085, SHZH98, and FY0805 or BrCr viruses in neonatal mice. Newborn ICR mice were infected with indicated viruse (106.5 CCID50/ml). Negative control mice were inoculated with MEM instead of virus. Survival rates and mean clinical scores were continually observed for 21 days after infection. The results are from three independent experiments, and each litter contained 8-10 mice. The mean clinical symptoms were scored as follows: 0, healthy; 1, lethargy or weakness; 2, wasting; 3, limb shake; 4, paralysis in hind limb; 5, moribund or dead
Lethal circulating EV-A71 viruses produce dose-dependent morbidity and mortality
Since the circulating EV-A71 viruses cause lethality in neonatal mice, we looked further into their virulence by measuring the LD50 of the viruses. Groups of one-day-old mice divided as described above were injected intracerebrally with 10-fold serial dilutions of CC063 (107.5 ~ 103.5 CCID50/ml), CC077 (107.5 ~ 104.5 CCID50/ml), CC080 (106.5 ~ 103.5 CCID50/ml), or FY0805 (107.5 ~ 103.5 CCID50/ml), with MEM as the negative control for each group (Fig. 2a-d). The mice that were infected with CC063 at titers of 107.5 ~ 105.5 CCID50/ml started to show symptoms on days 2, 3, and 4 post-infection, respectively; the symptoms were grade 1 and gradually became more severe until they reached grade 5 on days 9, 9, and 12, respectively (Fig. 2a). The three titers of CC063 (107.5 ~ 105.5 CCID50/ml) caused death beginning on days 5, 6, and 8, respectively, with a 100 % mortality on days 9, 9, and 12 post-infection, respectively (Fig. 2a). For CC063 at 104.5 CCID50/ml, the virus caused a 45 % mortality in the infected mice, with a reversible mean clinical score of grade 2 (Fig. 2a). CC063 at 103.5 CCID50/ml did not cause mortality, and it produced only a short period of grade 1 symptoms on days 8 ~ 10 (Fig. 2a).
Circulating CC063, CC077, CC080, and FY0805 resulted in dose-related disease and mortality. One-day-old ICR mice (n = 8 ~ 10 per litter) were intracerebrally challenged with 10-fold serial dosages of viruses. a EV-A71CC063 challenged with 103.5 CCID50/ml to 107.5 CCID50/ml. b EV-A71CC077 challenged with 104.5 CCID50/ml to 107.5 CCID50/ml. c EV-A71CC080 challenged with 103.5 CCID50/ml to 106.5 CCID50/ml. d FY0805 challenged with 103.5 CCID50/ml to 107.5 CCID50/ml. Control animals were given medium instead of virus. Mortality and clinical symptoms were monitored and recorded daily for 21 days after infection. One representative from three independent tests is shown. The LD50 values are 105.0 CCID50/ml, 107.0 CCID50/ml, 105.5 CCID50/ml, and 105.5 CCID50/ml, respectively
For CC077, the challenged mice given 107.5 CCID50/ml, 106.5 CCID50/ml, or 105.5 CCID50/ml began to sicken on days 4, 5, and 5, respectively, with a 0 %, 80 %, and 90 % survival rate (mean clinical score of grade 3), respectively (Fig. 2b). The challenged mice with 104.5 CCID50/ml began to sicken on day 7 (grade 1), and no deaths occurred during the period of observation (Fig. 2b).
The mice that were infected with CC080 in various titers all became sick on day 3 post-infection (grade 1); after an increased tropism in clinical grade, only the 106.5 CCID50/ml group reached grade 5 on day 12, whereas the 105.5 and 104.5 CCID50/ml groups had mean clinical scores of grade 3 during the whole test period (Fig. 2c). The challenged mice with 106.5 ~ 104.5 CCID50/ml began to die on days 5, 5, and 7, respectively (0 %, 60 %, and 80 % survival rate, respectively) (Fig. 2c). In the 103.5 CCID50/ml group, no obvious symptoms or death of the infected mice were observed (Fig. 2c).
The mice challenged with 107.5 CCID50/ml, 106.5 CCID50/ml, 105.5 CCID50/ml, or 104.5 CCID50/ml FY0805 began to sicken on days 4, 5, 5, and 6 (grade 1), with 100 %, 100 %, 60 %, and 10 % mortality rates by days 8, 9, 12, and 9, respectively. The mice that were infected with the lowest dosage of 103.5 CCID50/ml exhibited only mild symptoms (grade 1) on days 6 to 10 and no deaths (Fig. 2d).
In the aforementioned four experiments, no clinical symptoms or death were observed in the negative control group (Fig. 2a-d), indicating that the disease and death of the mice were specifically attributable to the EV-A71 viruses.
In conclusion, the symptoms and mortality rate produced by the four pathogenic virus strains increased in a dose-dependent manner. The median lethal doses (LD50) for CC063, CC077, CC080, and FY0805 were about 105.0 CCID50/ml, 107.0 CCID50/ml, 105.5 CCID50/ml, and 105.5 CCID50/ml, respectively. CC063 appeared to be the most pathogenic strain (Fig. 2). Thus, we have identified multiple circulating EV-A71 viruses that can induce strain-specific mortality and disease symptoms in newborn mice.
Pathology in the mice post-infected with a lethal dose of circulating CC063
The newborn mice challenged with any lethal dosage of EV-A71 virus all exhibited typical clinical symptoms such as wasting and hind-limb paralysis. To understand the pathological changes caused by CC strains in the neonatal mice, we selected the mice infected with the circulating CC063 strain, with strong virulence at the representative endpoint of grade 5 clinical score and conducted a series of pathologic analysis of multiple tissues to reveal changes that might be related to the death of the newborn mice. The results showed that the hind-limb muscle and spinal skeletal muscle fibers appeared severe necrosis, including muscle bundle fracture, muscle fiber swelling, and nuclear dissolution and shrinkage (Fig. 3b, d) when compared to those of the non-infected mice (Fig. 3a, c). However, no detectable pathological symptoms were found in the intestine, liver, spleen, kidney, or brain (data not shown). In addition, no obvious pathological changes were found in the heart or lungs (Fig. 3f, h). These results demonstrated that circulating EV-A71CC063 has a strong tropism cause severe lesions in the muscle tissues.
Pathological analysis of infected newborn mice after intracerebral challenge with a lethal dose (106.5 CCID50/ml) of EV-A71CC063. One-day-old ICR mice were intracerebrally inoculated with EV-A71CC063 (106.5 CCID50/ml) or medium (mock control). Representative images from the hind-limb muscle, spine muscle, cardiac muscle, and lung tissue at 6 days post-infection are shown. Infected mice (clinical grades 4-5) exhibited severe necrosis in the hind-limb muscle (b) and spinal skeletal muscle (d), but no obvious histological changes in cardiac muscle (f) or lung tissue (h) by HE staining. The results for non-infected mice were used as a control (a, c, e, and g). Magnification, 400 ×
To better understand the distribution of viral antigen in selected tissues of CC063-infected newborn mice, we performed IHC staining of the associated tissues of the infected mice. The results showed widespread expression of viral antigen in the tissues, including the hind-limb muscle (Fig. 4b), spinal muscle (Fig. 4d), cardiac muscle (Fig. 4f), lung (Fig. 4h), and intestine, liver, brain, spleen, and kidney (data not shown). In contrast to the pathological changes in the hind-limb and spinal muscles, which expressed widespread viral antigen, we did not detect high viral antigen expression in the other tissues. No viral antigen was detected in the non-infected mice (Fig. 4a, c, e, g), confirmed that the detection was specific. Our results suggested that even CC063 could lead to widespread infection, but only induced pathogenesis in the hind-limb and spinal muscles of the mice.
Immunohistochemical (IHC) staining results for infected mice after intracerebral challenge with a lethal dose (106.5 CCID50/ml) of EV-A71CC063. One-day-old ICR mice were intracerebrally inoculated with EV-A71CC063 (106.5 CCID50/ml) or medium (mock control). Representative sections from mouse tissues at 6 days post-infection (grades 4-5) are shown. The viral antigen (arrow) was detected in the hind limb muscle (b), spinal skeletal muscle (d), cardiac muscle (f), and lung tissue (h). In contrast, no viral antigens were detected in the corresponding tissues (a, c, e, and g) of the non-infected mice. Magnification, 400 ×
Viral loads in selected tissues of CC063-infected mice
To further characterized the viral replication of CC063 viruses in infected mice, we determined viral loads in various organs at various time points post-infection (Fig. 5). Viral loads were first detected in the heart (102.32 copies/mg), liver (102.23 copies/mg), brain (102.01 copies/mg), and blood (103.13 copies/ml) at 2 days post-infection in the CC063-infected mice. Four days after challenge, the virus was detected in all tested tissues. The viral loads in various tissues of the infected mice, including heart, liver, spleen, lung, kidney, brain, intestine, spine muscle, hind-limb muscle, and blood reached higher levels of 103.94 copies/mg, 103.46 copies/mg, 102.94 copies/mg, 103.39 copies/mg, 102.79 copies/mg, 103.11 copies/mg, 103.73 copies/mg, 104.87 copies/mg, 103.98 copies/mg, and 105.05 copies/ml, respectively, on day 4 post-infection. This result suggested that the virus had spread systemically by day 4 post-infection. By day 6, the viral loads in all the tissues except the intestine had steadily increased, and the virus copy numbers were 104.78 copies/mg, 104.54 copies/mg, 103.56 copies/mg, 104.23 copies/mg, 103.05 copies/mg, 103.53 copies/mg, 103.10 copies/mg, 106.01 copies/mg, 105.20 copies/mg, and 105.25 copies/ml, respectively. In the negative control mice, no viral loads were detected in selected tissues, indicating that our results were specific. The highest viral loads were found in the spinal muscle, hind-limb muscle, and blood in the later stages (4 ~ 6 days). The detection of viral loads was also consistent with our IHC analysis (Fig. 4), suggesting that CC063 has a strong tropism for spinal muscle and hind-limb muscle.
Kinetics of viral load levels in various tissues of EV-A71CC063-infected (106.5 CCID50/ml) one-day-old ICR mice. The viral RNA copies were detected by qRT-PCR at days 2, 4, and 6 post-infection, respectively. The results represent the mean virus loads [(log10copies)/mg of tissue or/ml of blood] ± SD (3 mice per group, repeated 3 times). RNA samples from the mock-infected mice and positive virus culture medium were run simultaneously in each qRT-PCR, and known copies of the DNA fragments were used as standards to calculate the copy numbers of the viral RNA in the infected tissues
EV-A71CC063 vaccine candidate showed strongest immunogenicity and broader cross-neutralization capacity
According to the standards of innovative vaccine development reported by Liang et al [40], we compared an analyzed the immunogenicity of these EV-A71 strains including the CC strains, SHZH98, and FY0805. Eight groups of seven mice were immunized with various EV-A71 viruses with equal inactivated virus antigen (10 μg/ml and 0.5 ml/mouse) according to the immunization procedure as Fig. 6a. The antibody cross-neutralization titer (NT) was determined by using 28 days serum in cellular cytopathogenic effect method in vitro. As shown in Fig. 6b, CC063 vaccine candidate generated highest NT, and the geometrical mean (GMT) value was 311. The mean cross-neutralization titer value (exclude CV-A16-CC024 and BrCr) of CC072, CC077, CC080, CC085, SHZH98, and FY0805 were 100, 83, 111, 98, 113, and 117 (GMT). Moreover, serum of CC063 generated higher NT value against other virus strains except itself than other vaccine candidate (Fig. 6b). Through the comprehensive analysis of immunogenicity and broad neutralized feature, CC063 with the strongest virulence was chosen as an eligible vaccine candidate.
Serum cross-neutralization titers generated by seven EV-A71 vaccine candidates. Eight groups (n = 8 per group) of femal adult ICR mice were immunized with EV-A71 vaccine candidates including CC063, CC072, CC077, CC080, CC085, SHZH98, FY0805, and MEM (negative control) twice at 2 week intervals: a Diagram of the experimental procedure. b CC063 vaccine candidate produced highest mean cross-neutralization titers (GMT = 311). Mean cross-neutralization titers of the other EV-A71 strains were 100, 83, 111, 98, 113, and 117 (exclude CV-A16-CC024 and BrCr) for CC072, CC077, CC080, CC085, SHZH98 and FY0805, separately. The values for the negative control and CV-A16-CC024 sera were all < 8
Maternal antibody against the CC063 vaccine candidate can broadly protect the neonatal mice from lethal virus challenge
We hypothesized that the animal model developed in the present study may be a useful model for EV-A71 vaccine evaluation. To examine this hypothesis, we immunized female mice (n = 10) with the EV-A71 inactivated vaccine candidate and mated these mice after vaccine injection (Fig. 7a). Serum samples of three immunized dams and their pups on day 1 were collected. Neutralizing antibody (NTAb) titers against various EV-A71 strains were measured. The NTAb titers of female mice immunized against CC063, CC072, CC077, CC080, CC085, SHZH98, FY0805, or BrCr reached an average of 1024, 421, 550, 487, 518, 128, 199, and 32 (GMT), respectively. The NTAb titers of non-immunized mice were not detectable (Fig. 7b). The serum NTAb titers of pups born to the immunized female mice averaged 499, 263, 271, 247, 245, 71, 80, and 14 (GMT), respectively. As expected, no NTAb was detected in those pups born to non-immunized mice (Fig. 7c).
Broadly neutralizing antibody in immunized dams and pups. a Experimental design. (b and c) Adult female mice were immunized with inactivated EV-A71 viral preparation and subsequently examined for serum neutralizing antibody titers as described in section of the Methods. The sera were collected from 3 dams and their pups (8 to 10 mice per group, mixed) after delivery as described in section of the Methods
Next, we determined whether our EV-A71 vaccine candidate could protect neonatal mice from challenge with various lethal viruses. After delivery, we intracerebrally challenged the pups were with lethal a dose of CC063 (200LD50), CC077 (100LD50), CC080 (100LD50), or FY0805 (100LD50) on day 1. The neonatal mice born to unimmunized mice (MEM) in the four groups started to die an average of 6 days after virus inoculation, and all were dead by day 10 post-infection (Fig. 8a-d). In contrast, newborn mice of maternal immunized with vaccine candidate showed a 100 % survival rate and expressed lower clinical scores (grade 1) in the early stage of the tests (Fig. 8a-d). These results suggested that the animal model developed in this study can be used to evaluate EV-A71 vaccine candidates and that our inactivated EV-A71 vaccine candidate offers high efficiency and broad-spectrum cross-protection against diverse EV-A71 viruses in a neonatal mouse model.
Broadly protection of maternal immunization with the inactivated EV-A71CC063 against lethal viruses challenge in neonatal mice. One-day-old ICR mice (n = 8 ~ 10 per litter) born to immunized mice were intracerebrally challenged with a CC063 (200LD50), b CC077 (100LD50), c CC080 (100LD50), or d FY0805 (100LD50) and had a 100 % survival rate during the test period of 21 days. The neonatal mice born to unimmunized mice (positive control) in the four groups all had a 100 % mortality rate and suffered increasing grades of clinical score
Animal models plays a critical role in the development and evaluation of potential EV-A71 vaccines. Since normal adult mice are insensitive to EV-A71 infection, some researchers have suggested using neonatal mice or transgenic mice challenged with single viruses to evaluate the protective efficacy of vaccines [27, 29, 33, 35]. In our study, we have identified multiple lethal circulating EV-A71 viruses in a neonatal mouse model and have analyzed the pathogenic features of the lethal and non-lethal strains. These circulating EV-A71 strains were isolated from different regions of China. Early genome analysis demonstrated that these EV-A71 strains are circulating recombinant viruses [38] that are distinct from the prototype EV-A71 (BrCr). Interestingly, although the prototype EV-A71 (BrCr) did not cause lethal infection in the neonatal mice, several primary EV-A71 isolates readily generated pathogenic infections in these mice. On the other hand, some primary EV-A71 isolates could not cause lethal infection, despite the fact that they are genetically closely related to the pathogenic primary EV-A71 viruses.
Primary circulating EV-A71 viruses cause many symptoms in neonatal mice, including wasting, limb-shake weakness, and hind leg paralysis. HE staining showed severe lesions in the hind limb and spinal skeletal muscles, but not in the cardiac muscles or lung tissues; these findings are very different from those in the neonatal mice infected with lethal CV-A16 viruses, which suffered from severe lung damage [41]. We could also detected viral antigen in the hind limb, cardiac, and spinal skeletal muscles. As expected, a large amount of viral antigen was detected in the brain tissue (data not shown), a point that has been emphasized in many previous studies [11, 14, 17, 19, 42, 43]. Virus loads in these organs were consistent with our results. However, the expression of viral antigen could also be detected by IHC (data not shown) in many other tissues of the infected mice that showed tissue-specific pathogenesis.
Several viruses, such as CC072, CC085, SHZH98, and BrCr, were unable to induce neonatal death in the mice (Figs. 1 and 2). Consistent with the findings, pathological changes and viral loads could not be detected in the organs of these infected mice (data not shown), suggestnig that the absence of viral replication in the neonatal mice might be the main reason that the virus could not induce animal death. Our mouse model should be useful for future identification of viral determinants of pathogenic infection.
Previous studies have demonstrated that 5'UTR and VP1 may be the major virulence associated regions of the EV-A71. We performed the 5'UTR and VP1 sequences alignment of the virulent and non-virulent EV-A71 viruses (Figs. 9 and 10). We found that the non-virulent EV-A71 contain mutations that are not detected in the virulent strains. Future studies will be required to determine whether these mutations influence virulence of EV-A71 in the mouse model. The eight EV-A71 strains evaluated in our mouse model have been associated with various disease symptoms in human. Only EV-A71 BrCr and CC063 have been associated with severe neurological diseases in people. Interestingly, BrCr is pathogenic in people but not virulent in the mouse model. It is possible that BrCr and CC063 could use different cellular receptors. The presence of certain cellular receptor in mouse allowed CC063 but not BrCr to replicate in the mouse model. Although the lethal mouse model may not be suitable for all EV-A71 strains, it is still useful for evaluating immune responses after EV-A71 vaccine immunization and protection of these immune responses against viral infection in this sensitive and convenient animal model.
Amino acid sequences alignment of VP1 region of virulent (first four) and non-virulent (last four) EV-A71 strains
Nucleotide sequences alignment of 5'UTR region of virulent (first four) and non-virulent (last four) EV-A71 strains
Widespread HFMD has raised serious public health concerns in the Asia-Pacific region [4, 8, 9]. Coxsackievirus 16 (CV-A16) and enterovirus 71 (EV-A71) can both cause HFMD, major studies are focused on the latter as EV-A71 infection often associated with severe complications, including various neurological symptoms. Interestingly, we observed that administration of only the higher dosage of the various EV-A71 strains (CC063 LD50 ≈ 105.0 CCID50/ml) could induce neonatal mouse death, as compared to circulating CV-A16 viruses (CC024 LD50 ≈ 101.65 CCID50/ml) [41]. The pathogenesis induced by EV-A71 and CV-A16 needs to be further investigated in the future.
Our study demonstrates that primary isolates of circulating EV-A71 viruses differ greatly in their ability to elicit broad-range neutralizing antibodies. Using these viruses isolated from diverse areas, we characterized EV-A71 vaccine candidates with the highest virulence and determined that these candidates, derived from virus from Northeast China, could protect neonatal mice from challenge with diverse lethal strains. Moreover, we found that vaccine candidate CC063 had the highest virulence (LD50 ≈ 105.0 CCID50/ml) (Fig. 2a), the strongest immunogenicity (Figs. 6 and 7), and the broadest cross-protection (Fig. 8). Therefore, using a sensitive mouse model and EV-A71 viruses, we have successfully studied the viral pathogenesis and identified a potential vaccine candidate.
Since no effective antiviral agents are available, EV-A71 vaccine will play an important role in controlling EV-A71-induced HFMD in children. The selection of a vaccine candidate strain is the crucial factor, especially for the EV-A71 vaccine, because of its high mutation rate and frequent recombination [44]. In general, the virus vaccine candidate strain should have higher virulence, better immunogenicity, broad cross-protection, and genetic stability [37]. For vaccine evaluation, with the exception of the cellular CPE method in vitro, animal model systems can serve directly evaluation to protective efficacy and immunogenicity of candidate vaccines. Indeed, we observed high immunogenicity of the EV-A71 vaccine candidate both in vitro and in vivo. Even more important is the fact that the EV-A71 candidate vaccine in our study demonstrated broad cross-protection against lethal challenge with multiple EV-A71 viruses from the Jilin and Anhui provinces of China. Our study therefore provides important guidance for future HFMD vaccine development and evaluation which may lead to the discovery of potential vaccine candidates for the prevention and control of HFMD.
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Wang SM, Lei HY, Huang KJ, Wu JM, Wang JR, Yu CK, et al. Pathogenesis of enterovirus 71 brainstem encephalitis in pediatric patients: roles of cytokines and cellular immune activation in patients with pulmonary edema. J Infect Dis. 2003;188(4):564–70.
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Yip CC, Lau SK, Woo PC, Yuen KY. Human enterovirus 71 epidemics: what's next? Emerging health threats journal. 2013;6:19780.
Chua KB, Kasri AR. Hand foot and mouth disease due to enterovirus 71 in Malaysia. Virol Sin. 2011;26(4):221–8.
Lin TY, Chang LY, Hsia SH, Huang YC, Chiu CH, Hsueh C, et al. The 1998 enterovirus 71 outbreak in Taiwan: pathogenesis and management. Clin Infect Dis. 2002;34 Suppl 2:S52–7.
Zeng M, El Khatib NF, Tu S, Ren P, Xu S, Zhu Q, et al. Seroepidemiology of Enterovirus 71 infection prior to the 2011 season in children in Shanghai. J Clin Virol. 2012;53(4):285–9.
Chia MY, Chiang PS, Chung WY, Luo ST, Lee MS. Epidemiology of enterovirus 71 infections in Taiwan. Pediatr Neonatol. 2014;55(4):243–9.
Wu WH, Kuo TC, Lin YT, Huang SW, Liu HF, Wang J, et al. Molecular epidemiology of enterovirus 71 infection in the central region of Taiwan from 2002 to 2012. PLoS One. 2013;8(12), e83711.
Chen H, Zhang Y, Yang E, Liu L, Che Y, Wang J, et al. The effect of enterovirus 71 immunization on neuropathogenesis and protein expression profiles in the thalamus of infected rhesus neonates. Virology. 2012;432(2):417–26.
Chen HL, Huang JY, Chu TW, Tsai TC, Hung CM, Lin CC, et al. Expression of VP1 protein in the milk of transgenic mice: a potential oral vaccine protects against enterovirus 71 infection. Vaccine. 2008;26(23):2882–9.
Chong P, Hsieh SY, Liu CC, Chou AH, Chang JY, Wu SC, et al. Production of EV71 vaccine candidates. Human vaccines & immunotherapeutics. 2012;8(12):1775–83.
Chung YC, Ho MS, Wu JC, Chen WJ, Huang JH, Chou ST, et al. Immunization with virus-like particles of enterovirus 71 elicits potent immune responses and protects mice against lethal challenge. Vaccine. 2008;26(15):1855–62.
Foo DG, Alonso S, Chow VT, Poh CL. Passive protection against lethal enterovirus 71 infection in newborn mice by neutralizing antibodies elicited by a synthetic peptide. Microbes and infection/Institut Pasteur. 2007;9(11):1299–306.
Lee MS, Tseng FC, Wang JR, Chi CY, Chong P, Su IJ. Challenges to licensure of enterovirus 71 vaccines. PLoS Negl Trop Dis. 2012;6(8), e1737.
Liang ZL, Mao QY, Wang YP, Zhu FC, Li JX, Yao X, et al. Progress on the research and development of inactivated EV71 whole-virus vaccines. Human vaccines & immunotherapeutics. 2013;9(8):1701–5.
Liu JN, Wang W, Duo JY, Hao Y, Ma CM, Li WB, et al. Combined peptides of human enterovirus 71 protect against virus infection in mice. Vaccine. 2010;28(46):7444–51.
Ong KC, Devi S, Cardosa MJ, Wong KT. Formaldehyde-inactivated whole-virus vaccine protects a murine model of enterovirus 71 encephalomyelitis against disease. J Virol. 2010;84(1):661–5.
Wu CN, Lin YC, Fann C, Liao NS, Shih SR, Ho MS. Protection against lethal enterovirus 71 infection in newborn mice by passive immunization with subunit VP1 vaccines and inactivated virus. Vaccine. 2001;20(5-6):895–904.
Li YP, Liang ZL, Gao Q, Huang LR, Mao QY, Wen SQ, et al. Safety and immunogenicity of a novel human Enterovirus 71 (EV71) vaccine: a randomized, placebo-controlled, double-blind, Phase I clinical trial. Vaccine. 2012;30(22):3295–303.
Zhu FC, Meng FY, Li JX, Li XL, Mao QY, Tao H, et al. Efficacy, safety, and immunology of an inactivated alum-adjuvant enterovirus 71 vaccine in children in China: a multicentre, randomised, double-blind, placebo-controlled, phase 3 trial. Lancet. 2013;381(9882):2024–32.
Wang X, Zhu C, Bao W, Zhao K, Niu J, Yu XF, et al. Characterization of full-length enterovirus 71 strains from severe and mild disease patients in northeastern China. PLoS One. 2012;7(3), e32405.
Reed LJMH. A simple method of estimating fifty per cent endpoints. The American Journal of Hygene. 1938;27(3):493–7.
Liang Z, Mao Q, Gao Q, Li X, Dong C, Yu X, et al. Establishing China's national standards of antigen content and neutralizing antibody responses for evaluation of enterovirus 71 (EV71) vaccines. Vaccine. 2011;29(52):9668–74.
Li J, Chang J, Liu X, Yang J, Guo H, Wei W, et al. Protection from lethal challenge in a neonatal mouse model by circulating recombinant form coxsackievirus A16 vaccine candidates. J Gen Virol. 2014;95(Pt 5):1083–93.
Nagata N, Iwasaki T, Ami Y, Tano Y, Harashima A, Suzaki Y, et al. Differential localization of neurons susceptible to enterovirus 71 and poliovirus type 1 in the central nervous system of cynomolgus monkeys after intravenous inoculation. J Gen Virol. 2004;85(Pt 10):2981–9.
Wang YF, Chou CT, Lei HY, Liu CC, Wang SM, Yan JJ, et al. A mouse-adapted enterovirus 71 strain causes neurological disease in mice after oral infection. J Virol. 2004;78(15):7916–24.
Li L, He Y, Yang H, Zhu J, Xu X, Dong J, et al. Genetic characteristics of human enterovirus 71 and coxsackievirus A16 circulating from 1999 to 2004 in Shenzhen, People's Republic of China. J Clin Microbiol. 2005;43(8):3835–9.
We thank Chunyan Dai, Xiuying Zhang, Qunying Mao, Zhenglun Liang, Panyong Mao, Qi Jin and Pangyong Mao for for critical reagents and Deborah McClellan for editorial assistance. This work was supported in part by funding from the Chinese Ministry of Science and Technology (2012CB911100 and 2013ZX10001-005), the Chinese Ministry of Education (IRT1016), the National Natural Science Foundation of China (No. 31270202), the Key Laboratory of Molecular Virology, Jilin Province (20102209), and the Health and Family Planning Commission of Jilin Province (2013Z066).
Institute of Virology and AIDS Research, First Hospital of Jilin University, No 519, East Minzhu Avenue, Changchun, Jilin, 130021, China
Junliang Chang
, Jingliang Li
, Xin Liu
, Guanchen Liu
, Jiaxin Yang
, Wei Wei
, Wenyan Zhang
& Xiao-Fang Yu
Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Baltimore, MD, 21205, USA
Wei Wei
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Correspondence to Wenyan Zhang or Xiao-Fang Yu.
XFY, WYZ, WW participated in the study design and the preparation of the manuscript, JC,JL,XL,GL,JY participated in the laboratory experimental work, JC and WW participated in the analysis of the data. All authors read and approved the final manuscript.
Junliang Chang and Jingliang Li contributed equally to this work
Chang, J., Li, J., Liu, X. et al. Broad protection with an inactivated vaccine against primary-isolated lethal enterovirus 71 infection in newborn mice. BMC Microbiol 15, 139 (2015). https://doi.org/10.1186/s12866-015-0474-9
Enterovirus 71
Mouse model
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Clinical microbiology and vaccines


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Commissioner Manfred: Once outbreak halts, Cardinals can still be a 'credible competitor' without complete season
Derrick Goold
Major League Baseball Commissioner Rob Manfred, right, and St. Louis Cardinals owner Bill DeWitt Jr. wait to be introduced before a baseball game between the Cardinals and the Chicago Cubs, Sunday, April 2, 2017, in St. Louis. (AP Photo/Jeff Roberson)
Although a substantial slice of the Cardinals' season has been postponed and their return to play is still unknown due to the novel coronavirus' spread through their roster, commissioner Rob Manfred believes there is time remaining for the Cardinals to play a "credible" season if not a complete 60-game season.
"I think whether you get all the way to 60 or not, that's difficult at this point," Manfred said in a phone interview Monday afternoon with the Post-Dispatch. "I think it's possible for them to play enough games to be credible, to be a credible competitor in this season."
The Cardinals continue to quarantine at their St. Louis residences and undergo daily testing for the COVID-19 virus. The tests the team took Sunday all came back negative, a source confirmed. That puts another day between them and their most recent positive tests – and a day closer to reaching Major League Baseball protocols for their return. The Cardinals and Major League Baseball have adopted a more conservative approach this week after requiring the team two consecutive days of negatives before returning from Milwaukee.
At the earliest the Cardinals could return to the field, they will have gone more than two weeks since their last game.
Asked if he sees an avenue for their return, Manfred said yes.
Cubs, Cardinals, and NL Central rivals are busy making moves, going nowhere
Cardinals finalize deals for Bader, Reyes, Hicks before deadline; exchange salary figures with Flaherty
"One hundred percent," the commissioner said. "I absolutely see a path back for the Cardinals. That is dependent on getting enough days with no positives that we're comfortable with, that we don't have a contagion risk. But, yes, 100 percent I see a path back."
The Cardinals have had 10 players and seven staff members test positive for the coronavirus in the past 12 days. The team's return to the field to face the Cubs this past weekend was derailed when less than 72 hours after their most recent positive test they had three more, including two players. That forced the team into quarantine for a second time in eight days and prompted additional concern and the team removing itself from the schedule for the entirety of the six-game home stand.
With Monday's postponement of the doubleheader Thursday in Detroit, the Cardinals have had 15 consecutive games postponed. To reach 60 games, the Cardinals would have to have to make up a fourth of the schedule in a third less of the time.
Major League Baseball will work to adjust their schedule, understanding that the calendar may run out before the scheduled games are complete. At that point, the commissioner's office has said that winning percentage will be used to determine playoff berths.
The schedule can wait while the focus is on the outbreak.
"Everything that has gone on right now is about controlling the outbreak," Manfred said. "We want to control the outbreak with the Cardinals and make sure we don't cross-contaminate. It's really that simple."
Much more from the Post-Dispatch's interview with the commissioner of Major League Baseball coming later today at STLtoday.com and in Tuesday's print edition.
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Bad news keeps coming for Cardinals: another positive test, another series postponed
Outfielder Lane Thomas becomes the 10th player and 17 member of team to test positive in past 11 days. Cardinals are unsure when they'll next play.
Derrick Goold: Read the complete Q-&-A from this week's Cardinals chat with subscribers
Post-Dispatch subscribers: See what our baseball writer had to say about the team during his live chat.
Derrick Goold: Bird Land
Cardinals series vs. Pirates postponed, team puts distance between new positive COVID-19 tests and next game
A complete home stand has been postponed due to the second wave of positive tests for COVID-19. The Cardinals schedule is in flux, and their next time on the field is uncertain.
COVID-19 symptoms sent some Cardinals team members to ER for treatment, 'clarity,' Shildt says
Cardinals are awaiting the results of Saturday tests and a discussion with Major League Baseball on their next step. Monday's game is unlikely, but series vs. Pirates is under review.

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Documentary America: Non-Fiction Writing
Documentary America: Non-Fiction Writing (Q3142)
The study of American fiction often precludes an examination of some of the best writing and forms of self-representation that America has produced. This includes political and photo-essays, social science publications, journalism and reportage.
This module explores the development of iconic non-fictional American literature and its relation with other forms of documentary representation (such as film and photography) from the 19th to 20th century.
We look at the style, content and circulation of non-fictional forms. We examine them within wider discourses of cultural, social and political representation.
You'll look at a variety of examples of documentary literary genres, including:
the insertion of the "I" in nineteenth century journalism and undercover reportage in the slums
Zora Neale Hurston's 'ethnographic' preservation of folk 'lies'
the use of documentary aesthetics in plays and poetry
the development of postmodern documentary aethetics.
You also examine how these forms intersect with the development of modernist and postmodernist literature in the US, and with the historical events they attempt to represent.
For this module, you read from a broad selection of materials that do not necessarily fit into conventional literary genres. We analyse why writers and artists represent events in the way they do and the wider cultural impact of those forms.
American Studies (with a study abroad year) BA
American Studies and English (with a study abroad year) BA
American Studies and Film Studies (with a study abroad year) BA
American Studies and History (with a study abroad year) BA
American Studies and Politics (with a study abroad year) BA
English BA
Philosophy and English BA


Come and enjoy a classic film for Valentine's Day. Never were two charming actors better displayed than Cary Grant and Audrey Hepburn in Charade. A thriller, a comedy and a romance, this genre-spanning classic film from 1963 holds up beautifully, thanks to a clever plot, witty dialogue and irresistible stars romping through Paris.


When training to improve agility and change of direction, most people think about different on-field or on-court running and cutting drills, often utilizing different lines or cones for things like the 5-10-5 drill or the pro agility. Cones set up in a square or different shape are often used with athletes performing specific running patterns through and around the cones. These would all be considered predetermined or closed change of direction drills. However, if you are using these drills or incorporating reactive agility drills, one factor that is often overlooked is the role of deceleration in that movement.
Deceleration is defined as a reduction in speed or the rate of decreasing velocity. Moreover, we know that being able to accelerate out of a cut or change of direction is extremely important. Hence, being able to decelerate quickly going into that cut is equally important. The higher the rate of deceleration and the faster the athlete can decelerate under control, the quicker that change of direction will be. With that, the ability to decelerate quickly under control depends largely on one thing, eccentric strength. Having the physical strength to quickly decelerate and absorb force is the basis of agility and change of direction.
There are two different ways that we for train for improving eccentric strength and deceleration. One is through the use of jumping and landing. The other is in the weight room by emphasizing the eccentric phase of lower body weight training movements. From there we progress to isometric pauses in those same exercises.
Below are some of the body weight jumping and landing drills that we incorporate, starting with Level 1 for beginning athletes and progressing from there to more advanced movements. The emphasis is always on soft landings and landing well by decelerating under control and absorbing the landing force. Most importantly, the drills basically progress from single jumps landing on both feet to more intense single leg, multiple jumps with a change in direction or height.
Again, the emphasis on these deceleration drills is on the landing aspect just as much as the jumping itself. Furthermore, the possibilities are endless when it comes to creating new ones, but that landing emphasis and having the patience to focus on that must always be there. These are a couple of the coaching cues that I use all the time with all of these types of deceleration drills.
When it comes to strength training for deceleration, we start with focusing on the eccentric phase of the exercise. With this approach, the athlete lowers the weight very slowly and under control for a specified length of time. Once the bottom of the range of motion is hit, fast concentric contractions take place. Secondly, lowering slowly increases the time under tension in the muscle while focusing on the same type of contraction used when decelerating to make a cut to change direction. Finally, following that with a fast concentric contraction helps to train the re-acceleration coming out of the cut.
Basically, the lighter the weight, the longer the eccentric time. The heavier the weight, the shorter the eccentric time, the lower the reps and sets.
Following the eccentric block is our isometric block. Once we have worked on developing eccentric strength, we focus on decelerating very quickly, slamming on the breaks, and pausing at the bottom of the range of motion. From there, it is again followed with a fast concentric contraction after holding the pause for a prescribed length of time. By utilizing pauses in this manner we are training rapid deceleration and force absorption, followed by rapid re-acceleration from a dead standstill. Both of these things are critical for improving agility and change of direction.
As with our eccentric block, the isometric training yields longer pauses with lighter weight. In comparison, our pauses get shorter as the weight increases.
Lastly, we finish with fast, full-speed training. We have gotten some great results by focusing on these two different training blocks combined with fast concentric contractions. It's very easy to do, can be incorporated into your weight training programs very easily and can be used for athletes of all ages. Once you have these strength aspects developed, you will quickly see the agility and the ability of your athletes to change direction improve dramatically.
Scott Meier is currently in his 19 year at Farmington High School (MN) where he is the Strength & Conditioning Coach. He is also a Physical Education Teacher at FHS and teaches Sports Conditioning, Weight Training, and 9th grade Fitness For Life classes. He coached Farmington's competitive weightlifting for 9 years, and in that time, the Tigers earned four state team titles, over 40 individual state champions, and multiple state record holders. Prior to that he was the head Strength and Conditioning Coach at Lakeville (MN) High School and worked as a personal trainer for 6 years. Scott is featured in the award-winning documentary "My Run" which tells the story of his 56-year-old client who ran 75 marathons in 75 days. He continues to compete in track & field at the masters level where he is a nationally ranked sprinter and holds several state age-group records. Scott is the current Minnesota state director of the National High School Strength Coaches Association.


A twist to the classic black jumper. This pretty sleeveless number allows u to pair with your favorite print top for an array of different looks. Go chic with a sleeveless black high neck crop or a stripped one for that preppy look. We love how versatile it is. Made of cotton poly blend. Side Zip.


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Home Campus Department of Library & Information Sciences
Department of Library and Information Science offers Bachelors & Masters degrees in Library and Information Science.
The Department has modern classrooms and a well-established computer lab with sufficient computers installed with the required software for teaching and practice.
Under Graduate Programs
Bachelor of Library and Information Sciences (B.L.I.S.)
Graduation or equivalent in any Stream with approx 50% marks.
INR 5,000/- Per semester
Syllabus for Bachelor of Library & Information Science (BLIS)
Master of Library and Information Sciences (M.L.I.S.)
Graduation or equivalent in any Stream with approx 50% marks & Bachelor degree in Library or Information Science.
2 Semester (1 Year)
Syllabus for Master of Library & Information Science (MLIS)
Name: Department of Library & Information Sciences


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The unemployment picture continues to brighten in Greater Victoria. Latest figures show regional unemployment at 6.3 per cent in November 2020. (Twitter/Greater Victoria Harbour Authority)
Unemployment picture in Greater Victoria continues to brighten in November
Rate drops to 6.3 per cent in November
Wolf Depner
The unemployment picture in Greater Victoria as well as Vancouver Island continues to improve.
New figures from Statistics Canada show the unemployment rate of Victoria Census Metropolitan Area (CMA) at 6.3 per cent in November, a drop of 1.3 per cent compared to October. Year-to-date, the unemployment rate is up 2.9 per cent compared to 3.4 per cent in November 2019.
Vancouver Island as a whole recorded an unemployment rate of 7.3 per cent in November, down one per cent from October. Year-to-date, the unemployment rate for Vancouver Island is up three per cent from 4.3 per cent in November 2019.
Compared to other CMAs in British Columbia, Victoria recorded the second-lowest unemployment, ahead of Vancouver and Abbotsford-Mission, with both recording a rate of 8.1 per cent in November. Kelowna CMA recorded the lowest rate among the four CMAs in British Columbia with a rate of 4.7 per cent.
RELATED: Unemployment in Greater Victoria continues to drop
Provincially, the unemployment rate stood at 7.1 per cent, down nearly a full per cent from eight per cent in October. Year-to-date, the provincial unemployment rate in November is up 2.1 per cent from five per cent in November 2019.
Nationally, the unemployment rate fell 0.4 per cent to 8.5 per cent in November, continuing the steady fall from the record high 13.7 per cent in May.
But these broad coordinates come with several caveats as new health restrictions have kicked into effect across the country. As Statistics Canada says, employment has fallen in sectors most directly affected by public health restrictions, notably in accommodation and food services.
"On the other hand, employment continued to approach or exceed pre-COVID levels in industries where working from home or physical distancing is more feasible, such as professional, scientific and technical services," it reads.
Accordingly, the number of Canadians working from home increased for the second consecutive month in November.
wolfgang.depner@peninsulanewsreview.com
Victoria's skateboarding community raises $18,000 for BIPOC counselling
Canadian millennials face a future of higher debt and lower incomes because of COVID-19


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Q: Query to check if an attribute was in the table from LAST YEAR Im trying to write a CASE statement to see if school_name with the school_form_status = completed, in the current year has been also completed the previous year.
   school_name | School_status | Report date|  FORM_PREVIOUS_YEAR
________________________________________________________________________________

     Paul Keys |    Completed  | 03/31/2016  |      YES

This is what I have but but not working , im a beginner
    select
    CASE REPORTED_DATE YEAR = REPORTED_DATE, YEAR,-1,DateAdd(Year,DATEDIFF(YEAR,0,getdate()),0))
    THEN 'Y'
     ELSE 'N'
    END AS FORM_PREV_YEAR,
    from
    TABLE1

A: Normally you would JOIN the table against itself:
SELECT this_year.school_name,
       this_year.school_status,
       this_year.reported_date,
       CASE WHEN last_year.school_status = 'Completed' THEN 'Y' ELSE 'N' END AS form_previous_year
FROM table1 AS this_year
LEFT OUTER JOIN table1 AS last_year ON last_year.school_name = this_year.school_name
WHERE this_year.reported_date >= TO_DATE('2017-01-01', 'YYYY-MM-DD')
  AND last_year.reported_date BETWEEN TO_DATE('2016-01-01', 'YYYY-MM-DD') AND TO_DATE('2016-12-31', 'YYYY-MM-DD')
  AND this_year.school_status = 'Completed'

The statement looks for all schools with the status completed and a reported_date from this year ad then finds the schools with the same name and the reported_date from last year if it exists (LEFT OUTER JOIN). If the last year's status is 'Completed' it will return 'Y' else 'N'.

A: Try this code:
select
CASE WHEN school_status = 'Completed' and EXTRACT(year from report_date) = EXTRACT(year from sysdate)-1
THEN 'Y'
ELSE 'N'
END AS FORM_PREV_YEAR
from table1;

redpajama_set_name: RedPajamaStackExchange


Mr. Brand has engaged in full time dispute resolution since 1983. A former negotiator and law professor, he has a varied national practice, ranging from arbitrating high profile individual disputes – including IHL and NFL players to mediating high-stakes public disputes – including the nation's first "pay for performance" contract in the Denver City School District. He has been named a Northern California "Super Lawyer" in ADR every year since 2006.
Mr. Brand has arbitrated and mediated cases involving complex issues of law and large dollar amounts. He has served as sole arbitrator in a $40 million labor-management case, a $50 million biotech case, a $65 million MPPAA case, and in many multi-million dollar pension cases. He has mediated multi-party disputes, including the model annual maintenance agreement for the Sacramento Delta, a Super Fund site, wage rates on the Bay Bridge construction project, and wage and hour class actions. He mediates and arbitrates executive compensation, ERISA, MPPAA, discrimination, statutory, and wrongful termination disputes involving Fortune 500 companies.
Mr. Brand has engaged in med-arb under statutory procedures involving state and federal entities, as well as in trust fund and trade secret cases. His primary practice is in labor and employment law, but he has also served on specialized panels requiring scientific literacy in biochemistry, medicine, and psychiatry, as well as an understanding of research and laboratory procedures. He has served on the Institutional Review Board of the University of California, San Francisco since 2008.
Mr. Brand is past President of the College of Labor and Employment Lawyers, a Fellow of the American College of Employee Benefit Counsel, past President of the California Dispute Resolution Council, a member of the National Academy of Arbitrators, and a Diplomate of the California Academy of Distinguished Neutrals. He is on the American Arbitration Association's Employment, ERISA/MPPAA, Commercial, Large Complex Case, and other panels. Among his publications are: Discipline and Discharge in Arbitration, 3rd ed. (Washington: BNA, 2015); Rev. of Toward a New Federal Law of Arbitration, by Thomas E. Carbonneau, Dispute Resolution Magazine (Summer 2015:24-27; Labor Arbitration: The Strategy of Persuasion, 2nd ed. (San Francisco: 2006); How ADR Works (Washington: BNA, 2002; and "When Experts Testify: Exploiting the Advantages of Arbitration," 4 ADR Currents 1 (Fall 1999). He is a contributor to "ADR and Settlement," in California Practice Guide – Employment Law (Rutter Group:2001) and Designing Integrated Conflict Management Systems, Cornell Studies in Conflict and Dispute Resolution, 2001).
Mr. Brand has a Ph.D. and JD. He has taught arbitration, negotiation, and mediation. He has taught neutrals for the American Arbitration Association, judges at the National Judicial College, and students as a Professor at Albany Law School and as an Adjunct Professor at Hastings College of the Law.


While I think this law is pretty ridiculous, the author of the article (and the man pushing the suit) misunderstand the law.
The First Amendment does not apply to the naming of corporations and LLCs. I can call myself "Bill Gates," stand on a box on the street-corner and tell everybody what life is like as the founder of Microsoft. Unless I'm libeling or defrauding, the 1st amendment protects me.
But the registration of corporations is very different. I can't register a corporation named "Microsoft." It's called "Trademark Infringement."
That's because when you register a corporation, the government is giving sanction, legitimacy, legal status, and protection to your company. You can't call yourself whatever you want. In Britain, for instance, you can't call yourself "The British lemonade stand company" without government approval.
When you register an LLC called "I Choose Hell Productions," the government is giving sanction and legitimacy to your company, and protection to that name.
Contrary to popular opinion, of course, the establishment clause prohibits the government from advancing or inhibiting the exercise of religion.
By providing government sanction and legitimacy to a name that insults a particular religion (for instance, 'All Christians need to be strung up and killed, Inc.' or 'Muslims are Morons, LLC' is a completely different case from expressing those opinions in a coffee shop with your friends. The First Amendment protects the behavior in the second case. It prohibits the behavior in the first case.
So is this law a good idea? Frankly, I don't particularly care. But criticisms of the law should rest of policy (like, 'This law is stupid') rather than constitutional law (like, 'the first amendment prohibits this') because if anything, the First Amendment supports the law.
Philadelphia is not like the rest of Pennsylvania. Overall the state is very conservative, so the fact that they have this law doesn't really surprise me at all.
Interesting point. But it depends on how blasphemy law in PA is actually written. If it includes a particular religion (or terms that apply to a particular religion) - then perhaps it will fall under the First Amendment.
I also think ungtss has an interesting point when it comes to the government conferring legitimacy on registered trademarks and corporate names, but in the case of this particular law it seems as if a particular religion is being singled out.
They could have kept it as a plain ol' controversial obscenity law, but "the Lord's name" pretty clearly signals a Jewish, Christian, or Islamic bias. If someone was interested in registering the equivalent of "I choose Hell" in Hindu terms, that clause of the law would probably have nothing to say about it. Hell, thinking of the conservative spirit in which the law was likely enacted, it seems unlikely that the religious synonymy would even be caught by legal watchdogs. I think there's a case to be made, but only in so much as the particular religious reference should be removed. Getting the whole anti-"obscenity" part of the law thrown out is something else entirely, and is made much more difficult in exactly the way you pointed out.


"Aisa Hi Hoon Main "
Mine was arranged...but we were in love by the time we got married...so i like to call it, arranged love marriage.
love marriage all the way.
Its a good question that love marriage and arrange marriage. But i will go with arrange marriage and i think arrange marriage is best because it is surprise and i think you don't know about your partner and you like to know about her or him. While in love marriage you know all about each other and there is less charm in knowing each other. So i prefer arrange marriage.
Are there any press release how much rumor of display off-well done, the popular and Mirage. I am sure, as long as the tickets are sold for at least a few more years, and the seats are full, will be around.
I prefer love marriage beacause in love marriage you know each other properly and other thing is you have proper time to know each other, what yor partner like or not and according to me this are basic thing to any relation.
Personally, I'd opt for a little bit of knowing each other before getting married. But but..
Lol! By both you mean two marriages? Or love + arranged marriage?


text:
cask "tempo" do
  arch arm: "release-arm64", intel: "release"

  version "6.0.0"
  sha256 arm:   "67260ec4260e30da28abc20d847282f08e09d8bce00730594c598caf3a63bdb7",
         intel: "acfe86aedce23077a34de27717f38920bf4eafb740841088ab31b685fec4e3af"

  url "https://download.yourtempo.co/#{arch}/Tempo-#{version}.dmg"
  name "Tempo"
  desc "Email client that delivers all email in batches"
  homepage "https://www.yourtempo.co/"

  auto_updates true

  app "Tempo.app"

  zap trash: [
    "~/Library/Application Support/Tempo",
    "~/Library/Logs/Tempo",
    "~/Library/Preferences/com.founders.mial.plist",
    "~/Library/Saved Application State/com.founders.mial.savedState",
  ]

  caveats do
    discontinued
  end
end

redpajama_set_name: RedPajamaGithub


text:
Kerala became first state to list Masala Bond in overseas market
Kerala became the first state to tap into masala bond market by listing the KIIFB masala bond amounting USD 312 million in London Stock Exchange (LSE)'s International Securities Market (ISM). The masala bond has tenor of 5-year .
KIIFB- Kerala Infrastructure Investment Fund Board
US launched new 'Merit-Based Immigration System'
US has launched a merit-based immigration system from which foreigners, waiting to get Green Cards or permanent legal residency stand to benefit. The reform proposals significantly increase the quota for skilled workers from existing nearly 12% to 57%.
This proposal was launched by US President, Donald Trump.
World Bank and CBA Partner to enable Secondary Bond Trading recorded on Blockchain
IBRD (World Bank) and Commonwealth Bank have enabled secondary market trading recorded on the blockchain for bond-i (blockchain operated new debt instrument), making it the first bond whose issuance and trading are recorded using distributed ledger tech.
International Bank for Reconstruction and Development -IBRD
BSNL partnered with Google for expanding WiFi footprint
State-run telecom major BSNL has partnered with Google to increase access to its WiFi footprint in India. Henceforth, Indians can use BSNL's free WiFi services. This initiative will allow customers to use BSNL's high-speed internet services on WiFi.
BSNL Director: Vivek Bansal
NSG Commandos summit Mount Everest in first attempt
NSG commandos created history as they scaled the Mount Everest, the world's highest peak(8848 m), in their maiden attempt.
The 7 member team was led by Lt Col J P Kumar.The team took the southeastern approach via South Col in Solukhumbhu region of Nepal.
NSG Director General: Sudeep Lakhtakia
Radha Venkataraman appointed as the next High Commissioner of India to the Kingdom of Eswatini
Ms. Radha Venkataraman, presently Director in the Ministry of External Affairs, has been appointed as the next High Commissioner of India to the Kingdom of Eswatini. She succeeds Rudra Gaurav Shresth.
Capital of Eswatini- Mbabane
Currency: Lilangeni
Rafael Nadal beats Novak Djokovic to win ninth Italian Open Title
Rafael Nadal defeated world's number 1 ranked player, Novak Djokovic to win a ninth Italian Open title and a record 34th Masters crown. World number two, Nadal, claimed the 81st title of his career.
Rafael Nadal is from Spain, while Novak Djokovic is from Serbia.
Naval drill , SIMBEX conducted by Navies of India and Singapore
The navies of India and Singapore conducted their bilateral naval drill SIMBEX in the South China Sea. Indian Navy ships Kolkata and Shakti took part in the exercise with Singapore, along with long-range maritime patrol aircraft Poseidon-8I (P8I).
SIMBEX -Singapore India Maritime Bilateral Exercise
Germany's Double Olympic biathlon champion Laura Dahlmeier retires aged 25
Germany's Double Olympic biathlon champion Laura Dahlmeier retires at the age of 25 owing to health issues. In the Winter Olympics 2018, she became the first female biathlete to win sprint as well as the pursuit at the same Olympics.
She took the overall biathlon World Cup in 2017.
Architect of Louvre Pyramid, Ieoh Ming Pei passed away at 102 in New York
The world-famous architect IM Pei, best known for his remarkable work, the Louvre pyramid in Paris, passed away at the age of 102. He was also the mastermind behind the landmark 72-story Bank of China tower in Hong Kong and Athens' Museum of Modern Art.
Pei won most awards in his field including the Pritzker Prize.


Parkway Flower Shop has many "flowers in a gift" that come in a beautiful and unique vase that can be used over and over again! The recipient will think of you every time they use the special vase you sent them! Parkway Flower Shop in Brooklyn, NY has Flowers in a Gift suitable for every occasion from birthdays to new babies to anniversaries! You can even send a modern bouquet, romantic roses or plants! Parkway Flower Shop can deliver your Flowers in a Gift locally to Brooklyn, NY or nationwide!


text:
Mechanism of Water and Sand Inrush Incident during Deep Buried Coal Seam Mining in Western China
Dong, Shuning (1,2); Guo, Xiaoming (1,2); Wang, Hao (1,2)
1: Xi'an Research Institute of China Coal Technology and Engineering Group Corp, China, People's Republic of; 2: Shaanxi Key Lab of Mine Water Hazard Prevention and Control,Xi'an,China
The water and sand inrush of the roof is showing water and sand flowing into underground working space, which caused considerable economic damage. It is common in shallow coal seam mining, however, a severe water and sand inrush of the roof happened in a deep buried working face of Zhaojin coal mine, Xunyao mining area, proposing a new type roof water hazard of deep buried coal seam mining.
In order to solve this problem, we investigated water source, material source and power source of the accident to study the formation mechanism of the water hazard in Zhaojin coal mine. Moreover, we established the water source identification model considering the evolution of water chemical composition by using hydrochemical, organic compounds in water, and rock dissolution test. This model can be used to determine that the source of inrush water (Cretaceous or Jurassic sandstone aquifers). In addition, we conducted underground drilling, rock mechanical, SEM, and X-ray diffraction test to analyze the source and original structural characteristics of the inrush sand. Furthermore, the breaking regular of the overburden in the soft roof of the coal seam was studied through analyzing and calculating discrete element numerical.
The results showed that the water of the Cretaceous sandstone aquifer flowed into the roof mudstone of the coal seam, which was easy to be muddy-soaked after touching water, resulting in the roof lost stability, then leading to water and sand inrush. Compared to water and sand inrush happening in shallow coal seam mining, the formation mechanism in deep coal seam mining is different: water and sand were separated rather than mixed together; and the incident were caused by mine pressure instead of water pressure.
The results explained the formation mechanism of water and sand inrush in deep coal seam mining. Also, this study provides further evidence for the risk assessment and the control of water and sand inrush in the deep Jurassic coal seam in western China.
Guo, Xiaoming


text:
Q: Mapping boolean with np.vectorize in Python I have this two columns in pandas df:
     seconds       min
0              52  0.866667
1               5  0.083333
2              10  0.166667
3               5  0.083333
4              16  0.266667
197           144  2.400000

I wanted to convert seconds in minutes in the ['min'] column only when ['seconds'] is greater than 60, otherwise it should be empty.
I tried to transform ['min'] column in boolean:
    x = df['min'] = np.array(df['seconds'].apply(lambda x: True if x >60 else False))

and I got the boolean values, then I tried to map it:
    mapping = [df['seconds']/60]
    result = np.vectorize(lambda i:mapping[i], x)

but I got in return:
TypeError: list indices must be integers or slices, not str
Is this correct way to do it or is there another simpler solution?
Output:
    seconds       min
0              52
1               5
2              10
3               5
4              16
197           144  2.400000

A: I'm not sure about how to use your vectorize approach, but the following will work
df.loc[df['seconds']<60,'min'] = 0
df.loc[df['seconds']>60,'min'] = df['seconds'][df['seconds']>60] /60

A: Your 'True' and 'False' are strings not boolean. Booleans have not quotes


text:
Google misled customers about use of location data, court rules
By Michaela Whitbourn
April 16, 2021 — 12.31pm
Google misled some consumers about the digital giant's collection and use of their location data, the Federal Court has ruled in a decision touted as a world-first by the consumer watchdog.
In a judgment on Friday, Justice Thomas Thawley said the Australian Competition and Consumer Commission (ACCC) had established Google misled some users of Android devices about how their location data could be obtained, retained and used when a customer used various Google apps including Google Maps.
The company is considering an appeal.
Google misled some consumers about how their location information could be accessed and used, the Federal Court said. Credit:Rob Homer
The ACCC alleged Google gave users the impression they could opt out of having location data collected by the company by switching off the "location history" setting on their accounts. The default setting for this function was "off".
In fact, location data would still be collected and used if another setting – "web & app activity" – was switched "on", as was the default position.
Justice Thawley said "these default settings meant that Google LLC could obtain, retain and use personal location data when a user was using various apps, including Google services such as Google Maps".
The watchdog alleged it was "misleading for Google to not properly disclose to consumers that both settings had to be switched off if consumers didn't want Google to collect, keep and use their location data".
Justice Thawley agreed and said the ACCC's case against Google was "partially made out". He found the company had contravened provisions of the Australian consumer law prohibiting misleading or deceptive conduct, including conduct liable to mislead the public about the performance characteristics, uses or benefits or services.
Penalties will be decided at a later date.
"Google's conduct would not have misled all reasonable users ... but Google's conduct misled or was likely to mislead some reasonable users," Justice Thawley said.
"The number or proportion of reasonable users who were misled, or were likely to have been misled, does not matter for the purposes of establishing contraventions [of Australian consumer law]."
ACCC chair Rod Sims said the decision was "the first ruling of its type in the world in relation to these location data issues" and the watchdog would seek penalties totalling millions of dollars.
Mr Sims said Google's business model was to offer services for free but to collect "as much data as it possibly can" for use in advertising.
He said that between January 2017 and December 2018 "consumers were led to believe that 'location history' was the only account setting that affected the collection of their personal location data, when that was simply not true".
"We think today's result is a very clear message to the digital platforms that they have to be upfront with consumers about what is actually happening with our data, how it is being used, and how consumers can protect their data," Mr Sims said.
But a Google spokesperson said the court "rejected many of the ACCC's broad claims".
"We disagree with the remaining findings and are currently reviewing our options, including a possible appeal," the spokesperson said.
"We provide robust controls for location data and are always looking to do more - for example we recently introduced auto delete options for location history, making it even easier to control your data."
The court heard that an Associated Press article published in August 2018 revealed that many Google services "store your location data even if you've used privacy settings that say they will prevent it from doing so".
Justice Thawley said an urgent meeting was convened in the wake of the article, which was "referred to internally as the 'oh shit' meeting".
"In response to the AP article and following the meeting, Google's director of communication and public affairs circulated a document entitled 'making location history simple', which referred to works being carried out to 'reduce user confusion re: how location is used across our products and services'," he said.
Catch all the day's headlines
At the end of each day, we'll send you the most important breaking news headlines, evening entertainment ideas and a long read to enjoy. Sign up to The Sydney Morning Herald's newsletter here, The Age's here, Brisbane Times' here, and WAtoday's here.
Michaela Whitbourn is a legal affairs reporter at The Sydney Morning Herald.Connect via Twitter, Facebook or email.


text:
Celiac Sufferers May Soon Have Better Bread Options Thanks to Genetically Modified Wheat
Researchers successfully removed 90 percent of the genes that code for the gluten proteins that trigger adverse symptoms
Brigit Katz
You can find plenty of tasty gluten-free products on supermarket shelves, but when it comes to bread, there's no substitute for the real thing. Gluten, the naturally occurring proteins found in wheat, rye and barley, affect the elasticity of dough and help give bread the chewy, delicious texture that is hard to find in gluten-free alternatives.
Fortunately for people with celiac disease, who experience severe immune reactions to gluten, a better bread option may be on the horizon. As Michael Le Page reports for New Scientist, researchers have genetically engineered a strain of wheat that is free of 90 percent of the types of gluten protein that triggers most adverse symptoms.
Not all types of gluten proteins cause problems for people with celiac. Most symptoms—which can include abdominal pain, vomiting, fatigue, malnutrition and joint pain—are triggered by a gluten protein called gliadin. So scientists at the Institute for Sustainable Agriculture in Cordoba, Spain set out to extract the genes that code for gliadin proteins from wheat using the CRISPR-Cas9 gene modification technology—a powerful tool that works like a pair of "molecular scissors," snipping away at DNA sequences.
But getting rid of of the genes has been a hefty task. There are 45 copies of the gene for the main gliadin protein that causes symptoms in people with celiac disease; as Kristen V. Brown reports for Gizmodo, the team has been able to remove 35 of them thus far.
More gliadin genes will need to be removed before the modified wheat is ready for testing. But according to the study, published in the journal Plant Biotechnology, the findings show "gluten immunoreactivity can be significantly reduced" using CRISPR.
The proof of the experiment's success is in the (bread) pudding. Jan Chojecki of the UK company PBL-Ventures, which is working to market products made with the new strain of wheat, told Le Page that while the modified wheat can't be used to make big, sliced loaves of goodness, it does produce decent baguettes and rolls.
"It's regarded as being pretty good, certainly better than anything on the gluten-free shelves," Chojecki said.
Celiac disease affects as many as 1 in 100 people worldwide, and medical researchers are not entirely sure what causes it. Those who suffer from the condition usually have to cut gluten out of their diet entirely. If the CRISPR-modified wheat proves successful in trials, adhering to a strict gluten-free diet may eventually be a little bit easier.
Brigit Katz | | READ MORE
Brigit Katz is a freelance writer based in Toronto. Her work has appeared in a number of publications, including NYmag.com, Flavorwire and Tina Brown Media's Women in the World.
Disease DNA Genetics Health


How to receive your LoL Preseason Update Rewards. Today's preseason update in League of Legends brought some of the biggest changes to the game to date, completely tossing out the old Rune and Mastery systems for a new more feature-rich Rune-only system.... Aw you're so kind! Thank you! And YES Keyozee is known for her stunning Mettaton!
In terms of the main path, Rune page for AP midlaners is similar to the one for AP toplaners, with more focus on mana regen and cooldown reduction. For bonus AP and CDR, Inspiration is a great second path to choose from. Depending on your playstyle, instead of Biscuit Delivery you can choose Hextech Flash. Summon Aerie is also a good keystone pick for mages.
Aw you're so kind! Thank you! And YES Keyozee is known for her stunning Mettaton!


text:
Washington-Watch > FDA General
FDA Expands Kisqali Approval for Breast Cancer
— New indications for ribociclib sped through under 'real-time' review program
by Elizabeth Hlavinka, Staff Writer, MedPage Today July 18, 2018
The FDA expanded the approval of ribociclib (Kisqali) on Thursday for the frontline treatment of late-stage, hormone receptor (HR)-positive, HER2-negative breast cancer.
Ribociclib -- a cyclin-dependent kinase (CDK) 4/6 inhibitor -- is now approved in combination with an aromatase inhibitor (AI) for treating pre- or postmenopausal women as upfront therapy, and in combination with fulvestrant for the treatment of postmenopausal women as upfront therapy or following progression on an endocrine therapy.
"The approval adds a new treatment choice for patients with breast cancer," Richard Pazdur, MD, director of the FDA's Oncology Center of Excellence, said in the agency's announcement. "We are committed to continuing to bring more treatment options to patients."
This is the first approval to be a part of two new FDA pilot programs, Real-Time Oncology Review and Assessment Aid. The programs aim to improve the efficiency and rigor of the approval process by having researchers submit study results on the safety and effectiveness of the product earlier, so the agency can review the data before it is officially submitted and troubleshoot any missing information, which often causes delays. Applicants will also now use a new structured template when officially submitting data, so the FDA can streamline its analysis approach.
"These new processes are good for patients, good for healthcare providers, good for product developers, and good for the FDA, by allowing our staff to have more time to engage with product developers and focus on the key aspects of drug reviews," FDA Commissioner Scott Gottlieb, MD, said in the statement. "We can improve efficiency and solidify our gold standard for review."
The expanded indications were based on a pair of phase III trials. In MONALEESA-7, premenopausal women who received ribociclib plus an AI had a higher median progression-free survival (PFS) than those that received an AI and placebo (27.5 versus 13.8 months).
In MONALEESA-3, postmenopausal women assigned to ribociclib plus fulvestrant reported a higher PFS than a control group that received fulvestrant and placebo (20.5 versus 12.8 months).
Ribociclib was originally approved in March of 2017 in combination with an AI to treat HR-positive, HER2-negative breast cancer in postmenopausal women with advanced cancer. Common adverse events associated with the the CDK 4/6 inhibitor include infections, leukopenia, neutropenia, headache, vomiting, and alopecia, among others.
Elizabeth Hlavinka covers clinical news, features, and investigative pieces for MedPage Today. She also produces episodes for the Anamnesis podcast. Follow


I'm Crystal, the mom behind Our Little Bunch. I am a stay-at-home, homeschooling mom to 3 awesome little people.
We are huge fans of Disney and love to vacation, whether it be Disneyland, Disney World or even a Disney Cruise!
Is there something you're struggling with as a mom? Let me know via any of my social media channels which you can find at very the top of this page.
Let's do this mommy thing together!


Ann qualified from the British School of Osteopathy in 1974.
Since coming to Cornwall and co-founding the practice in 1978, she has developed a wealth of experience in applying both Osteopathy and Cranial Osteopathy to a diverse range of conditions.
As she now works only part-time, Ann has decided to withdraw her name from the Register of Osteopaths, which means that she is no longer entitled to use the name "Osteopath". However, she is still fully entitled to use exactly the same treatment approaches that she has always used to such great effect.
She currently offers appointments on Wednesday and Saturday mornings.
I had been suffering neck and back ache for over a week and finally decided to seek professional help. I made an appointment with Ann Retallick and wish I had made it sooner. I came out feeling much better. Ann treated my good side and inflamed side. Next time I come to Cornwall I will make another appointment. As a visitor/tourist it is often hard to decide who to trust. I would definitely recommend.
I have been a regular patient of Ann's for about 7 years. I had a double hip replacement in 1993, another replacement in 2014 and … a shoulder replacement in 2015. I have always found Ann's treatments to be very helpful for pain relief and I have no hesitation in recommending her to other people.
I have been treated by Annie since a car accident. Thanks to her treatment, I made a full recovery. She gave me excellent advice and exercises to do myself, to maintain my fitness levels and reduce my pain.
I would recommend her (and have done) to anyone. She is an excellent practitioner and osteopath.


Dr. Quinlan has recently accepted a position as Associate Professor of Human Genetics and Biomedical Informatics at University of Utah, and will join the USTAR Center for Genetic Discovery as Associate Director in 2015. He is also a co-founder of Base2Genomics. Base2 Genomics is a Utah-based technology company creating state of the art software and cloud computing solutions for scalable, comprehensive genome processing.
He is an expert in computational genomics and the development of data integration tools. Dr. Quinlan has developed several popular software packages for large-scale genome analyses and human disease studies.
Dr. Quinlan's research group applies its methods to several human disease contexts and is currently focused on developing new strategies for extracting insight into disease from genetic variation observed among millions of human genomes.


You are one fine photographer!
-really nice shot with Bob and the mic cord.
-i saved the pic you took of dear Ashley in his green adidas zip up; your best work!
Feel free to send more pics to me by email.
Not bad mama wolf…not bad at all!
thanks for always capturing the vibe.
some cool photos! Good job, Alisa!
These shots are stunning Ailsa!! Just gorgeous! What a great job you did of capturing the burlesque gals in all their glory. Excellent.
heya Amber D. Great photos, missa you lotsa. Come home to visit soon. We will be in T.O. in August 28th at The Gladstone Hotel Ballroom for the Festival of Fear closing night gala.