text:
Execution Engines¶
Execution engine is for running Retiarii Experiment. NNI supports three execution engines, users can choose a specific engine according to the type of their model mutation definition and their requirements for cross-model optimizations.
Pure-python execution engine is the default engine, it supports the model space expressed by mutation primitives.
Graph-based execution engine supports the use of mutation primitives and model spaces represented by mutators. It requires the user's model to be parsed by TorchScript.
CGO execution engine has the same requirements and capabilities as the Graph-based execution engine. But further enables cross-model optimizations, which makes model space exploration faster.
Pure-python Execution Engine¶
Pure-python Execution Engine is the default engine, we recommend users to keep using this execution engine, if they are new to NNI NAS. Pure-python execution engine plays magic within the scope of inline mutation APIs, while does not touch the rest of user model. Thus, it has minimal requirement on user model.
Rememeber to add nni.retiarii.model_wrapper()
decorator outside the whole PyTorch model before using this engine.
You should always use super().__init__()
instead of super(MyNetwork, self).__init__()
in the PyTorch model, because the latter one has issues with model wrapper.
Graph-based Execution Engine¶
For graph-based execution engine, it converts user-defined model to a graph representation (called graph IR) using TorchScript, each instantiated module in the model is converted to a subgraph. Then mutations are applied to the graph to generate new graphs. Each new graph is then converted back to PyTorch code and executed on the user specified training service.
Users may find @basic_unit
helpful in some cases. nni.retiarii.basic_unit()
here means the module will not be converted to a subgraph, instead, it is converted to a single graph node as a basic unit.
is usually used in the following cases:
When users want to tune initialization parameters of a module using
, then decorate the module with@basic_unit
. For example,self.conv = MyConv(kernel_size=nn.ValueChoice([1, 3, 5]))
, hereMyConv
should be decorated.When a module cannot be successfully parsed to a subgraph, decorate the module with
. The parse failure could be due to complex control flow. Currently Retiarii does not support adhoc loop, if there is adhoc loop in a module's forward, this class should be decorated as serializable module. For example, the followingMyModule
should be decorated.@basic_unit class MyModule(nn.Module): def __init__(self): ... def forward(self, x): for i in range(10): # <- adhoc loop ...
Some inline mutation APIs require their handled module to be decorated with
. For example, user-defined module that is provided tonni.retiarii.nn.pytorch.LayerChoice
as a candidate op should be decorated.
Three steps are need to use graph-based execution engine.
if there is any in your model.Add
config.execution_engine = 'base'
. The default value ofexecution_engine
is 'py', which means pure-python execution engine.Add
when necessary following the above guidelines.
For exporting top models, graph-based execution engine supports exporting source code for top models by running exp.export_top_models(formatter='code')
CGO Execution Engine (experimental)¶
CGO (Cross-Graph Optimization) execution engine does cross-model optimizations based on the graph-based execution engine. In CGO execution engine, multiple models could be merged and trained together in one trial.
Currently, it only supports DedupInputOptimizer
that can merge graphs sharing the same dataset to only loading and pre-processing each batch of data once, which can avoid bottleneck on data loading.
To use CGO engine, PyTorch Lightning >= 1.6.1 is required.
To enable CGO execution engine, you need to follow these steps:
Create RetiariiExeConfig with remote training service. CGO execution engine currently only supports remote training service.
Add configurations for remote training service
Add configurations for CGO engine
exp = RetiariiExperiment(base_model, trainer, mutators, strategy)
config = RetiariiExeConfig('remote')
# ...
# other configurations of RetiariiExeConfig
config.execution_engine = 'cgo' # set execution engine to CGO
config.max_concurrency_cgo = 3 # the maximum number of concurrent models to merge
config.batch_waiting_time = 10 # how many seconds CGO execution engine should wait before optimizing a new batch of models
rm_conf = RemoteMachineConfig()
# ...
# server configuration in rm_conf
rm_conf.gpu_indices = [0, 1, 2, 3] # gpu_indices must be set in RemoteMachineConfig for CGO execution engine
config.training_service.machine_list = [rm_conf]
exp.run(config, 8099)
CGO Execution Engine only supports pytorch-lightning trainer that inherits nni.retiarii.evaluator.pytorch.cgo.evaluator.MultiModelSupervisedLearningModule
For a trial running multiple models, the trainers inheriting nni.retiarii.evaluator.pytorch.cgo.evaluator.MultiModelSupervisedLearningModule
can handle the multiple outputs from the merged model for training, test and validation.
We have already implemented two trainers: nni.retiarii.evaluator.pytorch.cgo.evaluator.Classification
and nni.retiarii.evaluator.pytorch.cgo.evaluator.Regression
from nni.retiarii.evaluator.pytorch.cgo.evaluator import Classification
trainer = Classification(train_dataloaders=pl.DataLoader(train_dataset, batch_size=100),
val_dataloaders=pl.DataLoader(test_dataset, batch_size=100),
max_epochs=1, limit_train_batches=0.2)
Advanced users can also implement their own trainers by inheriting MultiModelSupervisedLearningModule
Sometimes, a mutated model cannot be executed (e.g., due to shape mismatch). When a trial running multiple models contains a bad model, CGO execution engine will re-run each model independently in separate trials without cross-model optimizations.


text:
Cowboy Palace Saloon
Saves: 6
Check-ins: 8
The unlikely city of Chatsworth is home to this San Fernando Valley dive bar. Located in the northwestern part of the valley, Cowboy Palace Saloon caters to those with a taste for country music, the two-step, and the achy-breaky. If you need to brush up on your Western dancing, take one of their line-dancing classes. See the website for a calendar of events.
(Submitted by Christine Peeples)

subset: commoncrawl


text:
Global G48 G-48-7 inch 18cm Santoku Hollow Ground Knife 7" Stainless
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text:
Back to Journals » Journal of Multidisciplinary Healthcare » Volume 14
Psychological Status and Correlated Factors of Primary Medical Staff During the COVID-19 Outbreak in Hefei City, China
Authors Wang S, Gao RC, Li R, Wu GC
Received 4 January 2021
Accepted for publication 11 March 2021
Published 31 March 2021 Volume 2021:14 Pages 751—756
DOI https://doi.org/10.2147/JMDH.S289336
Checked for plagiarism Yes
Review by Single anonymous peer review
Peer reviewer comments 2
Editor who approved publication: Dr Scott Fraser
Song Wang, 1,* Rui-Chen Gao, 2,* Rui Li, 3,* Guo-Cui Wu 1
1Lianhua Community Health Service Center, The Second Affiliated Hospital, Anhui Medical University, Hefei, Anhui, People's Republic of China; 2School of Nursing, Anhui Medical University, Hefei, Anhui, People's Republic of China; 3Department of Hospital Infection Management, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, People's Republic of China
*These authors contributed equally to this work
Correspondence: Guo-Cui Wu
School of Nursing, Anhui Medical University, 15 Feicui Road, Hefei, 230601, Anhui, People's Republic of China
Email [email protected]
Objective: To investigate the mental illness and correlated factors of primary medical staff during the COVID-19 outbreak in Hefei city, China.
Methods: A total of 180 primary medical staff were randomly selected from seven community hospitals in Hefei Economic and Technological Development Zone as a study group. One hundred and eighty-two health people were recruited as the control group. The self-rating Anxiety Scale (SAS), self-rating Depression Scale (SDS) and Psychological questionnaire of public health emergencies were distributed to them for evaluation.
Results: The score of SAS, SDS in study group was higher than that in control group [(35.57± 10.39) vs (31.31± 7.98); (44.16± 8.46) vs (41.47± 9.47)] (t=4.371, P< 0.001; t=2.849, P=0.005). The fear subscale and total score in the psychological questionnaire of sudden public health events were negatively correlated with age (r=− 0.216, P=0.004; r=− 0.154, P=0.039). Marriage was negatively correlated with depression subscales in psychological questionnaires of SAS, SDS and sudden public health events (r=− 0.184, P=0.013; r=− 0.298, P< 0.001; r=− 0.161, P=0.031; r=− 0.147, P=0.049). Education level was positively correlated with the total score of a psychological questionnaire for sudden public health events (r=0.151, P=0.043); Logistic regression analysis showed that marital status was a protective factor of psychological abnormality.
Conclusion: It is necessary to pay attention to the psychological status of primary medical staff, especially the young unmarried medical staff.
Keywords: novel coronavirus pneumonia, doctor, nurse, anxiety, depressed
A Letter to the Editor has been published for this article.
The new coronavirus is a new strain of coronavirus found in humans in addition to the six coronaviruses currently known to infect humans. It is named "COVID-19" by the World Health Organization (WHO), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The virus strain spreads rapidly in a short time,1,2 and the doubling time is relatively shorter than severe acute respiratory syndrome (SARS). Currently, the disease has spread to other regions of China and around the world.3 The COVID-19 epidemic spreads very quickly and affects a large number of people. By February 15, 2020, a total of 26 countries had been affected by the epidemic, resulting in 51,857 laboratory-confirmed infections and 1669 deaths. In response to this serious situation, the WHO declared the COVID-19 epidemic a public health emergency of international concern on January 30, 2020 and called for collaborative efforts of all countries to prevent and control its rapid spread.4 However, the investigation from the early notification of the outbreak showed that many important information about the source of the virus and the ability to spread between the population, various clinical manifestations, treatment and epidemic characteristics is still unknown,2 and thus the prevention and control of COVID-19 and the implementation of effective measures has great uncertainty.
Currently, COVID-19 spread has become a major national public health event, threatening people's physical and mental health and even life safety, seriously affecting people's daily life and work, endangering social and economic development.5 Since the outbreak, response efforts by the China government have been swift, and three weeks into the epidemic, in an unprecedented move to retard the spread of the SARS-CoV-2 virus, a lockdown was imposed on Wuhan on 23 January, 2020, with travel restrictions. Within days, the quarantine was extended to additional provinces and cities, affecting more than 50 million people in total.6 However, COVID-19 patients were found in almost all provinces across China in the short term. There was no doubt that the medical workforce played an indispensable role in this major public health emergency.7 As of February 22, 2020, Anhui Province had a total of 989 confirmed cases, ranking in the top 10 in China. The primary medical staffs who are on the front line of prevention and control are under heavy work pressure and also suffer from huge psychological pressure. They feel lonely, isolated from the outside world, and unable to communicate face-to-face with their families. The pressure from all aspects cannot be effectively alleviated, making them feel more afraid and worried.8 In the present study, we selected primary medical staff during the first-level prevention and control period in Anhui Province as the study group to evaluate the mental health status and compared them with healthy people in Hefei city, China in the same period. In addition, we also compared the mental health differences between doctors and nurses groups.
Materials and Methods
Study Subjects
A total of 180 primary medical staff consisting of doctors, nurses and medical technicians from 7 community health service centers in the Economic Development Zone of Hefei City were selected as a study group. The average age was 37.96±9.81 years. Ninety two of 180 participants were male, and 88 were female. For marital status, 29 were unmarried, 139 were married, 10 were divorced, and 2 were widowed. For educational level, there were 85 cases with a bachelor degree or above, 38 cases with a junior college degree, 30 cases with higher vocational education and 27 cases with technical secondary school. The inclusion criteria of study subjects were as follows: (1) participated in first-line epidemic prevention; (2) voluntarily participated in this study; exclusion criteria: (1) complicated with physical and mental diseases; (2) those who have not completed all scale measurements. One hundred and eighty-two healthy citizens of Hefei City in the same period were selected as control group, and the inclusion criteria were as follows: (1) healthy people who did not suffer from COVID-19; (2) volunteered to participate in this study; (3) junior high school education level or above; exclusion criteria: (1) combined with other physical and mental diseases; (2) those who did not complete all scale measurements. There were no significant differences in age, gender, marriage, and education level (all P>0.05) between the two groups (Table 1).
Table 1 Baseline Characteristics of Study Subjects |
Ethics statement: The study protocol was approved by the Committee on Medical Ethics of the Second Affiliated Hospital of Anhui Medical University (approval number: YX2020-093), informed consent were received from the study participants and that the guidelines outlined in the Declaration of Helsinki were followed.
Research Tools
Wenjuanxing, an online crowdsourcing platform, was used for questionnaire survey (www.wjx.cn/). The scales used in this online survey included: (1) Self-rating Anxiety Scale (SAS):9 This scale contains a total of 20 items, using a 4-point scale of 1 to 4 points; there is anxiety in Chinese norm of depression of less than 50 points;9 (2) Self-rating Depression Scale (SDS):9 The scale has a total of 20 questions, all of which have a score of 1 to 4 points, and Chinese norm for depression is<50 points, there is depressive;9 (3) Impact of Event Scale-Revised (IES-R), a Psychological Questionnaire for Sudden Public Health Events:9 This scale was surveyed by residents in 2003 to be affected by SARS. After evaluation of reliability and validity, a total of 25 items were established, using 0 to 3 four-level scores, the higher the total score, the greater the psychological impact of public health emergencies.9
Statistical Analysis
SPSS 23.0 was used for statistical processing, measurement data was expressed as (mean ± sd), comparison of means between two groups was performed by two-sample t test, and comparison of category variables was performed by chi-square test. Correlation analysis was conducted by Pearson correlation coefficient, and risk factors wer analyzed by logistic regression analysis; P <0.05 means the difference is statistically significant.
Comparison of SAS, SDS and Psychological Questionnaires for Public Health Emergencies Between the Two Groups
The SAS and SDS scale scores of the study group were higher than those of the control group [(35.57±10.39) vs (31.31±7.98); (44.16±8.46) vs (41.47±9.47)] (t=4.371, P< 0.001; t=2.849, P=0.005) (Table 2).
Table 2 Comparison of SAS, SDS and IES-R Scores Between Two Groups |
Comparison of Abnormal SAS and SDS Between the Two Groups
In the study group, there were 11 cases with abnormal scores on the SAS scale, 46 cases with abnormal scores on the SDS scale, and 52 persons with abnormal scores on the SAS and SDS scales. In the control group, there were 4 cases with abnormal scores on the SAS scale and 23 cases with abnormal scores on the SDS scale; the total number of abnormal scores on the SAS and SDS scale was 27. There was no significant difference in the proportion of the number of abnormal SAS scores between the study group and the control group (χ2 = 3.489, P = 0.062); the study group had a greater proportion of SDS population than the control group (χ2= 9.789, P = 0.002; χ2 = 10.477, P = 0.001).
Analysis of Related Factors of Mental Health Status of the Study Group
Age was negatively correlated with the fear subscale and total score in the psychological questionnaire of sudden public health events (r=−0.216, P=0.004; r=−0.154, P=0.039). Marriage was negatively correlated with SAS, SDS, depression scale in Psychological Questionnaire of Sudden Public Health Events, and total score of the psychological questionnaire of sudden public health events (r=−0.184, P=0.013; r=−0.298, P<0.001; r=−0.161, P=0.031; r=−0.147, P=0.049). The educational level was positively correlated with the total score of the psychological questionnaire of sudden public health events (r=0.151, P=0.043) (Table 3).
Table 3 Analysis of the Relevant Factors of Mental Health Status (r Values) |
Logistic Regression Analysis of Related Factors Affecting the Mental Health of Primary Medical Staff
Mental state was taken as the dependent variable (Y = 1 means "mental abnormality" and 0 means "title mental status"), the above four factors were regarded as independent variables, and the forward LR method was adopted to incorporate the above factors into the logistic regression model. The analysis showed that marriage was a protective factor for psychological abnormalities (OR = 0.015, 95% CI 0.003–0.070) (Table 4).
Table 4 Logistic Regression Analysis of Influencing Factors of Mental State Among Primary Medical Staff |
It has been reported that during the epidemic of influenza A (H1N1) in 2009 high-risk medical staff had a certain degree of psychological disorders, mainly manifested as emotional and sleep disorders, among which the medical staff in fever clinic were the most severe, especially at the early stage and the outbreak stage of H1N1.10 Liu et al11 found that there were problems with the mental health status of medical staff in military hospitals during the SARS outbreak. The scores of somatization, depression, anxiety and obsession were all increased in the psychological questionnaire evaluation, and the scores of somatization and depression were significantly increased. During the outbreak of the Ebola virus outbreak, an analysis of the psychological status of medical staff in Africa found that front-line medical staff had more prominent psychological problems in terms of compulsive behavior, interpersonal sensitivity, depressive symptoms and partial execution behaviors.8 A study from Arab countries showed that during the outbreak of Middle East respiratory syndrome (MERS), medical staff's anxiety scores were relatively high.12 During the outbreak of influenza A in Japan, a survey of the psychological status of 1625 medical staff showed that medical staff in high-risk departments showed varying degrees of anxiety and mental fatigue13. Chan et al14 conducted a survey on the psychological status of medical staff in Singapore two months after the SARS outbreak, their results showed that approximately 20% of medical staff had stress disorders. At the same time, this psychological disorder was related to health, family relations, friends or colleagues. There is a correlation between the relationship, work, and mental appearance of the medical staff, and the emotions of medical staff after the SARS outbreak were affected and hurt to varying degrees. Not only during the epidemic outbreak, medical staff suffered severely from the stress of the epidemic, the anxiety symptoms, depression symptoms and post-traumatic stress of high-risk medical staff remained at a relatively high level for 1–2 years or more after the SARS outbreak.15,16
Previous studies were mostly concentrated in general hospitals and were mostly limited to cross-sectional investigations.8,11 The present study suggested that the mental health status of primary medical staff was lower than that of the healthy control group. Age was negatively correlated with the fear subscale and total score in the psychological questionnaire for public health emergencies; Marriage was negatively correlated with the subscale and total score of depression in the psychological questionnaire of SAS, SDS and sudden public health events. Educational level was positively correlated with the total scores of psychological questionnaires for public health emergencies; these findings indicated that young and unmarried medical care groups had higher scores on the scale and worse mental health status; logistic regression analysis showed that marriage was a risk factor for psychological abnormalities. This study suggests that during the epidemic period, attention should be paid to the psychological status of primary care workers, especially young unmarried health care workers. Studies on the psychological status of and its influencing factors in health care workers (HCWs) during the coronavirus disease 2019 (COVID-19) outbreak indicated that married people were 1.639 times than single (OR = 1.639, 95% CI 1.355–1.922). The main reason for married people's stress might be that their work increased the risk of infection among family members.17
At present, there are relatively fewer researches on the mental state of medical staff during the outbreak of COVID-19, and most of the related studies were in Wuhan18,19 or the tertiary hospitals.20 There are few researches on the psychological changes among primary medical staff under long-term stress. As the first line of epidemic prevention and control, primary-level medical institutions need to participate in pre-examination and triage, key personnel investigation, and follow-up work of people returned from epidemic areas, fever residents and close contacts.21 Because there is a certain gap between the medical knowledge, skills and protective equipment of the primary medical staff and the general hospital above the second level, the psychological problems may be more prominent. However, due to the COVID-19 epidemic, the questionnaire survey could only be conducted through an online platform, and the sample size is limited. In the future, further studies with a large sample size are still needed to provide a more precise reference for the psychological health interventions among primary medical staff in public health emergencies.
Song Wang, Rui-Chen Gao and Rui Li should be considered co-first authors.
Humanities Research Center Project of Anhui Medical University (No. YJSK202017). Doctoral Research Project of the First Affiliated Hospital of Anhui Medical University (No.BSKY2019037).
The authors report no conflicts of interest in this work.
1. Munster VJ, Koopmans M, van Doremalen N, et al. A novel coronavirus emerging in china – key questions for impact assessment. N Engl J Med. 2020;382(8):692–694. doi:10.1056/NEJMp2000929
2. Zhu N, Zhang D, Wang W, et al. A novel coronavirus from patients with pneumonia in China. N Engl J Med. 2020;382(8):727–733. doi:10.1056/NEJMoa2001017
3. Hong KH, Lee SW, Kim TS, et al. Guidelines for Laboratory Diagnosis of Coronavirus Disease 2019 (COVID-19) in Korea. Ann Lab Med. 2020;40(5):351–360. doi:10.3343/alm.2020.40.5.351
4. Zhong BL, Luo W, Li HM, et al. Knowledge, attitudes, and practices towards COVID-19 among Chinese residents during the rapid rise period of the COVID-19 outbreak: a quick online cross-sectional survey. Int J Biol Sci. 2020;16(10):1745–1752. doi:10.7150/ijbs.45221
5. Chen Y, Jin YL, Zhu LJ, et al. The network investigation on knowledge, attitude and practice about Novel coronavirus pneumonia of the residents in Anhui Province. Chin J Prevent Med. 2020;54(4):367–373. doi:10.3760/cma.j.cn112150-20200205-00069
6. Wang C, Pan R, Wan X, et al. Immediate psychological responses and associated factors during the initial stage of the 2019 Coronavirus Disease (COVID-19) Epidemic among the General Population in China. Int J Environ Res Public Health. 2020;17:5.
7. Lu W, Wang H, Lin Y, et al. Psychological status of medical workforce during the COVID-19 pandemic: a cross-sectional study. Psychiatry Res. 2020;288:112936. doi:10.1016/j.psychres.2020.112936
8. Chan-Yeung M. Severe acute respiratory syndrome (SARS) and healthcare workers. Int J Occup Environ Health. 2004;10(4):421–427. doi:10.1179/oeh.2004.10.4.421
9. Zhang ZJ. Behavioral Medicine Scale Manual [M/CD]. China Med Electronic Audiovisual Press. 2005;
10. Wu JS, Yan T, Peng S. A retrospective analysis of the cognition and psychological state of medical staff during A-flow. Chine J Health Psychol. 2011;19(11):1324–1327.
11. Liu JH, Wang W, Gao WB, et al. The effect of SARS on the mental health of medical staff in the hot hospitals of military hospitals. Southern J Nurs. 2004;11(4):9–10.
12. Alsubaie S, Temsah MH, Al-Eyadhy AA, et al. Middle East Respiratory Syndrome Coronavirus epidemic impact on healthcare workers' risk perceptions, work and personal lives. Infect Dev Countr. 2019;13(10):920–926. doi:10.3855/jidc.11753
13. Matsuishi K, Kawazoe A, Imai H, et al. Psychological impact of the pandemic (H1N1) 2009 on general hospital workers in Kobe. PSYCHIAT CLIN NEUROS. 2012;66(4):353–360. doi:10.1111/j.1440-1819.2012.02336.x
14. Chan AOM, Huak CY. Psychological impact of the 2003 severe acute respiratory syndrome outbreak on health care workers in a medium size regional general hospital in Singapore. Occup Med (Chic Ill). 2004;54(3):190–196. doi:10.1093/occmed/kqh027
15. McAlonan GM, Lee AM, Cheung V, et al. Immediate and sustained psychological impact of an emerging infectious disease outbreak on health care workers. Can J Psychiatry. 2007;52(4):241–247. doi:10.1177/070674370705200406
16. Su TP, Lien TC, Yang CY, et al. Prevalence of psychiatric morbidity and psychological adaptation of the nurses in a structured SARS caring unit during outbreak: a prospective and periodic assessment study in Taiwan. J Psychiatr Res. 2007;41(1–2):119–130. doi:10.1016/j.jpsychires.2005.12.006
17. Yao Y, Tian Y, Zhou J, et al. Psychological status and influencing factors of hospital medical staff during the COVID-19 Outbreak. Front Psychol. 2020;11.
18. Shen W, Qin Y, Tao MY, et al. Investigation on mental health level of first-line medical personnel in prevention and control of new coronavirus pneumonia in Hunan province. General Care. 2020;18(8):957–962.
19. Wang F, Shu C. Psychological stress response and intervention measures of first-line medical staff in the new coronavirus pneumonia epidemic. Gen Nurs. 2020;18(7):837–838.
20. Wu JJ, Rong X, Chen F, et al. Sleep quality investigation and influencing factors of clinical first-line nurses in the fight against the new coronavirus pneumonia epidemic. Nurs Res. 2020;34(4):558–562.
21. Chi CH, Wu H, Yang T, et al. Do a good job in the primary prevention and control of new coronavirus infection. Chine J Gen Pract. 2020;19. doi:10.3760/cma.j.issn.1671-7368.2020.0002
© 2021 The Author(s). This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution - Non Commercial (unported, v3.0) License. By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms.


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How To figure out What New Video Games Are Coming out |
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Is the sport you're taking part in have you ever stumped? Whereas it's not always easy to do so, step away from the sport. If you'll distance your self, take a break and come again in a short while, you will have a transparent mind and be ready to play successfully.
To help keep your youngster protected whereas playing video games, discover out concerning the multiplayer options of the video games they are taking part in. Set age applicable limits to gaming interactions with others on-line. Some games have parental control options that you should use to find out the kind of interaction your child can have with strangers.
Simplifying Tips To ASSIST YOU TO Understand Video Game Cheat Codes can improve your life in ways you never thought doable. You do not even have to depart your living room to learn how to talk Italian or lose these last 10 pounds! Hopefully this text has given you loads to consider with gaming and how to make the best of your video play.
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The beautiful City Hall of Vianen was built around 1425 and has been awarded as a national monument. The beautiful facade with battlements, built from Namur stone, is striking. On that facade you can see the coat of arms of Lexmond, Meerkerk, Hei- and Boeicop, Ameide, Vianen and Tienhoven.
Originally, the town hall consisted of a civic hall, which was also used as a market hall and a smaller space from which the city was administered and where justice was administered. At the beginning of the 21st century, the town hall is renovated, combining the old building with a modern design.
City Hall Vianen
Voorstraat 30
Contact details
E: firstname.lastname@example.com
W: https://www.vrijstadvianen.nl/de-vrijstad/historische-parels/het-stadhuis


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Great Rates for MotorHome Rental in Alghero, Sardinia
Campervan rental in Alghero
Alghero is a fantastic destination to start a self-exploration of the stunning island of Sardinia. Consisting of beaches, World Heritage sites, hills and mountains, the island is best seen by hiring a self contained motorhome. The road network is great and there are plenty of camping van sites to be found.
Sardinia has always been popular among locals but is fast becoming an international destination. Self-drive is one of the best ways to see the island and witness all that is has to offer.
What a wonderful trip in Italy with a camper van from motorhome Italy. We had 30 days days traveling across that beautiful country and the van became our temporary home that we now really miss . We found Kindness everywhere from the people of Italy and the country itself was just beautiful. It was sad returning the van on the last day. We will return next year and will rent a van with you as it is easy and secure. Jon Van Leeuwen
See more comments or add your comments here
Recommended campervan drives in Alghero
This drive will take you to the awe-inspiring Cappo Gracia, where Neptune's Grotto is located. Visit some lovely fishing villages along the coast as well as the famous beaches at La Pelosa. Rest at camper sites such as campsite Torre del Porticciolo or camping Village Li Nibari.
Drive along this stunning coast near Arbus to visit breathtaking beaches. From Alghero, head south along the SP49 and stop at camping Village Bella Sardinia to visit the medieval town of Bosa. Then continue along the SS-292, which joins the SS-126. You can break the journey up by stopping at campsite S'Ene Arrubia.
This is a lovely drive through the interior of the island, ending at the popular resort areas of San Teodoro and Budoni. There are some great camper sites along this route, including campsite La Pineta and Campsite Tavolara, among others.
The roads throughout the island are great; some of the smaller roads are slower but scenic. There are petrol stations along most routes.
Urban roads: 50kph unless otherwise posted
Provincial highways: 90kph
National roads and motorways: 110-130kph
0.5 per cent, fines are quite high, with possible imprisonment.
Driving on the right is customary here, and be aware that Italians love to drive close to the vehicle in front of them. Most routes are patrolled and police do not tolerate speeding.
High due to transportation costs.
Compare Camping Van Hire rates with RVRentaCampervan
Touring van hire is available at the airport, Alghero harbour and Alghero city centre, the latter being the best option. There is a wide selection of touring vans in various Nugget and Sky models, with the best rates easily compared right here.


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Table of Contents
Are you tired of the same old chai tea lattes and flavors? Want to try something new that will tantalize your taste buds and invigorate your senses?
Look no further than Chai Tea Citrus – a recipe that combines the spicy warmth of chai with the zesty freshness of citrus.
This unique recipe is perfect for anyone who loves bold, complex flavors that awaken the palate. Whether you're a fan of the tea or just looking to try something new, Chai Tea Citrus is the perfect way to add delight to your daily routine.
Not only is this recipe delectable, but it also has health benefits. It is known to boost metabolism, aid digestion, and reduce inflammation, while citrus is packed with vitamin C and antioxidants. So, you can indulge in this recipe knowing it's good for you too.
Imagine the aroma of cinnamon, cloves, and cardamom wafting through your kitchen, blended with the tangy scent of orange and lemon. Picture yourself sipping a warm, frothy cup of Chai Tea Citrus, feeling the smooth texture of the tea and the bright burst of citrus on your tongue.
This recipe has its roots in Indian culture, where chai is a beloved beverage enjoyed in households and street corners alike. By adding citrus to the mix, we've created a modern twist on a classic recipe that will delight your taste buds and make you move to a different time and place.
Are you ready to feel the magic of Chai Tea Citrus for yourself? Follow this article, and you'll be sipping on this delicious brew in no time.
What is Chai Tea and Why Choose Chai Tea Citrus?
Chai tea is a traditional Indian beverage that has been enjoyed for centuries. The word "chai" actually means tea in Hindi, and the drink is made by brewing black tea with a mix of aromatic spices such as cinnamon, cardamom, cloves, and ginger.
Chai tea is known for its warming and soothing properties, making it popular during colder months. However, Chai Citrus offers a unique and refreshing twist on this classic beverage by incorporating citrus fruits' bright, zesty flavors such as lemon and orange. The combination of warm, spicy flavors and bright citrus notes creates a delicious and invigorating drink that can be enjoyed any time of year.
Ingredients for Chai Tea Citrus
To prepare it, you'll need the following ingredients:
- Orange zest
- Lemon zest
- Cinnamon stick
- Fresh ginger
- Cardamom pods
- Whole cloves
- Used vanilla bean
- Black tea bag
- Lemon juice
- Orange juice
- Sweetened condensed milk
One of the key differences between traditional Chai tea and Chai tea citrus is the addition of citrus fruits and juices. These ingredients give the drink a bright, zesty flavor that complements the warm, spicy flavors of the traditional Chai blend. Additionally, using whole spices rather than ground spices can enhance the flavor of the tea and create a richer, more complex taste.
How To Make Chai Tea Citrus
Preparing this delicious drink is a simple and straightforward process. Here's what you'll need:
Prep Time: 5 min
Cooking Time: 2 hr
Total Time: 2 hr 5 min
Makes 8 servings
For Aromatics
- 1 ea. Orange zest, save rest for juice
- 1 ea. Lemon zest, save rest for juice
- 3 tsp. Cinnamon stick
- 1 tsp. Knob of fresh ginger
- 1 tsp. Cardamom pods
- 1 tsp. Whole cloves
- 1 ea. Used vanilla bean, or sub 1 tsp extract
- 5 cup Water
For 1 cup of tea
- 2 ea. Black tea bag
- 1/2 ea. Lemon juice
- 1/2 ea. Orange juice
- 1 tbsp. Sweetened condensed milk (as an alternative, use almond milk)
- In a medium saucepan, place all aromatic ingredients together. Bring to a simmer over low heat. Additionally, you can place it in a slow cook on the lowest setting. The aromatics can simmer for up to 2 hours, adding additional water as needed.
- To make aromatic chai tea, strain a cup of aromatic liquid into a mug. Squeeze as much citrus juice as desired. Steep black tea for 2 minutes and then remove. Add sweetened condensed milk (or almond milk).
- Enjoy your tea
Tips For The Perfect Cup
To ensure that your Chai tea citrus is the perfect balance of warm, spicy, and zesty flavors, here are a few tips to keep in mind:
- Use whole spices whenever possible. Whole spices will release their flavors more slowly, resulting in a more full and complex taste.
- Don't let the tea steep for too long. Steeping longer than needed can result in a bitter taste, so remove the tea bag after 3-5 minutes.
- Adjust the sweetness and acidity to your liking. If you prefer a sweeter or more tart flavor, adjust the amount of sweetened condensed milk and citrus juices accordingly.
- Experiment with different variations. Citrus Chai is a versatile drink that can be customized to your taste. Try adding different fruits, spices, or sweeteners to create your own unique version.
Chai Lattes Variations
While Chai tea latte citrus is delicious on its own, there are many variations of chai tea lattes that you can try to mix things up. Here are a few chai lattes to get you started:
- Apple Chai: Replace the citrus fruits with apple juice and add a sliced apple to the pot while simmering the spices.
- Pumpkin Chai: Add pumpkin puree and pumpkin pie spice to the pot for a cozy fall-inspired drink.
- Vanilla Chai: Add a vanilla bean or a few drops of vanilla extract to the pot for a creamy and indulgent flavor.
- Chocolate Chai: Add a few tablespoons of cocoa powder to the pot for a rich and chocolatey twist on Chai tea.
If you're a fan of chai tea lattes and want to prepare them easily at home, you have a few options to consider. Here are three ways to make a delicious chai tea latte: chai tea bag, chai concentrate, or Starbucks chai.
If you have chai tea bags at home, you can easily make a chai tea latte by making a strong cup of chai tea and adding steamed milk and sweetener.
Another way to make a chai tea latte at home is to use chai concentrate. Chai concentrate is a highly concentrated chai sold in most grocery stores.
If you're a Starbucks chai tea lattes fan, you can also make them at home using Starbucks chai tea concentrate. This concentrate can be purchased at most Starbucks locations or online.
How To Serve Chai Tea Citrus
Chai tea citrus can be served hot or iced, depending on your preference. Simply pour the tea into a mug for a hot drink and enjoy. For an iced chai tea latte, pour the tea into a glass filled with ice and decorate it with a slice of lemon or orange. Add a spoonful of whipped cream or a sprinkle of cinnamon on top for an extra indulgent treat.
Why is chai tea latte good for you?
Chai tea latte is often made with a combination of black tea and various spices such as cinnamon, cardamom, ginger, and cloves, which can offer a range of potential health benefits. Black tea, the primary ingredient in chai, contains antioxidants that could help reduce the risk of chronic diseases like heart disease and cancer. Additionally, some of the spices commonly used in chai, such as ginger and cinnamon, have anti-inflammatory properties and may aid in digestion.
Is there any caffeine in chai tea?
Yes, it typically contains caffeine, as it is made with black tea leaves. But, the exact amount of caffeine can vary based on the type of tea and the brewing method. On average, an 8-ounce serving contains about 25-50 mg of caffeine, less than a cup of coffee.
Which is healthier, chai tea or coffee?
It's difficult to directly compare the health benefits of chai tea and coffee as they have different compositions and effects on the body. However, chai tea may offer some potential health benefits due to its antioxidant content and the anti-inflammatory properties of some of its spices. On the other hand, coffee has several health benefits, including a lower risk of certain types of cancer, type 2 diabetes, and Parkinson's disease. Ultimately, the healthfulness of any of them will depend on individual factors and dietary preferences.
Does a chai tea latte taste better with milk or water?
Traditionally, a chai tea latte is made with milk, as it helps to balance out the spiciness and bitterness of the tea. The milk also adds creaminess and sweetness to the drink. However, some people may prefer to use water or a combination of water and milk to make their chai tea latte. Ultimately, it comes down to personal preference, so you should try both ways of chai tea lattes and see which one you like better.
Why is chai latte so delicious?
Chai latte is a delicious beverage because it combines the bold and warming flavors of chai tea with creamy milk and a touch of sweetness. The spices in chai, such as cinnamon, cardamom, ginger, and cloves, add a complex and aromatic flavor profile to the tea. When combined with the creamy texture of steamed milk and a sweetener like honey or sugar, it creates a comforting and indulgent drink that appeals to many people's taste buds. Additionally, the warmth of the drink can provide a soothing and relaxing effect, making chai lattes a popular choice for cozy and comforting moments.
What is a dirty chai latte?
A dirty chai latte is a variation of a traditional chai tea latte recipe that includes a shot of espresso. To make it, add a shot of espresso to a cup of chai tea, then mix in steamed milk and any desired sweeteners or spices. Adding espresso adds a strong coffee flavor and boosts caffeine content. It's a great choice for those who enjoy the spicy and sweet flavors of a chai latte but also want an energy boost with coffee.
Is Starbucks chai tea latte good for you?
Starbucks chai tea latte contains a mix of black tea, spices, and milk, offering potential health benefits. However, it is also high in sugar. Consuming high amounts of added sugar regularly can lead to several health problems, such as weight gain, type 2 diabetes, and heart disease. Therefore, while a Starbucks chai tea latte can be enjoyed in moderation as a treat, it is not a particularly healthy beverage choice.
In summary, the Chai Tea Citrus recipe is a delicious and healthy beverage option that is easy to make and can be enjoyed anytime. With its unique blend of spices, fresh citrus flavors, and antioxidant-rich tea, this recipe offers many health benefits and is a great alternative to sugary drinks.
If you're looking to experiment with the recipe, try adding different types of citrus fruits or alternative sweeteners such as honey or agave nectar. Depending on your preference, you can also try serving the beverage hot or cold.
As someone who has tried and enjoyed this recipe, I can attest to its flavor and health benefits. It's a great way to start your day or to enjoy a refreshing afternoon pick-me-up.
I invite you to share your experience with this recipe in the comments section or on social media. I'm excited to hear your thoughts and ideas.
If you're looking for more healthy beverage options, check out our other recipes on our website. I wish you success in the kitchen and hope you enjoy this Chai Tea Citrus recipe!
Thank you for reading, and happy cooking!


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Saudi Arabian Prince Shuns Nigeria Jollof & Orders Ghana Jollof for Entire Mosque to Break Ramada
Saudi Arabian Prince-Mohammed bin Salman treated a full mosque yesterday in Riyadh, the capital of Saudi Arabia to a delicious Ghana Jollof to break a long day of fasting—and according to reports, the mosque received a record attendance as regulars called friends and family to come and taste the delicious coloured rice they were enjoying.
Apparently, Mohammed bin Salman chanced on Ghana Jollof in London last year during an African Charity Ball, organised by footballer-Didier Drogba and since then he has hired a Ghanaian in his country as one of his maids, who regularly serves him and his family the the African dish.
And on his return to Riyadh from United States, Prince Mohammed bin Salman decide to initially treat his close friends and family to a special Jollof dinner which he somehow extended to an entire mosque, the US Today reports.
After the dinner, Mohammed bin Salman who is an active twitter user, and has in the past ran into twitter exchanges with Donald Trump tweeted the above photo—saying, "for the first time, we broke today's fasting with Ghana Jollof and everyone is talking about the tasty spicy food."
Over 100,000 people have since retweeted Mohammed bin Salman's tweet—with some Ghanaian twitter users making fun of Nigerians, that Ghana Jollof rocks.


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Did you come here from a link on another website? See page bottom for latest version of this page. This site is not associated with any ski area. Use of any page means you accept the SkiMyBest Site Use Agreement or EXIT NOW.
"SKIING IS A SLIDING SPORT": Tactics for Terrains and Snow Textures and Racing-- Skiing Narrow Trails by Bill Jones, Ski Instructor Certified Professional Ski Instructor (Registration #110478), Level III How To Reserve Private Ski Lessons with Bill Jones
Narrow ski trails where there is not width enough for the classic round ski turn are sometimes encountered where less steep routes are cut through the forest to avoid steeper slopes or as runouts below open bowls. At times they may be no more than skinny paths winding among trees. And they may follow valley bottoms along snow-filled streams, adding to the challenge..
Speed management and continual turning in these situations are musts, lest one fly off the side of the trail into a tree or over a bank or go somewhere else you don't want to at a speed you don't want..
The tactic to emphasize in these situations is turning by leg steering with lots of skidding to tighten arcs of turns and manage speed with the friction generated. Sometimes there will not be room or time enough for turns, and then lengthy sideslipping or forward sideslipping are good choices. And keep watching for others who might not manage their speed and turning as well as you, or maybe just want to pass--let them and then they will be out of the way.
With good tactics, narrow trails can be pleasing pathways, providing more intimate looks inside the winter woods than the expansive vistas that may be seen on the more typical open ski slopes. One might even encounter non-hibernating tree squirrels, rabbits, or even an ermine. Snow-draped trees may remind of a scene Walt Disney might have created, and if along a stream, patches of open water may gurgle at you with dashing white water that then disappears below the white blanket.
A final word: Eventually, after selecting the tactic to apply, you must "Point the skis down the hill; let them buck; the mountain will teach you!"
Contents of "TACTICS FOR TERRAINS and SNOW TEXTURES and RACING": Overview Skiing Groomed Snow Skiing Hard Snow/Ice Skiing Among Trees Skiing Narrow trails--you are on this page. Skiing Moguls Skiing Powder Snow Skiing Cold Snow-Warm Snow/New Snow-Old Snow Skiing "Spring" Snow Skiing Steeps Skiing Gates/Racing "SKIING IS A SLIDING SPORT"--a skiing web manual: Skiing Web Manual Contents Why Read This Skiing Web Manual That First Skiing Lesson A Little Skiing History A Little Skiing History Motion inSkiing Conventional Skiing Wisdoms Skier Excuses Fear in Skiing Conditioning for Skiing How Skis Work Equipment and Technique Skiing Equipment How to Develop Balance on Skis A Skiing Turn Simplified The Final Skiing Skill: pressure management TACTICS FOR TERRAINS and SNOW TEXTURES and RACING Skiing Tips and Tales--a potpourri Exercises for Developing Skiing Skills Children and Skiing Age and Skiing Gender & Skiing Culture & Skiing Skiing Ethics and Slope Survival Slope Safety Skiing Environment Videos and Apps Glossary Acknowledgements SkiMyBest Website Contents
This "Skiing Narrow Trails" page last modified January 9, 2022. Did you come here from a link on another website? For latest version of this page, copy to your browser: http://www.SkiMyBest.com/skinartr.htm. Copyright © 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021, 2022. William R Jones.


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It only takes a day or two for zucchini to develop to the scale of a bowling pin or a rabbit to interrupt through your fence and finish off your peas. And, finally, there may be The Gardenettes, a neighborhood and account dedicated to gardening, DIY, and cooking movies. Run by Chloe Thomson and Melissa King, you'll completely remember to discover tons of inspo for your personal garden.
The gardening expertise may involve visiting a few of the world's nice gardens at completely different seasons to see the relation of individual teams of crops, timber, and shrubs to the whole design; to review the positioning of plants by way of their color, texture, and weight of leaf or blossom; and to appreciate using special features corresponding to ponds or watercourses, pavilions, or rockeries. Garden visiting on a world scale provides an opportunity to grasp the broad cultural influences, as well as the variations in climate and soil, that have resulted in so many various approaches to backyard making. People dwelling in these so-called "blue zones" have certain components in frequent – social help networks, day by day train habits and a plant-based food regimen, for starters.
In addition, rich ancient Egyptians grew vineyards, as wine was a sign of the upper social lessons. Roses, poppies, daisies and irises might all even be found within the gardens of the Egyptians.
This is a really intently related problem to starting out too large. Small seedlings correctly spaced can go away the garden looking barren.
The goal is to create a backyard in concord with, and adapted to a given area. This kind of gardening sometimes reduces water usage, maintenance, and fertilization prices, while increasing native faunal interest. They surrounded the gardens by partitions to guard them from animals and to offer seclusion. During the subsequent two centuries, Europeans began planting lawns and raising flowerbeds and trellises of roses.
Deal Alert: Succulents and different indoor crops are 25 p.c off at present
There are helpful bugs which might be your companions in the garden. They kill off pests or keep vegetation cross-pollinated. And always ensure of what you are spraying for earlier than your spray something.
Make certain the water penetrates the soil versus just placing somewhat bit on the floor." Newer plants will must be watered extra regularly because their root systems aren't fully developed. As for the best time of day, Lambton suggests early morning earlier than it gets too warm so the plant can really soak up the water.
They will ripen and rot or bolt to seed. Better to start small than to waste each your effort and your vegetables. Two different issues are sited it by a source of water and in a spot that will get at least six hours of solar a day. Vegetables that don't get sufficient sunlight will wrestle all season, set fewer fruits, and develop less tasty sugars than nicely-sited greens.


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The case of a Colorado 4-year-old girl who was strip searched by a government social worker could prompt the U.S. Supreme Court to take a new look at its "qualified immunity" doctrine that lets officials off the hook in some circumstances when they violate an individual's civil rights.
At the court's May 16 private conference, the justices will consider whether to grant review in the case of I.B. v. Woodard. The U.S. Court of Appeals for the Tenth Circuit in January dismissed the girl's claim that case worker April Woodard violated the Fourth Amendment by stripping and photographing her without a warrant or parental consent. The search came after allegations that the child had been abused.
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HTML Full TextION EXCHANGERS: A USEFUL TOOL FOR SEPARATION AND SIMULTANEOUS PURIFICATION OF LYSOSOMAL CYSTEINE PROTEINASES, CATHEPSINS B, H AND L.
N. Raghav*, M. Singh, S. Garg, R. Kaur, S. Jangra and I. Ravish
Department of Chemistry, Kurukshetra University, Kurukshetra-136119, Haryana India
ABSTRACT: Lysosomes of mammalian tissues contain a number of cysteine proteinases such as Cathepsins B, H and L, which participate in intracellular protein degradation and are involved in various pathological disorders. These proteinases have been purified to ~ 1600 fold with ~25% yields from goat liver. These Cathepsins having similar molecular weight (~25,000 Da) were obtained as a single pool after initial purification steps i.e. homogenization of acetone powder, acid autolysis at pH 4.0, 30-70 % (NH4)2SO4 fractionation and molecular sieve chromatography on Sephadex G-100, were simultaneously separated and purified on exchange chromatographies. Cathepsin L was completely separated and purified from Cathepsin B & H at CM- Sephadex C-50 column at pH 5.6 and was eluted as bound protein at 0.62 M NaCl with a purification fold ~1629 and 22 % yield. Cathepsin B was eluted as bound protein in ~1545 fold with ~45% yield at 0.40 M concentration. Complete separation of Cathepsin H from Cathepsin B was achieved on DEAE-Sephadex A-50 at pH 6.0 where the former was obtained as unbound protein and the latter; Cathepsin H thus obtained was purified to ~ 1601.11 fold with ~29.18% yield. Cathepsin B, H & L were thus purified using ion-exchange chromatography.
Keywords: |
Cathepsin B,
Cathepsin H, Cathepsin L,
ion-exchange chromatography
INTRODUCTION: Mammalian tissues contain a number of lysosomal proteinases 1-4 which belongs to papain super family. Besides their role inside lysosomes, these cysteine proteinases also degrade proteins outside lysosomes 5. The cathepsins alone are involved in protein breakdown in lysosomes, antigen presentation, proteolytic processing of proenzymes and prohormones, fertilization, cell proliferation, differentiation and apoptosis 6-8. Cathepsins B (EC 184.108.40.206), Cathepsins H (EC 220.127.116.11) and Cathepsins L (EC 188.8.131.52), are lysosomal proteolytic enzymes belonging to the cysteine protease family.
They are widely distributed in almost all mammalian cells, being mainly responsible for intracellular protein degradation 9 and protein turnover 10-13. When secreted lysosomal cysteine proteinases result in pathological conditions, they are observed to be involved in a number of diseases such as muscular dystrophy 14, malignant melanoma 15-16, cancer 17-19, rheumatoid arthritis 20, osteoarthritis21-22, inflammatory and bone and joint disorders 23, Alzheimer's disease 24-25, multiple sclerosis 26 and pancreatitis 27.
In many of these diseases lysosomal enzymes were found to be present in extracellular / extralysosomal environment in the proforms, which are substantially more stable than the mature enzymes 28. The physiological, pathological role of these important protein processors necessitates their identification, isolation, purification and characterization at the required site. The present work focuses on Cathepsins B, H & L having similar molecular weight 29-31, how ion exchangers can be efficiently employed for simultaneous purification of Cathepsins B, H & L from a single source i.e. goat liver.
All the chemicals were of analytical grade. Fast Garnet GBC (o-aminoazotoluene diazonium salt, α-N – benzoyl - D, L-arginine - 2-naphthylamide (BANA), Z-Arg-Arg-NNapOMe, Z-Phe-Arg-NNapOMe and Leu-2NNap were purchased from Bachern Feinchemikalien AG, Switzerland. Sephadex G-100, CM-Sephadex C-50 and DEAE- Sephadex-A-50 were obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. The protein sample was concentrated using Amicon stirred cells with YM 10 membrane under nitrogen pressure of 4–5 psi. The source of enzyme was fresh goat liver obtained from local slaughter house. Glass double distilled water was used in all the experiments.
Enzyme Assay:
Cathepsin B activity was determined using BANA or Z-Arg-Agr-NNapOMe as substrate 29 at pH 6.0. The activity of Cathepsin H was assayed as described previously 30 using Leu-2-NNap as substrate at pH 7.0 and also by BANA at pH 6.5. Cathepsin L activity was assayed 31 using Z-Phe-Agr-NNapOMe at pH 6.0.
Protein assay:
The acid soluble proteins were quantitated in the supernatant using Bradford method 32.
Preparation of acetone powder:
Fresh goat liver was washed thoroughly with cold saline solution to remove blood capillaries and was minced properly with ten volumes of chilled acetone in a mixer cum grinder to disintegrate the tissue. The precipitate thus obtained, were filtered, using a Buchner funnel. The residues were resuspended in three volumes of chilled acetone, homogenized, and filtered. Finally, the filtrates were collected and a stream of nitrogen was flushed through it and dried in vacuum over concentrated sulphuric acid and the acetone powder was stored over anhydrous calcium chloride at 4oC until use.
Separation and purification of Cathepsins B, H and L: All the purification steps were carried out at 4°C unless otherwise stated. The initial steps of purification i.e., Goat liver acetone powder homogenization in cold 0.1 M sodium acetate buffer pH 5.5 containing 0.2 M NaCl and 1mM EDTA, Acid-autolysis at pH 4·0 and 30-80% ammonium sulphate fractionation. Fractionation of proteases based on molecular weight on Sephadex G-100 column chromatography according to the method described by Kamboj et.al29-31. 2-Mercaptoethanol (2mM) was included in all these steps, except during acetone powder preparation. 50 ml of enzyme solution was applied to a column of Sephadex G-100 equilibrated with 0.1M sodium acetate buffer pH 4.76 containing 0.2mM NaCl, 1mM disodium EDTA.
The column was eluted with the same buffer-EDTA solution at a flow rate of 40 mlh, and the fractions of 10 ml were collected and stored at 4oC till the absorbance of effluent buffer at 280nm was nil against the column buffer. The enzyme activities were measured using BANA. Thereafter BANA active fractions were analysed for Z-Arg-Arg-NNapOMe, Leu-2-NNap and Z-Phe-Arg-NNapOMe hydrolysing activities to check the presence of Cathepsin B, H and L (Fig. 1).
FIG.1: MOLECULAR SIEVE CHROMATOGRAPHY ON SEPHADEX G-100 COLUMN SHOWING PROTEIN, BANA, Leu-βNA, Z-Arg-Arg-4-m βNA, Z-Arg-Arg-4-m βNA HYDROLYZING ACTIVITIES.
The fractions active against BANA, Leu-2-NNap Z-Arg-Arg-NNapOMe and Z-Phe-Arg-NNapOMe obtained from Sephadex G-100 column were pooled, concentrated and dialysed against 20mM sodium acetate buffer pH 5.6 containing 1mM disodium EDTA and 2mM 2-mercaptoethanol for 24 h. Cathepsin B, H and L having molecular weights in the same range29-31 (~25,000) were eluted as one peak from Sephadex G-100 column and further purified using ion-exchange chromatography.
Ion Exchange Chromatography on CM-Sehadex C-50:
The dialyzed enzyme sample was applied to a column of CM-Sephadex C-50 equilibrated with 20 mM sodium acetate buffer pH 5.6 containing 1 mM disodium EDTA and 2mM 2-mercaptoethanol. The column was washed with same buffer-EDTA-mercaptoethanol solution. The fractions of 5ml were monitered for their protein content by measuring the absorbance at 280nm. When the absorbance of effluent buffer for unbound protein at 280nm was found nil, the bound proteins were eluted with a linear NaCl (0.0-0.8M) gradient and the fractions of 5ml were collected and were analysed for their protein content by measuring the absorbance at 280nm.
The unbound and salt-gradient eluted fractions were analyzed for their activities against BANA, Leu-2-NNap, Z-Arg-Arg-NNapOMe and Z-Phe-Arg-NNapOMe. The Z-Arg-Arg-NNapOMe and Z-Phe-Arg-NNapOMe hydrolyzing activities were pooled separately. The fractions containing both BANA as well as Leu-2-NNap hydrolyzing activities were pooled together and processed for separation of Cathepsin B from H (Fig. 2).
FIGURE 2 ION EXCHANGE CHROMATOGRAPHY ON CM-SEPHADEX C-50 COLUMN SHOWING PROTEIN, BANA, Leu-βNA, Z-Arg-Arg-4-m βNA, Z-Arg-Arg-4-m βNA HYDROLYZING ACTIVITIES
Anion Exchange Chromatography on DEAE-Sephadex A-50:
The above obtained combined pool of Cathepsin B and Cathepsin H were dialyzed against 20mM sodium phosphate buffer pH 6.0 containing 1mM disodium EDTA and 2mM 2-mercaptoethanol for 24h and were applied on DEAE-Sephadex A-50 column equilibrated with 20mM sodium phoshphate buffer (pH 6.0) containing 1mM disodium EDTA. After removing unbound proteins active against Leu-2-NNap, the fractions of bound proteins were eluted with NaCl linear (0.0-0.5M) gradient and were analysed for their activities against Z-Arg-Arg-NNapOMe (Fig. 3).
Z-Arg-Arg-NNapOMe hydrolyzing activity obtained after CM-Sephadex C-50 and DEAE-Sephadex A-50 were pooled together. The separated pools of Cathepsins B, H and L were pooled, concentrated to 15 ml and dialyzed against 50 mM sodium acetate buffer pH 4.6 containing 1 mM disodium EDTA, 0.2M NaCl were concentrated and stored at 4oC for further use. The preparations of Cathepsins B and H contained 0.1mM HgCl2 whereas in cathepsin H 2mM 2- mercaptoethanol was added.
FIG.3: ION EXCHANGE CHROMATOGRAPHY ON DEAE-SEPHADEX A-50 COLUMN SHOWING PROTEIN, Leu-βNA, Z-Arg-Arg-4-m βNA HYDROLYZING ACTIVITIES.
RESULT AND DISCUSSION: Fig. 1 shows the elution profile of different enzymes on Molecular sieve chromatography on Sephadex G-100 column after ammonium sulphate fractionation. It helped in separation and isolation of Cathepsins B, H and L on the basis of their molecular weight (~ 25000) from cathepsin D and DPPI. The results correlate with earlier work 29-31, 34. Some workers 36-41 didn't include this step at an early stage of purification and hence could not accomplish simultaneous isolation of these proteases. Further fractionation of Sephadex G-100 pool on cation exchange chromatography at pH 5.6 resulted in complete separation of cathepsin L from B and L (Fig. 2). Cathepsin B was eluted as bound protein at 0.62M NaCl exclusively as Z-Phe-Arg-NNapOMe hydrolyzing activity. The separation of cathepsin L from cathepsin B and H on CM-Sephadex cation exchanger has earlier been reported 42-43. Cathepsin B was present in unbound (~ 40%) as well as in bound fractions (~ 60%) as reported45-46 at pH 5.0. the bound Cathepsin H was eluted at 0.48M NaCl alongwith Cathepsin B.
Although at cation exchanger Cathepsin H showed a tendency to separate as indicated by a shoulder peak of BANA having Leu-2-NNap activity, complete separation of Cathepsin B and H was achieved on DEAE-Sephadex A-50 at pH 6.0, where Cathepsin H was eluted unbound and Cathepsin B was eluted at 0.2M NaCl. Earlier studies also report the separation of Cathepsin H from B on DEAE-Sephadex A-5042-44. Further purification of Cathepsin H and L was done on organomercuric affinity column to extensive inactivation of the enzymes.
Table 1 depicts the purification factor achieved and % yield obtained after each step of purification. The % yield obtained in all the three enzymes ranges between ~25% whereas ~1600 fold purification was achieved in all the three proteinases. The results are quite encouraging in comparison to earlier reports37-38 in case of Cathepsin H. these workers have not reported simultaneous separation and purification of Cathepsins B, H and L. For the first time, cation and anion exchangers have been successfully used for simultaneous purification of three cysteine proteinases. We are working in the identification of small molecular weight compounds as inhibitors to endogenous proteolytic activities 46-51.
S.No. | Purification Steps | Cathepsins B | Cathepsins H | Cathepsins L | |||
Purification Factor | % Yield | Purification Factor | % Yield | Purification Factor | % Yield | ||
1. | Crude Extract Supernatant, S1 | 1.0 | 100.0 | 1.0 | 100.0 | 1.0 | 100.0 |
2. | Acid Fractionation Supernatant, S2 | 1.75 | 99.31 | 1.40 | 79.84 | 1.72 | 98.52 |
3. | 30-80% (NH4)2SO4 Pellet, P4 | 30.65 | 97.52 | 22.92 | 73.30 | 30.53 | 97.86 |
4. | Sephadex G-100 Pool | 69.61 | 75.61 | 61.78 | 67.61 | 68.51 | 74.58 |
5. | CM-Sephadex C-50 Bound Pool | 1545.99 | 45.71 | 1154.61 | 55.08 | 1629.96 | 22.22 |
7. | DEAE-Sephadex A-50 Pool | - | - | 1601.11 | 29.18 | - | - |
The results presented here are of a single enzyme preparation that resulted in simultaneous separation and purification of Cathepsin B, H and L using different steps as detailed above. Cathepsin B, H and L activities were assayed using BANA, Leu-2-NNap, Z-Phe-Arg-NNapOMe at pH 6.0, 7.0 and 6.0, respectively. Cathepsin B, H and L has emerged out as targets for development of anticancer drugs. Therefore it becomes relevant to evaluate inhibitory potency of compounds of different classes known to possess anticancer activities on these purified enzymes. The enzymes separated using these steps have been used for studies of different compounds.52-57
CONCLUSION: From the literature study, it is clear that cathepsin B, H and L are important targets in cancer and other pathological disorders such as arthritic conditions and tissue generative disorders and the development of selective inhibitors to these enzymes are a rational approach in the search for chemotherapeutic agents. In this paper, we have reported simultaneous separation and purification of Cathepsins B, H and L using cation and anion exchangers.
ACKNOWLEDGEMENT: Among the authors, Mamta Singh and Suman Jangra are thankful to CSIR New Delhi, India and Shweta Garg and Indu Ravish are thankful to UGC- New Delhi, India for award of SRF and Ravinder Kaur is thankful to Kurukshetra University, Kurukshetra for providing URS. And the authors are thankful to Kurukshetra University, Kurukshetra for providing necessary research laboratory facilities. The authors have declared no conflict of interest.
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How to cite this article:
Raghav N, Singh M, Garg S, Kaur R, Jangra S and Ravish I : Ion Exchangers: A Useful Tool for Separation and Simultaneous Purification of Lysosomal Cysteine Proteinases, Cathepsins B, H and L.. Int J Pharm Sci Res 2015; 6(7): 2944-49.doi: 10.13040/IJPSR.0975-8232.6(7).2944-49.
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Article Information
N. Raghav*, M. Singh, S. Garg, R. Kaur, S. Jangra and I. Ravish
Department of Chemistry, Kurukshetra University, Kurukshetra, Haryana India
12 November, 2014
12 January, 2015
, 21 March, 2015
01 July, 2015


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Flintshire landlord given 28 days to pay £11,000 fine after being found guilty nine offences
A Flintshire landlord has been fined over £11,000 after leaving an elderly Shotton tenant without adequate heating and bathing facilities.
Steven John Gleave from Prestatyn, the owner of four rental properties in Flintshire, was found guilty of failing to comply with an Improvement Notice and eight other housing offences, including: failing to register his rental properties with Rent Smart Wales and failing to obtain a licence to operate as a private landlord in Wales both legal requirements since November 2014.
The property subject to the Improvement Notice, issued under the Housing Act 2004, was a ground floor flat in a two storey terrace in Shotton occupied by an elderly tenant.
The property had not been maintained by Gleave over a number of years.
Subsequently, the occupier had to suffer with penetrating dampness, black mould growth, a lack of adequate heating, poor kitchen and bathing facilities, poor fire safety provisions and slates falling off the roof.
Gleave also refused to pay off the electricity bill for another flat in the same building and refused to get the electricity meter for both flats separated.
This meant the elderly occupier had no choice but to pay off the arrears (over £1, 400) through the shared meter.
Gleave had also refused to register all four of his rental properties in Flintshire with Rent Smart Wales and failed to carry out mandatory training in order to become licensed to operate as a landlord in Wales.
Flintshire County Council's Cabinet Member for Planning and Public Protection, Councillor Christopher Bithell, said:
"This successful prosecution reflects Flintshire County Council's commitment to ensuring homes in the private rented sector meet all the legal standards required for private housing in Wales.
We believe everyone has the right to live in a home which is in good repair, has ready access to all necessary amenities and is free from physical hazards.
Whilst we aim to reduce the health effects of poor housing conditions through a combination of advice and financial assistance, occasionally we deal with matters that are so serious a prosecution is necessary.
This successful prosecution sends out a clear message to other private landlords, that non-compliance with current housing standards and non compliance with the Rent Smart Wales laws – is completely unacceptable."
Gleave, who did not attend the court hearing, was fined £11,081 and ordered to pay the total within 28 days or face further court appearances
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Manchester United are chasing highly rated Benfica youngster Umaro Embalo but the teenager will only be available for a huge fee – according to reports from the Manchester Evening News.
Umaro Embalo is one of the most highly rated youngsters in European football at the moment, with many already regarding the teen to become the real deal even at such a young age.
This huge potential means there are many suitors for the forward and one of those are Premier League giants Manchester United, who are currently plotting a January move for the attacker.
However despite such interest, landing Embalo will not come easy for United as his club Benfica have attached a massive asking price of £16million – which is certainly a lot for a 16-year-old.
This ridiculous asking price is believed to be due to the surge in interest for the teenager and while United can afford the fee, it will be interesting to see if the Red Devils will be ready to take such a huge leap for the unproven youngster.
Embalo certainly has huge potential and if indeed United are able to sign him, he could prove to be a very good signing for the future.