Survival Rate and Enzyme Activities ofLactobacillus acidophilusFollowing Frozen Storage

The ability of two strains of Lactobacillus acidophilus, CRL 640 and CRL 800, to survive and retain their biological activities under frozen storage was determined. Freezing and thawing, as well as frozen storage, damaged the cell membrane, rendering the microorganisms sensitive to sodium chloride and bile salts. Both lactic acid production and proteolytic activity were depressed after 21 days at -20 degreesC, whereas beta-galactosidase activity per cell unit was increased. Cell injury was partially overcome after repair in a salt-rich medium. Copyright 1998 Academic Press.     

An ultrasensitive high-throughput electrochemiluminescence immunoassay for the Cdc42-associated protein tyrosine kinase ACK1

Several drugs inhibiting protein kinases have been launched successfully, demonstrating the attractiveness of protein kinases as therapeutic targets. Functional genomics research within both academia and industry has led to the identification of many more kinases as potential drug targets. Although a number of well-known formats are used for measuring protein kinase activity, some less well-characterized protein kinases identified through functional genomics present particular challenges for existing assay formats when there is limited knowledge of the endogenous substrates or activation mechanisms for these novel kinase targets. This is especially the case when a very sensitive assay is required to differentiate often highly potent inhibitors developed by late-stage medicinal chemistry programs. ACK1 is a non-receptor tyrosine kinase that has been shown to be involved in tumorigenesis and metastasis. Here we describe the development of an extremely sensitive high-throughput assay for ACK1 capable of detecting 240 fmol per well of the kinase reaction product employing a BV-tag-based electrochemiluminescence assay. This assay is universally applicable to protein tyrosine kinases using a BV-tag-labeled monoclonal antibody against phosphotyrosine. Furthermore, this assay can be extended to the evaluation of Ser/Thr kinases in those cases where an antibody recognizing the phospho-product is available.     

Diurnal variation in temperature, mental and physical performance, and tasks specifically related to football (soccer)

Football (soccer) training and matches are scheduled at different times throughout the day. Association football involves a variety of fitness components as well as psychomotor and game-related cognitive skills. The purpose of the present research, consisting of two separate studies, was to determine whether game-related skills varied with time of day in phase with global markers of both performance and the body clock. In the first study, eight diurnally active male association football players (19.1+/-1.9 yrs of age; mean+/-SD) with 10.8+/-2.1 yrs playing experience participated. Measurements were made on different days at 08:00, 12:00, 16:00, and 20:00 h in a counterbalanced manner. Time-of-day changes in intra-aural temperature (used as a marker of the body clock), grip strength, reaction times, flexibility (markers of aspects of performance), juggling and dribbling tasks, and wall-volley test (football-specific skills) were compared. Significant (repeated measures analysis of variance, ANOVA) diurnal variations were found for body temperature (p<0.0005), choice reaction time (p<0.05), self-rated alertness (p<0.0005), fatigue (p<0.05), forward (sit-and-reach) flexibility (p<0.02), and right-hand grip strength (p<0.02), but not left-hand grip strength (p=0.40) nor whole-body (stand-and-reach) flexibility (p=0.07). Alertness was highest and fatigue lowest at 20:00 h. Football-specific skills of juggling performance showed significant diurnal variation (p<0.05, peak at 16:00 h), whereas performance on the wall-volley test tended to peak at 20:00 h and dribbling showed no time-of-day effect (p=0.55). In a second study, eight diurnally active subjects (23.0+/-0.7 yrs of age) completed five test sessions, at the same times as in the first study but with a second session at 08:00 h. Test-re-test comparisons at 08:00 h for all components indicated good reliability. Intra-aural temperature showed a significant time-of-day effect (p<0.001) with mean temperature at 16:00 h (36.4 degrees C) higher than at 08:00 h (35.4 degrees C). There was no significant effect of chronotype on the temperature acrophase (peak time) (p>0.05). Diurnal variation was found for performance tests, including sit-and-reach flexibility (p<0.01) and spinal hyper-extension (p<0.05). Peaks occurred between 16:00 and 20:00 h and the daytime changes paralleled the temperature rhythm. Diurnal variation was also found for football-specific tests, including dribbling time (p<0.001, peak at 20:00 h) and chip test performance (p<0.01), being more accurate at 16:00 h (mean error=0.75 m) than at 08:00 h (mean error=1.01 m). Results indicate football players perform at an optimum between 16:00 and 20:00 h when not only football-specific skills but also measures of physical performance are at their peak. Body temperature peaked at a similar time, but positive mood states seemed to peak slightly earlier. While causal links cannot be established in these experiments, the results indicate that the diurnal variation of some aspects of football performance is affected by factor(s) other than body temperature alone.     

Template switching within exons 3 and 4 of KV11.1 (HERG) gives rise to a 5' truncated cDNA

K(V)11.1 (HERG) channels contribute to membrane potential in a number of excitable cell types. We cloned a variant of K(V)11.1 from human jejunum containing a 171 bp deletion spanning exons 3 and 4. Expression of a full-length cDNA clone containing this deletion gave rise to protein that trafficked to the cell membrane and generated robust currents. The deletion occurred in a G/C-rich region and identical sequence elements of UGGUGG were located at the deletion boundaries. In recent studies these features have been implicated to cause deletions via template switching during cDNA synthesis. To examine this possibility we compared cDNAs from human brain, heart, and jejunum synthesized at lower (42 degrees C) and higher temperatures (70 degrees C). The 171 bp deletion was absent at the higher temperature. Our results suggest that the sequence and secondary structure of mRNA in the G/C rich region leads to template switching producing a cDNA product with a 171 bp deletion.     

High-throughput screening of cellulase F mutants from multiplexed plasmid sets using an automated plate assay on a functional proteomic robotic workcell

Background  

The field of plasmid-based functional proteomics requires the rapid assay of proteins expressed from plasmid libraries. Automation is essential since large sets of mutant open reading frames are being cloned for evaluation. To date no integrated automated platform is available to carry out the entire process including production of plasmid libraries, expression of cloned genes, and functional testing of expressed proteins.     

Synthesis and optimization of substituted furo[2,3-d]-pyrimidin-4-amines and 7H-pyrrolo[2,3-d]pyrimidin-4-amines as ACK1 inhibitors

Two classes of ACK1 inhibitors, 4,5,6-trisubstituted furo[2,3-d]pyrimidin4-amines and 4,5,6-trisubstituted 7H-pyrrolo[2,3-d]pyrimidin-4-amines, were discovered and evaluated as ACK1 inhibitors. Further structural refinement led to the identification of potent and selective dithiolane inhibitor 37.     

TNF-α is associated with loss of lean body mass only in already cachectic COPD patients

Background

Systemic inflammation may contribute to cachexia in patients with chronic obstructive pulmonary disease (COPD). In this longitudinal study we assessed the association between circulating C-reactive protein (CRP), tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 levels and subsequent loss of fat free mass and fat mass in more than 400 COPD patients over three years.

Methods

The patients, aged 40–76, GOLD stage II-IV, were enrolled in 2006/07, and followed annually. Fat free mass and fat mass indexes (FFMI & FMI) were calculated using bioelectrical impedance, and CRP, TNF-α, IL-1ß, and IL-6 were measured using enzyme immunoassays. Associations with mean change in FFMI and FMI of the four inflammatory plasma markers, sex, age, smoking, FEV₁, inhaled steroids, arterial hypoxemia, and Charlson comorbidity score were analyzed with linear mixed models.

Results

At baseline, only CRP was significantly (but weakly) associated with FFMI (r = 0.18, p < 0.01) and FMI (r = 0.27, p < 0.01). Univariately, higher age, lower FEV₁, and use of beta2-agonists were the only significant predictors of decline in FFMI, whereas smoking, hypoxemia, Charlson score, and use of inhaled steroids predicted increased loss in FMI. Multivariately, high levels of TNF-α (but not CRP, IL-1ß or IL-6) significantly predicted loss of FFMI, however only in patients with established cachexia at entry.

Conclusion

This study does not support the hypothesis that systemic inflammation is the cause of accelerated loss of fat free mass in COPD patients, but suggests a role for TNF-α in already cachectic COPD patients.     

'Positive biology' as a new paradigm for the medical sciences. Focusing on people who live long, happy, healthy lives might hold the key to improving human well-being

The nearly exclusive focus on understanding and treating chronic disease might not be the most efficient way to improve public health, especially as an effective alternative strategy exists.On 27 April 2009, during a speech at the National Academy of Sciences, US President Barack Obama pledged to invest more than 3% of US GDP in scientific research and development—the amount represented the largest ever investment in research and innovation. However, even a financial investment of such magnitude does not ensure that science is ''well-ordered'' [1], in the sense that the scientific research that is prioritized aspires to address the most significant challenges and problems for humanity.Among the many issues facing society that research must address, improving human health and tackling disease rank high, if not first, on the agenda. Accordingly, a huge fraction of research funding is spent on basic and applied research to further our understanding of the causes of disease and to find new cures and therapies. But is this focus on pathology the most efficient way to conduct research with the aim to improve human health and well-being?…a huge fraction of research funding is spent on basic and applied research to further our understanding of the causes of disease and to find new cures and therapiesMost of today''s medical research could be called ''negative biology''. It is conducted in an intellectual framework that presumes that the most important question to answer is: what causes pathology? Disease is its central focus and this explains why medical research and research funding is mainly concerned with trying to understand, prevent and treat specific diseases. The design of the US National Institutes of Health, which is largely composed of individual institutes dedicated to specific diseases such as cancer, mental illness or infectious diseases, reflects this prevalence of pathology-oriented negative biology.Positive biology, by contrast, focuses on a different set of questions and priorities. Rather than making pathology and disease the central focus of intellectual efforts and financial investments, positive biology seeks to understand positive phenotypes: why do some individuals live more than a century without ever suffering from the chronic diseases that afflict most humans much earlier in their lives? Why are some individuals more happy, optimistic, talented, or have a better memory than most people? The paradigm of positive biology is based on the insight that the process of evolution by natural selection does not create a perfect organism in terms of life expectancy, resistance to disease or other abilities. Observations of exceptional longevity or superior cognition therefore present fascinating puzzles for positive biology: which biological mechanisms would explain these exemplars of health and well-being? The goal of understanding positive phenotypes is that such knowledge might lead to new interventions that generally improve human well-being. This might be achieved by modulating the rate of ageing or by increasing opportunities for play and joy at all stages of the human lifespan, or by developing pharmaceuticals that safely enhance cognition or positive emotions, and so on.The goal of understanding positive phenotypes is that such knowledge might lead to new interventions that generally improve human well-beingThis is distinct from negative biology, which focuses on the proximate causes of specific diseases, rather than on the evolutionary causes of positive phenotypes. It presumes that health, survival and happiness are the default states and aims to explain the deviations: why do we develop cancer? Why do we suffer from depression? Why do we develop hypertension? Negative biology therefore faces the laudable but insurmountable task of trying to prevent or cure all disease. This is a costly and ultimately futile endeavour. Eliminating all types of cancer would increase life expectancy in the USA by approximately only three years [2]. Even eliminating cancer as a cause of death would not prevent any of the other chronic diseases of ageing—cardiovascular disease, Alzheimer and Parkinson disease, diabetes and so on—from afflicting the elderly. Moreover, the more than 40 years of ''war against cancer'' has not defeated a single type of cancer: we still have a long way to go before we can realistically expect to reap the three-year increase in life expectancy that eliminating all cancers could yield.In fact, negative biology has not yet developed a single cure for any one of the hundreds of chronic diseases that afflict millions of people living today. Of course, it has made significant advances to help prevent and treat chronic disease, but the fixation on pathology has meant that other potential avenues for research have been neglected.Indeed, a better understanding of exemplars of health and happiness—the goal of positive biology—could create more benefits for humans more quickly and more easily. A drug that would safely mimic the effects of caloric restriction, for instance, might delay, simultaneously, most diseases and afflictions of ageing. It would generate a much greater health dividend for ageing populations than defeating any one specific disease of ageing because slowing down the rate of ageing by seven years would reduce the age-specific risk of death, frailty and disability by about half at every age [3].Scientists are already making good progress on the project of positive biology, even if the intellectual framework is not yet clearly defined and their topics are rather piecemeal. Richard Miller, for example, a professor of pathology at Michigan University, USA, studies the genetics of ageing in mice and participates in the National Institute of Aging''s multi-institutional programme that evaluates the effects of drugs and nutriceuticals on the ageing process in mice. David Sinclair from Harvard University, USA, and others found that the plant compound resveratrol, which is found in the skin of grapes, can modulate the ageing process. Nir Barzilai and colleagues at the Albert Einstein School of Medicine in New York, USA, have conducted genetic research on more than 500 healthy elderly people between the ages of 95 and 112 years. Michael Rose from the University of California, Irvine, USA, has quadrupled the lifespan of fruit flies by delaying the age of reproduction. Finally, the biologist Cynthia Kenyon demonstrated that in Caenorhabditis elegans, a single gene can control the ageing process. Any of these research projects could eventually lead to the development of a new drug that retards the ageing process and diminishes the onslaught of chronic diseases that typically afflict humans after their sixth decade of life.Similarly, a lot of pioneering work is being undertaken in the burgeoning field of ''positive psychology''. Rather than studying why people suffer from mental illnesses such as depression, schizophrenia or ADHD (attention deficit hyperactivity disorder), positive psychology is primarily interested in how to improve the happiness of the ''average'' person. Martin Seligman, a psychologist at the University of Pennsylvania, USA, and a pioneer in the field of positive psychology, distinguishes different kinds and levels of happiness [4]. Hedonists who pursue immediate rewards such as the pleasure of buying something or receiving a compliment seek momentary happiness or what Seligman calls ''the pleasant life''. But these pleasures fade quickly and do not leave a lasting impact on subjective well-being. Enduring happiness, by contrast, is realized when we lead a meaningful life. After years spent studying what makes people happy, Seligman contends that it is rooted in attachment to something larger, and the larger the entity to which you attach yourself, the more meaning your life has [4].Eliminating all types of cancer would increase life expectancy in the USA by approximately only three yearsThis is clearly illustrated by the role of wealth. People often assume that being richer will mean being happier, yet surveys in many countries indicate that global levels of life satisfaction or happiness have not changed much during the past four decades despite large increases in real income per capita [5]. Most disposable income is spent on consumer goods that do little to actually enhance our well-being.In a recent study of the daily behaviour of happy people, researchers used an electronically activated recorder to record, and then later classify, participants'' daily conversations with others as either ''small talk'', that is banal conversations, and ''substantive talk'', where meaningful information was exchanged. They found that higher well-being was associated with less small talk and more substantive conversations [6]. While such a study does not establish the truth of Socrates'' famous claim that “the unexamined life is not worth living”, it does suggest that our need to feel attached to something larger is important to our happiness and well-being. This hypothesis is supported by recent studies on how people spend their money. Researchers from the University of British Columbia and Harvard Business School found that when individuals spend more money on prosocial goals, such as charity, they actually experience greater happiness than when they spend money on consumer products for themselves [7]. Similarly, the psychologist Barbara Fredrickson''s research on positive emotions—joy, serenity and gratitude—suggests that these expand cognition and behavioural tendencies [8].Finally, research on exemplars of resilience, that is, the ability of some people to cope and manage with tragic and traumatic events, could lead to the development of drugs that would increase people''s resilience. Avshalom Caspi and colleagues found that individuals with one or two copies of the short allele of the promoter of the 5-HTT serotonin receptor experience more depressive symptoms, diagnosable depression and suicidal thoughts in response to stressful events compared with individuals who are homozygous for the long allele [9].Cognitive functioning is another central topic of positive biology. What are the genetic and environmental determinants of high IQ, exceptional memory or social intelligence? Barbara Sahakian and colleagues found that the analeptic drug modafinil significantly enhanced performance tests of digit span, visual pattern recognition memory, spatial planning and stop-signal reaction time in healthy volunteers [10]. These findings of positive biology will eventually give us a better understanding of our human nature than the very limited focus on disease and pathology of negative biology and might then lead to new interventions, environments and attitudes that improve human well-being and happiness.Negative biology dominates medical research, from the questions research scientists tackle to the education of physicians and government regulation of health interventions. The dominance of this approach to the medical sciences presumes that the most important questions concern the causes of pathology rather than the causes of exemplar health and happiness. Positive biology takes a different approach: it does not limit the moral duty to apply knowledge and technology to improve human welfare to only treating specific diseases or impairments. Rather, it works under the assumption that if knowledge and research can improve people''s lives, there is a moral duty to advance that knowledge and promote well-being. Nor is positive biology predicated on a sharp distinction between therapy and enhancement. Instead, as the bioethicist John Harris has argued, “the overwhelming moral imperative for both therapy and enhancement is to prevent harm and confer benefit. Bathed in that moral light, it is unimportant whether the protection or benefit conferred is classified as enhancement or improvement, protection or therapy” [11].Generally, the medical system as a whole could be much more efficient if it concentrated its efforts on making people healthier and happier in the first place instead of its current focus on understanding and treating disease. Advancing the paradigm of positive biology should therefore help the medical sciences transcend the limited perspectives and aspirations of negative biology. Such a paradigm could help the world''s population to reap the benefits that new knowledge and technologies can offer in terms of making people healthier and happier. Societies and individuals already seek to achieve these goals: we educate our children to eat healthily and exercise and to develop their social goals to find fulfilment in life. The paradigm of positive biology simply encourages us to make use of the full range of options to realize these goals.…the medical system as a whole could be much more efficient if it concentrated its efforts on making people healthier and happier in the first place…In conclusion, positive biology is not contrary to the goals and aspirations of negative biology. Indeed the two paradigms are often complementary. For example, understanding why some high-risk individuals, such as sex workers, seem to have an intrinsic resistance to HIV-1 might spur the development of an HIV vaccine [12]. Similarly, understanding human brains with exceptional cognitive functioning might lead to new avenues for developing drugs and therapies against severe cognitive impairment. Understanding exemplars of health could create real benefits for those who are more vulnerable to disease and disability.