A strain-specific real-time PCR assay was developed for quantification of a probiotic Lactobacillus reuteri (DSM 16350) in poultry feed and intestine. The specific primers were designed based on a genomic sequence of the strain derived from suppression subtractive hybridization with the type strain L. reuteri DSM 20016. Specificity was tested using a set of non-target strains from several sources. Applicability of the real-time PCR assay was evaluated in a controlled broiler feeding trial by using standard curves specific for feed and intestinal matrices. The amount of the probiotic L. reuteri was determined in feed from three feeding phases and in intestinal samples of the jejunum, ileum, and caecum of three, 14, and 39 day old birds. L. reuteri DSM 16350 cells were enumerated in all feeds supplemented with the probiotic close to the inclusion rate of 7.0×103 cfu/g, however, were not detected in L. reuteri DSM 16350 free feed. In three day old birds L. reuteri DSM 16350 was only detected in intestinal samples from probiotic fed animals ranging from 8.2±7.8×105 cfu/g in the jejunum, 1.0±1.1×107 cfu/g in the ileum, and 2.5±5.7×105 cfu/g in the caecum. Similar results were obtained for intestinal samples of older birds (14 and 39 days). With increasing age of the animals the amount of L. reuteri signals in the control animals, however, also increased, indicating the appearance of highly similar bacterial genomes in the gut microbiota. The L. reuteri DSM 16350 qPCR assay could be used in future for feeding trials to assure the accurate inclusion of the supplement to the feed and to monitor it's uptake into the GIT of young chicken.

/pdf/development-of-a-strain-specific-real-time-pcr-assay-for-3gk3e7p1id.pdf

Development of a strain-specific real-time PCR assay for enumeration of a probiotic Lactobacillus reuteri in chicken feed and intestine.

Suppression subtractive hybridisation and real-time PCR for strain-specific quantification of the probiotic Bifidobacterium animalis BAN in broiler feed.

Dear Sirs, A deficiency of leucine has been associated with diabetes, headaches, dizziness, fatigue, depression, confusion, irritability, et cetera. Some of these symptoms may be partly associated with altered proton traffic. Given the lack of g-methyl group, leucine is devoid of hyperconjugation and electron delocalization in b-carbon. The weak van der Waals interaction between the side group and the carbonyl group generates a short C1⁄4O bond length, allowing only weak secondary chemical bonding on the carbonyl oxygen atom. Therefore, leucine is virtually insoluble in water, and plays important roles in the formation of hydrophobic cores in proteins. The risk factors for rheumatic heart disease are characterized by the presence of a high content of amino acids with potent hydrogen bonding. A modest supplement of leucine, in addition to a plantbased diet or a carbohydrate-based diet, could generate a higher amount of proteins which are inherently hydrophobic than without the supplement in rheumatic heart disease patients. This measure could change the protein expression profiles and relieve the symptoms by reducing the cellular concentrations of the pertinent risk factors. Clinical trials must first be conducted to confirm the efficacy and safety of the regimen. Adverse reactions should be monitored closely as potent antimicrobial peptides usually possess a number of leucine residues, and protein fragments rich in leucine and basic amino acids could also be stressful on mammalian cells.

/pdf/modest-leucine-supplement-for-prevention-of-rheumatic-heart-1vwrrsz53l.pdf

Modest leucine supplement for prevention of rheumatic heart disease.

Carbohydrate-based diet such as rice does not produce much essential amino acids and creates bottlenecks in protein synthesis, consequently reducing the cellular levels of causative factors. Modest lysine supplement combined with food rich in weak organic acids can change the proteome and solubilize insoluble and stiff salts. Warm temperature minimizes the generation of oxalate. Integration of these strategies can be favorable in the treatment of numerous diseases but cancer. The aforementioned diet may be suitable for human subpopulation with primary constipation, one of the manifestations of many disorders such as heart disease, Alzheimer’s disease, amyotrophic lateral sclerosis, muscular dystrophy, Rabies and Mad Cow disease. 

Food regimen to treat heart disease, Alzheimer’s disease and amyotrophic lateral sclerosis

Dear Sirs, Some viral virulence factors are capable of lysing human cells and releasing nutrients which allow bacterial growth and spawn secondary infections. Carbohydrate diet or intermittent fasting do not give rise to essential amino acids or nutrients respectively, consequently the dietary regimens may reduce the risks of secondary bacterial infections. Drugs enhancing autophagy help recycle protein wastes and reduce cell debris, and are beneficial to the patient to curb secondary infections. The use of antibiotics or cocktails of antibiotics is necessary when the situation becomes dire. Infections may increase the levels of oxalate or calcium oxalate, which worsen the conditions of heart disease patients. Excessive intake of valine, glycine and basic amino acids needs to be avoided as they augment the buildup of oxalate or calcium oxalate. These protein building blocks are either essential or semi-essential amino acids, whose intake can be minimized by the aforementioned dietary regimens.

/pdf/how-to-prevent-secondary-infections-by-bacteria-in-heart-39rlm23pte.pdf

How to prevent secondary infections by bacteria in heart disease patients.

Dear Sirs, Reported risk factors for rheumatic heart disease include bone morphogenetic protein receptor type II, glucokinase, interleukin 10 and brain natriuretic peptide. These proteins possess over 5% certain amino acids with potent hydrogen bonding, including asparagine, glutamine, serine, proline, threonine, et cetera. This feature enables hydrogen bonding of amino acid residues with water and protons as well as other substances. Potent hydrogen bonding also promotes the manifestation of disease symptoms, such as hypersensitivity to high humidity, heart pain and breathing difficulties. Cell volume changes and cell swelling are triggered by water influx into the cells and cause breathing and heart problems. As these amino acids are non-essential, alternative strategies are required to treat the disorder. Non-essential amino acid-based diet with supplement of vitamins can be formulated, excluding asparagine, glutamine, serine, proline, threonine and isoleucine. It is preferable for the diet to be taken for a short period of time. Transient low expression of risk factors alleviates the disease symptoms at least temporarily after long-term buildup of pathogenic factors or oxalate in the patients. Methionine, an essential amino acid, is excluded from the formula as its metabolite homocysteine is associated with atherosclerosis, a major cause of coronary heart disease. To further enhance the effects of the aforementioned diet, short chain organic acids, such as acetic acid, lactic acid, butyric acid, can be supplemented to potentially partially inhibit the biogenesis of certain non-essential amino acids in vivo via their structural similarities to metabolic intermediates. The risk factors mentioned above also possess high basic amino acid content, augmenting the traffic of negatively charged oxalate, which is proposed as one of the major causes of heart disease. Therefore, lysine, arginine and histidine are excluded from the diet. Animal experiments and clinical trials should be first conducted to confirm the efficacy of the regimen. A teaspoon of sugar may be provided to increase the palatability of the regimen. Parenteral formulation can be designed to deliver the necessary nutrients to the patients via injections.

/pdf/how-to-design-non-essential-amino-acid-based-diet-for-4383mdf5j5.pdf

How to design non-essential amino acid-based diet for rheumatic heart disease patients.

A 10 min plasmid extraction method by the direct boiling of Escherichia coli cells, which avoids isopropanol/ethanol precipitation and drying steps used in standard plasmid preparations, is described. Plasmid DNA thus isolated can be used for restriction characterization, chemical transformation, polymerase chain reaction (PCR) amplification as well as cloning purposes.



PRACTICAL APPLICATIONS

This method requires minimum experimental setup, and plasmid DNA prepared can be subjected to restriction digestions without visible degradation. It will enable researchers to process hundreds of cell cultures in a single day. Terrific Broth can be adopted for high cell density growth to yield more DNA with our protocol.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1745-4581.2008.00113.x

A rapid plasmid preparation method by the direct boiling of escherichia coli cells

Screening recombinant DNA clones from cDNA library or transformation experiments is a regular task in molecular biology research; the routine alkaline extraction procedure is tedious and time consuming (90–120 min for 12 samples), and unsuitable for screening large numbers of clones from these experiments, especially when no blue-white indicator (α-complementation of lacZ gene) exists in recombinant clones. Some commercial plasmid preparation kits are more rapid (20–60 min for 12 samples) and simple in plasmid extraction; however, they are costly on a daily basis. Therefore, a simple, rapid and economic method for screening recombinant DNA clones is needed. Here we present an extremely simple, rapid and economic procedure of plasmid DNA preparation for screening recombinant clones from cDNA library or transformation experiments based on a modified alkaline lysis method. In this method, bacterial colonies or cultures were directly lysed in 2% NaOH solution, and then recombinant plasmids in the supernatant were visualized quickly by agarose gel electrophoresis. The entire process, from Escherichia coli colonies or cultures to plasmid DNA, takes only about 12–15 min for 12 samples, which is the quickest method for plasmid preparation in our best knowledge. Hundreds of recombinant clones with no easy screening method can be rapidly identified in several hours using this method.



PRACTICAL APPLICATIONS

Our approach is extremely rapid and simple to screen large numbers of recombinant clones from bacterial colonies or bacterial cultures of cDNA library or transformation experiments. It is especially suitable for identifying those high copy recombinant vectors without blue-white color reaction such as pDNR-LIB, pPICZαA, etc. In addition, this protocol is also suitable for extracting plasmids and screening recombinant DNA clones from bacterial cultures. Our data have indicated that plasmids extracted by our method could meet the criteria for restriction enzyme digestion after neutralization.

A rapid and simple method for screening large numbers of recombinant dna clones

Dear Sirs, Acupuncture has been successfully used in treating numerous human diseases in the practices of traditional Chinese medicine. However, the mechanism underlying its effects is not well understood. Proton traffic and ion traffic may be abnormal in many patients, including heart disease patients, resulting in higher or lower than normal osmotic pressures. The changed cell volumes or/and cell swelling have negative consequences on human health. Acupuncture may be able to alter osmotic pressures via the channels created by a needle, leading to the adjustment of cell volumes and improvement of cell swelling. The changed anion traffic by acupuncture may help dissolve insoluble and rigid salts such as calcium oxalate in the body, improving the conditions of the heart. The combination of acupuncture with western medicine may be necessary to bring about the best clinical outcomes and maximize health benefits.

Acupuncture for heart disease patients.

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How to design non-essential amino acid-based diet for rheumatic heart disease patients.

A rapid plasmid preparation method by the direct boiling of escherichia coli cells

A rapid and simple method for screening large numbers of recombinant dna clones

Acupuncture for heart disease patients.