Indirect estimation of biomass by rapid ribonucleic acid determination

Summary Microbial ribonucleic acid (RNA) was estimated by determinations of nucleotides formed by complete hydrolysis. The steps involved in this procedure comprise the alkaline hydrolysis of RNA (5 N NaOH; 100°C; 5 min) to a mixture of 2-and 3-mononucleotides and successive separation and quantification by means of high performance liquid chromatography (HPLC). Since guanylic acid (GMP) shows adequate stability during RNA degradation and may be separated well and rapidly from other compunds, it is used as an equivalent measure of RNA. The assay is performed within 15 min having a lowest detection limit of 4 mg RNA/l. It was applied to isolated yeast cells and fermentation broths of mycelial organisms. Linear correlation to mycelial dry weight was established for unlimited growth (trophophase), while in idiophase determination of RNA may serve as a measure of active biomass.     

Quantitative cytochemical aspects of a combined Feulgen-Naphthol Yellow S staining procedure for the simultaneous determination of nuclear and cytoplasmic proteins and DNA in mammalian cells

The simultaneous cytophotometric determination of nuclear and cytoplasmic proteins and DNA by means of a combined Feulgen-Naphthol Yellow S (NYS) staining procedure was investigated. According to this procedure Feulgen staining is performed prior to NYS staining. The following main results were obtained:

1. 1. After NYS staining alone, the amount of NYS bound to the cell was found to be closely correlated to the cellular dry mass. The correlation coefficient was 0.99 in ethanol-acetone fixed cells and 0.95 in formaldehyde-fixed cells. This close correlation was not significantly altered by the Feulgen staining procedure and was 0.92 in ethanol-acetone and 0.94 in formaldehyde-fixed cells. However, the absolute amount of NYS bound per unit dry mass was affected by the method of fixation and type of Feulgen hydrolysis.

2. 2. The cells lose material during the Feulgen procedure, particularly during the acid hydrolysis stage. The type of hydrolysis most suitable for the Feulgen procedure (5 N HCl, 22 °C, 60 min) resulted in a considerable loss of dry mass in ethanol-acetone fixed cells. This loss was smaller in formaldehyde-fixed cells (15%) and was in addition closely correlated (correlation coefficient 0.99) to the dry mass of the cells prior to hydrolysis. In formaldehyde-fixed cells the dry mass after the Feulgen procedure is thus a good measure of the true cellular dry mass of the fixed cells. This is further demonstrated by the close correlation between NYS binding to Feulgenstained cells and the dry mass of these cells prior to the Feulgen procedure (correlation coefficient 0.95).

3. 3. When using the combined Feulgen-NYS staining procedure under standardized conditions (formaldehyde fixation and acid hydrolysis in 5 N HCl, 22 °C, 60 min) a constant amount of NYS was found to be bound per unit dry weight to nuclear and cytoplasmic proteins in various types of mammalian cells with different proliferative activity.

4. 4. The Feulgen DNA determination was not found to be quantitatively affected by the subsequent NYS staining.

From the results of the present study it seems that, under standardized conditions, the combined Feulgen-NYS staining procedure can be used as a reliable quantitative method for the determination of nuclear and cytoplasmic proteins and DNA in mammalian cells.     

An adaptation of the filter paper disk technique for the differential estimation of DNA and RNA of rat liver

A method is described for the differential estimation of DNA and RNA from rat liver on filter paper disks. To differentiate between DNA and RNA, samples are subjected to alkaline hydrolysis in microtitration plates prior to absorption on the disks. This procedure eliminates the loss of DNA which is observed if the disks are subjected to RNA hydrolysis after the sample has been absorbed on disks. The procedure is directly comparable to standard methods for the measurement of nucleic acids.     

Acid hydrolysis of fibers from dairy manure

Concentrated acid hydrolysis of lignocellulosic materials is a conventional treatment process for the production of mono-sugars. However, this method has been proved ineffective and undesirable for the treatment of dairy manure due to the high nitrogen content of dairy manure and the environmental issues caused by the use of highly concentrated acid solution. In an effort to overcome these barriers, a modified acid hydrolysis process with short reaction time was introduced that involved a nitrogen-removing pretreatment followed by decrystallization with concentrated acid and then hydrolysis using dilute acid. The effects of nitrogen, acid concentration, reaction time, and temperature were investigated. A pretreated manure with a low nitrogen content of 1.3% was used as the substrate. The results indicated that the optimal conditions for fiber decrystallization were 75% acid concentration, 3:5 sample to acid ratio (weight basis), and 30 min of reaction time; while the optimal conditions for acid hydrolysis were 12.5% acid and 10% dry sample at 135 degrees C for 10 min. These conditions produced 26 g/L glucose at a yield of 84% and 11 g/L hemicellulose-sugars at a yield of 80%.     

Influence of manufacturing variables on the characteristics and effectiveness of chitosan products. I. Chemical composition,viscosity, and molecular-weight distribution of chitosan products

Ten chitosan products were manufactured from dry shrimp hulls under differing process conditions and compared to a commercially available product. Manufacturing variables tested were: alkali versus enzymatic deproteination; acid demineralization versus no treatment; air versus nitrogen atmosphere; 5 min vs. 15 min deacetylation period: and varying the particle size of the dry starting material. Deproteination by alkali of enzymatic extraction did not substantially affect the nitrogen and ash compositions of dry chitosan samples. However, the viscosity was reduced in samples deproteinated by enzymatic hydrolysis. Elimination of the demineralization step resulted in products having 31–36% ash, as expected. Some differences in viscosity were observed between deminiralized and undemineralized samples, but on important differences in the molecular-weight distribution of these samples were evident. Purging the reaction vessel with nitrogen resulted in chitosan preparations having higher viscosities and molecular-weight distributions than those prepared in an air atmosphere. The degradative effect of air became more proshrimp hulls to 1 mm prior to any treatment resulted in a chitosan product of both higher viscosity and molecular weight than when ground to either 2 or 6.4 mm. Viscosity was not always a direct indicator of molecular weight, for although the presence of colloidal particles increased the viscosity of some samples, the molecular-weight distribution after filtration was essentially the same as in other less viscous samples.     

Challenges in the Testing of Non-Heart-Beating Cadavers for Viral Markers: Implications for the Safety of Tissue Donors

Natural changes that occur in blood and tissue after death may result in false positive results in antigen and antibody detection tests performed to identify markers of viral infection in potential tissue donors. Such tissue, which might otherwise be acceptable for therapeutic purposes, would not meet current standards for safe tissue banking. This is especially important in the context of insufficiency in the tissue supply. In this study, a series of blood samples collected during routine post-mortem examination was assayed using a range of commercially available kits for the detection of HBsAg, anti-HCV and anti-HIV 1 + 2 antibody/antigen. Results of tests on 104 samples collected from 97 individuals indicate that some kits result in a higher number of initial reactive samples than others. Approximately 40% of samples were reactive in one or more HBsAg assay, less than 10% in at least one anti-HIV kit and only 1 sample at low level on an anti-HCV kit. Liver or lymph node samples from individuals whose serum sample gave reactive results in antigen/antibody assays were tested for viral nucleic acid in the corresponding nucleic acid amplification test. Only one individual’s sample was confirmed to test positive for HBsAg in a confirmatory neutralisation test and by nucleic acid amplification technology, and a second individual whose serum was scored reactive for anti-HCV, but negative for HBsAg, had a liver sample which was HBV DNA positive and HCV RNA negative. The results of the study indicate that antibody/antigen assays are not as specific as NAT using state of the art DNA extraction techniques. Both types of assay complement each other and used together will help assure the safety of tissues for transplantation.     

Nonradioactive assay for cellular dimethylallyl diphosphate

A sensitive, nonradioactive method was developed to measure cellular levels of dimethylallyl diphosphate (DMAPP), a central intermediate of isoprenoid metabolism in nature. The assay is based on the hydrolysis of DMAPP in acid to the volatile hydrocarbon isoprene (2-methyl-1,3-butadiene), with subsequent analysis of isoprene by headspace gas chromatography with reduction gas detection. In the assay, cell samples are directly acidified with 4 M H(2)SO(4) in sealed reaction vials. Therefore, there is no need to extract metabolites, purify them, and keep them stable prior to analysis, and degradative enzymatic activities are destroyed. DMAPP levels of 23 +/- 4 nmol (g fresh weight)(-1) [ca. 85 nmol (g dry weight)(-1)] and 80 +/- 14 nmol (g fresh weight)(-1) [ca. 296 nmol (g dry weight)(-1)] were measured in dark- and light-adapted leaves of Populus deltoides (Eastern cottonwood), respectively. Evidence is presented to show that DMAPP is the major leaf metabolite giving rise to isoprene following acid hydrolysis. DMAPP levels in Bacillus subtilis and Saccharomyces cerevisiae were determined to be 40.8 +/- 16.7 pmol (OD(600))(-1) [ca. 638 pmol (mg dry weight)(-1)] and 6.3 +/- 3.7 pmol (OD(600))(-1) [ca. 139 pmol (mg dry weight)(-1)], respectively. The method should be suitable for any cell or tissue type and isolated cellular organelles.     

Occurrence of coenzyme F420 and its gamma-monoglutamyl derivative in nonmethanogenic archaebacteria.

Analysis of the fluorescent compounds extracted from six different species of halobacteria and one species each of Sulfolobus and Thermoplasma revealed the universal occurrence of coenzyme F420, (N-[N-[O-[5-(8-hydroxy-5-deazaisoalloxazin-10-yl)-2,3,4-trihydroxy -4-pentoxyhydroxyphosphinyl]-L-lactyl]-L-gamma-glutamyl]-L -glutamic acid), or its gamma-monoglutamyl derivative or both. The total amount (approximately 100 pmol/mg [dry weight]) of these compounds found in the halobacteria studied was approximately 5% of the amount previously reported for methanogenic bacteria. The amount of F420 found in the Sulfolobus and Thermoplasma strains was approximately 1% of that found in the halobacteria. The major compound in all but one of the examined strains was the gamma-monoglutamyl derivative of F420; one strain of halobacteria contained only F420. For the halobacterium-derived samples, the additional glutamic acid was shown to be linked by a gamma-glutamyl peptide bond to the terminal glutamic acid of the F420 core structure by enzymatic hydrolysis of the samples with three different gamma-glutamyltranspeptidases. The product of this enzymatic hydrolysis was F420 with one less glutamic acid in the side chain.     

Combined procedure for assays of poly-β-hyroxybutyric acid and inorganic polyphosphate

A combined procedure has been developed for the sequential assay of poly-β-hydroxybutyric acid and inorganic polyphosphate employing a single 10–15 ml sample containing 1–25 mg dry weight of bacterial cells. The procedure is based on the insolubility of both polymers in alkaline hypochlorite. The sample may consist of freshy harvested cells washed in a phosphate-free suspending medium, or frozen washed cells. The combined procedure is capable of quantitative assay of either polymer when at least 0.1 μg/ml is present in the cell suspension. Assay values obtained with the combined procedure were consistent with the physiological condition of the cells (Caulobacter crescentus) and with the size, number and identity of intracellular polymer granules discernible by electron microscopy.     

Sugar recovery and fermentability of hemicellulose hydrolysates from steam-exploded softwoods containing bark

The hemicellulose sugar recovery and ethanol production obtained from SO2-catalyzed steam explosion of a mixed white fir (70%) and ponderosa pine (30%) feedstock containing bark (9% dry weight/dry weight) was assessed. More than 90% of the available hemicellulose sugars could be recovered in the hydrolysate obtained after steam explosion at 195 degrees C, 2.38 min, and 3.91% SO2, with 59% of the original hemicellulose sugars detected in a monomeric form. Despite this high sugar recovery, this hydrolysate showed low ethanol yield (64% of theoretical yield) when fermented with a spent sulfite liquor-adapted strain of Saccharomyces cerevisiae. In contrast, most hydrolysates prepared at higher steam explosion severity showed comparable or higher ethanol yields. Furthermore, the hydrolysates prepared from bark-free feedstock showed better fermentability (87% of theoretical yield) despite containing higher concentration of known inhibitors. The ethanol yield from the hydrolysate prepared from a bark-containing wood sample could be improved to 81% by an extra stage acid hydrolysis (121 degrees C for 1 h in 3% sulfuric acid). This extra stage acid hydrolysis and steam explosion at higher severity conditions seem to improve the fermentability of the hydrolysates by transforming certain inhibitory compounds present in the hydrolysates prepared from the bark-containing feedstock and thus lowering their inhibitory effect on the yeast used for the ethanol fermentation.     

Real-time optical analysis of a colorimetric LAMP assay for SARS-CoV-2 in saliva with a handheld instrument improves accuracy compared with endpoint assessment

Controlling the course of the Coronavirus Disease 2019 (COVID-19) pandemic will require widespread deployment of consistent and accurate diagnostic testing of the novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Ideally, tests should detect a minimum viral load, be minimally invasive, and provide a rapid and simple readout. Current Food and Drug Administration (FDA)-approved RT-qPCR–based standard diagnostic approaches require invasive nasopharyngeal swabs and involve laboratory-based analyses that can delay results. Recently, a loop-mediated isothermal nucleic acid amplification (LAMP) test that utilizes colorimetric readout received FDA approval. This approach utilizes a pH indicator dye to detect drop in pH from nucleotide hydrolysis during nucleic acid amplification. This method has only been approved for use with RNA extracted from clinical specimens collected via nasopharyngeal swabs. In this study, we developed a quantitative LAMP-based strategy to detect SARS-CoV-2 RNA in saliva. Our detection system distinguished positive from negative sample types using a handheld instrument that monitors optical changes throughout the LAMP reaction. We used this system in a streamlined LAMP testing protocol that could be completed in less than 2 h to directly detect inactivated SARS-CoV-2 in minimally processed saliva that bypassed RNA extraction, with a limit of detection (LOD) of 50 genomes/reaction. The quantitative method correctly detected virus in 100% of contrived clinical samples spiked with inactivated SARS-CoV-2 at either 1× (50 genomes/reaction) or 2× (100 genomes/reaction) of the LOD. Importantly, the quantitative method was based on dynamic optical changes during the reaction and was able to correctly classify samples that were misclassified by endpoint observation of color.     

Storage of environmental samples for guaranteeing nucleic acid yields for molecular microbiological studies

The purpose of this study is to evaluate whether sample preservation can affect the yield of nucleic acid extracts from environmental samples. Storage of microbial samples was studied using three sediment types of varying carbon contents (10–57% carbon of dry weight). Four different storage solutions were tested at three temperatures. Freezing of samples at ?20 °C or ?80 °C, either without preservative or in phenol–chloroform solution, retained nucleic acid quantities very efficiently. Storage of samples in phenol–chloroform solution at +4 °C also gave good yields except for sediment with extremely high-carbon content. Ethanol and RNAlater® preservation decreased nucleic acid yields drastically at all temperatures. To study how sample preservation may affect the result of microbial community analysis, one type of sediment was selected for length heterogeneity-PCR analysis and PCR cloning of the 16S rRNA genes. Ethanol and RNAlater® preservation caused a slight bias towards certain microbial types in the community analyses shown by underrepresentation of Bacteroidetes, Betaproteobacteria and Gammaproteobacteria-affiliated peak sizes and overrepresentation of Actinobacteria, Chloroflexi and Alphaproteobacteria-affiliated peak sizes. Based on the results of this study, preservation in phenol–chloroform solution can be recommended as an alternative storage method when freezing is not possible such as during extended field sampling; however, ethanol and RNAlater® may cause serious problems when used as preservatives for environmental samples containing humic acids.     

The determination of 2'-O-methylnucleosides in RNA

A rapid and sensitive procedure is described for determining the 2′-O-methylnucleoside methylnucleoside composition of an RNA sample. The RNA is enzymatically hydrolyzed to nucleosides and the 2′-O-methylnucleoside fraction is isolated by DEAE-cellulose (borate) column chromatography. Boric acid is removed as its methyl ester and the 2′-O-methylnucleosides are resolved by liquid chromatography in the presence of ethylene glycol. The sensitivity of this method is sufficient to distinguish RNA samples which differ only 2–3% in 2′-O-methylnucleoside composition.     

Optimizing dilute acid hydrolysis of hemicellulose in a nitrogen-rich cellulosic material--dairy manure

Effective dilute acid hydrolysis of dairy manure which contains roughly 12% hemicellulose on a dry matter basis can produce a variety of mono-sugars such as arabinose, xylose and galactose, as well as to further benefit utilization of cellulose in the manure. To enhance the effectiveness of this dilute acid hydrolysis, the effect of manure nitrogen content was studied because some reactions such as the browning reaction between amino acids and reducing sugars and acid-base reactions involving ammonia and acid interfere with the hydrolysis. Two dairy manure samples were used to study this nitrogen effect; the original manure and the pretreated manure derived from a solid/liquid separation pretreatment. The pretreated manure had a total nitrogen content of 1.3% dry matter (DM) while the original dairy manure had twice that amount with a total nitrogen content of 2.6% DM. Results found that the optimal conditions for hydrolysis of manure hemicellulose were 2 h reaction time, 1% sulfuric acid concentration, 135 degrees C, and 10% sample concentration using the pretreated dairy manure as raw material. Under these conditions the corresponding sugar yield from hemicellulose was 111% and sugar concentration in the solution reached 16.5 g/l. At the same time, the hydrolyzed solid had 43% DM of cellulose, which was much higher than both the original manure containing 22% and the pretreated manure with 32%.     

Characterization of enzymatic hydrolyzed snow crab (Chionoecetes opilio) by-product fractions: A source of high-valued biomolecules

Snow crab (Chionoecetes opilio) constitutes valuable and nutritional sources of components, such as proteins, lipids and chitin. The present investigation was undertaken to evaluate the feasibility of applying a pilot scale enzymatic hydrolysis process of snow crab by-products, followed by fractionation, in order to recover enriched high-valued compounds. The yield of snow crab by-products recovered after manual processing; on a dry weight was 87.4%. The by-products (raw materials) were mainly moist (approximately 78%), and contained 42.9% proteins, 14.8% lipids, 25.7% minerals, 16.2% chitin, all expressed on a dry weight. The fatty acid profile of snow crab by-products and all fractions obtained following processing showed a higher content in mono-unsaturated fatty acids (MUFAs; approximately 50%), followed by polyunsaturated fatty acids (PUFAs; approximately 20%) and saturated fatty acids (SFAs; approximately 15%). The n − 3/n − 6 ratio was approximately 10 and represents a good index of nutritional value for snow crab oil by-products. Most protein enriched fractions demonstrate a well-balanced amino acid composition, notably the most essential amino acids. These protein fractions are characterized by biomolecules having a relatively low molecular weight (35 kDa and less) range. The enzymatic hydrolysis process developed in this study shows that snow crab by-products should to be viewed as having the potential of being identified as high-valued products. Even though the process could be optimized, it is controllable, and depending on hydrolyses conditions, the products obtained are reproducible and well defined. Results presented in this study indicate that snow crab by-products may serve as excellent nutritional components for future applications in the health and food sectors.     

A bone biopsy procedure for neonatal pigs

Neonatal pigs were used to develop a surgical biopsy procedure to remove bone tissue from the ilium of small animals, with potential application for infants and small children. While the neonatal pig was under general anaesthesia, a scalpel was used to make a punch incision down to the ilium. Then a Craig Biopsy Trephine was used to remove a core sample of the bone. The samples ranged from 5 to 15 mm in length and 2 to 3 mm in diameter, with an average dry weight of 34.4 mg. The samples were adequate for mineral (calcium and zinc) analysis in our laboratory and may be equally suitable for histological or biochemical analyses. Surgical trauma was minimal, which permitted each pig to be biopsied every 7 days for 5 weeks without adverse consequences.     

Some methods for processing of single-cell protein

Methods for the production of protein concentrates, with a low content of nucleic acid, in kilogram quantities from yeast have been studied with the aid of equipment designed for operation on pilot-plant scale. The influence of drum drying and mechanical disintegration on the nutritive value of the yeast was also investigated. Drum drying and mechanical disintegration improved the nutritive value of the yeast but high extractability of protein and nucleic acid was only obtained after mechanical disintegration. Protein concentrates without and with cell walls were produced from mechanically disintegrated yeast. The different fractions which were obtained when separating cell walls and precipitating protein by heating at alkaline pH, were analyzed. After protein precipitation, about 90% of the RNA could be precipitated from the supernatant by addition of acid, giving a product containing 50% RNA of the dry weight. The protein precipitate obtained after cell wall separation had an RNA content of less than 2% and contained 70–l75% of the amino acids in the starting yeast material. Protein concentrates containing cell walls were produced by precipitating protein by heating at alkaline pH directly after mechanical disintegration. The content of RNA was about 2% and the yield of amino acids was 70–80%. It was found that the nutritive value of the protein concentrate was higher than that of the starting yeast material. To produce such a protein concentrate on a large scale, the process described can probably be employed.     

A rapid TRIzol-based two-step method for DNA-free RNA extraction from Arabidopsis siliques and dry seeds

Extraction of high-quality RNA from Arabidopsis seeds has been a challenge. Here we report a two-step TRIzol-based procedure for RNA extraction from Arabidopsis siliques and dry seeds. This procedure employs a modified, high pH (pH 9.5) extraction buffer. High pH plus the addition of either DTT or β-mercaptoethanol in the extraction buffer effectively inhibits RNase activity during the extraction, and removes most polysaccharides, polyphenols and other insoluble material. TRIzol reagent was subsequently used to purify the RNA. Using this procedure we isolated high-quality DNA-free RNA samples without DNase I treatment from Arabidopsis seeds or siliques in less than 3 h.     

Effect of malformin on the major constituents of Phaseolus vulgaris

Malformin inhibits wet and dry weight, nitrogen accumulation,and cell wall, RNA, DNA and protein synthesis in Phaseolus vulgaris.The relative proportion of dry matter and nitrogen in malformedtissues is increased in the ethanol soluble fraction and decreasedin the residue remaining after hydrolysis with 0.5 N HCl. Inhibitionof cell wall and protein synthesis was generally greater thaninhibition of nitrogen accumulation and RNA and DNA synthesis.The effects of malformin on the composition of P. vulgaris aresimilar to alterations in composition reported for ethylene,and opposite to those reported for gibberellic acid. ¹This research was supported by grant GB-7158 from the NationalScience Foundation and grant E-146-F from the American CancerSociety. ²Journal Paper No. 3509 of the Purdue Agricultural ExperimentStation. (Received October 23, 1968; )