Comprehensive Benchmarking of CITE-seq versus DOGMA-seq Single Cell Multimodal Omics

The recently developed transcription, epitopes, and chromatin accessibility by sequencing (TEA-seq) and similar DOGMA-seq single-cell trimodal omics assays provide unprecedented opportunities for understanding cell biology, but independent optimization, benchmarking and evaluation are lacking. We explored the utility, pros and cons of DOGMA-seq compared to the bimodal cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) assay in activated and stimulated human peripheral blood T cells. We identified an optimal incubation time and concentration of digitonin (DIG) for cell permeabilization and found that single-cell trimodal omics measurements after DIG permeabilization were generally better than after an alternative low-loss lysis (LLL) permeabilization condition. Next, we found that DOGMA-seq with optimized DIG permeabilization and its ATAC library provides more information, even though its mRNA and cell surface protein antibody-derived tag (ADT) libraries have slightly inferior quality, compared to CITE-seq. Finally, we recognized the additional value of DOGMA-seq for studying lineage-specific T helper cells.

Experimental studies to increase the natural resources of brine shrimp Artemia in hyperhaline reservoirs

A brief analysis of the available technologies for growing Artemia in the world and in Russia is given in the paper. The conditions for production of Artemia in natural reservoirs with a local Artemia population are shown. The results of laboratory experiments on reducing the incubation time of cysts to 2-23 hours (instead of the standard 24-48 hours) and the inoculation of nauplii and non-hatched cysts into the brine of natural lakes with salinity of 101, 125, 225 and 333‰ are given in details. The following indicators are analyzed: the rate of hatching during incubation, the rate of hydration of cysts during incubation and dehydration in brine; the survival rate of nauplii in brine, the possibility of hatching nauplii from cysts in brine with different salinity. The results of long-term observations of the survival of Artemia crustaceans in the brine of the lake in the absence of feeding are also presented. It is possible to reduce the incubation time to 6-20 hours is concluded in the paper. The dependence of the duration of incubation of cysts on the temperature, quality of cysts and salinity of natural brine was noted. To determine the optimal incubation time of cysts the formulas for calculating T90 according to the temperature and salinity of brine for cysts of different quality are given.

Incubation temperature and physiological aging in the zebra finch

In birds, incubation temperature has received increased attention as an important source of phenotypic variability in offspring. A lower than optimal incubation temperature may negatively affect aspects of nestling physiology, such as body growth and energy metabolism. However, the long-term effects of sub-optimal incubation temperature on morphology and physiology are not well understood. In a previous study, we showed that zebra finches from eggs incubated at a low temperature (35.9°C) for 2/3 of the total incubation time suffered a lower post-fledging survival compared to individuals that had been incubated at higher temperatures (37.0 and 37.9°C). In the present study, we investigated whether these variations in incubation temperature could cause permanent long-lasting differences in body mass, body size, or basal metabolic rate. Furthermore, we tested whether the observed differences in survival between treatment groups would be reflected in the rate of physiological deterioration, assessed through oxidative damage and decreased metabolic rate with age (i.e. ‘metabolic aging’). Incubation temperature did not significantly affect embryonic or nestling body growth and did not influence final adult body mass or body size. Nor was there any long-term effect on basal metabolic rate. Birds from eggs incubated at the lowest temperature experienced an accumulation of oxidative damage with age, although this was not accompanied by an accelerated rate of metabolic aging. The present results suggest that the low survival in these birds was possibly mediated by increased oxidative stress, but independent of body growth and the basal metabolic rate.

Application and characterization of crude fungal lipases used to degrade fat and oil wastes

Aspergillus niger MH078571.1 and A. niger MH079049.1 were identified previously as the two highest Aspergillus niger strains producing lipase. Biochemical characterizations of lipase activity and stability for these two strains were examined and revealed that the optimal temperature is 45 °C at pH 8for A. niger MH078571.1 and 55 °C for MH079049.1. The lipase production of both strains was studied on medium contains waste oil, as a cheap source to reduce the industrial cost, showed that the optimal incubation period for the enzyme production is 3 days. Moreover, an experiment on lipase activates in organic solvents demonstrated that 50% of acetone is the best solvent for the two strains. In the presence of surfactants, 0.1% of tween 80 surfactant showed the best lipase activities. Furthermore, Mg2+ and Zn2+ ions enhanced the lipase activity of A. niger MH078571.1, while Na2+ and Cu2+ enhanced the enzyme activity of A. niger MH079049.1. Lipase activity was also tested for industrial applications such as integrating it with different detergents. Maximum lipase activity was obtained with 1% of Omo as a powder detergent for both strains. In liquid detergent, 0.1% of Fairy showed maximum lipase activity in A. niger MH078571.1, while the lipase in A. niger MH079049.1 was more effective in 1% of Lux. Moreover, the degradation of natural animal fat with crude enzyme was tested using chicken and sheep fats. The results showed that more than 90% of fats degraded after 5 days of the incubation period.

Optimization of Fermentation Conditions for Carrageenase Production by Cellulophaga Species: A Comparative Study

Carrageenases appear in various species of marine bacteria and are widely used for the degradation of carrageenans, the commercially significant sulphated polysaccharides. The carrageenase production ability of six different Cellulophaga species was identified, with ι-carrageenase being the most abundant carrageenolytic enzyme. C. algicola was the most potent strain, followed by C. fucicola and C. geojensis, whereas C. pacifica was the least effective carrageenase producer among the studied strains. The enzyme production was maximized using the one-factor-at-a-time optimization method. The optimal incubation temperature was identified as 25 °C and the incubation time was set as 48 h for all tested species. The optimal medium composition for Cellulophaga strains was determined as 30 g/L sea salt, 1.4 g/L furcellaran, and 3 g/L yeast extract. An ultrafiltered enzyme extracted from C. algicola had the highest activity at around 40 °C. The optimal pH for enzymatic degradation was determined as 7.8, and the enzyme was fairly stable at temperatures up to 40 °C.

The Effect of Incubation Temperature On Semen Parameters Before Intra-Uterine Insemination

Objectives To evaluate the effect of different incubation temperature between room (26–28˚c) and body (37 ˚c) temperature on percentage of progressive sperm motility and the optimal incubation period before intrauterine insemination. Methods Seventy-one normal semen samples under WHO 2010 criteria were recruited. All semen was prepared with Density Gradient Centrifugation technique (DGC) and divided into two groups to evaluate sperm motility at 60 min of incubation time. First group: prepared semen was incubated in room temperature (26˚-28˚c) and second group: prepared semen was incubated in body temperature (37°c). Moreover, each group is divided into 4 items for compare sperm motility between both groups in same of incubation time and evaluate the optimal incubation time between the items in the same groups. Results Spermatozoa incubated at body temperature had a significantly higher percentage of progressive sperm motility than those incubated at room temperature (89.62 ± 8.02 vs 85.97 ± 9.42; p < 0.01). The optimal incubation time at room temperature was 30 minutes and at body temperature was 60 minutes. These results suggest that spermatozoa incubated at 37°C for 60 minutes were more likely to have better sperm motility functions for IUI. Conclusion These results suggest that spermatozoa incubated at 37°C for 60 minutes were more likely to be effective for use in IUI in terms of sperm motility functions.

Shortening the Time of the Identification and Antimicrobial Susceptibility Testing on Positive Blood Cultures with MALDI-TOF MS

The current processes used in clinical microbiology laboratories take ~24 h for incubation to identify the bacteria after the blood culture has been confirmed as positive and fa further ~24 h to report the results of antimicrobial susceptibility tests (ASTs). Patients with suspected bloodstream infection are treated with empiric broad-spectrum antibiotics but delayed targeted antimicrobial therapy. This study aimed to develop a method with a significantly shortened turnaround time for clinical application by identifying the optimal incubation period of a subculture. A total of 188 positive blood culture samples obtained from Nov. 2019 to Aug. 2020 were included. Compared to the conventional 24-h incubation for bacterial identification, our approach achieved 96.1% and 97.4% identification accuracy after shortening the incubation time to 4.5 and 3.5 h for gram-positive (GP) and gram-negative (GN) bacterial samples, respectively. Samples from short-term incubation without any intermediate step or process were directly subjected to analysis with the Phoenix M50 AST. Compared to the conventional disk diffusion AST, the category agreements for GP (excluding Streptococcus spp.), Streptococcus spp., and GN bacterial samples were 91.8%, 97.5%, and 92.7%, respectively. Our approach significantly reduced the average turnaround time from 48 h to 28 h for reporting bacterial identity and decreased average AST from 72 h to 50.3 h compared to the conventional methods. Accordingly, this approach allows a physician to prescribe the appropriate antibiotic(s) ~21.7 h earlier, thereby improving patient outcomes.

RPAcan3990: an ultrasensitive recombinase polymerase assay to detect Angiostrongylus cantonensis DNA

Angiostrongylus cantonensis (Ac) is one of the leading causes of eosinophilic meningitis worldwide. A field deployable molecular detection method could enhance both environmental surveillance and clinical diagnosis of this emerging pathogen. Accordingly, RPAcan3990, a recombinase polymerase assay (RPA) was developed to target a region predicted to be highly repeated in the Ac genome. The assay was then adapted to produce a visually interpretable fluorescent readout using an orange camera lens filter and a blue light. Using Ac genomic DNA, the limit of detection was found to be 1fg/μl by both fluorometer measurement and visual reading. All clinical samples known to be positive for Ac from various areas of the globe were positive by RPAcan3990. Cerebrospinal fluid samples from other etiologies of eosinophilic meningitis (i.e. Toxocara sp., Gnathostoma sp.) were negative in the RPAcan3990 assay. The optimal incubation temperature range for the reaction was between 35-40°C. The assay successfully detected 1fg/μl of Ac genomic DNA after incubation at human body temperature (in a shirt pocket). In conclusion, these data suggest RPAcan3990 is potentially a point of contact molecular assay capable of sensitively detecting Ac by producing visually interpretable results with minimal instrumentation.

Evaluation of Optimal Blood Culture Incubation Time to Maximize Clinically Relevant Results from a Contemporary Blood Culture Instrument and Media System

Timely diagnosis of microorganisms in blood cultures is necessary to optimize therapy. Although blood culture media and systems have evolved for decades, the standard interval for incubation prior to discard as negative has remained five days. Here, we evaluated the optimal incubation time for the BACT/ALERT VIRTUO blood culture detection system (bioMérieux) using FA Plus (aerobic) and FN Plus (anaerobic) resin culture bottles in routine clinical use. Following IRB approval, a retrospective review evaluated the outcomes of 158,710 bottles collected between November 2018 and October 2019. The number of positive blood bottles was 13,592 (8.6%); 99% of positive aerobic and anaerobic bottles flagged positive by 91.5 h and 108 h, respectively. The mean (median) time-to-positivity for Staphylococcus aureus was 18.4 h (15.6 h), Escherichia coli 12.3 h (9.5 h), Pseudomonas aeruginosa 22.2 h (15.9 h), and Candida spp. 48.9 h (42.9 h). Only 175 bottles (0.1% of all bottles) flagged positive after four days of incubation; 89 (51%) of these bottles grew Cutibacterium (Propionibacterium) species. Chart review of blood cultures positive after four days (96 h) rarely had clinical impact, and sometimes had a negative impact on patientcare. Finally, a seeded study of the HACEK group, historically associated with delayed blood culture positivity, demonstrated no benefit to extended incubation beyond four days. Collectively, these findings demonstrated that a four-day incubation time was sufficient for the VIRTUO system and media. Implementation of the four-day incubation time could enhance clinically relevant results by reducing recovery of contaminants and finalizing blood cultures one day earlier.

New Deoxyribozymes for the Native Ligation of RNA

Deoxyribozymes (DNAzymes) are small, synthetic, single-stranded DNAs capable of catalyzing chemical reactions, including RNA ligation. Herein, we report a novel class of RNA ligase deoxyribozymes that utilize 5′-adenylated RNA (5′-AppRNA) as the donor substrate, mimicking the activated intermediates of protein-catalyzed RNA ligation. Four new DNAzymes were identified by in vitro selection from an N40 random DNA library and were shown to catalyze the intermolecular linear RNA-RNA ligation via the formation of a native 3′-5′-phosphodiester linkage. The catalytic activity is distinct from previously described RNA-ligating deoxyribozymes. Kinetic analyses revealed the optimal incubation conditions for high ligation yields and demonstrated a broad RNA substrate scope. Together with the smooth synthetic accessibility of 5′-adenylated RNAs, the new DNA enzymes are promising tools for the protein-free synthesis of long RNAs, for example containing precious modified nucleotides or fluorescent labels for biochemical and biophysical investigations.