Impact of the Purification Process on the Spray-Drying Performances of the Three Families of Lipopeptide Biosurfactant Produced by Bacillus subtilis

Lipopeptides produced by Bacillus subtilis display many activities (surfactant, antimicrobial, and antitumoral), which make them interesting compounds with a wide range of applications. During the past years, several processes have been developed to enable their production and purification with suitable yield and purity. The already implemented processes mainly end with a critical drying step, which is currently achieved by freeze-drying. In this study, the possibility to replace this freeze-drying step with a spray-drying one, more suited to industrial applications, was analyzed. After evaluating their thermal resistance, we have developed a spray-drying methodology applicable for the three lipopeptides families produced by B. subtilis, i.e., surfactin, mycosubtilin (iturin family), and plipastatin (fengycin family). For each lipopeptide, the spray-drying procedure was applied at three steps of the purification process by ultrafiltration (supernatant, diafiltered solution, and pre-purified fraction). The analysis of the activities of each spray-dried lipopeptide showed that this drying method is not decreasing its antimicrobial and biosurfactant properties. The methodology developed in this study enabled for the first time the spray-drying of surfactin, without adjuvants’ addition and regardless of the purification step considered. In the case of fengycin and mycosubtilin, only diafiltered solution and purified fraction could be successfully spray-dried without the addition of adjuvant. Maltodextrin addition was also investigated as the solution for the direct drying of supernatant. As expected, the performances of the spray-drying step and the purity of the powder obtained are highly related to the purification step at which the product was dried. Interestingly, the impact of mycosubtilin concentration on spray-drying yield was also evidenced.

Two extraction-free reverse transcription loop-mediated isothermal amplification assays for detection of SARS-CoV-2

Background Current assays for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rely on time consuming, costly and laboratory based methods for virus isolation, purification and removing inhibitors. To address this limitation, we propose a simple method for testing RNA from nasopharyngeal swab samples that bypasses the RNA purification step. Methods In the current project, we have described two extraction-free reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for the detection of SARS-CoV-2 by using E gene and RdRp gene as the targets. Results Here, results showed that reverse transcription loop-mediated isothermal amplification assays with 88.4% sensitive (95% CI: 74.9–96.1%) and 67.4% sensitive (95% CI: 51.5–80.9%) for E gene and RdRp gene, respectively. Conclusion Without the need of RNA purification, our developed RT-LAMP assays for direct detection of SARS-CoV-2 from nasopharyngeal swab samples could be turned into alternatives to qRT-PCR for rapid screening.

A Simple, Cost-Effective, and Automation-Friendly Direct PCR Approach for Bacterial Community Analysis

Understanding bacterial interactions and assembly in complex microbial communities using 16S rRNA sequencing normally requires a large experimental load. However, the current DNA extraction methods, including cell disruption and genomic DNA purification, are normally biased, costly, time-consuming, labor-intensive, and not amenable to miniaturization by droplets or 1,536-well plates due to the significant DNA loss during the purification step for tiny-volume and low-cell-density samples.

A two-hour synthesis of anti-Parkinson drug Safinamide Methanesulfonate

The critical moment of COVID-19 outbreak requires a real time supply of therapeutic agents. Thus, the time economy in the synthesis of biologically active compounds has been increasingly decisive. In this work, we have developed a two-hour synthesis of anti-Parkinson drug safinamide methanesulfonate in 4 steps, with 64% overall yield. Microwave irradiation was used in the first three steps in a one-pot fashion. In fact, this presented protocol can provide Safinamide free base in one hour without chromatographic purification step. Also, green solvents such as methanol and ethyl acetate were applied.

Assessment of Successful qRT-PCR of SARS-CoV-2 Assay in Pool Screening Using Isopropyl Alcohol Purification Step in RNA Extraction

The study is aimed at establishing the optimal parameters for RNA purification of pooled specimens, in SARS-CoV-2 assay. This research work evaluates the difference of extracted RNA purity of pooled samples with and without treatment with isopropyl alcohol and its effect on real-time RT-PCR. As per the protocol of the Indian Council of Medical Research (ICMR), 5 sample pools were analysed using qRT-PCR. A total of 100 pooled samples were selected for the study by mixing 50 μL of one COVID-19 positive nasopharyngeal/oropharyngeal (NP/OP) specimen and 50 μL each of 4 known negative specimens. Pool RNA was extracted using the column-based method, and 1 set of pooled extracted RNA was tested as such, while RNA of the second set was treated additionally with chilled isopropyl alcohol (modified protocol). Further, the purity of extracted RNA in both the groups was checked using Microvolume Spectrophotometers (Nanodrop) followed by RT-PCR targeting E-gene and RNaseP target. The results showed that the purity index of extracted RNA of untreated pooled specimens was inferior to isopropyl alcohol-treated templates, which was observed to be 85% sensitivity and 100% specificity. The average Cq (E gene) in the unpurified and purified pool RNA group was 34.66 and 31.48, respectively. The nanodrop data suggested that purified RNA concentration was significantly increased with an average value of 24.73 ± 1.49 ng / uL , which might be the reason for high sensitivity and specificity. Thus, this group testing of SARS-CoV-2 cases using pools of 5 individual samples would be the best alternative for saving molecular reagents, personnel time, and can increase the overall testing capacity. However, purity of RNA is one of the important determinants to procure unfailing results, thus, this additional purification step must be included in the protocol after RNA has been extracted using commercially available kit before performing qRT-PCR.

Strategy For Cellulase Immobilization And Its Partial Purification And Characterization

Conversion of cellulose to glucose units by cellulases, called hydrolysis, is a very complex step in ethanol production. It requires the mixing of aqueous suspensions of cellulose/cellulases so that cellulases (majority composed of the active site domain and the binding site domain) can attach to cellulose chains, cut or hydrolyze ß(1-4) glycosidic bonds between glucose units, de-attach and move to another location. Mixing extent (insufficient or excessive agitation) might influence the attachment of cellulases and possibly lead to lower glucose yields. A long-term goal of this research is to determine the strength of mixing required to be applied during the cellulose-cellulase mixing cycle. For that purpose, one of the objectives was to purify CBH I exocellulase from the commercial cellulase mixture. A partial purification of the CBH I that was performed on a much smaller scale with uncontrolled flow rate was successful. Another objective was to propose a scheme that would covalently immobilize CBH I exoceullase via its active site domain (ASD) on an atomic force microscopy-compatible support, a silicon support. A theoretically-developed hypothetical scheme was constructed (with the provided detailed procedure). The approach of immobilizing the inhibitor specific to the ASD of CBH I enzyme led to the possibility that no purification of CBH I could be required. Skipping CBH I purification step would save time and hassle associated with purification step. Once the ASD of CBH I is immobilized on a silicon support, the AFM force profile between the free-floating CDB and substrate cellulose could be established.

Strategy For Cellulase Immobilization And Its Partial Purification And Characterization

Conversion of cellulose to glucose units by cellulases, called hydrolysis, is a very complex step in ethanol production. It requires the mixing of aqueous suspensions of cellulose/cellulases so that cellulases (majority composed of the active site domain and the binding site domain) can attach to cellulose chains, cut or hydrolyze ß(1-4) glycosidic bonds between glucose units, de-attach and move to another location. Mixing extent (insufficient or excessive agitation) might influence the attachment of cellulases and possibly lead to lower glucose yields. A long-term goal of this research is to determine the strength of mixing required to be applied during the cellulose-cellulase mixing cycle. For that purpose, one of the objectives was to purify CBH I exocellulase from the commercial cellulase mixture. A partial purification of the CBH I that was performed on a much smaller scale with uncontrolled flow rate was successful. Another objective was to propose a scheme that would covalently immobilize CBH I exoceullase via its active site domain (ASD) on an atomic force microscopy-compatible support, a silicon support. A theoretically-developed hypothetical scheme was constructed (with the provided detailed procedure). The approach of immobilizing the inhibitor specific to the ASD of CBH I enzyme led to the possibility that no purification of CBH I could be required. Skipping CBH I purification step would save time and hassle associated with purification step. Once the ASD of CBH I is immobilized on a silicon support, the AFM force profile between the free-floating CDB and substrate cellulose could be established.

A protocol for the detection of genetic markers in saliva by polymerase chain reaction without a nucleic acid purification step: examples of SARS-CoV-2 and GAPDH markers.

Background: The objective of this study was to develop a protocol for direct use of saliva in tests for genetic markers, without purification of nucleic acids. Currently, diagnostic tests use purified nucleic acids from clinical samples. This purification step adds time, cost, and affects the quality of testing. Multiple attempts to remove the purification step were reported. Results: We report a protocol for the direct detection of genetic markers in saliva. The protocol is based on collection of saliva in a solution containing a detergent and ethanol, and is compatible with isothermal amplification (LAMP), real-time RT-PCR and RT-PCR. SARS-CoV-2 and GAPDH markers were used as reference markers. We observed that mild detergents allow efficient detection of markers (e.g. GAPDH and SARS-CoV-2), while strong detergent, e.g. sodium dodecyl sulfate, inhibited the PCR reaction. Under these conditions, saliva samples can be stored for 24 h at +40C or -180C with preservation of the markers. Storage at room temperature led to deterioration of marker detection. Snap heating of saliva samples at the time of collection, followed by a storage at the room temperature, provided partial protection.Conclusions: The protocol presented in this report describes collection and storage of saliva for direct detection of genetic markers and is compatible with PCR and LAMP tests.