Efficient and reliable protoplasting, regeneration, and fusion techniques were established for the prototrophic strain Bacillus stearothermophilus NUB36. Auxotrophic mutants were isolated, and protoplast fusion was used to construct isogenic mutant strains and for chromosomal mapping. Markers were mapped using two-, threeP., and four-factor crosses. The order of the markers was hom-1-thr-1-his-1(gly-1 or gly-2)-pur-1-pur-2. These markers may be analogous to hom, thrA, hisA, glyC, and purA markers on the Bacilus subtilis chromosome. No analogous pur-l marker has been reported in B. subtlis. The relative order of three of the markers (hom-1-thr-1-gly-1) was independently confirmed by transduction.Insights into the molecular mechanisms of thermophily have been hampered because virtually nothing is known about the genetic basis of thermophily. This gap in our knowledge is related to the lack of an efficient genetic exchange system in a thermophilic bacterial species.Research on thermophile genetics has been confined mostly to Bacillus stearothermophilus and related thermophilic bacilli. Of the various traditional modes of gene transfer, DNA-mediated transformation was used for screening experiments because conjugation has not been demonstrated in the genus Bacillus and transduction occurs at too low a frequency to be useful for preliminary studies.Over the past 20 years, a majority of the standard strains of B. stearothermophilus have been screened for genetic transformation by using procedures that were developed for B. subtilis. To our knowledge however, there are only two published reports of genetic transformation in this organism (17,30). The low frequency of transformation and the lack of reproducibility precluded the use of these systems for genetic studies.The lack of success encountered in these studies underlines the relevance of certain practical considerations in the development of a genetic exchange system in a strain of interest. The first consideration is that the standard strains of B. stearothermophilus are most likely noncompetent. Only a few bacterial strains have evolved a mechanism for the uptake and expression of DNA, and even in the bestcharacterized bacterial transformation systems, many wildtype strains are noncompetent. In others, only certain auxotrophic mutants are competent. A second consideration is the need for a battery of auxotrophic markers to facilitate the genetic analysis of B. stearothermophilus. A few mutants of B. stearothermophilus have been isolated, but a majority of these are either antibiotic-resistant mutants or mutants lacking a particular enzyme activity (19). There is only one report on the isolation of auxotrophic mutants (25). This is not surprising since most standard strains of B. stearothermophilus have relatively complex nutritional requirements (3, 5). In the absence of prototrophic strains that grow well in a minimal medium, it is not possible to isolate auxotrophic * Corresponding author. (12) for Bacillus licheniformis were used to screen the strains for tr...

A novel coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) emerged in late 2019, causing an outbreak of pneumonia [coronavirus disease 2019 (COVID-19)] globally. Although the use of ready-made reaction mixes can enable more rapid PCR-based diagnosis of COVID-19, the need to transport and store these mixes at low temperatures presents challenges to already overburdened logistics networks. Methods: Here, we present an optimized freeze-drying procedure that allows SARS-CoV-2 PCR mixes to be transported and stored at ambient temperatures, without loss of activity. Additive-supplemented PCR mixes were freeze-dried. The residual moisture of the freeze-dried PCR mixes was measured by Karl-Fischer titration. Results: We found that the freeze-dried PCR mixes with~1.2% residual moisture are optimal for storage, transport, and reconstitution. The sensitivity, specificity, and repeatability of the freeze-dried reagents were similar to those of freshly prepared, wet reagents. The freeze-dried mixes retained activity at room temperature (18~25°C) for 28 days, and for 14 and 10 days when stored at 37°C and 56°C, respectively. Conclusion: The uptake of this approach will ease logistical challenges faced by transport networks and make more cold storage space available at diagnosis and hospital laboratories.

A spoken language understanding (SLU) system includes two main tasks, slot filling (SF) and intent detection (ID). The joint model for the two tasks is becoming a tendency in SLU. But the bi-directional interrelated connections between the intent and slots are not established in the existing joint models. In this paper, we propose a novel bi-directional interrelated model for joint intent detection and slot filling. We introduce an SF-ID network to establish direct connections for the two tasks to help them promote each other mutually. Besides, we design an entirely new iteration mechanism inside the SF-ID network to enhance the bi-directional interrelated connections. The experimental results show that the relative improvement in the sentence-level semantic frame accuracy of our model is 3.79% and 5.42% on ATIS and Snips datasets, respectively, compared to the state-of-the-art model.

19A novel coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) : bioRxiv preprint 23 19, the need to transport and store these mixes at low temperatures presents challenges 24 to already overburdened logistics networks. Here, we present an optimized freeze-25 drying procedure that allows SARS-CoV-2 PCR mixes to be transported and stored at 26 ambient temperatures, without loss of activity. Additive-supplemented PCR mixes were 27 freeze-dried. The residual moisture of the freeze-dried PCR mixes was measured by 28 Karl-Fischer titration. We found that freeze-dried PCR mixes with ~1.2% residual 29 moisture are optimal for storage, transport, and reconstitution. The sensitivity, 30 specificity, and repeatability of the freeze-dried reagents were similar to those of freshly 31 prepared, wet reagents. The freeze-dried mixes retained activity at room temperature 32 (18~25℃) for 28 days, and for 14 and 10 days when stored at 37℃ and 56℃, 33 respectively. The uptake of this approach will ease logistical challenges faced by 34 transport networks and make more cold storage space available at diagnosis and 35 hospital laboratories. This method can also be applied to the generation of freeze-dried 36 PCR mixes for the detection of other pathogens. 37 38

A theoretical analysis of the stress state in specimen of the SHPB experiment was performed in consideration of lateral inertia effect. The nonuniformity of lateral stress in specimen and variety of deforming velocity in the loading process were taken into account in the analysis. A formula to correcting the lateral inertia effect was obtained. The force and deformation velocity of specimen-bar interfaces during the loading process got from a numerical simulation of SHPB were used to verify the theoretical analysis formula. It shows that the deviation of reconstructed curve from the inputted relationship can be brought down with the correction formula.