Targeted editing of transcriptional activator MXR1 on the Pichia pastoris genome using CRISPR/Cas9 technology.

A highly efficient and targeted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing system was constructed for Pichia pastoris (syn Komagataella phaffii). Plasmids containing single guide RNA and the methanol expression regulator 1 (MXR1) homology arms were used to precisely edit the transcriptional activator Mxr1 on the P. pastoris genome. At the S215 amino acid position of Mxr1, one, two, and three nucleotides were precisely deleted or inserted, and S215 was also mutated to S215A via a single-base substitution. Sequencing of polymerase chain reaction (PCR) amplicons in the region spanning MXR1 showed that CRISPR/Cas9 technology enabled efficient and precise gene editing of P. pastoris. The expression levels of several of the Mxr1-targeted genes, AOX1, AOX2, DAS1, and DAS2, in strains containing the various mutated variants of MXR1, were then detected through reverse transcription PCR following induction in methanol-containing culture medium. The frameshift mutations of Mxr1 led to almost zero transcription of AOX1, DAS1, and DAS2, while that of AOX2 was reduced to 60%. For the Mxr1 S215A mutant, the transcription of AOX1, AOX2, DAS1, and DAS2 was also reduced by nearly 60%. Based on these results, it is apparent that the transcription of AOX1, DAS1, and DAS2 is exclusively regulated by Mxr1 and serine phosphorylation at Mxr1 residue 215 is not critical for this function. In contrast, the transcription of AOX2 is mainly dependent on the phosphorylation of this residue. CRISPR/Cas9 technology was, therefore, successfully applied to the targeted editing of MXR1 on the P. pastoris genome, and it provided an effective method for the study of this transcription factor and its targets.

[An analysis of etiologies of fever of unknown origin in 372 patients].

OBJECTIVE: To analyze the etiology of fever of unknown origin (FUO). METHODS: A total of 372 patients with FUO who hospitalized in Capital Medical University Affiliated Beijing Friendship Hospital were retrospectively analyzed from January 2003 to August 2013. All the patients were divided into two groups: group A (January 2003 - December 2007) and group B (January 2008-August 2013). Diagnosis rate, duration of hospitalization (days) and time to diagnosis between the two groups were artificially compared. RESULTS: Of the 372 FUO cases, 336 were positively diagnosed with a diagnosis rate of 90.3%. Infectious diseases were still the primary causes of FUO (60.2%), including 72 cases (32.1%) of tuberculosis. Connective tissue diseases accounted for 12.9% of the FUO cases, malignancies were 8.3%, and miscellaneous diseases were 8.9%. Yet thirty six patients (9.7%) could not be confirmed until they were discharged from hospital. The duration of fever in patients with malignancies was longer than that with infectious diseases [60.0 (30.0, 90.0) days vs 30.0 (20.0, 60.0) days, P = 0.003]. Time to diagnosis of connective tissue disease and malignancies was longer than infectious diseases [(12.0(7.3, 18.8) days and 11.0 (7.0, 18.0) vs 5.0 (3.0, 8.0) days, both P values = 0.000]. The duration of hospitalization in group A was longer than that of group B [17.0(12.0, 30.0) days vs 14.0(10.0, 20.0) days, P = 0.000]. The diagnosis rate and time to diagnosis of group A were similar with those of group B. The proportion of connective tissue diseases in group A was higher than group B (18.1% vs 9.2%,χ(2) = 6.201, P = 0.013) . The proportion of infectious disease, malignancies and miscellaneous diseases was not significantly different between the two groups. CONCLUSIONS: Infectious diseases are the major causes of FUO, and the most common cause is tuberculosis. Connective tissue diseases and malignancies are the second and third causes of FUO. The duration of fever and time to diagnosis are significantly different between the different origins.

[Determination of artemisinic acid in Artemisia annua at different growth stages based on spot area].

OBJECTIVE: Based on the important medicinal applications of artemisinic acid and the superiority of Thin Layer Chromagraphy (TLC), the spot area method of TLC was presented to determine the content changes of artemisinic acid of Artemisia annua at different growing stages. METHODS: The separation conditions including chromatographic solutions and chromogenic agent were optimized. The detection limit and the linear concentration range were analyzed. And the content changes of artemisinic acid of Artemisia annua at different growing stages were detected. RESULTS: The results showed that artemisinic acid extracted from Artemisia annua could be separated completely by the chromatographic solutions composed by petroleum ether,acetone and ethyl acetate (80: 19: 1). The artemisinic acid was clearly colored using the chromogenic agent consisting by ethanol, bromophenol blue and sulfuric acid. The detection limit of TLC was 0.05 mg/mL. The spot area of TLC had a good linear relationship within the range of 0.05-0.6 mg/mL, accorded with regression equation of y = 11.162 x + 0.0823. The results showed that the content of artemisinic acid at 0.041 mg/g in April which below the detection limit of TLC had no color spot. Contrarily, the spots of artemisinic acid were obvious in materials growing from May to September, and content was about 0.7, 1.2, 2.1, 2.4 and 2.7 mg/g, respectively corresponding to results by HPLC. CONCLUSION: The method can be applied to the quantitative analysis of artemisinic acid in Artemisia annua.

[Variation of content of artemisic acid in different growth stages of Artemisia annua].

OBJECTIVE: To study the variation of content of Artemisic acid of Artemisia annua from eight areas of four provinces around Wuling Mountain. METHODS: Artemisic acid of plants were extracted by organic solvent method. Qualitative and quantitative analysis of Artemisic acid were measured by thin layer chromatography (TLC) and high performance thin layer chromatography (HPLC), respectively. RESULTS: The results showed the average levels of Artemisic acid in May and August changed from 0.964 to 2.288 mg/g and from 1.837 to 3.737 mg/g, respectively. The average level in August was 1.5 times as that in May. The Artemisic acid in cultured plants was higher than the levels in wild plants, and Artemisic acid in plant collected below 300 m altitude was higher than that of the plant collected above 300 m altitude. CONCLUSION: The biosynthesis of Artemisic acid depends on the plant growth stage,which is mainly accumulated in plant at the mature stage.

Deep learning radiomic analysis of DCE-MRI combined with clinical characteristics predicts pathological complete response to neoadjuvant chemotherapy in breast cancer.

Objective: The aim of this study was to develop and validate a deep learning-based radiomic (DLR) model combined with clinical characteristics for predicting pathological complete response (pCR) to neoadjuvant chemotherapy (NAC) in breast cancer. For early prediction of pCR, the DLR model was based on pre-treatment and early treatment dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) data. Materials and methods: This retrospective study included 95 women (mean age, 48.1 years; range, 29-77 years) who underwent DCE-MRI before (pre-treatment) and after two or three cycles of NAC (early treatment) from 2018 to 2021. The patients in this study were randomly divided into a training cohort (n=67) and a validation cohort (n=28) at a ratio of 7:3. Deep learning and handcrafted features were extracted from pre- and early treatment DCE-MRI contoured lesions. These features contribute to the construction of radiomic signature RS1 and RS2 representing information from different periods. Mutual information and least absolute shrinkage and selection operator regression were used for feature selection. A combined model was then developed based on the DCE-MRI features and clinical characteristics. The performance of the models was assessed using the area under the receiver operating characteristic curve (AUC) and compared using the DeLong test. Results: The overall pCR rate was 25.3% (24/95). One radiomic feature and three deep learning features in RS1, five radiomic features and 11 deep learning features in RS2, and five clinical characteristics remained in the feature selection. The performance of the DLR model combining pre- and early treatment information (AUC=0.900) was better than that of RS1 (AUC=0.644, P=0.068) and slightly higher that of RS2 (AUC=0.888, P=0.604) in the validation cohort. The combined model including pre- and early treatment information and clinical characteristics showed the best ability with an AUC of 0.925 in the validation cohort. Conclusion: The combined model integrating pre-treatment, early treatment DCE-MRI data, and clinical characteristics showed good performance in predicting pCR to NAC in patients with breast cancer. Early treatment DCE-MRI and clinical characteristics may play an important role in evaluating the outcomes of NAC by predicting pCR.

Simple, rapid, sensitive, and versatile SWNT-paper sensor for environmental toxin detection competitive with ELISA.

Safety of water was for a long time and still is one of the most pressing needs for many countries and different communities. Despite the fact that there are potentially many methods to evaluate water safety, finding a simple, rapid, versatile, and inexpensive method for detection of toxins in everyday items is still a great challenge. In this study, we extend the concept of composites, impregnated porous fibrous materials, such as fabrics and papers, by single-walled carbon nanotubes (SWNTs), toward very simple but high-performance biosensors. They utilize the strong dependence of electrical conductivity through nanotubes percolation network on the width of nanotube-nanotube tunneling gap and can potentially satisfy all the requirements outlined above for the routine toxin monitoring. An antibody to the microcystin-LR (MC-LR), one of the common culprits in mass poisonings, was dispersed together with SWNTs. This dispersion was used to dip-coat the paper rendering it conductive. The change in conductivity of the paper was used to sense the MC-LR in the water rapidly and accurately. The method has the linear detection range up to 10 nmol/L and nonlinear detection up to 40 nmol/L. The limit of detection was found to be 0.6 nmol/L (0.6 ng/mL), which satisfies the strictest World Health Organization standard for MC-LR content in drinking water (1 ng/mL) and is comparable to the detection limit of the traditional ELISA method of MC-LR detection, while drastically reducing the time of analysis by more than an order of magnitude, which is one of the major hurdles in practical applications. Similar technology of sensor preparation can also be used for a variety of other rapid environmental sensors.

Ultrasensitive detection of trace protein by Western blot based on POLY-quantum dot probes.

In this study, we describe an ultrasensitive quantum dots (QDs)-based Western blot. With the high affinity of avidin-functionalized POLY-QDs and simplification of the detection process, this enabled the quantitative analysis of protein by Western blotting. To prepare the POLY-QDs, CdTe quantum dots were first coated with biotinylated denatured bovine serum albumin and then, via the effect of the biotin-avidin system, the biotinylated denatured bovine serum albumin-coated QDs, which had strong fluorescence, were linked together. With this series of modifications, the fluorescence intensity of CdTe QDs was significantly increased. Using the POLY-QDs as labels, the signal of Western blotting was more sensitive in tracing the protein than traditional dyeing. In the present study, trace protein A was applied to POLY-QDs-based Western blotting as a model. The linearity of this method was from 30 pg to 1.5 ng, and the sensitivity was up to low pictogram values. The final fluorescence signal on the polyvinylidenedifluoride (PVDF) membrane was retained for at least 40 min. The results of this study indicate that the POLY-QDs-based Western blot is an excellent quantitative analytical method for trace protein analysis.

Fabricated aptamer-based electrochemical "signal-off" sensor of ochratoxin A.

An ultrasensitive and rapid electrochemical platform for the specific detection of ochratoxin A (OTA) was developed. In this method, three single-stranded DNA molecules, including the aptamer, were immobilized on the surface of an electrode. Binding of the OTA target analyte to the aptamer changed the redox current of methylene blue (MB), which was used as the electrochemical probe, in a manner that was dependent on OTA concentration. With signal enhancement from gold nanoparticle-functionalized DNA, the sensitivity of this method for OTA was as low as 30 pg/mL, and the effective sensing range was from 0.1 to 20 ng/mL. To investigate the sensing process, the conformational switch of the aptamer was studied by circular dichroism (CD), which confirmed the recognition of the aptamer by the target OTA. Given its sensitivity and rapid detection, we believe this approach has the potential to be a main technology for the detection of toxins in the field of food safety, and in other areas.

Rapid and sensitive detection of microcystin by immunosensor based on nuclear magnetic resonance.

A stable and sensitive toxin residues immunosensor based on the relaxation of magnetic nanoparticles was developed. The method was performed in one reaction and offered sensitive, fast detection of target toxin residues in water. The target analyte, microcystin-LR (MC-LR) in Tai lake water, competed with the antigens on the surface of the magnetic nanoparticles and then influenced the formation of aggregates of the magnetic nanoparticles. Accordingly, the magnetic relaxation time of the magnetic nanoparticles was changed under the effect of the target analyte. The calibration curve was deduced at different concentrations of the target analyte. The limit of detection (LOD) of MC-LR was 0.6 ng g(-1) and the detection range was 1-18 ng g(-1). Another important feature of the developed method was the easy operation: only two steps were needed (1) to mix the magnetic nanoparticle solution with the sample solution and (2) read the results through the instrument. Therefore, the developed method may be a useful tool for toxin residues sensing and may find widespread applications.

Simultaneous and sensitive determination of multiplex chemical residues based on multicolor quantum dot probes.

Biotinylated denatured bovine serum albumin (Bt-dBSA)-coated cadmium telluride (CdTe) quantum dot (QD) conjugates were prepared and used to develop the multiplexed fluoroimmunoassay for the simultaneous determination of five chemical residues. An immune complex was formed using avidin as the bridge to link the Bt-dBSA-QDs with the antibodies. Primarily, individual quantitative determinations of five representative chemical residues were carried out based on the different emission properties of the QDs. Five antibodies were then conjugated with the corresponding QDs to establish the indirect competition fluorescent-linked immunosorbent assay (ic-FLISA) for the simultaneous detection of five chemicals in one well of a microplate. The linear range for dexamethason (DEX) was from 0.33 microg/kg to 10 microg/kg, 0.28 microg/kg to 10 microg/kg for gentamicin (GM), 0.16 microg/kg to 25 microg/kg for clonazepam (CZP), 0.17 microg/kg to 10 microg/kg for medroxyprogesterone acetate (MPA) and 0.32 microg/kg to 25 microg/kg for ceftiofur (CEF), respectively. The limit of detection (LOD) for the simultaneous determination of DEX, GM, CZP, MPA and CEF were as low as 0.13 microg/kg, 0.16 microg/kg, 0.07 microg/kg, 0.06 microg/kg and 0.14 microg/kg, respectively. This detection method was used to analyze samples of pork muscle and the recoveries ranged from 61.3% to 80.3% for DEX and from 74.0% to 87.2% for MPA. Further more, good correlation between the novel ic-FLISA and traditional ELISA was demonstrated during the determination of DEX and MPA residues in real samples. The QD-based protocol described here is less time consuming than the classical method and it may be sufficiently flexible to be used in other systems for the simultaneous multicolor detection of the drugs.