high throughput assay
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2022 ◽  
pp. 114547
Author(s):  
Hang-Qin Zhu ◽  
Wen-Ye Hu ◽  
Xiao-Ling Tang ◽  
Ren-Chao Zheng ◽  
Yu-Guo Zheng

2021 ◽  
Author(s):  
Qiting Wan ◽  
Li Hu ◽  
Lu Yao ◽  
Jiuan Chen ◽  
Jie Sun ◽  
...  

The demand for genetic testing for breast cancer susceptibility genes is increasing for both breast cancer patients and healthy individuals. Here we established a novel high-throughput assay to detect germline pathogenic variants in breast cancer susceptibility genes. In general, up 10 to 50 individual genomic DNA samples were mixed together to create a mixed DNA sample and the mixed DNA sample was subjected to a next-generation multigene panel. Germline pathogenic variants in breast cancer susceptibility genes could be found in the mixed DNA sample; next, site-specific Sanger sequencing was performed to identify individuals who carried he pathogenic variant in the mixed samples. We found that the recall and precision rates were 89.9% and 92.9% when twenty individual genomic samples were mixed. Therefore, our new assay can increase an approximately 20-fold of efficacy to identify the pathogenic variants in breast cancer susceptibility genes in individuals when compared with current assay.


Author(s):  
Mengqiao Li ◽  
Stefan Gaussmann ◽  
Bettina Tippler ◽  
Julia Ott ◽  
Grzegorz M Popowicz ◽  
...  

Human pathogenic trypanosomatid parasites harbor a unique form of peroxisomes termed glycosomes that are essential for parasite viability. We and others previously identified and characterized the essential Trypanosoma brucei ortholog TbPEX3, which is the membrane-docking factor for the cytosolic receptor PEX19 bound to the glycosomal membrane proteins. Knockdown of TbPEX3 expression leads to mislocalization of glycosomal membrane and matrix proteins, and subsequent cell death. As an early step in glycosome biogenesis, the PEX3–PEX19 interaction is an attractive drug target. We established a high-throughput assay for TbPEX3–TbPEX19 interaction and screened a compound library for small-molecule inhibitors. Hits from the screen were further validated using an in vitro ELISA assay. We identified three compounds, which exhibit significant trypanocidal activity but show no apparent toxicity to human cells. Furthermore, we show that these compounds lead to mislocalization of glycosomal proteins, which is toxic to the trypanosomes. Moreover, NMR-based experiments indicate that the inhibitors bind to PEX3. The inhibitors interfering with glycosomal biogenesis by targeting the TbPEX3–TbPEX19 interaction serve as starting points for further optimization and anti-trypanosomal drug development.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Anusha Dravid ◽  
Brad Raos ◽  
Darren Svirskis ◽  
Simon J. O’Carroll

AbstractNeuronal models are a crucial tool in neuroscientific research, helping to elucidate the molecular and cellular processes involved in disorders of the nervous system. Adapting these models to a high-throughput format enables simultaneous screening of multiple agents within a single assay. SH-SY5Y cells have been widely used as a neuronal model, yet commonly in an undifferentiated state that is not representative of mature neurons. Differentiation of the SH-SY5Y cells is a necessary step to obtain cells that express mature neuronal markers. Despite this understanding, the absence of a standardised protocol has limited the use of differentiated SH-SY5Y cells in high-throughput assay formats. Here, we describe techniques to differentiate and re-plate SH-SY5Y cells within a 96-well plate for high-throughput screening. SH-SY5Y cells seeded at an initial density of 2,500 cells/well in a 96-well plate provide sufficient space for neurites to extend, without impacting cell viability. Room temperature pre-incubation for 1 h improved the plating homogeneity within the well and the ability to analyse neurites. We then demonstrated the efficacy of our techniques by optimising it further for neurite outgrowth analysis. The presented methods achieve homogenously distributed differentiated SH-SY5Y cells, useful for researchers using these cells in high-throughput screening assays.


2021 ◽  
Author(s):  
◽  
Richard Laurence Campen

<p>Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), is the leading cause of death and disease by a bacterial pathogen worldwide. The growing incidence of drug resistant TB, especially multi-drug resistant TB highlights the need for new drugs with novel modes of action. Current treatment of TB involves a multi-drug regimen of four drugs including isoniazid and rifampicin, both of which were discovered over 40 years ago. Bedaquiline is one of the first novel TB drugs to enter clinical trials since the discovery of rifampicin, and has shown excellent activity against drug resistant TB. Although isoniazid and rifampicin are well established anti-TB drugs, significant gaps in knowledge regarding their modes of action exist. Furthermore, little information on the mode of action of the novel drug bedaquiline is known beyond its primary target. Characterisation of drug mode of action facilitates rational modifications of drugs to improve the treatment of TB.  The aim of this study was to identify novel aspects of the modes of action of isoniazid, rifampicin, and bedaquiline by characterising drug hypersensitive mutants of M. smegmatis mc²155. A sub-saturated M. smegmatis mc²155 transposon mutant collection with 1.1-fold genome coverage (7680 mutants) was constructed, with this collection estimated to contain mutations in 73.2% of all genes capable of maintaining a transposon insertion (non-essential genes). A high-throughput assay was developed for screening the collection, and mutants related to known drug mode of action were identified for isoniazid (ahpC and eccCa₁) and bedaquiline (atpB). Additionally, known mechanisms of drug inactivation were identified for isoniazid (nudC), rifampicin (arr and lspA), and bedaquiline (mmpL5). The finding that transposon mutants of nudC are hypersensitive to isoniazid independently validated the recent discovery of the role of NudC in basal isoniazid resistance by Wang et al. (2011). The remaining genes identified in this thesis represent potentially novel aspects of the modes of action or resistance mechanisms of these drugs.  Cross-sensitivity to other drugs indicated that the mechanism of sensitivity was drug specific for the mutants examined. Differential-sensitivity testing against drug analogues revealed that Arr is involved in resistance to the rifampicin analogue rifapentine as well, indicating that Arr can detoxify rifapentine similar to rifampicin. The nudC mutant showed increased sensitivity to a range of isoniazid analogues, indicating that it can detoxify these analogues similar to the parent compound. Interestingly six analogues were found to be less active against the nudC mutant than expected. A number of overexpression strains were tested against these six analogues; a nudC overexpression strain, and a strain overexpressing inhA, the primary target for isoniazid. Overexpression of nudC as well inhA increased the resistance of WT to isoniazid, but failed to increase resistance to three of the analogues, NSC27607, NSC33759, and NSC40350. Isoniazid is a prodrug and is activated by the peroxidase/catalase enzyme KatG. Overexpression of katG resulted in increased isoniazid sensitivity, as well as increased sensitivity to NSC27607, NSC33759, and NSC40350. Together these results suggest that NSC27607, NSC33759, and NSC40350 are activated by KatG, but that InhA is not the primary target. Additionally the inability of NudC overexpression to confer resistance suggests these analogues are not acting via a NAD adduct, the mechanism by which isoniazid inhibits InhA. These results suggest that there are other toxic metabolites being produced by KatG activation of these three analogues.  In conclusion, characterisation of mutants identified in a high-throughput assay for drug hypersensitivity identified genes involved in the modes of action or resistance mechanisms for isoniazid, rifampicin, and bedaquiline. Additionally, a number of novel genes were identified that have no known connections to the known modes of action or resistance mechanisms for these drugs. Further testing of a nudC mutant revealed three isoniazid analogues that appear to inhibit growth of M. smegmatis mc²155 independent of InhA, the primary target of isoniazid. This study has successfully demonstrated that screening for drug hypersensitivity can generate novel information on drug mode of action and resistance mechanisms. This information can ultimately be used to help drive the development of new drugs, and improve treatment of TB.</p>


2021 ◽  
Author(s):  
◽  
Richard Laurence Campen

<p>Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), is the leading cause of death and disease by a bacterial pathogen worldwide. The growing incidence of drug resistant TB, especially multi-drug resistant TB highlights the need for new drugs with novel modes of action. Current treatment of TB involves a multi-drug regimen of four drugs including isoniazid and rifampicin, both of which were discovered over 40 years ago. Bedaquiline is one of the first novel TB drugs to enter clinical trials since the discovery of rifampicin, and has shown excellent activity against drug resistant TB. Although isoniazid and rifampicin are well established anti-TB drugs, significant gaps in knowledge regarding their modes of action exist. Furthermore, little information on the mode of action of the novel drug bedaquiline is known beyond its primary target. Characterisation of drug mode of action facilitates rational modifications of drugs to improve the treatment of TB.  The aim of this study was to identify novel aspects of the modes of action of isoniazid, rifampicin, and bedaquiline by characterising drug hypersensitive mutants of M. smegmatis mc²155. A sub-saturated M. smegmatis mc²155 transposon mutant collection with 1.1-fold genome coverage (7680 mutants) was constructed, with this collection estimated to contain mutations in 73.2% of all genes capable of maintaining a transposon insertion (non-essential genes). A high-throughput assay was developed for screening the collection, and mutants related to known drug mode of action were identified for isoniazid (ahpC and eccCa₁) and bedaquiline (atpB). Additionally, known mechanisms of drug inactivation were identified for isoniazid (nudC), rifampicin (arr and lspA), and bedaquiline (mmpL5). The finding that transposon mutants of nudC are hypersensitive to isoniazid independently validated the recent discovery of the role of NudC in basal isoniazid resistance by Wang et al. (2011). The remaining genes identified in this thesis represent potentially novel aspects of the modes of action or resistance mechanisms of these drugs.  Cross-sensitivity to other drugs indicated that the mechanism of sensitivity was drug specific for the mutants examined. Differential-sensitivity testing against drug analogues revealed that Arr is involved in resistance to the rifampicin analogue rifapentine as well, indicating that Arr can detoxify rifapentine similar to rifampicin. The nudC mutant showed increased sensitivity to a range of isoniazid analogues, indicating that it can detoxify these analogues similar to the parent compound. Interestingly six analogues were found to be less active against the nudC mutant than expected. A number of overexpression strains were tested against these six analogues; a nudC overexpression strain, and a strain overexpressing inhA, the primary target for isoniazid. Overexpression of nudC as well inhA increased the resistance of WT to isoniazid, but failed to increase resistance to three of the analogues, NSC27607, NSC33759, and NSC40350. Isoniazid is a prodrug and is activated by the peroxidase/catalase enzyme KatG. Overexpression of katG resulted in increased isoniazid sensitivity, as well as increased sensitivity to NSC27607, NSC33759, and NSC40350. Together these results suggest that NSC27607, NSC33759, and NSC40350 are activated by KatG, but that InhA is not the primary target. Additionally the inability of NudC overexpression to confer resistance suggests these analogues are not acting via a NAD adduct, the mechanism by which isoniazid inhibits InhA. These results suggest that there are other toxic metabolites being produced by KatG activation of these three analogues.  In conclusion, characterisation of mutants identified in a high-throughput assay for drug hypersensitivity identified genes involved in the modes of action or resistance mechanisms for isoniazid, rifampicin, and bedaquiline. Additionally, a number of novel genes were identified that have no known connections to the known modes of action or resistance mechanisms for these drugs. Further testing of a nudC mutant revealed three isoniazid analogues that appear to inhibit growth of M. smegmatis mc²155 independent of InhA, the primary target of isoniazid. This study has successfully demonstrated that screening for drug hypersensitivity can generate novel information on drug mode of action and resistance mechanisms. This information can ultimately be used to help drive the development of new drugs, and improve treatment of TB.</p>


2021 ◽  
Author(s):  
◽  
Victoria Catherwood

<p>The serotonin (5-HT) transporter (SERT) and the kappa opioid receptor (KOPr) are important brain proteins. Currently, it is known that KOPr modulates 5-HT concentrations but the biochemical mechanisms responsible remain enigmatic. The 5-HT and KOPr relationship is thought to involve SERT because both SERT and KOPr co-localise in the dorsal raphe nucleus (DRN), a brain region involved in drug addiction, affective disorders, and stress homeostasis. Thus, elucidating the KOPr and SERT relationship will clarify biomedical targets for these disorders. To investigate this relationship the effect of KOPr activation on SERT function was examined in HEK-293 cells co-expressing myc-rKOPr and eGFP-tagged hSERT. We found the KOPr agonists U50,488H, Salvinorin A (Sal A) and DS-3-240 had no acute (5 min) effect on SERT function as measured by rotating disc electrode voltammetry (RDEV) incubated with 1’-diethyl-2,2’-cyanine iodide (D-22; 1.5 mM) and by confocal microscopy. Interestingly, SERT function significantly decreased with chronic (30 min) exposure to U50,488H (**p<0.01) and Sal A (**p<0.01) as measured with a novel high-throughput assay; this decrease was also attenuated with 5 min pre-treatment of the SERT inhibitor, fluoxitene (***p<0.001). Furthermore, this novel high-throughput assay also replicated our laboratories recent finding that chronic (30 min) exposure to U50,488H (**p<0.01) and Sal A (**p<0.01) significantly increase dopamine transporter (DAT) function in HEK-293 cells co-expressing myc-rKOPr and eYFP-tagged DAT. Collectively, these findings suggest in this cell model that chronic (30 min) KOPr activation by U50,488H and Sal A decreases SERT function.</p>


2021 ◽  
Author(s):  
◽  
Victoria Catherwood

<p>The serotonin (5-HT) transporter (SERT) and the kappa opioid receptor (KOPr) are important brain proteins. Currently, it is known that KOPr modulates 5-HT concentrations but the biochemical mechanisms responsible remain enigmatic. The 5-HT and KOPr relationship is thought to involve SERT because both SERT and KOPr co-localise in the dorsal raphe nucleus (DRN), a brain region involved in drug addiction, affective disorders, and stress homeostasis. Thus, elucidating the KOPr and SERT relationship will clarify biomedical targets for these disorders. To investigate this relationship the effect of KOPr activation on SERT function was examined in HEK-293 cells co-expressing myc-rKOPr and eGFP-tagged hSERT. We found the KOPr agonists U50,488H, Salvinorin A (Sal A) and DS-3-240 had no acute (5 min) effect on SERT function as measured by rotating disc electrode voltammetry (RDEV) incubated with 1’-diethyl-2,2’-cyanine iodide (D-22; 1.5 mM) and by confocal microscopy. Interestingly, SERT function significantly decreased with chronic (30 min) exposure to U50,488H (**p<0.01) and Sal A (**p<0.01) as measured with a novel high-throughput assay; this decrease was also attenuated with 5 min pre-treatment of the SERT inhibitor, fluoxitene (***p<0.001). Furthermore, this novel high-throughput assay also replicated our laboratories recent finding that chronic (30 min) exposure to U50,488H (**p<0.01) and Sal A (**p<0.01) significantly increase dopamine transporter (DAT) function in HEK-293 cells co-expressing myc-rKOPr and eYFP-tagged DAT. Collectively, these findings suggest in this cell model that chronic (30 min) KOPr activation by U50,488H and Sal A decreases SERT function.</p>


2021 ◽  
Author(s):  
Shuchi Anand ◽  
Maria E Montez-Rath ◽  
Jialin Han ◽  
Pablo Garcia ◽  
LinaCel Cadden ◽  
...  

Background: Patients receiving dialysis are a sentinel population for groups at high risk for death and disability from COVID-19. Understanding correlates of protection post-vaccination can inform immunization and mitigation strategies. Methods: Monthly since January 2021, we tested plasma from 4791 patients receiving dialysis for antibodies to the receptor-binding domain (RBD) of SARS-CoV-2 using a high-throughput assay. We qualitatively assessed the proportion without a detectable RBD response and among those with a response, semiquantitative median IgG index values. Using a nested case-control design, we matched each breakthrough case to five controls by age, sex, and vaccination-month to determine whether peak and pre-breakthrough RBD IgG index values were associated with risk for infection post-vaccination. Results: Among 2563 vaccinated patients, the proportion without a detectable RBD response increased from 6.6% [95% CI 5.5-8.1] in 14-30 days post-vaccination to 20.2% [95% CI 17.1-23.8], and median index values declined from 92.7 (95% CI 77.8-107.5) to 3.7 (95% CI 3.1-4.3) after 5 months. Persons with SARS-CoV-2 infection prior-to-vaccination had higher peak index values than persons without prior infection, but values equalized by 5 months (p=0.230). Breakthrough infections occurred in 56 patients, with samples collected a median of 21 days pre-breakthrough. Peak and pre-breakthrough RBD values <23 (equivalent to <506 WHO BAU/mL) were associated with higher odds for breakthrough infection (OR: 3.7 [95% CI 2.0-6.8] and 9.8 [95% CI 2.9-32.8], respectively). Conclusions: The antibody response to SARS-CoV-2 vaccination wanes rapidly, and in persons receiving dialysis, the persisting antibody response is associated with risk for breakthrough infection.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Gal Manella ◽  
Dan Aizik ◽  
Rona Aviram ◽  
Marina Golik ◽  
Gad Asher

AbstractCircadian clocks are self-sustained and cell-autonomous oscillators. They respond to various extracellular cues depending on the time-of-day and the signal intensity. Phase Transition Curves (PTCs) are instrumental in uncovering the full repertoire of responses to a given signal. However, the current methodologies for reconstructing PTCs are low-throughput, laborious, and resource- and time-consuming. We report here the development of an efficient and high throughput assay, dubbed Circadian Single-Cell Oscillators PTC Extraction (Circa-SCOPE) for generating high-resolution PTCs. This methodology relies on continuous monitoring of single-cell oscillations to reconstruct a full PTC from a single culture, upon a one-time intervention. Using Circa-SCOPE, we characterize the effects of various pharmacological and blood-borne resetting cues, at high temporal resolution and a wide concentration range. Thus, Circa-SCOPE is a powerful tool for comprehensive analysis and screening for circadian clocks’ resetting cues, and can be valuable for basic as well as translational research.


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