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Dual‐layered hydrogels allow complete genome recovery with nucleic acid cytometry
Targeting specific cells for sequencing is important for applications in cancer, microbiology, and infectious disease. Nucleic acid cytometry (NAC) is a powerful approach for accomplishing this because it allows specific cells to be isolated based on sequence biomarkers that are otherwise impossible to detect. However, existing methods require specialized microfluidic devices, limiting adoption. Here, a modified workflow is described that uses particle-templated emulsification (PTE) and flow cytometry to conduct the essential steps of cell detection and sorting normally accomplished by microfluidics. Our microfluidic-free workflow allows facile isolation and sequencing of cells, viruses, and nucleic acids and thus provides a powerful enrichment approach for targeted sequencing applications
DNA-Linked Enzyme-Coupled Assay for Probing Glucosyltransferase Specificity
Traditional enzyme characterization
methods are low-throughput
and therefore limit engineering efforts in synthetic biology and biotechnology.
Here, we propose a DNA-linked enzyme-coupled assay (DLEnCA) to monitor
enzyme reactions in a high-throughput manner. Throughput is improved
by removing the need for protein purification and by limiting the
need for liquid chromatography mass spectrometry (LCMS) product detection
by linking enzymatic function to DNA modification. We demonstrate
the DLEnCA methodology using glucosyltransferases as an illustration.
The assay utilizes cell free transcription/translation systems to
produce enzymes of interest, while UDP-glucose and T4-β-glucosyltransferase
are used to modify DNA, which is detected postreaction using qPCR
or a similar means of DNA analysis. OleD and two glucosyltransferases
from <i>Arabidopsis</i> were used to verify the assay’s
generality toward glucosyltransferases. We further show DLEnCA’s
utility by mapping out the substrate specificity for these enzymes
What to expect from a third step in treatment resistant depression: A prospective open study on escitalopram
Objectives. Only few studies investigated treatment strategies for
treatment resistant depression (TRD). The objective of this multicentre
study was to evaluate TRD patients who did not respond to at least two
antidepressants. Methods. A total of 417 patients, who failed to respond
to a previous retrospectively assessed antidepressant (AD1), were
firstly included in a 6-week venlafaxine treatment (AD2); secondly,
those who failed to respond were treated for further 6 weeks with
escitalopram (AD3). Results. Out of 417 patients who had failed to
respond to previous treatment (AD1), 334 completed treatment with
venlafaxine to prospectively define TRD. In the intent to treat (ITT)
population in the first phase of the trial (AD2), responders to
venlafaxine were 151 (36.21%) out of which remitters were 83 (19.90%).
After phase one, 170 non-responders, defined as TRD, were included in
the second phase and 157 completed the course. Of the 170 ITT entering
the second phase (AD3), responders to escitalopram were 71 (41.76%) out
of which remitters were 39 (22.94%). After the third treatment,
patients showed a dropout rate of 7.65% and a rate of presence of at
least one serious adverse event of 19.18%. Conclusions. Relevant rates
of response and remission may be observed after a third line treatment
in patients resistant to two previous treatments. A relevant limitation
of this study was represented by the design: naturalistic,
non-randomized, open-label, without a control sample and with unblinded
raters