c********6
发帖数: 693 | 1 无意中看到一个老外写的，用了下面这篇文章里边介绍的使用荧光标签检控mitophagy。
Tools and techniques to measure mitophagy using fluorescence microscopy
DOI: 10.4161/auto.24001
FP-based biosensors for mitophagy
The field of FP engineering is accelerating with a plethora of new variants
described each year. These proteins show differen- tial properties, many of
which may provide useful tools to study mitophagy. For example, FP-based
biosensors such as a pH- sensitive mitochondrial-targeted FP chimera has
been recently developed to monitor mitochondrial accumulation in the yeast
vacuole.42,64 This approach utilized the pH-sensitive GFP variant pHluorin65
in a tandem fusion with the pH-insensitive DsRed which has an acidic pK .45
When degraded, yeast mitochondria localize to the acidic vacuole and the
emission from pHluorin is lost leaving only the DsRed fluorescence emission
and an indication of mitophagy. Outside the vacuole, however, the
mitochondrial-targeted tandem-FP biosensor is in a neutral pH environment
and therefore fluorescence emission is derived from both DsRed and pHluorin.
This new probe represents a particularly useful tool to study mitophagy;
however, employing the dual FP (DsRed and pHluorin) approach in Rosella may
have the caveats associated with other tandem FP biosensors, where the FPs
may undergo FRET,48 exhibit fluorescence emission overlap, or differential
bleaching kinetics and brightness.47 An alternative strategy that
circumvents many of the potential problems associated with tandem FP
biosensors is to use a single FP-based biosensor with spectral properties
that shift with changes in pH. The Miyawaki laboratory48 used the FP Keima
to construct a mitophagy probe. Keima exhibits a pH-dependent shift in its
peak excitation wave- length. At a high pH, Keima absorbs light at 440 nm,
whereas at acidic pH Keima absorbs light at a peak wavelength of 586 nm.
Keima was successfully targeted to mitochondria48 yielding a FP-based
biosensor for mitophagy. Under basal conditions the mitochondrial-targeted
Keima is in a neutral environment and absorbs 440 nm light. Upon induction
of mitophagy, the probe accompanies mitochondria sequestered into
autophagosomes and on to the lysosome. In the acidic lysosomal environment
Keima becomes ionized and the peak absorption shifts to 586 nm. The authors
show eloquent, quantitative imaging of mitophagy。。。
然后根据这篇文章里的方法构建了细胞系，检测了一下几个带有特异突变基因的细胞系
，有点差异，然后就说他自己建立了一套筛选autophagy或者mitophagy的平台系统。
Here we developed a novel high throughput phenotyping platform with
automated high content image analysis to assess autophagy and mitophagy
alterations in a polygenetic neurodegenerative disease cohort. As a proof of
concept we outlined the autophagy and mitophagy alterations in the
neurodegenerative disease associated mutations 。然后投nature子刊。我真没看
出来相比原始的文章，他的东西有任何创新的地方。难道是我太外行。 | c********6
发帖数: 693 | 2
mitophagy。
variants
of
pHluorin65
【在 c********6 的大作中提到】
: 无意中看到一个老外写的，用了下面这篇文章里边介绍的使用荧光标签检控mitophagy。
: Tools and techniques to measure mitophagy using fluorescence microscopy
: DOI: 10.4161/auto.24001
: FP-based biosensors for mitophagy
: The field of FP engineering is accelerating with a plethora of new variants
: described each year. These proteins show differen- tial properties, many of
: which may provide useful tools to study mitophagy. For example, FP-based
: biosensors such as a pH- sensitive mitochondrial-targeted FP chimera has
: been recently developed to monitor mitochondrial accumulation in the yeast
: vacuole.42,64 This approach utilized the pH-sensitive GFP variant pHluorin65
| x*****e
发帖数: 309 | | c********6
发帖数: 693 | 4 太具讽刺意味了
【在 x*****e 的大作中提到】
: 又发明了轮子, 这次是其他颜色的,呵呵.
| c********6
发帖数: 693 | 5 我真是看不出来他做的这个在原有技术之上有任何的improvement | c********6
发帖数: 693 | 6 没人感兴趣吗 为什么别人认为能够发nature，而我却认为啥玩意儿都不是呢
【在 c********6 的大作中提到】
: 我真是看不出来他做的这个在原有技术之上有任何的improvement
| c********6
发帖数: 693 | 7 无意中看到一个老外写的，用了下面这篇文章里边介绍的使用荧光标签检控mitophagy。
Tools and techniques to measure mitophagy using fluorescence microscopy
DOI: 10.4161/auto.24001
FP-based biosensors for mitophagy
The field of FP engineering is accelerating with a plethora of new variants
described each year. These proteins show differen- tial properties, many of
which may provide useful tools to study mitophagy. For example, FP-based
biosensors such as a pH- sensitive mitochondrial-targeted FP chimera has
been recently developed to monitor mitochondrial accumulation in the yeast
vacuole.42,64 This approach utilized the pH-sensitive GFP variant pHluorin65
in a tandem fusion with the pH-insensitive DsRed which has an acidic pK .45
When degraded, yeast mitochondria localize to the acidic vacuole and the
emission from pHluorin is lost leaving only the DsRed fluorescence emission
and an indication of mitophagy. Outside the vacuole, however, the
mitochondrial-targeted tandem-FP biosensor is in a neutral pH environment
and therefore fluorescence emission is derived from both DsRed and pHluorin.
This new probe represents a particularly useful tool to study mitophagy;
however, employing the dual FP (DsRed and pHluorin) approach in Rosella may
have the caveats associated with other tandem FP biosensors, where the FPs
may undergo FRET,48 exhibit fluorescence emission overlap, or differential
bleaching kinetics and brightness.47 An alternative strategy that
circumvents many of the potential problems associated with tandem FP
biosensors is to use a single FP-based biosensor with spectral properties
that shift with changes in pH. The Miyawaki laboratory48 used the FP Keima
to construct a mitophagy probe. Keima exhibits a pH-dependent shift in its
peak excitation wave- length. At a high pH, Keima absorbs light at 440 nm,
whereas at acidic pH Keima absorbs light at a peak wavelength of 586 nm.
Keima was successfully targeted to mitochondria48 yielding a FP-based
biosensor for mitophagy. Under basal conditions the mitochondrial-targeted
Keima is in a neutral environment and absorbs 440 nm light. Upon induction
of mitophagy, the probe accompanies mitochondria sequestered into
autophagosomes and on to the lysosome. In the acidic lysosomal environment
Keima becomes ionized and the peak absorption shifts to 586 nm. The authors
show eloquent, quantitative imaging of mitophagy。。。
然后根据这篇文章里的方法构建了细胞系，检测了一下几个带有特异突变基因的细胞系
，有点差异，然后就说他自己建立了一套筛选autophagy或者mitophagy的平台系统。
Here we developed a novel high throughput phenotyping platform with
automated high content image analysis to assess autophagy and mitophagy
alterations in a polygenetic neurodegenerative disease cohort. As a proof of
concept we outlined the autophagy and mitophagy alterations in the
neurodegenerative disease associated mutations 。然后投nature子刊。我真没看
出来相比原始的文章，他的东西有任何创新的地方。难道是我太外行。 | c********6
发帖数: 693 | 8
mitophagy。
variants
of
pHluorin65
【在 c********6 的大作中提到】
: 无意中看到一个老外写的，用了下面这篇文章里边介绍的使用荧光标签检控mitophagy。
: Tools and techniques to measure mitophagy using fluorescence microscopy
: DOI: 10.4161/auto.24001
: FP-based biosensors for mitophagy
: The field of FP engineering is accelerating with a plethora of new variants
: described each year. These proteins show differen- tial properties, many of
: which may provide useful tools to study mitophagy. For example, FP-based
: biosensors such as a pH- sensitive mitochondrial-targeted FP chimera has
: been recently developed to monitor mitochondrial accumulation in the yeast
: vacuole.42,64 This approach utilized the pH-sensitive GFP variant pHluorin65
| x*****e
发帖数: 309 | | c********6
发帖数: 693 | 10 太具讽刺意味了
【在 x*****e 的大作中提到】
: 又发明了轮子, 这次是其他颜色的,呵呵.
| c********6
发帖数: 693 | 11 我真是看不出来他做的这个在原有技术之上有任何的improvement | c********6
发帖数: 693 | 12 没人感兴趣吗 为什么别人认为能够发nature，而我却认为啥玩意儿都不是呢
【在 c********6 的大作中提到】
: 我真是看不出来他做的这个在原有技术之上有任何的improvement
| c********6
发帖数: 693 | | c********6
发帖数: 693 | |
|
