Pik3c3 expression profiling in the mouse kidney and its role in proximal tubule cell physiology.

The class 3 phosphatidylinositol 3-kinase (Pik3c3) plays critical roles in regulating autophagy, endocytosis, and nutrient sensing, but its expression profile in the kidney remains undefined. Recently, we validated a Pik3c3 antibody through immunofluorescence staining of kidney tissues from cell type-specific Pik3c3 knockout mice. Immunohistochemistry unveiled significant disparities in Pik3c3 expression levels across various kidney cell types. Notably, renal interstitial cells exhibit minimal Pik3c3 expression. Further, coimmunofluorescence staining, utilizing nephron segment- or cell type-specific markers, revealed nearly undetectable levels of Pik3c3 expression in glomerular mesangial cells and endothelial cells. Intriguingly, although podocytes exhibit the highest Pik3c3 expression levels among all kidney cell types, the renal proximal tubule cells (RPTCs) express the highest level of Pik3c3 among all renal tubules. RPTCs are known to express the highest level of the epidermal growth factor receptor (EGFR) in adult kidneys; however, the role of Pik3c3 in EGFR signaling within RPTCs remains unexplored. Therefore, we conducted additional cell culture studies. The results demonstrated that Pik3c3 inhibition significantly delayed EGF-stimulated EGFR degradation and the termination of EGFR signaling in RPTCs. Mechanistically, Pik3c3 inhibition surprisingly did not affect the initial endocytosis process but instead impeded the lysosomal degradation of EGFR. In summary, this study defines, for the first time, the expression profile of Pik3c3 in the mouse kidney and also highlights a pivotal role of Pik3c3 in the proximal tubule cells. These findings shed light on the intricate mechanisms underlying Pik3c3-mediated regulation of EGFR signaling, providing valuable insights into the role of Pik3c3 in renal cell physiology. NEW & NOTEWORTHY This is the first report defining the class 3 phosphatidylinositol 3-kinase (Pik3c3) expression profile in the kidney. Pik3c3 is nearly absent in renal interstitial cells, glomerular mesangial cells, and endothelial cells. Remarkably, glomerular podocytes express the highest Pik3c3 level in the kidney. However, the proximal tubule exhibits the highest expression level among all renal tubules. This study also unveils the pivotal role of Pik3c3 in regulating EGFR degradation and signaling termination in RPTCs, furthering our understanding of Pik3c3 in renal cell physiology.

Facility for spectral radiance calibration at low light level.

The spectral radiance measurement at daytime level can be realized with high accuracy, while it's difficult when the spectral radiance is at nighttime level. We design a spectral radiance calibration facility which has the characteristics of completely unchanged spectrum over 3 orders of magnitude and approximately unchanged spectrum for about 6 orders of magnitude. It combines a spectral radiance light source, a precision aperture and a white diffuser together, make it easy to reproduce the spectral radiance at 380 nm from 4 × 10-9 W/(m2·sr·nm) to 4 × 10-3 W/(m2·sr·nm). The facility can be easily used to calibrate a spectroradiometer at nighttime level. When the spectral radiance from 380 nm to 780 nm is around 1 × 10-7W/(m2·sr·nm), the calibration uncertainty of the spectroradiometer is 0.87%∼1.0% (k = 1).

pH biosensors based on hydrogel optical fiber.

This paper presents a hydrogel optical fiber fluorescence pH sensor doped with 5(6)-carboxyfluorescein (5(6)-FAM). The hydrogel optical fiber was fabricated with 2-hydroxy-2-methylpropiophenone as a photoinitiator, with different concentrations of polyethylene glycol diacrylate (PEGDA) for the core and cladding. A pH-sensitive fluorescence indicator 5(6)-FAM was doped into the core of the fiber. The prepared hydrogel optical fiber pH sensor showed good response within the pH range of 5.0-9.0. The linear range of the pH sensor is 6.0 to 8.0, with R 2=0.9904; within this range, the sensor shows good repeatability and reversibility, and the resolution is 0.07 pH units. The pHs of pork tissues soaked in different pH buffers were detected by the hydrogel optical fiber pH sensor; the linearity is 0.9828 when the pork tissue pH is in the range of 6.0-7.5. Due to the good ion permeability and biocompatibility of the hydrogel, this hydrogel optical fiber pH sensor is expected to be used in biomedical applications.

Approximation of a melting plateau of large area HTFP cells used for spectral irradiance realization.

This paper adopted an approximation of a melting plateau to solve the problem that temperature data cannot be monitored continuously when measuring the spectral irradiance of a large area tungsten carbide-carbon high-temperature fixed-point blackbody at each measured wavelength. Tests with fully measured curves showed that the method has a rather small deviation from the measured data of 0.017 K maximum, which corresponds to the spectral irradiance deviation of 0.005% at 500 nm. The maximum relative deviation between the Akima fitting method and the measured temperature in terms of spectral irradiance was 0.002%, which was better than -0.067% of a single temperature of 3020.11 K method and 0.026% of a linear interpolation method.

Cre/loxP approach-mediated downregulation of Pik3c3 inhibits the hypertrophic growth of renal proximal tubule cells.

Nephron loss stimulates residual functioning nephrons to undergo compensatory growth. Excessive nephron growth may be a maladaptive response that sets the stage for progressive nephron damage, leading to kidney failure. To date, however, the mechanism of nephron growth remains incompletely understood. Our previous study revealed that class III phosphatidylinositol-3-kinase (Pik3c3) is activated in the remaining kidney after unilateral nephrectomy (UNX)-induced nephron loss, but previous studies failed to generate a Pik3c3 gene knockout animal model. Global Pik3c3 deletion results in embryonic lethality. Given that renal proximal tubule cells make up the bulk of the kidney and undergo the most prominent hypertrophic growth after UNX, in this study we used Cre-loxP-based approaches to demonstrate for the first time that tamoxifen-inducible SLC34a1 promoter-driven CreERT2 recombinase-mediated downregulation of Pik3c3 expression in renal proximal tubule cells alone is sufficient to inhibit UNX- or amino acid-induced hypertrophic nephron growth. Furthermore, our mechanistic studies unveiled that the SLC34a1-CreERT2 recombinase-mediated Pik3c3 downregulation inhibited UNX- or amino acid-stimulated lysosomal localization and signaling activation of mechanistic target of rapamycin complex 1 (mTORC1) in the renal proximal tubules. Moreover, our additional cell culture experiments using RNAi confirmed that knocking down Pik3c3 expression inhibited amino acid-stimulated mTORC1 signaling and blunted cellular growth in primary cultures of renal proximal tubule cells. Together, both our in vivo and in vitro experimental results indicate that Pik3c3 is a major mechanistic mediator responsible for sensing amino acid availability and initiating hypertrophic growth of renal proximal tubule cells by activation of the mTORC1-S6K1-rpS6 signaling pathway.

The expression level of class III phosphatidylinositol-3 kinase controls the degree of compensatory nephron hypertrophy.

Excessive compensatory nephron hypertrophy (CNH) has been implicated in setting the stage for progressive nephron damage. Lack of a class III phosphatidylinositol 3-kinase (Pik3c3) inhibitor suitable for using in animals and lack of a Pik3c3-deficient animal model preclude the possibility of conclusively defining a role for Pik3c3 in CNH in previous studies. Here, we report that insertion of an Frt-flanked PGK-Neo cassette into intron 19 of the mouse Pik3c3 gene resulted in a hypomorphic allele. This allowed us to create a unique mouse model and provide the first definitive genetic evidence demonstrating whether Pik3c3 is essential for the regulation of CNH. Our results indicate that homozygous Pik3c3 hypomorphic (Pik3c3Hypo/Hypo) mice express significantly low levels of Pik3c3 than heterozygous Pik3c3 hypomorphic (Pik3c3Hypo/WT) littermates, which already express a lower level of Pik3c3 than wild-type (Pik3c3WT/WT) littermates. Interestingly, after unilateral nephrectomy (UNX), Pik3c3Hypo/Hypo mice develop a significantly lower degree of CNH than Pik3c3WT/WT mice and Pik3c3Hypo/WT mice, as revealed by measurement of kidney weight, kidney-to-body weight ratio, renal protein-to-DNA ratio, and morphometric analysis of proximal tubular and glomerular size. Mechanistically, UNX-induced mammalian target of rapamycin complex 1 (mTORC1) signaling to phosphorylation of ribosomal protein S6 (rpS6) in the remaining kidney was markedly inhibited in Pik3c3 hypomorphic mice. In conclusion, the present study reports a Pik3c3 hypomorphic mouse model and provides the first definitive evidence that Pik3c3 controls the degree of compensatory nephron hypertrophy. In addition, our signaling data provide the first definitive in vivo proof that Pik3c3 functions upstream of the mTORC1-S6 kinase 1-rpS6 pathway in the regulation of compensatory nephron hypertrophy.

Role of vergence eye movements in the visual recognition of long time duration.

When viewing dichoptic stimuli in long time duration, visual percepts are always the alternation between the left and right eye inputs, while not the combination. This is known as binocular rivalry. An efficient coding theory reported that binocular visual inputs can be combined into binocular summation (S+) and difference (S-) channels in V1 brain area. In this study, we used specially designed stimuli as the previous study, in which monocular inputs caused ambiguous percepts, but S+ and S- channels had unambiguous percepts. We aim to investigate whether the visual percepts alter between S+ and S- channels in long time duration and whether vergence eye movements are involved in the process. To do so, the stimuli were presented in 300-s time duration in a trial, and a binocular eye tracker was used to record eye information. Participants' real-time behavioral responses about the visual percepts and binocular information were recorded simultaneously. The results show there are perceptual flips between S+ and S- channels in both central and long time viewing conditions. More importantly, in central vision there are vergence eye movements before perceptual flips, suggesting the involvement of high level visual attention; the time of a perceptual flip from S+ is shorter than that of a flip from S-, which might be due to different involvements of visual attention, indicating a bias of feedback connection from higher brain areas for visual attention to S+ channel. Since S+ and S- dominated signals can be carried by different types of binocular neurons, our results provide new insights into high level visual attention and binocular neurons in V1 brain area by using specially designed dichoptic stimuli and eye vergence as measuring tools.

A method for optimizing the reference temperature in the effective emissivity calculation of nonisothermal blackbody cavities.

For a nonisothermal blackbody cavity, different reference temperatures have influence on the calculation of effective emissivity. Previous studies proposed a weighted average method which can be indicated by a priori to calculate the reference temperature. However, these studies did not mention how to define the weight function but used some arbitrary temperature or the temperature of a fixed position like the central bottom of the cavity as the reference temperature. In this study, a quantitative analysis and calculation method, which is implemented in the Monte Carlo method based optical simulation software Tracepro, is proposed to define the weight coefficients and optimize the reference temperature. To do so, in the Tracepro software, a surface source is placed in front of the cavity opening and emits radiation to the blackbody cavity. The radiation from this surface source can be absorbed or reflected many times in the cavity, and finally the incident radiation distribution in the cavity can be obtained. According to the principle of light path reversibility, the normalized incident radiation can be considered as the contribution of its position to the effective emissivity. In the experiment, the actual temperatures of two different-shaped blackbody cavities are measured with the non-contact method in 873 K temperature. By dividing the inner surface of each blackbody cavity into several regions based on the positions of the actually measured temperature points, the incident radiation from the surface source to each segmented region is calculated and normalized to the total incident radiation across all regions as its weight coefficient; the reference temperature is the sum of the weighted temperature (by multiplying each weight coefficient with its measured temperature) in each region. Different from previous studies, this study optimizes the reference temperature by considering the contribution of the whole cavity to the effective emissivity, which should be more consistent with the actual situation. Moreover, the influences of different shapes of the blackbody cavities, different radiation characteristics of the inner surface materials and different viewing conditions of the effective emissivity on the reference temperature are discussed and compared. The results suggest that the optimization of reference temperature has close link with above factors and thus should be calculated individually.

A method for spectral irradiance measurement based on a large area WC-C fixed point blackbody.

We have proposed a novel method for the spectral irradiance measurement of the standard lamp. The measurement has been realized by using national primary scale based on a large area WC-C fixed point blackbody for the first time. The wavelength range is from 450 nm to 1000 nm. The spectral irradiance of the standard lamp has been measured based on the traditional variable temperature blackbody and large area WC-C fixed point blackbody. The results of the two methods agree within the uncertainties. Compared with the traditional measurement method, the fixed point method can reduce the major component of the spectral irradiance measurement uncertainty related to the temperature measurement.

Analysis and improvements of effective emissivities of nonisothermal blackbody cavities.

The emissivity of the blackbody is a very important parameter in spectral radiance measurement systems. In the conventional method, the emissivity is calculated based on the isothermal model. However, the actual temperature distribution in the blackbody cavity is always nonisothermal; the emissivity calculated based on the isothermal model may not accurately present the radiation characteristic of the blackbody. In this study, the actual temperature distributions of two blackbodies (one has an extended cone shape, and the other a 65-mm diameter cylindrical shape) are measured, and the emissivities are calculated accordingly based on the nonisothermal model at a certain temperature (873 K). The results show there are different tendencies of temperature distributions in the two blackbodies. When compared with the isothermal model, the emissivities in the 873 K temperature and 2.0-20.0 µm wavelength condition are about 1.75% and 0.18% lower at the nonisothermal model for the extended cone shape and cylindrical blackbodies, respectively. To improve the emissivity, different types of apertures are customized for the two blackbodies. For the extended cone-shaped blackbody, the emissivity in the 873 K temperature and 2.0-20.0 µm wavelength condition increases by 1.12% when using a ring-shaped graphite aperture in the cavity, whereas for the cylindrical-shaped blackbody, the emissivity in the same condition increases by 0.09% when using a high-reflective aperture in front of the cavity opening. Different from previous studies, this study provides new insight in calculating and improving the effective emissivity of blackbodies by using the measured temperature in the cavity based on the nonisothermal model.

Spectral irradiance primary scale realization and characterization of deuterium lamps from 200 to 400 nm.

To meet the increasing metrology demand of spectral irradiance in the short UV spectral range, a new spectral irradiance scale from 200 to 400 nm was realized at National Institute of Metrology (NIM) based on a high-temperature blackbody BB3500M, and a group of stable deuterium lamps are used as the transfer standards. Accurate real-time temperature of a blackbody is derived to reduce the temperature drift during the measurement period. A combination of an absolute and relative measurement system is designed to reduce repeatability uncertainty, and a selective optical filter method is used to remove fluorescence with a peak at 330 nm. A seven-point bandwidth novel correction method based on differential quadrature formula is put forward to correct the bandwidth error of the monochromator. The expanded uncertainties of the new spectral irradiance scale are 5.3% at 200 nm, 1.8% at 250 nm, 1.9% at 330 nm, and 3.6% at 400 nm, respectively. In the overlap wavelength from 250 to 400 nm, the average deviation between two types transfer standards, deuterium lamps and tungsten halogen lamps, is verified to be 0.39%, which are consistent with the associated measurement uncertainties.

[Seven-Point Correction Approach for Spectrum Measurement of Light Source with a Narrow Bandwidth].

The finite bandwidth of spectroradiometers always causes significant errors when the measured light source has a narrow bandwidth compared to that of spectroradiometers. In order to solve this problem, an improved correction approach which is called seven-point correction approach is proposed. Firstly, the seven-point correction formula is obtained with Taylor's series and related derivative formula. Secondly, the effect of seven-point formula is validated through a simulated spectrum with a sine function shape. Considering the sine function as true spectrum, we calculate the measured spectrum with the bandpass function of spectroradiometers. We also correct the measured spectrum with the seven-point formula. At last, we validate the seven-point formula experimentally with a LED lamp whose center wavelength is 365 nm. Using a double grating monochromator, we measure the irradiance of LED lamp when the bandwidth of spectroradiometer is 5 and 0.5 nm. We also obtain the corrected spectrum by applying seven-point formula to measured spectrum. The simulated results show that, the corrected value at the center wavelength could be above 99% of the true value. The experimental results show that, the corrected value at the center wavelength could reach above 95% of the true value. Above all, the proposed seven-point approach has an improved correction effect compared with three-point and five point approach. This correction approach could be widely applied in the field of spectrum measurement.

Autonomous regulation of retinal insulin biosynthesis in diabetes.

Diabetic retinopathy (DR) is a neurodegenerative disease that results as a complication of dysregulated glucose metabolism, or diabetes. The signaling of insulin is lost or dampened in diabetes, but this hormone has also been shown to be an important neurotrophic factor which supports neurons of the brain. The role of local insulin synthesis and secretion in the retina, however, is unclear. We have investigated whether changes in local insulin synthesis occur in the diabetic retina and in response to stressors known to initiate retinal neurodegenerative processes. The expression of insulin and its cleavage product, c-peptide, were examined in retinas of a Type I diabetes animal model and human postmortem donors with DR. We detected mRNAs for insulin I (Ins1), insulin II (Ins2) and human insulin (Ins) by quantitative real-time polymerase chain reaction (qRT-PCR) and in situ hybridization. Using an ex-vivo system, isolated neuroretinas and retinal pigmented epithelium (RPE) layers were exposed to glycemic, oxidative and inflammatory environments to measure insulin gene transcripts produced de novo in the retina under disease-relevant conditions. The expression of insulin in the retina was altered with the progression of diabetes in STZ mice and donors with DR. Transcription factors for insulin, were simultaneously expressed in a pattern matching insulin genes. Furthermore, de novo insulin mRNA in isolated retinas was induced by acute stress. RPE explants displayed the most pronounced changes in Ins1 and Ins2. This data reveals that the retina, like the brain, is an organ capable of producing local insulin and this synthesis is altered in diabetes.

The injury and cumulative effects on human skin by UV exposure from artificial fluorescence emission.

The injury and cumulative effects of UV emission from fluorescence lamp were studied. UV intensity from fluorescence lamp was measured, and human skin samples (hips, 10 volunteers) were exposed to low-dose UV irradiation (three times per week for 13 consecutive weeks). Three groups were examined: control group without UV radiation; low-dose group with a cumulative dose of 50 J cm(-2) which was equivalent to irradiation of the face during indoor work for 1.5 years; and high-dose group with 1000 J cm(-2) cumulative dose equivalent to irradiation of the face during outdoor activities for 1 year. Specific indicators were measured before and after UVA irradiation. The findings showed that extending the low-dose UVA exposure decreased the skin moisture content and increased the transepidermal water loss as well as induced skin color changes (decreased L* value, increased M index). Furthermore, irradiated skin showed an increased thickness of cuticle and epidermis, skin edema, light color and unclear staining collagen fibers in the dermis, and elastic fiber fragmentation. In addition, MMP-1, p53 and SIRT1 expression was also increased. Long-term exposure of low-dose UVA radiation enhanced skin photoaging. The safety of the fluorescent lamp needs our attention.