As a programmed necrotic cell death, necroptosis has the intrinsic initiators, including receptor-interacting serine/threonine-protein kinase 1 (RIPK1), RIPK3 and mixed-lineage kinase domain-like protein (MLKL), which combine to form necroptotic signaling pathway and mediate necroptosis induced by various necroptotic stimuli, such as tumor necrosis factor (TNF). Although chemical inhibition of RIPK1 blocks TNF-induced necroptosis, genetic elimination of RIPK1 does not suppress but facilitate necroptosis triggered by TNF. Moreover, RIPK3 has been reported to mediate the RIPK1-independent necroptosis, but the involved mechanism is unclear. In this study, we found that TRADD was essential for TNF-induced necroptosis in RIPK1-knockdown L929 and HT-22 cells. Mechanistic study demonstrated that TRADD bound RIPK3 to form new protein complex, which then promoted RIPK3 phosphorylation via facilitating RIPK3 oligomerization, leading to RIPK3-MLKL signaling pathway activation. Therefore, TRADD acted as a partner of RIPK3 to initiate necroptosis in RIPK1-knockdown L929 and HT-22 cells in response to TNF stimulation. In addition, TRADD was critical for the accumulation of reactive oxygen species (ROS), which contributed to RIPK1-independent necroptosis triggered by TNF. Collectively, our data demonstrate that TRADD acts as the new target protein for TNF-induced RIPK3 activation and the subsequent necroptosis in a RIPK1-independent manner. It is well known that programmed cell death has been classified into several distinct forms, including apoptosis, necroptosis, pyroptosis and ferroptosis . As a reAs the firstly identified target protein in initiating necroptosis, receptor-interacting serine/threonine-protein kinase 1 (RIPK1) binds TNF receptor (TNFR) to form complex I via death domain upon TNFR ligation , 1996. IIt has been reported that RIPK1 transfers necroptotic signals through binding TNFR1 or RIPK3 to form different protein complexes, such as protein Complex I and necrosome . MoreoveIn the current study, TRADD was identified as the target protein for TNF-induced necroptosis in RIPK1-knockdown L929 and HT-22 cells. Mechanistic study discovered that TRADD bound RIPK3 to form new protein complex and activated RIPK3 via facilitating RIPK3 oligomerization and the subsequent autophosphorylation, leading to RIPK3-MLKL signaling pathway activation. Moreover, TRADD was also essential for ROS accumulation which also contributed to RIPK1-independent necroptosis in response to TNF stimulation. Therefore, our data demonstrate that TRADD acts as the partner of RIPK3 to initiate RIPK1-independent necroptosis upon TNFR ligation.As the first target protein identified for necroptosis induction, RIPK1 binds its partner RIPK3 to form necrosome and initiates necroptosis in response to TNF stimulation . HoweverThough RIPK3 could mediate RIPK1-dependent necroptosis, artificial activation of RIPK3 initiates necroptosis in a RIPK1-independent manner . TherefoCollectively, our results demonstrate that RIPK3 and MLKL mediate RIPK1-independent necroptosis triggered by TNF.Though RIPK3 mediates RIPK1-independent necroptosis, it is unclear how to transduce the necroptotic signal from TNFR1 to RIPK3 because RIPK3 has no death domain to directly bind TNFR1. In this study, we explored the role of some adaptor proteins, including TRADD, FADD and TRAF2, in RIPK1-independent necroptosis. Firstly, we explored the effect of TRADD, FADD, and TRAF2 knockdown on TNF-induced necroptosis. As shown in In conclusion, our results demonstrate that TRADD functions as a critical target protein for RIPK1-independent necroptosis induction.As TRADD is essential for RIPK1-independent necroptosis initiating by RIPK3, we next explored the role of TRADD in RIPK3-MLKL signaling transduction. As shown in Because phosphorylated MLKL oligomerizes via disulfide bonds , we nextIn conclusion, TRADD is critical for RIPK3 and MLKL phosphorylation and oligomerization in RIPK1-knockdown L929 cells in response to TNF stimulation.As TRADD is essential for RIPK3 activation in RIPK1-independent necroptosis, we next determined whether TRADD could directly bind RIPK3. As shown in In conclusion, our results demonstrate that RIPK1 knockdown facilitates the formation of TRADD and RIPK3 heterodimer upon TNF stimulation.It has been reported that RIPK3 phosphorylation facilitates cellular ROS accumulation, which is critical for TNF-induced L929 cell necroptosis . As TRADIn conclusion, ROS accumulate in a TRADD-dependent manner and contribute to RIPK1-independent necroptosis in response to TNF stimulation.–/– animals, it is sufficient to reduce the systemic cell death and inflammation . 293TN cells were obtained from System Biosciences . The cells were cultured in Dulbecco’s modified Eagle medium  containing 10% fetal bovine serum . Necrostatin-1  and Z-VAD-FMK (20 μM) were purchased from Medchem Express . GSK’872 was obtained from Biovision . Tumor necrosis factor (100 ng/mL) was obtained from GeneScript . Butyl hydroxyl anisd , β-mercaptoethanol (β-ME) and propidium iodide (PI) were purchased from Sigma-Aldrich .Cell death analysis was performed according to the method described by Western blotting experiment was described previously , and theImmunoprecipitation was described previously . Flag  to express Flag-tag TRADD, and verified by DNA sequencing. HA-tagged RIPK3 plasmid (MG51069-NY) was purchased from Sino biological . The plasmids were transfected into 293T cells using Chemifect transfection reagent (Fengrui Biotechnology) to overexpress the target genes with different tag sequences. The protein levels of Flag and HA tags were measured by western blot analysis. The pLV-Myc-TRADD plasmid was obtained from Cyagen Biosciences , and co-transfected into 293TN cells (SBI) with second-generation packaging systems to produce lentivirus, which mediates TRADD ectopical expression after infecting L929 cells.Duolink proximity ligation assay was described previously . The antCellular ROS were measured with a Reactive Oxygen Species Assay Kit (Beyotime Institute of Biotechnology). Briefly, cells were collected by trypsinization and washed with PBS, then incubated with 5 μM DCFH-DA for 30 min. After washing with PBS, the cells were analyzed by flow cytometry  at an excitation wavelength of 480 nm and an emission wavelength of 525 nm. Twenty thousand stained cells were analyzed with flow cytometry for each measurement. The ROS fold changes were normalized by comparing the mean geometry fluorescence in all groups with that in the group treated with DMSO (the negative control of TNF plus Z-VAD stimulation).t-test or one-way ANOVA test and defined as P < 0.01. All experiments were repeated at least three times, and the data are expressed as the mean ±SD from representative experiments.GraphPad prism 7 software was used to analyze the data and construct statistical graphs. Statistical significance was analyzed by performing a two-tailed Student’s The datasets generated for this study can be found in the NCBI Reference Sequences: NM_001033161.2, NM_009068.3, and NM_019955.2.LW, XC, and JF prepared all the figures. JY collected some data. GC designed all the experiments and wrote the manuscript. All authors reviewed the manuscript.JY was employed by company Beijing Zhendandingtai Biotechnology Co., Ltd.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In-vitro characterization demonstrated that the dermascope is capable of quantifying changes in flow across a physiologically relevant range even when used in a handheld manner with clinic lighting and dermascope LEDs on. In a small pilot clinical study, we demonstrated the dermascope’s ability to detect flow differences between two distinct lesion types.Dermascopes are commonly utilized for the qualitative visual inspection of skin lesions. While automated image processing techniques and varied illumination strategies have been used to aid in structural analysis of lesions, robust quantification of functional information is largely unknown. To address this knowledge gap, we have developed a compact, handheld dermascope that enables real-time blood flow measurements of skin during conventional visual inspection.  Timely and accurate diagnosis of such lesions is critical due largely to their particularly high incidence rate and potential morbidity. Non-melanoma skin cancer is the most common cancer in the US with an estimated 3.5 million cases each year, and although mortality rates are relatively low, it represents a significant burden to the healthcare system5. Melanoma has an estimated five-year mortality rate as high as 23%6 depending on lesion stage. Although dermoscopy aids inspection of lesions, the subjectivity and difficulty in interpreting magnified lesion images commonly necessitates biopsy of suspicious lesions for histopathological inspection, and this practice is still viewed as a diagnostic gold standard.Dermoscopy, the use of a handheld device with a magnifying lens and light source, is commonly used in the field of dermatology to aid in the visual inspection of suspicious skin lesions. Dermoscopy has been shown to improve diagnostic accuracy of melanocytic and nonmelanocytic pigmented lesions compared to the unaided eye, and can specifically aid the diagnosis of melanoma, basal cell carcinoma, and squamous cell carcinoma9. Although these tools offer a degree of quantification and objectivity to lesion assessment, their accuracy and general applicability has been questioned11. Several commercial dermascopes have been developed to aid in objective and quantitative assessment of acquired images, including MoleMax and SolarScan. These devices use an embedded camera to acquire images and software to aid in image analysis12.To help overcome the reliance on biopsies, diagnostic methodologies which attempt to standardize the analysis of dermascope images have been used to assess pigmented lesions. These include the ABCD rule and the seven-point check list, which rely on color and structural information to characterize lesions13. To assist in the visualization of tissue structures within lesions that exhibit varying spectral characteristics (predominantly absorption), multispectral dermascopes have also been investigated. These devices inspect a region using different spectral bandwidths of light that can improve visualization of lesion texture, blood vessel morphology, and melanin location17. Commercial embodiments include the SIAscope and MelaFind, which use multispectral information to aid in identifying morphological features19.Dermascopes with functionality beyond reliance on simple white-light illumination have also been explored to aid in diagnosis. Most commonly, cross-polarized illumination dermascopes have been used to reject superficially reflected and scattered light and thus aid in visualization of subsurface features21. It has been shown histologically that the density of microvasculature is increased significantly in squamous cell and basal cell carcinomas relative to normal skin22. This finding has been further supported using laser Doppler perfusion imaging, which has demonstrated that blood flow in skin tumors is significantly greater than the surrounding skin and that varying degrees of skin blood flow may aid in differentiating tumor type24.Despite these advancements in dermoscopy, the techniques and devices described above, all rely solely on structural information to aid in diagnosis. Functional information, such as tissue perfusion, may supplement this structural information to provide a more robust characterization or diagnosis of inspected lesions. For example, it is well known that tumor progression is closely accompanied by angiogenesis as additional blood flow is needed to meet the metabolic demands of neoplastic tissue26. We have designed the LSCI-dermascope to overcome these limitations and facilitate the practical integration of perfusion assessment into a typical dermatology workflow.To that end, we have developed a handheld dermascope that enables the user to perform standard magnified analysis of skin lesions simultaneously with skin blood flow measurements. This was achieved by integrating a compact laser speckle contrast imaging (LSCI) system into a custom-built dermascope . While LSCI is a flexible technique for quantifying cutaneous blood flow, clinical adoption is hindered by the fact that commercial LSCI systems are relatively large and bulky, do not provide simple registration of inspected lesions with collected blood flow maps, and suffer from measurement artifact due to movementTo determine whether room light and the white LED lights used for visual inspection of the skin would interfere with laser speckle contrast imaging (LSCI) measurements during LSCI-dermascope use, the average speckle contrast in a static tissue phantom was measured with and without these light sources on. The results can be seen in Fig. LSCI measurements are prone to artifact due to user movement. To quantify whether user movement significantly altered LSCI measurements performed using the LSCI-dermascope, average speckle contrast was measured from a static tissue phantom with the LSCI-dermascope either mounted or used handheld. The results can be seen in Fig. To characterize the response of LSCI-dermascope to varying degrees of simulated blood flow, LSCI measurements were acquired from a tissue phantom with an embedded tube through which Intralipid was flowed at known rates. A diagram of the measurement setup and plot of the average speckle contrast in a dynamic region above the tube and static region away from the tube for each volumetric flow are shown in Fig. The ability of the LSCI-dermascope to quantify differences in blood flow between different skin lesions was validated by acquiring LSCI measurements from lesions expected to have increased blood flow relative to the surrounding tissue , and from skin lesions not expected to have increased blood flow relative to the surrounding tissue . Representative color images of each lesion type along with a corresponding blood flow map are shown in Fig. Dermascopes are one of the most frequently used tools by dermatologist for the inspection of suspicious skin lesions. However, while improvements to dermascopes over the past several decades have offered additional structural information, little to no functional information can be measured. Since abnormalities in functional tissue properties such as blood flow often accompany skin pathologies, the inability to measure such properties represents a potentially significant gap in diagnostic or prognostic capability. LSCI has been used extensively to measure tissue blood flow; however, its practical use in the dermatology clinic is limited by the fact that most measurement systems are relatively large and bulky, do not provide simple co-registration of inspected lesions with collected blood flow, and suffer from measurement artifact due to movement. We have described a LSCI-dermascope which addresses each of these limitations.To address the size limitation of conventional LSCI systems, we utilized compact laser illumination and imaging hardware to create a device that is comparably sized to a conventional dermascope and equally simple to use. To address the difficulty in registering LSCI flow maps with visually inspected lesions, the LSCI-dermascope spectrally separates laser illumination from white-light illumination. We demonstrated that dermoscopy, even with the room lights on, could be performed simultaneously with LSCI measurements without significant effects on the collected data. To address the potential for measurement errors due to motion, we have developed a probe which uses minimal but direct contact with the subject to help stabilize the device. This resulted in LSCI measurements that could be acquired by hand without significant motion artifact.29. As such, utilizing an internal control for measurements is potentially useful for collecting data that can be compared from subject to subject.Beyond the limitations above, we additionally addressed the relative nature of LSCI measurements by presenting blood flow information from measured lesions relative to that in the surrounding tissue. Acquiring LSCI data from both regions is relatively simple, because simultaneous imaging with white light streamlines identification of lesions and their borders. By utilizing the surrounding tissue of an inspected lesion as an internal control, factors that can affect LSCI measurements but unrelated solely to lesion blood flow can be minimized or eliminated. These include changes in laser coherence, imaging angle, tissue curvature, room temperature, subject position, and others34.Finally, in a small pilot clinical study, we demonstrated the ability of the LSCI-dermascope to measure significant blood flow differences between two distinct skin lesion types: cherry angiomas and solar lentigos. This result is consistent with expected results. Cherry angiomas are papules with an abnormally high density of superficial capillaries that are expected to exhibit increased perfusion relative to normal skin. Alternatively, solar lentigos are macules of the epidermis characterized by increased basal layer pigmentation without alterations in the skin microvasculature and therefore are not expected to exhibit altered perfusion relative to the surrounding skinIn summary, we have presented a novel dermascope that is capable of providing functional tissue information in combination with standard structural information. By addressing many of the shortcomings associated with typical LSCI measurements, the LSCI-dermascope enables relatively quick and simple measurements perfusion measurements from lesions. Very little additional time is required for LSCI-dermascope measurements compared to conventional dermoscopy, facilitating practical use in the clinic. Future study will entail determining whether lesion blood flow information provided by the LSCI-dermascope can be used adjunctively with standard lesion diagnostic assessment to improve diagnostic accuracy without necessitating tissue biopsy.A photograph of the LSCI-dermascope and a diagram illustrating the internal components is shown in Fig. Conventional dermascope functionality was performed by illuminating the tissue under inspection with four white LEDs  wired in series with a 20 mA current limiter  and mounted inside of a light diffusing cone that was placed in direct contact with the skin during imaging. LED light remitted from the tissue entered a 50:50 beam splitter  that directed approximately half of the light into the handle of the LSCI-dermascope and approximately half of the light toward the eye of the user. LED light directed into the handle was collimated using a 35 mm focal length lens  and then attenuated using a 780 nm longpass dielectric filter . LED light directed toward the eye of the user was magnified using a 60 mm focal length lens  and then passed through a 700 nm shortpass filter .35. Laser light remitted from the same region of tissue being imaged during conventional dermascope measurements was also separated using the 50:50 beam splitter. Laser light directed toward the eye of the operator was attenuated via the shortpass filter. Laser light directed toward the handle of the device was collimated using a 35 mm focal lens and then directed through a 780 nm longpass filter and into a 35 mm camera lens mounted to a CMOS camera . The 3.3 V power supply on the CMOS camera was used to power both the LEDs and laser diode during imaging. The camera lens was set to an f/# of 8, as this aperture enabled sampling of the speckle pattern which satisfied the Nyquist criterion36. The resulting dual-mode imaging system enabled acquisition of co-registered visual and camera-based (LSCI) images.To perform LSCI simultaneously with conventional dermascope imaging, a 50 mW 785 nm laser diode  was affixed to the side of the diffusing cone. The diffusing cone was machined from an acetal polymer whose optically scattering properties created a more homogenous illumination field for LSCI measurements than would be achieved by illuminating the tissue directly by the laser diode41. The technique is based on the principle of quantifying the spatiotemporal changes in the speckle pattern that result from the constructive and destructive interference of the laser light used to illuminate the tissue. Movement of blood within the tissue causes changes in the speckle pattern and blurring in collected images, which can be quantified using a metric called speckle contrast to create full-field maps of tissue blood flow. Speckle contrast, K, is conventionally computed as:σ and <I> represent the standard deviation and average number of the pixel counts in a local neighborhood of pixels. LSCI images acquired with the LSCI-dermascope were acquired at a rate of 30 frames per second and at a pixel resolution of 640 × 480. Speckle contrast maps were computed from raw images using a 7 × 7 spatial sliding window algorithm that implemented Equation , 10 measurements were acquired and averaged, each of which was derived from an average of 30 speckle contrast maps.The presence of LED or room light within images collected for LSCI measurements can potentially introduce measurement error. Room light which travels through the lens of the LSCI-dermascope and white LED light projected directly onto the region of interest are relatively broadband sources incoherent light, which if not adequately filtered from the signal detected by the camera system, will result in an artificial decrease in speckle contrast. To quantify this effect on the LSCI-dermascope, LSCI measurements were acquired from a tissue-simulating phantom with and without the LEDs and fluorescent room lights turned on. The phantom was fabricated from polydimethylsiloxane with titanium dioxide added to achieve a reduced scattering coefficient of 1 mmMovement during LSCI can cause significant artifacts and measurement error. To quantify the effect of motion artifact imparted from using the LSCI-dermascope in a handheld fashion as opposed to mounted, LSCI measurements were acquired under these conditions from the tissue phantom used above. During handheld measurements, the LSCI-dermascope was held in direct contact with the tissue phantom for five seconds, during which 30 images were collected. Between measurements, the LSCI-dermascope was removed from the tissue phantom and then replaced. The same protocol was followed for mounted measurements but with the LSCI-dermascope rigidly mounted during image acquisition. For each condition, 10 measurements were acquired and averaged, each of which was derived from an average of 30 speckle contrast maps.−1 at 785 nm46. The embedded tube was made of Tygon and had an inner diameter of 0.79 mm. A syringe pump was used to flow lipid emulsion  at volumetric flow rates ranging from 0.01 to 0.09 mL/min. This range was chosen to simulate the approximate speed of blood flow in skin capillaries47. Intralipid was diluted to a concentration of 0.54% to result in a reduced scattering coefficient of 0.6 mm−1, which is comparable to that of human blood at 785 nm51. For each volumetric flow rate, the LSCI-dermascope was placed at the location shown in Fig. The response of the LSCI-dermascope to known fluid flow rates was characterized by acquiring data from a tissue phantom with an embedded tube to simulate a subsurface blood vessel which was located 0.5 mm below the surface. The phantom was fabricated from polydimethylsiloxane with titanium dioxide added to achieve a scattering coefficient of 1 mmTo assess the LSCI-dermascope’s ability to differentiate quantitatively between skin lesions with suspected blood flow differences, LSCI data were acquired from 13 cherry angiomas and 10 solar lentigos. Cherry angiomas were expected to exhibit increased blood flow relative to the surrounding skin, whereas solar lentigos were not. Diagnosis of each lesion type was performed by a trained dermatologist (KMK) at the University of California, Irvine Health Gottschalk Medical Center. During LSCI measurements, the dermascope was placed over the lesion of interest and proper location confirmed visually using the magnified white-light image. Fifty images were acquired from each lesion. During processing, each image was converted to a SFI map and then averaged together. The median SFI was computed within a manually-selected region of interest corresponding to the lesion and the surrounding normal skin. A blood flow ratio was then computed for each lesion by dividing the median SFI in the lesion by the median SFI within the surrounding skin. The average of these ratios was computed for all cherry angiomas and for all solar lentigos. All human data collection was collected in accordance with methods approved by the University of California Institutional Review Board (HS#2008-6307). Informed consent was obtained from all subjects.Statistical differences among populations were analyzed using either ANOVA or the Mann-Whitney test. p-values less than 0.05 were considered significant.


The α4β7 integrin is a non-essential HIV-1 adhesion receptor, bound by the gp120 V1V2 domain, facilitating rapid viral dissemination into gut-associated lymphoid tissues. Antibodies blocking this interaction early in infection can improve disease outcome, and V1V2-targeted antibodies were correlated with moderate efficacy reported from the RV144 HIV-1 vaccine trial. Monoclonal α4β7-blocking antibodies recognise two slightly different helical V2 conformations, and current structural data suggests their binding sites are occluded in prefusion envelope trimers. Here, we report cocrystal structures of two α4β7-blocking antibodies from an infected donor complexed with scaffolded V1V2 or V2 peptides. Both antibodies recognised the same helix-coil V2 conformation as RV144 antibody CH58, identifying a frequently sampled alternative conformation of full-length V1V2. In the context of Envelope, this α-helical form of V1V2 displays highly exposed α4β7-binding sites, potentially providing a functional role for non-native Envelope on virion or infected cell surfaces in HIV-1 dissemination, pathogenesis, and vaccine design. Antibodies blocking the V1V2 domain of HIV Envelope from binding integrin are associated with positive disease outcomes. Here, Wibmer et al. determine the structure of full length V1V2 bound to these antibodies, revealing an alternative fold of V1V2 with exposed integrin-binding sites that functions on non-native Envelope. The resulting gp120 and gp41 subunits remain noncovalently associated and assemble with two other cleaved gp160 protomers to form a metastable trimer of heterodimers that mediates HIV-1 entry and infection. Interprotomer interactions are found between the three gp41 molecules, as well as at the apex of gp120 where three V1V2 and V3 loop regions form the trimer association domain5. When the gp120 receptor binding subunit interacts with the host cellular receptor CD4, large conformational rearrangements reposition V1V2, expose the V3 loop along with the associated CCR5 coreceptor binding site, and free the fusion peptide, thus activating the gp41 membrane fusion subunit7. Infectious HIV-1 particles display only 10–14 properly folded and post-translationally processed, entry-competent, prefusion Env trimers per virion8. A portion of the remaining viral surface is coated with aberrant forms of Env, also erroneously referred to as “junk” Env, formed from uncleaved or inappropriately processed gp160 precursors, non-trimeric protomers, or Env that has prematurely sampled the CD4-bound state11. These proteins appear to act as immunological decoys, thwarting the neutralising antibody response by exposing several highly immunogenic non-neutralising antibody epitopes such as the V3 loop. However, antibodies bound to these non-neutralisable epitopes may still play an important role in countering HIV-1 infection and improving disease outcome by engaging Fc-mediated effector functions12.The HIV-1 envelope glycoproteins (Env) are the only virally encoded proteins presented on virion membrane surfaces. They are expressed from the 15. The gp120 subunit has also been implicated in direct binding of other host cell surface proteins such as the dendritic cell specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) or the gut mucosal homing integrin α4β717. Although not essential for virus entry, these adhesion receptors play an important role in viral dissemination and pathogenesis19. The α4β7 integrin colocalizes with CD4, defining a highly HIV-1 susceptible T-cell population that is infected and depleted from gut-associated lymphoid tissues (GALT) within the first two weeks of infection20. The α4β7 integrin binds to a highly-conserved tripeptide motif in the V2 region of gp120 (at positions 179–181) that mimics a binding loop in its natural ligands17. More recently, an additional determinant of α4β7 binding has been suggested upstream of this site at V2 residues 170–17321. Monoclonal antibodies (mAbs) that block α4β7 binding limit the early loss of CD4 T cells, or the establishment of certain viral reservoirs. In the non-human primate model, α4β7 binding antibodies can reduce mucosal transmission from repeated low-dose challenges22, and early co-administration of antiretroviral therapy with α4β7 binding antibodies has been shown to significantly improve disease outcome by supressing viral rebound23. Similarly, antibodies that bind to V1V2 were identified as a correlate of reduced infection risk in the RV144 HIV-1 vaccine trial, which showed 31% efficacy in the absence of neutralising antibodies24. Antibodies isolated from these RV144 vaccine recipients were shown to block the binding of V2 to α4β7. Altogether these data suggest a potentially beneficial role for the early induction of antibodies to the V2 region of HIV-1 gp120 capable of blocking α4β7 binding. However, in the context of the native, prefusion trimer, the α4β7 binding site is occluded in a 5-stranded β-barrel by intraprotomer interfaces with gp12026, and a mechanism through which such antibodies might act against intact virions is lacking.In addition to binding the entry receptors, HIV-1 also binds to target cells through various host proteins that have been incorporated onto viral surfaces to promote cell invasion and syncytium formation through colocalization of Env and the entry receptors27. The 5-stranded β-barrel form of V1V2 that is found on native, prefusion Env is bound by quaternary structure preferring V2 neutralising antibodies (V2q mAbs)34, as well as by conformation dependent non-neutralising V2 antibodies that recognise the primary α4β7 integrin binding residues 179-181 in the context of non-trimeric Env (V2i mAbs)27. A third group, also comprised of non-neutralising V2 antibodies, recognises short V2 peptides (V2p mAbs)35. These V2p mAbs are the types of antibodies that were initially isolated from RV144 vaccine recipients and bind to an epitope that overlaps both the primary and potential secondary α4β7 binding determinants in V2. In accordance, an analysis of the Env sequences from breakthrough infections in RV144 identified two genetic signatures in V2 that were associated with vaccine efficacy at positions 169 and 181, near the secondary and primary α4β7 binding determinants, respectively36. Cocrystal structures of RV144 vaccine antibodies CH58 and CH59 showed how these two sites colocalise in V2p antibody paratopes35. However, these data also revealed an unexpected degree of conformational plasticity for V2 peptides, which exhibited helical structures that were vastly different from the 5-stranded β-barrel bound by V2q and V2i mAbs37. Molecular dynamics simulations, in silico modelling, and Nuclear Magnetic Resonance studies have all suggested that unconstrained V2 could adopt more helical alternative conformations similar to those observed in the CH58 and CH59 cocrystal structures38. The biological significance of helical V2 variants and whether these epitopes exist in the context of HIV-1 virions remained unclear but has important implications for understanding the role of α4β7 blocking V2 antibodies in pathogenesis and vaccination.Primary sequence elements of the V1V2 domain can be divided into interspersed conserved or variable regions. The conserved terminal strands A and D connect gp120 to V1V2, and also form part of the ‘bridging sheet’ that stabilises gp120 in the CD4-bound conformation. Between them, the hypervariable loop V1 (residues 132 and 156) precedes V2 and is bound at its base by a disulphide bond between C131 and C157. The subsequent N terminal region of V2 (residues 157–181) is relatively conserved and forms the primary target of all cross-reactive V2 antibodies, while the C terminal portion of V2 is a hypervariable loop (residues 182 – 189). Antibodies targeting V1V2 can be grouped into three broadly defined binding modalities+ T cells.We recently isolated two mAbs from an HIV-1 infected donor (CAP228) that bind to V2 peptides, confirming that V2p epitopes are in fact presented during HIV-1 infection . Here, we first determined the cocrystal structures of these new antibodies bound to V2 peptides, identifying a common class of V2p mAbs that share a conserved α-helix-coil epitope with the RV144 mAb CH58. We subsequently determined the cocrystal structure of one mAb (CAP228-16H) bound to the entire V1V2 domain, while still N and C terminally constrained as a scaffolded protein. This revealed an alternatively folded V1V2 exhibiting a highly exposed α4β7 binding site on Envelope. These data provide a potentially functional role for aberrant Env on the surface of HIV-1 virions and further elucidate a mechanism by which V2p mAbs can mediate antiviral activities by blocking the interaction with α4β7 on CD439. Both the CAP228 antibodies also shared the same IGHV5-51 germline heavy chain gene as CH58, suggesting that V2p antibodies might be grouped by common modes of V2 recognition. To investigate this, we screened the CAP228 antibodies for binding to various 1FD6 scaffolded V1V2 proteins and short V2 peptides  derived from different light chain genes, IGVλ6-57 for CAP228-3D and IGVλ3-21 for CAP228-16H . This was similar to the RV144 vaccine elicited antibody CH58 which also uses the IGVλ6-57 gene, as well as CH59 which uses the IGVλ3-10 geneH/VL packing and substantial sequence variation between antigens derived from genetically diverse strains , or from epitopes recognised as part of a 5-stranded β-barrel by V2i (Cα RMSD of 5.4 Å2 for the CH58/830 A comparison) or V2q (Cα RMSD of 6.8 Å2 for the CH58/PG9 comparison) mAbs . Individual IGHV5-51 heavy chain signatures, together with commonalities at the heavy-light chain interface, describe a common class of V2p binding antibodies with a characteristic mode of antigen recognition.In contrast to these similarities, analysis of the CDR-H3 loops from all three antibodies revealed no correlation in charge distribution, predicted D-gene usage or loop conformation  inhibition by CAP228-16H or CH58 was comparable, but at four-fold lower concentrations CAP228-16H still showed substantial inhibition of α4β7 adhesion while CH58 lacked any blocking activity  to a cyclic V2 peptide from the RV144 vaccine strain 92TH023, or the clade A strain BG505 Fig. . At highity Fig. . When te −1 Fig. . Altoget41. These constrain the V1V2 N and C termini in a hairpin structure that facilitates appropriate disulphide bond formation. However, these same 1FD6-scaffolded V1V2s were also bound by the CAP228 antibodies (and to a lesser extent CH58) in ELISA 2 in the mAb paratope, similar to the ~940 Å2 of surface area buried in the V2 peptide interaction  at 3.2 Å resolution Fig. . DespiteVisualised here, the V1 and V2 cystine bonds appeared correctly paired, despite substantial domain reorganisation , V2q  and V2i  mAbs to full length V1V2, using the 1FD6-scaffold to orient the interaction Fig. . Since V42. Thus, unlike the epitopes recognised by V2i mAbs in the 5-stranded β-barrel, the α-helical conformation of V1V2 results in substantial exposure of V2p associated epitopes relative to the rest of gp160.Both V2p and V2q mAb epitopes share an overlapping region that includes a cationic patch (residues 168-171) in the α-helix or C-strand of V1V2, respectively  reposition 180° relative to each other, forming more stable antiparallel strand interactions between β2 and β21 known as the bridging sheet  or CMU06 envelopes. Both envelope strains were from HIV-1 clade AE and had the H173 V2 immunotype important for CH58/CH59 binding. The infected CD4+ T cells were then allowed to proliferate for several days before being stained with the RV144 V2p mAbs CH58 and CH59  or an irrelevant influenza-specific antibody could not be detected. Thus, V2p mAb epitopes are readily detectable on both infected cell surfaces and HIV-1 virions and represent a potential mechanism through which V2p mAbs might exert an α4β7 blocking antiviral effect.To provide more information about the physiological relevance of V2p mAbs, we next sought to confirm the presence of α-helical V1V2 within membrane associated gp160. Primary CD4H59 Fig. . In contH59 Fig. . C1080 i+ T cells by HIV-1, but can serve as an adhesion receptor that is being increasingly shown to play an important role in HIV-1 pathogenesis20. Antibodies that block this interaction by targeting α4β7 can reduce infection or substantially affect disease outcome in acutely SIV infected non-human primates, while V1V2-targeted antibodies have been implicated as a correlate of reduced risk of HIV-1 infection in the RV144 vaccine trial24. Thus far, the mechanism whereby V2p antibodies might contribute to this effect against HIV-1 viruses has been unclear, but may include Fc-mediated effector functions44. Here, we use X-ray crystallography to define a class of V2p mAbs that all derive from a common IGHV5-51 heavy chain gene and are able to block the binding of HIV-1 to α4β7 by recognising a common helix-coil epitope. Additionally, we show that V2p helix-coil epitopes are presented as alternatively folded forms of V1V2 which exist on infected cell surfaces and virion particles. These likely represent aberrantly misfolded, incompletely glycosylated, non-trimeric or CD4-triggered gp160 protomers that display a highly exposed α4β7 binding site. Overall, these data provide a structural explanation for how V2p antibodies may mediate antiviral activities.The α4β7 integrin is non-essential for the infection of CD435. In contrast, the monoclonal antibodies from HIV-1 infected donor CAP228 reveal a common V2 helix-coil conformation that is recognised by several different antibody lineages both from HIV-1 infection or vaccination. This helix-coil conformation was reproducibly adopted by monomeric V1V2 from several clade AE and clade C strains, independent of V1 or V2 loop length or levels of V1V2 glycosylation. Thus, despite some conformational plasticity, the common mode of antigen recognition described herein suggests that the V2 region adopts at least two reproducible folds: the 5-stranded β-barrel recognised by V2q and V2i antibodies31, and the cyclical helix-coil conformation recognised by CH58-like IGHV5-51 class V2p antibodies. The functional significance of the β-barrel conformation has been known for some time, but an understanding of the role of helical V1V2 has been limited by the absence of V2p antibodies that contact appropriate immunotypes, particularly at position 173. The CAP228 antibodies have therefore provided an opportunity to explore this alternative conformation, and revealed that these helix-coil structures are present on infection relevant cell/virion membrane surfaces.V2p mAbs can be broadly categorised by the requirement for an anionic light chain motif, critical for the binding of V2 residues 169–173 . Previo45. A comparison between the published crystal structures of IGHV5-51 antibodies bound to V346, and IGHV5-51 V2p mAbs from this study, revealed several overlapping features of V2p and V3 recognising antibodies . The codon for position 28 spans two activation induced deaminase (AID enzyme) hotspots and is the most mutable amino acid in the VH5-51 gene, making R28 relatively easy to select through vaccination/infection. In addition to this mechanism of maturation, the presence of R97 in the CDR-H3 of CAP228-16H may have also contributed to D180 binding in the context of the initial CAP228 B cell activation event. Historically IGHV5-51 heavy chain antibodies paired with lambda light chains were identified as being preferentially selected for by the HIV-1 V3 loop antigen49. These data suggest that some of the antiviral functions performed by V2p antibodies are V2 conformation specific, and therefore cannot be recapitulated by β-strand preferring V2i or V2q antibodies. Thus far, helical V2p epitopes have only been characterised in the context of short V2 peptides35. Computational V1V2 modelling and Nuclear Magnetic Resonance studies of slightly longer, unconstrained V2 peptides suggested that the conserved portion of V2 has a propensity for adopting an alternate α-helical fold, but it remained unclear how this conformation was related to full length Env38. The 1FD6 scaffold used here to characterise the entire N-terminally and C-terminally constrained V1V2 domain in the V2p helix-coil reveal how an alternatively folded α-helical V1V2, that was not the result of aberrant disulphide formation, forms V2p epitopes through a 180° relocation of the V2 exit loop (strand D) relative to V1 (and strand A). We hypothesise that the refolded α-helical form of V1V2 is more compatible with the CD4-bound conformation of gp120 (and the formation of the bridging sheet), which has been freed of some oligomeric constraint. This circular conformation likely resembles the structures adopted by cyclic V2 peptides (C157-C196) commonly used to characterise V2p antibody responses. The 16-19 amino acid CDR-H3 lengths normally exhibited by IGHV5-51 V2p antibodies may be explained by their position at the centre of this cyclic epitope, where they contribute to removing internal solvent molecules and reducing entropy to stabilise the interaction. Refolding of V1V2 is inhibited in-part by the steric constraints imposed by glycosylation, and it has been shown that deglycosylated V1V2 binds far better to α4β751. However, the V1V2 scaffolded antigens, viral particles, and infected cells described here were all bound by V2p antibodies without any deglycosylation required, suggesting an inherent propensity for monomeric V1V2 to adopt this conformation.The CAP228 antibodies were able to efficiently block the binding of V2 to α4β7 integrin. Importantly RV144 mAbs CH58 and CH59 were elicited by clade AE antigens with an H173 immunotype, which is less globally prevalent, limiting their reactivity. This is particularly true for CH59, which fails to bind to most Y173 containing V2 antigens. The CAP228 antibodies are more tolerant of sequence variation at this position, recognising both common and rare V2 immunotypes, and therefore have a greater breadth of α4β7 blocking activity. Interestingly, while both V2i and V2p antibodies have been shown to mediate effective ADCC of HIV-1 infected cells , engineered to include an HRV-3C protease cleavage site (GLEVLFQGP) between the Fab and Fc fragments, in Expi293F cells (ThermoFisher Scientific) using PEI-MAX 40,000 (Polysciences). Cell culture supernatants were harvested seven days after transfection and purified using protein A (BioVision). The antibodies were digested with HRV-3C (Merck Millipore) at 25 °C for four hours to release the Fab fragments. Fab-peptide complexes were then incubated with a 10-fold molar excess of linear V2 peptides (synthesised by Genscript) before purification by size exclusion chromatography using a superdex 200 column .Monoclonal antibodies were expressed as IgG1 subclass from separate heavy and light chain encoding CMV/R plasmidsGalanthus nivalis affinity (Sigma) and Q sepharose ion exchange (ThermoFisher Scientific) chromatography.ConC gp120 was similarly expressed in adherent HEK293T/17 cells  seeded into hyperflasks (Corning), and purified from cell culture supernatants by sequential The V1V2 scaffolded proteins were expressed in HEK293S cells genetically altered to lack N-acetylglucosaminyltransferase I , in suspension culture for seven days using 293Freestyle media supplemented with 2% Foetal bovine serum (ThermoFisher Scientific), and fed with Hyclone™ SFM4HEK293  24 h post-transfection. The V1V2 scaffolds were captured from the cell culture supernatant with Ni sepharose™ excel , and further purified using size exclusion chromatography. Following this initial purification, V1V2 scaffolds were captured with CAP228 mAbs bound to protein A to obtain the antigen-antibody complexes. These Fab-scaffold complexes were deglycosylated on-column with Endo H (New England BioLabs), eluted following HRV-3C digestion overnight at 4 °C (to cleave off their Fc fragments and any His8 tags), and then further purified using size exclusion chromatography on a superdex 200 column . All cell lines were negative for mycoplasma testing.−1. V2 peptides were C-terminally biotinylated and coated onto streptavidin ELISA plates. The plates with blocked with PBS  for 2 h, before being probed sequentially with five-fold serially diluted monoclonal antibodies (starting at 10 µg mL−1) and an anti-Fc HRP conjugate used a 1:10,000 dilution  in PBS  for 1 h each. The plates were developed using 1-step ultra TMB-ELISA solution (ThermoFisher Scientific) followed by 1 M sulphuric acid and read at 450 nm.V1V2 protein scaffolds were directly coated onto high binding ELISA plates (Corning) overnight at 4 °C at 4 µg mLg, resuspended in PBS, and coated directly onto high binding ELISA plates. Plates were washed and probed as above in PBS .Envelope pseudotyped viruses (PSV) were grown in HEK293T/17 cells by co-transfecting the C1080 envelope plasmid and a pSG3ΔEnv backbone. Cell culture supernatants were collected after two days and filtered through 0.45 µm. PSVs were purified through a 20% sucrose cushion at 40,000×−1, aliquoted, and flash frozen in liquid nitrogen. Thawed aliquots were hand-screened by sitting drop vapour diffusion in a humidified chamber using a 96-well deep block plate format (corning), that included the preformulated Wizard Precipitant Synergy and Wizard Classic 1–4 crystallisation screens (Rigaku). Crystal hits were identified in 1 µL drops containing 50% mother liquor, and further hand-optimised in 15-well hanging drop plates at 25 °C. Crystals of the CAP228-3D Fab complexed with a CAP45 V2 peptide (gp120 residues 164–182) were initially obtained in drops containing 0.1 M imidazole HCl (pH6.5), 20% PEG8000, 3% 2-methyl-2,4-pentanediol (MPD) or 0.1 M HEPES NaOH (pH7.5), 20% PEG8000, 0.2 M ammonium sulphate, 10% isopropanol. Larger crystals were obtained by mixing both conditions in a 1:3 ratio, supplemented with 25% MPD as a cryoprotectant. Crystals of CAP228-16H in complex with the CAP225-V1V2 1FD6-scaffold were obtained in 0.1 M HEPES NaOH (pH7.5), 20% PEG8000, 0.2 M ammonium sulphate, 10% isopropanol and cryoprotected with 20% glycerol, while crystals of CAP228-16H in complex with the autologous CAP228-V1V2 1FD6-scaffold were obtained in 0.1 M Tris HCl (pH8.5), 5% PEG4000, 2 M sodium chloride, further optimised to include 10% butanol, and cryoprotected in 25% 2,3-Butanediol. Diffraction data was collected at SER-CAT ID-22 beamline , at a wavelength of 1.00 Å, 100 K and processed with HKL2000. Model building and refinement was handled with winCOOT v0.8 and PHENIX v1.9-1692, using 5% of the data as an R-free cross validation test set, and with refined hydrogens to minimise clashes.Fab-peptide and Fab-V1V2 scaffold complexes were concentrated to 10–20 mg mL54, while antibody sequences were numbered by the Kabat convention55. All graphs were generated with Graphpad, while structure figures were created in PyMol, and the RMSD, approximate charge, and BSA were estimated using PyMol and PISA.All sequence alignments were created in BioEdit. HIV-1 Env sequences were numbered according to the HXB2 convention57. HEK293T/17 cells were co-transfected with α4 and β7 encoding plasmids (Origene), cultured for two days, and then gently moved into solution with 1 mM EDTA/PBS. Co-expression of α4β7 was confirmed by flow cytometric staining with CD49d (anti-α4) phycoerythrin  and anti-human/mouse integrin β7 fluorescein isothiocyanate. Cells were washed into HBS  in order to activate the integrin, and then incubated with or without mAb (five-fold titrations starting at 20 µg mL−1) for 15 min at 4 °C, followed by biotinylated ConC gp120 (5 µg mL−1) for 25 min. Cells were washed 3 times in HBS buffer, and stained with phycoerythrin linked streptavidin for 30 min in order to detect gp120.The flow cytometry based α4β7 blocking assay was adapted from previously described methodologies58 as follows. Flat bottom 96-well polypropylene plates (Greiner Bio-One) were coated in triplicate overnight at 4 °C with NeutrAvidin. The NeutrAvidin-coated plates were then incubated with a biotinylated cyclic V2 peptide from HIV-1 strains BG505 or 92TH023 (5 μg mL−1 in bicarbonate buffer) for 1 h at 37 °C. The supernatants were then removed and the plates blocked with 25 mM Tris, 2.7 mM potassium chloride, 150 mM sodium chloride, 0.5% BSA, 4 mM manganese chloride, pH 7.2 for 1 h at 37 °C. The solution was discarded and plates were manually washed 4 times with the same blocking buffer. After this 2 × 105 RPMI8866 α4β7+ cells, which had been pre-incubated for 40 min at 37 °C in 50 μL sample buffer in the absence or presence of anti-V2 or anti-α4 mAb at 10 μg mL−1, were added at 37 °C (5% CO2) for 1 h. The wells were then washed 5 times with PBS followed by the addition of 100 μl of RPMI-1640 containing 1% FBS, 1% pen/strep/glutamine, 25 mM HEPES with 10 μL/well of AlamarBlue® dye. Fluorescence (excitation 560 nm and emission 590 nm) was measured immediately after the addition of the AlamarBlue® dye for 8 h.This assay was adapted from previously described work+ T-cells were isolated from fresh, healthy donor PBMCs by negative selection using magnetic beads (StemCell Technologies). Cells were cultured in RPMI-1660 media supplemented with 10% FBS and 2% Penicillin/Streptomycin/L-Glutamine and activated with OKT3, IL-2 (20 U mL−1) and retinoic acid (10 nM) for 4 days. Cells were then infected or mock-infected with HIV-1 strains CMU06, TH023, or a media-only control. Viral stocks were generated as infectious molecular clones using an NL4.3 HIV-1 backbone and a separately cloned Env gene from either CMU06 (Genbank accession #AY669771) or TH023 (Genbank accession #KU562843) transfected into HEK293T cells and passaged once through activated PBMCs. Infected CD4+ T-cells were stained six days post infection using 2 × 105 cells and 1ug of either CH58 or CH59 anti-gp120 mAb per test. Data were acquired using a BD FACS Canto II and all flow data were analysed using FlowJo.CD4Peer Review FileSupplementary Information


Background: Cadmium (Cd) is an environmental pollutant that leads to nephrotoxicity. However, the mechanisms of Cd-induced glomerular injury have not been fully clarified. Von Willebrand factor (vWF) and occludin are important endothelial cell markers in renal vasculature. In this study, the effects of Cd on the vWF and occludin expression in mouse glomeruli was investigated.Objectives: The goal of this study was to analyze the expression of von Willebrand factor and occludin in glomerular endothelial cells of tumor necrosis factor-α−/− (TNF-α−/−) mice after treatment with Cd.Material and methods: C57BL6/J wild-type (WT) mice and TNF-α−/− mice (n = 6) were treated with Cd, and the kidney tissues were collected. The expression of von Willebrand factor and occludin was detected by using quantitative real-time PCR, immunofluorescence, and immunohistochemistry. In vitro, Human umbilical vascular endothelial cells (HUVECs) were used to examine the regulatory role of TNF-α on expression of von Willebrand factor and occludin.Results: We found that Cd significantly increases mRNA and protein expressions of von Willebrand factor and occludin in TNF-α−/− mice, but not in WT mice. In vitro, Cd significantly increased mRNA and protein expression of von Willebrand factor and occludin in HUVECs with TNF-α small interfering RNA (siRNA) transfection.Conclusions: These results suggest that TNF-α acts to balance homeostasis of glomerular endothelium after Cd treatments. Cadmium (Cd) is a heavy metal that is widely used in industry and extensively disseminated in the environment . Occupatvon Willebrand factor (vWF) is a plasma protein almost exclusively expressed by endothelial cells . Before Occludins and zonula occludens (ZO) proteins are important components of tight junctions (TJs) . OccludiCd promotes secretion of proinflammatory cytokines, such as IL-6, IL-8, IL-1β, and tumor necrosis factor-α (TNF-α) . It has −/− mice, we determined the effects of Cd on the expression of vWF and occludin is dependent on TNF-α. In HUVECs with TNF-α siRNA transfection, Cd significantly increased mRNA transcription and protein expression of vWF and occludin. This study provided insights for better understanding the mechanisms of Cd injury on glomerular endothelium in the kidneys.To date, the mechanism of Cd injury of renal vasculature has not yet been fully clarified ,23. In t−/−) (C57BL6/J background) were purchased from the Jackson Laboratory . Wild-type mice (WT) (C57BL6/J background) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All mice were 6–8-week-old males. TNF-α−/− mice or WT mice were randomly and equally divided into a control group and a trial group, with six mice in each group. Trial group mice were fed a standard chow diet and received treatment with CdCl2 (50 mg/L) in distilled water for three consecutive days. The control group was provided water without addition of CdCl2 [2 was purchased from Sigma Aldrich . All the procedures were approved by the Animal Care Committee of Shandong Provincial Qianfoshan Hospital, Shandong University.TNF-α-deficient mice (TNF-αof CdCl2 . The mic−/− mice (n = 6) and WT mice (n = 6) were injected intraperitoneally with lipopolysaccharide  and were bled 6 h later. The levels of TNF-α in the serum were determined with an ELISAs Kit  according to the manufacturer’s instructions. LPS was purchased from Sigma Aldrich .TNF-αqRT-PCR was performed as previously described ,26. TotaImmunofluorescence was performed as previously described . Frozen The kidney tissue samples from mice were fixed in PBS-buffered 4% paraformaldehyde, dehydrated and embedded in paraffin. The paraffin sections were then dewaxed and dehydrated using alcohol gradient. After thermal remediation, sections were blocked with hydrogen peroxide at room temperature. Then, sections were incubated with primary antibody vWF  and Occludin  overnight at 4 °C. After rinsing with PBS, sections were incubated with secondary antibody Max-Vision TMHRP-Polymer anti-Mouse/Rabbit IHC Kit .2 at 37 °C.Human umbilical vascular endothelial cells (HUVECs) were purchased from American Type Culture Collection  and cultured in MV2 media (Promo Cell) with 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco BRL). Cells were maintained in a humidified atmosphere of 5% CO2 were added after 24 h. Cells were collected for RT-PCR after 48 h and for Western blot after 72 h. The qRT-PCR primer sequences for HUVECs are summarized in HUVECs were seeded 24 h prior to transfection in 60-mm-diameter dishes with culture media. Transfection was performed with 20 nanomol (nM) TNF-α siRNA and negative control  using Lipofectamine 3000  according to the manufacturer's protocol. The next day, media were changed and fresh media containing 4 μM CdClHUVECs were washed with PBS and lysed in RIPA buffer . Protein concentration was determined using the BCA assay . Equal amounts of protein were separated by 10% SDS-PAGE gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. PVDF membranes were blocked with nonfat dry milk in PBS containing 0.05% Tween-20 and incubated overnight with a primary antibody in PBS-T at 4 °C. Primary antibodies were rabbit anti-human vWF antibody , anti-occludin , and anti-β-actin . Immunoreactivity was visualized with HRP-conjugated secondary antibodies and chemiluminescence .t-test (2-tailed). All statistical analyses were performed using SPSS 17.0 statistical software . A p values <0.05 was considered significant.The data are expressed as the mean ± standard error. The difference between the two groups was evaluated using the −/− mice, the TNF-α−/− mice and WT controls were treated with LPS. The blood samples were then collected and TNF-α level was determined using ELISA. −/− mice was almost undetectable (p < 0.0001). This suggests that TNF-α expression was successfully eliminated in the TNF-α−/− mice.To assure that TNF-α was not produced from the TNF-α−/− mice and WT mice were treated with Cd, and the kidney tissues were collected and detected with quantitative real-time PCR. After treatment with Cd, the expression of vWF mRNA was not significantly changed in the WT mice (0.89 ± 0.16-fold, p = 0.143, −/− mice (2.36 ± 0.72-fold, p = 0.0057, p = 0.0537, −/− mice (3.75 ± 0.56-fold, p = 0.0002, To clarify the effect of Cd stimulation on gene expression of vWF and occludin in the kidneys, the TNF-α−/− mice and WT mice were treated with cadmium and kidney tissues were collected and assessed with immunofluorescence and immunohistochemistry. After treatment with Cd, the expression of vWF was not significantly changed in the glomeruli in the WT mice (−/− mice (−/− mice (To further validate the effects of Cd treatment on protein expression of vWF and occludin in the kidneys, TNF-α WT mice , whereas−/− mice . In addi−/− mice , while i−/− mice . Immunohin vivo study showed that the effects of Cd stimulation on gene expression of vWF and occludin might be regulated by TNF-α. In vitro, HUVECs were used to validate the regulatory role of TNF-α on expression of vWF and occludin. As shown in p = 0.00007, p = 0.0883, p = 0.00009, p = 0.722, p = 0.00008, The To further validate the effect of Cd stimulation on protein expression of vWF and occludin in HUVECs, the treated cells were collected and examined by Western blotting. After treatment of Cd, the protein expression of vWF was unchanged in HUVECs, whereas it was significantly increased in TNF-α siRNA-transfected HUVECs . Consist−/− mice were used to investigate whether Cd affects the expression of vWF and occludin in the kidney in a TNF-α-dependent manner. The TNF-α−/−mice did not produce TNF-α after LPS stimulation, suggesting it as a successful model for TNF-α deletion. Kidney is the primary target of Cd toxicity [Cd is a toxic metal that affects mammalian organs and cells ,28. Givetoxicity . Previoutoxicity , ascorbitoxicity , Potentitoxicity , and Alptoxicity . Our stu−/− mice and in TNF-α siRNA-transfected HUVECs, suggesting that TNF-α is involved in inhibition of expression of occludin in the glomerulus. In addition, TNF-α inhibits vWF expression [It has been reported that renal damage is associated with decreased renal expression of occludin . Cd incrpression ,37. In t−/− mice but not in WT mice. In addition, vWF expression was significantly elevated by Cd treatment in TNF-α siRNA-transfected HUVECs. Together, these results suggest that TNF-α regulates vascular homeostasis by decreasing vWF expression during Cd-induced endothelial cell injury.Endothelial cells synthesize and secrete vWF, a key regulator in hemostasis . However−/− mice and in TNF-α siRNA-transfected HUVECs, suggesting the role of TNF-α in mediating Cd induced toxicity. Our study provides a new insight for understanding the mechanisms of Cd-related effects in glomerular endothelium.In summary, we found that Cd significantly increased expression of vWF and occludin in TNF-α


Caenorhabditis elegans, but the effects appear to be complex and the underlying mechanism for lifespan regulation by mitochondrial respiratory genes is still not fully understood. Here, we investigated the role of Y82E9BR.3, a worm homolog of the ATP synthase subunit C, in modulating longevity in C. elegans. We found that the Y82E9BR.3 protein is localized in mitochondria and expressed in various tissues throughout development. RNAi knockdown of Y82E9BR.3 extends lifespan, decreases the accumulation of lipofuscin, and affects various physiological processes, including development delay, reproduction impairment and slow behavior. Further tissue-specific RNAi analysis showed that the intestine is a crucial organ for the longevity effects conferred by Y82E9BR.3 RNAi. Moreover, we demonstrated that lifespan extension by Y82E9BR.3 RNAi is associated with reduced mitochondrial function, as well as the suppression of complex I activity in mitochondria. Unexpectedly, Y82E9BR.3 RNAi knock down did not influence the whole-worm ATP level. Our findings first reveal the crucial role of Y82E9BR.3 in mitochondrial function and the underlying mechanism of how Y82E9BR.3 regulates lifespan in C. elegans.Mild inhibition of mitochondrial respiration leads to longevity. Disruption of mitochondrial respiratory components extends lifespan in  Although complete disruption of mitochondrial function is detrimental or even lethal for many eukaryotes, including humans, accumulating evidence has revealed that partial inhibition of mitochondrial function tends to increase lifespan5. In C. elegans, mutations in various mitochondrial electron transport chain (ETC) genes can greatly extend lifespan; these include mutations in isp-1 (encoding the iron sulfur protein of Complex III)6 and clk-1 (encoding the hydroxylase protein necessary for the biosynthesis of the ETC electron transporter coenzyme Q)8. In addition, RNAi knockdown of various mitochondrial ETC genes prolongs lifespan in yeast, worms and fruit flies11.Mitochondria play an essential role in many important physiological processes, including aging. Mitochondrial function has been thought to gradually decline with age, while oxidative damage and mitochondrial DNA mutations accumulateC. elegans and Drosophila. For example, mutations in mev-1, which encodes a subunit of the succinate dehydrogenase cytochrome b of complex II, causes a short lifespan in worms12. In addition, the underlying causes for lifespan regulation by ETC genes remain incompletely understood. For example, the roles of reactive oxygen species (ROS) production and mitochondrial function in aging and lifespan of ETC mutants can be opposite ways4. One model interpreting these opposite effects is that moderate mitochondrial impairments increase lifespan until a threshold is reached, beyond which animals display wide-spread damage, shortened lifespan, or even death13. Nevertheless, how mitochondrial genes modulate lifespan and whether they function in modulating lifespan in other species remain incompletely elucidated.The effects of mitochondrial ETC genes on modulating lifespan appear to be complex. Inhibition of some ETC genes increases lifespan, whereas inhibition of others decreases or does not alter lifespan in 15. ATP synthase is composed of a proton channel F0 and a peripheral catalytic domain F1. Proton transport through the F0 domain drives the release of ATP product on F1 through long-range conformational changes16. In mammals, ATP synthase deficiency is one of the rarer mitochondrial oxidative phosphorylation deficiencies, covering only 1%16. Selective deficiency of the human mitochondrial ATP synthase of nuclear origin caused by altered biosynthesis of the enzyme results in heart failure17.ATP synthase, also known as complex V of the mitochondrial respiratory chain, is the primary cellular energy-generating machineryatp-2 gene, which encodes the beta-subunit of F1 domain in complex V, leads to developmental delay and increased lifespan18. Additionally, a genome-wide RNAi screen revealed that RNAi knockdown of atp-3 (encoding O subunit of F1 domain), atp-4 (encoding ATP synthase coupling factor 6 of F0 domain), atp-5 (encoding ATP synthase D chain of F0 domain) or asb-2 (encoding ATP synthase B homolog of F0 domain) prolongs worm lifespan20. However, the underlying mechanism for lifespan extension due to inhibition of these subunits in the ATP synthase remains unclear. As ATP synthase is highly conserved throughout evolution15, understanding the role of the ATP synthase in lifespan regulation can lead to untangling of the complexity of mitochondrial ETC genes in modulating lifespan.ATP synthase is also intimately linked to aging. In worms, genetic inhibition of the C. elegans Y82E9BR.3 is a worm homolog of the ATP synthase subunit C. We previously reported that knockdown of Y82E9BR.3 using RNA interference promoted longevity in worms21, which is also observed in another report22. However, the role of Y82E9BR.3 in mitochondrial function and lifespan regulation remained uncharacterized. Here, we found that the Y82E9BR.3 protein is localized in mitochondria and expressed in various tissues throughout development. Genetic inhibition of Y82E9BR.3 lengthens lifespan associated with reduced mitochondrial function as well as a decrease in complex I activity in mitochondria. In addition, the intestine proved a crucial organ for the longevity effects conferred by Y82E9BR.3 RNAi. Collectively, our study demonstrated the important role of Y82E9BR.3 in mitochondrial function and the underlying mechanism involving Y82E9BR.3 in aging.24. We found that the accumulation of lipofuscin was remarkably decreased by Y82E9BR.3 RNAi   . Y82E9BR.3 RNAi-treated worms exhibited a smaller body size than the wild type 48 h after hatching Fig. .We also 01) Fig. , exhibit01) Fig. , exhibitY82E9BR.3p::gfp). We found that the strongest fluorescence was observed in the pharyngeal muscles, and the signal was widely distributed in the whole body of adult animals, including the intestine, body wall muscles and hypodermis .We then visualized tissues that expressed Y82E9BR.3 by generating and examining a Y82E9BR.3 promoter-driven GFP-expressing transgenic animal . Y82E9BR.3::gfp was predominantly expressed in the pharyngeal muscle, intestine and more faintly in the hypodermis   activity  in Y82E9BR.3 RNAi-treated animals. We also used short-lived ity Fig. . We founity Fig. . Consistnts Fig. . ComplexC. elegans by inhibiting complex V, ATP synthase27. As we showed that Y82E9BR.3 RNAi reduced the activity of complex V  promotes longevity in x V Fig. , we wondx V Fig. . These dp < 0.05, Fig. To examine the effects of Y82E9BR.3 RNAi on the MRC components in animal physiology, we measured the oxygen consumption rates and ATP production using whole animals. We found that oxygen consumption rates were significantly reduced in intact young adult worms treated with Y82E9BR.3 RNAi by 37.5% compared to the control  concentrations of FUdR, which is much lower than the concentration of FUdR previously reported to have effects on mitochondria function. However, we still cannot completely exclude the possibility that FUdR might have affected mitochondrial function. Importantly, we found that the extended lifespan of Y82E9BR.3 RNAi-treated animals without FUdR was similar to that performed with FUdR  knockdown prolongs lifespan, but does not affect locomotor activity in Drosophila37. This might be attributable to differences in gene expression in various tissues, and the integrated response from these tissues represents the final phenotype. Notably, swimming and feeding in Y82E9BR.3 RNAi-treated worms decline with age in different manners, in accordance with a previous report38. This might be related to the difference of Y82E9BR.3 RNAi efficiency in different tissues.In addition to life extension, Y82E9BR.3 RNAi-treated worms exhibited a typical “Mit phenotype”, including a development delay, small adult body size, altered brood size and slow behavior39. In our study, Y82E9BR.3 is expressed in tissues of pharynx, hypodermis and intestine, and Y82E9BR.3 RNAi successfully knocked down the Y82E9BR.3 expression in all these tissues. The tissue-specific RNAi lifespan assay further showed that among those tissues, the intestine was required for the lifespan regulation. Our finding is in line with the study of the cco-1 subunit of complex IV in which intestinal- or neuronal-specific RNAi knockdown prolongs worm lifespan39. Our data further support the notion that key tissues transmit longevity signals to modulate aging processes in a whole organism. Here, long lifespan conferred by intestine-specific Y82E9BR.3 RNAi is shorter than that of Y82E9BR.3 RNAi in wild-type worms, which is probably related to the possibilities that the genetic backgrounds of those strains are different and lifespan results vary among sets. In addition, our data also suggest that Y82E9BR.3 expression does not change with age.It has been suggested that the RNAi targeting ETC genes in specific tissues is essential for establishing and maintaining a prolongevity cueC. elegans, but the mechanism varies, even when targeting the same genes40. In our study, we found that Y82E9BR.3 knockdown significantly decreased the activity of complex I, as well as ATP synthase activity. This result suggests that the complex I-associated pathway is involved in the effects of ATP synthase subunit C on lifespan. The interdependence among the individual components of ETC has been reported previously. For example, long-lived clk-1 mutants have a severely defective complex I-dependent metabolism due to differences in complexes I and II in their ability to use quinone pools41. In addition, long-lived isp-1 mutants have a decreased complex I activity due to allosteric interaction between ETC components42. Complex IV knockdown shortens nematode lifespan, and decreases complex I enzymatic activity due to interdependence among individual components of the ETC supercomplex43. These findings show that defects in a single ETC component can elicit wide ranging effects on the function of various ETC complexes. Additionally, polyphenolic ATP synthases inhibitors have been shown to inhibit complex I activity, but did not significantly inhibit the activity of complexes II and III45, indirectly supporting our data. Therefore, we speculate that the disruption of the ATPsyn subunit C in the tightly coupled MRC would affect multiple enzymatic steps of electron transfer and may disturb proton gradient and electron carriers, resulting in reduced complex I activity. However, specific mechanisms still require further investigation.Genetic inhibition of mitochondrial ETC genes both by mutation and RNAi prolongs lifespan in atp-5), in which ATPsyn-d knockdown does not significantly alter the activities of complex III and complex IV or the formation of five oxidative phosphorylation complexes in Drosophila37.We observed that the activity of complex III or complex IV was not significantly decreased in Y82E9BR.3 RNAi-treated worms. This result is in agreement with the effect of ATPsyn-d  activity but have severely reduced succinate-cytochrome c oxidoreductase (complex II plus III) activity. Here, we report for the first time that complex V activity significantly decreased in mev-1 mutants, in support of the notion of intimate interdependence among MRC components. Our data also suggest that ATP synthase might play a role in the aging phenotypes of mev-1 mutants. However, ATP levels are normal in mev-1 mutants46, further representing the complexity between mitochondrial function and aging.The C. elegans, α-KG and other metabolic products with similar structures to α-KG are enriched47. Additionally, α-KG binds the b-subunit  of complex V and extends nematode lifespan27. In our study, we exogenously treated Y82E9BR.3 knockdown worms with α-KG, and did not observe an additive lifespan-increasing effect, supporting the notion that Y82E9BR.3 regulates lifespan by affecting MRC. Notably, α-KG decreased the lifespan of Y82E9BR.3 RNAi-treated worms. One possible explanation for this is the antioxidant effect of α-KG48. Long-lived mitochondrial respiration mutant C. elegans have been reported to contain higher levels of ROS than wild-type animals49. Recent studies demonstrated that the increased ROS levels promote longevity in mitochondrial respiration mutant C. elegans and antioxidant treatment abolishes the long lifespan caused by the inhibition of mitochondrial respiration51. Therefore, it seems likely that Y82E9BR.3 RNAi increased ROS levels, and this in turn probably promotes the longevity conferred by Y82E9BR.3 RNAi. α-KG may have counteracted the ROS and thereby abolish the long lifespan conferred by Y82E9BR.3 RNAi. This also suggests the potential role of ROS in the mechanism by which Y82E9BR.3 knockdown leads to longevity.In mitochondrial mutants or starved atp-2, atp-3, atp-4, atp-5 and asb-2, which encode different subunits of ATP synthase, prolongs nematode lifespan20, the underlying mechanisms remain mostly uncharacterized. In particular, a recent study showed that knockdown of ATPsyn-d (atp-5) interacts with TOR signaling to modulate protein homeostasis and lifespan in Drosophila, showing a conserved role for ATP synthase in modulating lifespan37. Moreover, α-KG inhibits ATP synthase, likely by binding to ATPsyn-b (atp-2), and reduces TOR signaling to extend lifespan in C. elegans27. It seems likely that mTOR signaling plays an important role in the longevity induced by the inhibition of ATP synthase subunits. Inhibition of TOR signaling also extends lifespan in several model organisms and the longevity conferred by inhibition of TOR signaling is associated with mitochondrial activity4. For example, in Drosophila, dietary restriction upregulates 4E-BP, a translational repressor in TOR signaling, to increase the expression of genes essential for mitochondrial function, including mitochondrial complexes I and IV. Lifespan extension induced by dietary restriction is suppressed by knocking down the ETC genes regulated by 4E-BP52. In addition, TOR signaling increases mitochondrial respiration through YY1 (yinyang 1), a key mediator of the interaction between TOR and PGC-1α in mammalian cells53. These lines of evidence suggest a possible correlation between ATP synthase, TOR signaling and mitochondrial complex I in aging, indirectly supporting our finding. It further implies that the mTOR pathway might mediate ATPsyn-subunit C knockdown-induced longevity.Although mutation or RNAi knockdown of the genes 54. Alternatively, we assume that Y82E9BR.3 RNAi-treated worms might have decreased energy expenditure, thereby maintaining ATP at a normal level. In a previous report, deficiency in clk-1 decreases mitochondrial function with normal or increased ATP levels, likely due to their decreased energy utilization55. Another possibility is glycolysis can be upregulated given that cellular levels of ATP are tightly regulated.ATP generation can be achieved through the coupling of glycolysis to oxidative phosphorylation in mitochondria, which is oxygen dependent. Therefore, the oxygen consumption rate of an organism is an important indicator of normal cellular function. In our study, we used the Clark electrode instrument to monitor the continuous decrease in oxygen concentration using whole worms. In agreement with the idea that the disruption of the ETC component genes would result in altered patterns of oxygen consumption, Y82E9BR.3 knockdown reduced mitochondrial respiration. Surprisingly, Y82E9BR.3 knockdown did not alter ATP levels. One possibility might be the initiation of a compensatory response to low levels of ATP synthesis. In worms, a glyoxylate pathway serves as an alternative energy source that can bypass mitochondrial MRC dysfunctionatp-2 knockdown decreases ATP level and oxygen consumption and prolongs lifespan in worms27, whereas ATPsyn-d (atp-5) knockdown does not significantly change or even increases ATP levels in Drosophila37. Apparently, Y82E9BR.3 influences animal physiology in a different way. It is more likely that ATPsyn-c (Y82E9BR.3), ATPsyn-b (atp-2) and ATPsyn-d (atp-5) affect ATP synthase and lifespan through different mechanisms. This also suggests that lifespan extension is not necessarily correlated with reduced ATP levels, as supported by studies using both C. elegans and Drosophila, which have shown that disruption of mitochondrial genes that extends longevity does not necessarily impact ATP levels55.For other genes encoding ATPsyn subunits, C. elegans28. In our study, worms treated with Y82E9BR.3 RNAi displayed an increased mtDNA levels, consistent with the previous observations using clk-1 and isp-1 mutants28. Our data further support the possible notion that animals with the defects in mitochondrial respiration initiate a compensatory response to produce more mitochondria.Impaired mitochondrial respiration elicits retrograde signalling in + transport, and structurally possesses a conserved acidic residue in the transmembrane helix, but differs in the surrounding residues among species56, potentially serving as a drug target for certain chemicals57. Multiple deficiencies in the subunits of ATP synthase have been reported in patients. In particular, a patient with conditions such as neonatal onset, lactic acidosis, 3-methylglutaconic aciduria and mild mental retardation has a mutation in ATP synthase subunit epsilon, with a remarkable accumulation of subunit c58. It was suggested that the compensatory responses to counteract mitochondrial mutations in the Mit mutants are very likely the same ones initiated to slow disease appearance in many human mitochondrial associated diseases (HMADs)59. Thus, the mechanism presented in our study may also be relevant to the search for potential preventative therapies for ATP synthase deficiency related to HMADs.ATP synthase is highly conserved and widely distributed in bacteria, eukaryotic mitochondria and chloroplasts. The C subunit functions in HC. elegans.In conclusion, we explored the role of the mitochondrial respiratory chain gene Y82E9BR.3 in aging in this study for the first time. We demonstrated that RNAi targeting Y82E9BR.3 extends lifespan by reducing mitochondrial respiration as well as suppression of complex I activity. Our findings uncover the mechanism of the regulation of Y82E9BR.3 on lifespan and reveal the importance of the Y82E9BR.3 protein in determining aging and lifespan in rde-1(ne219) V., NR222 rde-1(ne219) V.; kzls9, NR350 rde-1(ne219) V.; kzls20, VP303 rde-1(ne219) V.; kbls7, NL3321 sid-1(pk3321) V., TU3401 sid-1(pk3321) V.; uIs69, JM43 rde-1(ne219) V.; Is[wrt-2::RDE-1], and TK22 mev-1(kn1) III.The following strains were analyzed in this study: N2 wild type, WM27 21, was cultured overnight in LB broth containing 50 μg/ml ampicillin . To induce double-stranded RNA, 1 mM isopropyl β-D-1-thiogalactopyranoside  was added to plates for incubation at room temperature for 1 day before usage.The Y82E9BR.3-specific RNAi clone, which was prepared previously51. Quantitative PCR using the cDNA was executed in a StepOne Real Time PCR System  and analyzed using a comparative CT method. Average mRNA levels for ama-1 (the large subunit of RNA polymerase II) were used for normalization. The average of two technical repeats was used for each biological data point.Approximately 500–1000 synchronized RNAi-treated young adult worms were used for quantitative RT-PCR analysis. Extraction, purification and reverse transcription of RNA were performed as previously describedSequences of primers used for quantitative RT-PCR are listed below:Y82E9BR.3-F-CCTCCTCGCCTCGAGAGCCCCACTC;Y82E9BR.3-R-CGGCTCCAGCTCCGATGTACTTGGC;ama-1-F-TGGAACTCTGGAGTCACACC;ama-1-R-CATCCTCCTTCATTGAACGG.60. Single late-L4 stage worms were placed on individual RNAi-bacteria plates (10 worms per condition). The animals were transferred to new RNAi-bacteria-containing plates every 24 hours until they stopped producing progeny. Trials involving worms that bagged, ruptured, burrowed or crawled off were censored. All plates containing progeny were placed in a 20 °C incubator until the progeny reached L4 or adulthood, and the number of worms that developed was counted. Total brood size was determined by adding the numbers of progeny produced by a single worm each day.Experiments were performed as described previouslyhttp://rsb.info.nih.gov/ij/).The autofluorescence of intestinal lipofuscin was measured as an index of senescence at day 18 of adulthood. Day-18 worms from the RNAi-bacteria NGM plates were picked and mounted onto a 2% agarose pad coated with 10 mM sodium azide  in M9 buffer to induce anaesthesia. Lipofuscin autofluorescence images were captured using a DAPI  channel from an AxioCam HRc CCD digital camera (Zeiss Corporation) with a Zeiss Axio Scope A1 compound microscope (Zeiss Corporation). The fluorescence intensity of the animals was quantified by using ImageJ software , 50 μg/ml ampicillin , and 1 mM isopropyl β-D-1-thiogalactopyranoside . To measure body bending in liquid, day-1 and day 9 worms were transferred into each well of the 24-well plates containing 1 ml of M9 buffer. After 30 seconds for equilibration, worm movements were recorded using a DIMIS-M  camera, and the body bends of individual worms were counted for 30 seconds. The number of bend per minute were calculated.Locomotion was measured as described previously with modifications61. Briefly, gravid adults were placed on plates containing RNAi bacteria to synchronize the worms, and progeny were allowed to develop to young adults and were then transferred onto RNAi plates containing 10 μM 5-fluoro-2′-deoxyuridine , 50 μg/ml ampicillin , and 1 mM isopropyl β-D-1-thiogalactopyranoside . Pharyngeal pumping (feeding) rates were counted for 15 seconds by observing the pharynx of a day-1 and day 9 worm under a dissecting microscope using a Zeiss SteREO Discovery.V8 stereomicroscope , and converted to the number of pump per minute.Feeding rate measurements were performed as described previously with some modificationsY82E9BR.3p::gfp DNA construct, a DNA fragment that contained 2.0 kb upstream of the start codon of Y82E9BR.3 from C. elegans genomic DNA was PCR amplified. The DNA fragment was inserted into the pPD95.75 vector (Fire lab C. elegans vector kit), which contains unc-54 3′UTR and GFP and was constructed using the In-Fusion HD Cloning Kit  following the manufacturer’s instruction. The constructs were transformed into DH5a competent cells. The resulting plasmid (25 ng/μL) was microinjected into the gonad of day 1 adult worms with the coinjection marker odr-1p::rfp (75 ng/μL)62.To generate the http://rsb.info.nih.gov/ij/, 1997–2009).Transformed offspring were selected by picking the RFP-expressing F1 larvae. Stable transformants were picked among the F2 generation worms. In these transformants, the transcriptional activity of the Y82E9BR.3 promoter was reported by green fluorescence. The fluorescence and bright-field images were captured using an AxioCam HRc CCD digital camera (Zeiss Corporation) with a Zeiss Axio Scope A1 epifluorescence microscope (Zeiss Corporation). Images were merged using ImageJ software  were cloned into the pPD95.75 vector using the In-Fusion HD Cloning Kit  following the manufacturer’s instructions. Constructs were transformed into DH5a competent cells. The construct was microinjected into the gonad of day 1 adult worms with the coinjection marker odr-1p::rfp. The transgenes showed an apparent genotoxicity in the process: the Y82E9BR.3::GFP fusion construct at an injection concentration of 25 ng/μl showed strong genotoxic effects, and we were not able to obtain transformants; the construct at 5 ng/μl showed decreased genotoxicity and we obtained only transient transformants; when the construct concentration decreased to 1 ng/μl, we acquired transmitted lines. For imaging of Y82E9BR.3p::Y82E9BR.3::gfp animals, animals were synchronized on OP50-seeded plates, and the fluorescence and bright field images were captured using a two-photon confocal fluorescence microscope (Leica TCS SP8 MP). Confocal images were obtained using a 488-nm excitation laser and GFP image was captured at the emission wavelength of 500–560 nm.63. MitoTracker CMXRos (Invitrogen) was dissolved in dimethylsulfoxide (DMSO) to a stock concentration of 10 mM and was added to animals on the plates to a final concentration of 10 μM for 6 hours before analyses. The images were photographed by using the Nikon A1si/Ni-E upright confocal microscope with 100×, 1.4 NA oil-immersion objective lens. Confocal microscopy data were acquired at the Brain Research Core Facilities in Korea Brain Research Institute (KBRI).MitoTracker staining was performed as described previously, with some modificationshttp://rsb.info.nih.gov/ij/).Y82E9BR.3p::Y82E9BR.3::GFP (IJ1392) worms were synchronized on the control or Y82E9BR.3 RNAi bacteria-seeded plates. When worms were reached L2 – L3 stages, the worms were anesthetized by using 100 mM sodium azide and imaged by using AxioCam HRc camera mounted on a Zeiss Axioscope A.1 microscope. The GFP intensity was quantified by using ImageJ software  at 37 °C and seeded onto nematode growth media (NGM) plates containing 50 μg/mL ampicillin. RNAi bacteria seeded on plates were treated with 1 mM isopropylthiogalactoside  to induce dsRNA expression for 1 d at room temperature.Lifespan assays were performed as described previously, with some modifications65 was used for statistical analysis. P values were calculated using the log-rank (Mantel-Cox) method.Gravid adults were placed on plates containing RNAi bacteria to synchronize the worms. The progeny were allowed to develop to young adults. Young adults were then transferred onto 5 μM 5-fluoro-2′-deoxyuridine -treated RNAi-bacteria NGM plates; or young adult worms were transferred onto 5 μM FUdR -treated RNAi-bacteria NGM plates with or without 8 mM α-ketoglutarate (α-KG). Approximately 120 worms per condition were examined for death every 2 or 3 days until all animals were dead. Animals that ruptured, bagged, burrowed, or crawled off the plates were censored but used as censored subjects for statistical analysis. Lifespan assays were performed at 20 °C. Online application of survival analysis 60. Briefly, approximately 5000 synchronized day 1 adult worms were collected, quickly washed three times in M9 buffer, resuspended in 1 ml of M9, and transferred into the chamber. Oxygen concentration was monitored with a Clark electrode in a closed chamber for ~10 min. Worms were collected, pelleted, and kept at −80 °C for protein quantification using a Bradford Protein Assay kit (Bio-Rad). Oxygen consumption rates were normalized to protein contents. The experiments were repeated five times.Oxygen consumption rates were measured using the Oxytherm , a Clark-type oxygen electrode as described previously40. Briefly, synchronized young adult worms fed on Y82E9BR.3 RNAi bacteria or control RNAi bacteria were collected and washed three times in M9 buffer. The worm pellets were treated with three freeze/thaw cycles and boiled for 15 min to release ATP and destroy ATPase activity. Worms were centrifuged at 12,000 × g for 10 min at 4 °C. Aliquots of the supernatant were used for the ATP assay following the manufacturer’s instructions. Luminescence was measured with a microplate reader . The total protein concentration of the samples was determined using Bradford  assay. ATP content was normalized against the protein level. Three independent experiments were performed.ATP levels were quantified using the luciferin-luciferase method based ATP Determination Kit 66. Briefly, wild type worms were fed Y82E9BR.3 RNAi bacteria or control RNAi bacteria, and mev-1(kn1) worms were cultured on NGM plates until reaching a young adult stage. Approximately 300,000 age-synchronized animals were harvested from the cultures, cleaned and washed with M9 buffer and suspended in mitochondrial isolation buffer (MSME)67. All subsequent treatments were performed at 4 °C. The worm suspension was ruptured using a homogenizer . An equal volume of MSME containing 0.4% bovine serum albumin (BSA) was added, and the suspension was thoroughly mixed and centrifuged for 5 min at 380 × g. The supernatant was centrifuged repeatedly. The resulting supernatant containing crude mitochondria was centrifuged for 5 min at 4500 × g, and the resulting mitochondrial pellet was resuspended in MSME. Aliquots were used to determine total protein contents using the Bradford  assay. The remainder was frozen at −80 °C for quantification of mitochondrial respiratory chain (MRC) enzymatic activities.Mitochondria were isolated following the previously described method68: rotenone-sensitive NADH-ubiquinone oxidoreductase (complex I), succinate dehydrogenase (complex II), antimycin A-sensitive decylubiquinol cytochrome c oxidoreductase (complex III), cytochrome c oxidase (complex IV) and ATP synthase (complex V). Briefly, frozen (−80 °C) samples of mitochondria preparations were thawed on ice and solubilized with 2% CHAPS, and aliquots were used for enzymatic activity assay following the protocol of the manufacturer. The reactions used for the determination of complexes I-V were monitored at 340, 600, 550, 550 and 340 nm using a spectrophotometer  respectively, and the activities of complexes I-V were calculated according to the formula given in the protocols. The activities of Complex I-V were normalized to total protein contents, measured as described above, and reported in nmol substrate/min/mg protein. Six independent experiments were performed.The activities of the following MRC enzymes from the isolated mitochondria were assayed spectrophotometrically using a Mitochondria Complex I-V Activity Assay Kit (Genmed Scientifics)28. Synchronized worms were cultured on the corresponding RNAi plates until reaching young adult stage. Single worm was lysed by using proteinase K. qPCR reaction was performed with SYBR green. Mito1 primer set was used for the mtDNA measurement. Sequences of primers used for quantitative RT-PCR are listed below:mtDNA contents were measured as described previously with some modificationsMito-1-F-GTTTATGCTGCTGTAGCGTG;Mito-1-R-CTGTTAAAGCAAGTGGACGAG.ama-1-F-TGGAACTCTGGAGTCACACC;ama-1-R-CATCCTCCTTCATTGAACGG.Supplementary information