In this study, we monitored NF-κB and caspase signalling in human cancer cells treated with a short pulse of Tumour Necrosis Factor (TNF). TNF is an inflammatory cytokine that can induce both the pro-survival NF-κB-driven gene transcription pathway and the pro-apoptotic caspase pathway. We find that a few seconds of exposure to TNF is sufficient to activate the NF-κB pathway in HeLa cells and induce apoptotic cell death in both HeLa and Kym-1 cells. Strikingly, a 1-min pulse of TNF can be more effective at killing than a 1-hour pulse, indicating that in addition to TNF concentration, duration of exposure also coordinates cell fate decisions.In tissues and tumours, cell behaviours are regulated by multiple time-varying signals. While in the laboratory cells are often exposed to a stimulus for the duration of the experiment,  In vivo, TNF released after lipopolysaccharide (LPS) exposure is rapidly cleared, and the half-life of injected TNF in the blood is a few minutes1235678910Although many experiments characterize cellular responses to continuous exposure to a stimulus, stimuli in normal tissues and tumours are often time-varying. One such stimulus is Tumour necrosis factor (TNF). TNF regulates diverse cellular behaviours such as migration, differentiation and apoptosis567161719Experiments in mouse embryonic fibroblasts have shown that NF-κB activation long outlasts treatment with a 1-, 2- or 5-min pulse of TNF and that, compared to continuous treatment, NF-κB activation is nearly indistinguishable in amplitude or timing when quantified by electromobility shift assaysHere, we set out to determine the TNF pulse duration required for NF-κB activation in single human cancer cells, and study how pulse duration affects TNF-induced apoptosis. Using single-cell data, we quantified the threshold of NF-κB nuclear translocation required for inducing early gene transcription. We show that for a high TNF concentration, a 10-sec pulse was sufficient for NF-κB activation but a longer pulse is required at a lower TNF concentration. Extrinsic apoptosis was also strongly activated by a short TNF pulse. In HeLa cells, a 1-min TNF pulse induced apoptosis with a potency similar to that of a 10-hr treatment whereas a 30- or 60-min pulse was less effective in cell killing. For Kym-1 cells, a 30-sec pulse of TNF induced as much death as continuous stimulation. Our study reveals that the duration of TNF exposure influences the TNF-induced cell fate decision.We designed and built a simple microfluidic system that uses laminar flowTo monitor TNF-induced NF-κB activation, we seeded the device with EGFP-RelA-expressing HeLa cells  and quan2225262728IL8 and TNFAIP3, two NF-κB-inducible genes with no or few transcripts in unstimulated cells , as well as in the endogenous RelA distributions, for IFNγ-pre-treated cells exposed to a 1-min TNF pulse versus cells exposed to TNF continuously . This isOur simulations of TNF-TNFR1 binding suggest that at low TNF concentrations much longer exposures might be required to achieve binding of TNF to a large fraction of TNFR1 . TherefoTaken together, our data show that a short pulse of high-concentration TNF is sufficient to induce substantial cell death in both HeLa and Kym-1 cells. The duration of this pulse in turn affects the efficacy and timing of the pro-apoptotic effect of TNF – cells treated with a brief pulse tend to die earlier than cells treated continuously. Although the extent of TNF-induced cell death after a 1-min pulse is comparable with continuous treatment, longer pulse durations are less effective in HeLa cells suggesting that the duration of cytokine exposure is an important mediator of TNF-induced signalling and cell fate.In a tissue, cell may experience transient exposure to inflammatory cytokines as these are released in bursts by migrating tissue-resident immune cells and can be rapidly cleared. Using a simple microfluidic system, we characterized the cellular responses to different durations of exposure to TNF in single-cells. Our results demonstrate that the minimum pulse required for TNF-induced signal transduction is concentration-dependent, and that at high TNF concentrations, a pulse as short as 10–30 sec can induce significant NF-κB translocation and a pulse of 30 sec or 1 min can induce caspase activation and apoptosis. A similar concentration-dependence was recently reported for the treatment duration required for LPS-induced NF-κB activation in mouse embryonic fibroblasts and PDGF-induced PDGFR phosphorylation in NIH-3T323in vivo, in a tissue or tumour, should therefore experience a “puff”, or pulse, of TNF at local concentrations that far exceed those measured in blood. Therefore a locally concentrated short TNF pulse, which we showed to be sufficient to initiate cell fate decisions, is also likely to be biologically relevant.We note here that although the maximal TNF concentration used in our study exceeds serum concentrations from patient samples, cytokine secretion in intact tissue is spatially restricted and efficiently captured by neighbouring cells46Based on the current understanding of TNF-induced signalling, cell-to-cell differences in the formation of Complex I and Complex II should alter the relative strength of pro-survival and pro-apoptotic signals. TNF dissociates slowly from TNFR1 ref. , over a 1920in vivo. The study of the interplay between intracellular and extracellular signals in the context of a TNF response will require experimental paradigms that integrate measurements and manipulations of both types of signals.Nevertheless, our results do not exclude the possibility that TNF-treated cells may also integrate additional pathways downstream of Complex I and Complex II to arrive at a duration-specified cell fate decision. Specifically, TNF-induced autocrine signals are known to contribute to the fate decisionOverall, our results show that treatment duration is an important mediator of TNF-induced signalling and cell death decision and that in certain contexts reaching the highest fractional kill may not require maximizing the duration of exposure to a pro-death stimulus. These findings meet a growing body of work showing that signalling dynamics as well as the timing and sequence of drug addition can all influence cell fate decisions3649505152532. Kym-1 cells2. For experiments in microfluidic devices, TNF treatments are described below. For experiments in 96-well imaging plates , on day 1, HeLa cells were seeded at ~4000 cells/well, Kym-1 cells were seeded at ~6000 cells/well. On day 2, culture medium was replaced with medium with 200 U/ml IFNγ  to sensitize cells to TNF-induced apoptosis . IFNγ is, like TNF, an inflammatory cytokine and it sensitizes many cancer cell lines to TNF-induced apoptosis, partly via increased initiator caspase-8 expression35363738HeLa cells  stably expressing EGFP-RelA . Medium was replaced then and again 24 hrs and 4 hrs before an experiment.® 647 conjugated-BSA . The device was securely mounted on a custom stage for a BD Pathway 855 BioImager and flow allowed to reach a steady state in the pre-pulse mode  on the BD Pathway 855 BioImager  using a UAPO/340 20x objective , capturing images at 3 min intervals for EGFP-RelA HeLa and at 10 min intervals for IC-RP HeLa. Data was extracted from flat-field and background corrected time-lapse images using ImageJ. Mean fluorescence intensity (MFI) of nuclear EGFP-RelA was collected for each cell at each time point using custom scripts. For caspase activity measurements, the ratios of MFI in the CFP and YFP channels were calculated. For Kym-1 cells, the time of cell death was determined by finding for each cell the first frame where membrane blebbing, a hallmark of apoptosis, was evident. For all experiments, cells that overlapped, left the imaging field or divided within an hour of TNF treatment were excluded from analysis.For all live-cell experiments, HeLa cells stably expressing EGFP-RelA or IC-RP were imaged for 30 min before addition of TNF. Wide-field epifluorescence and transmitted light live-cell imaging was done in an environmentally controlled chamber  which we defined as the minimum number of transcripts expected from a cell that has undergone a transcriptional response to TNF. We thus assumed that a cell with transcriptcell ≥ transcriptcutoff would have been likely to be transcriptionally activated in response to a stimulus. The second parameter is the fold-change threshold (FCthresh), a parameter that defines the value of ‘nuclear fold change’ at which significant transcriptional activation should occur. Because our dataset contains a pool of paired values that describe the maximum fold change (FCcell) as well as the transcript number (transcriptcell) for each of hundreds of single cells exposed to a range of TNF concentrations, we evaluated the total error as follows:transcriptcutoff correctly partitions the dataset into transcriptionally responsive and non-responsive cells. From this it follows that we assumed that a cell with transcriptcell ≥ transcriptcutoff mRNAs has undergone a transcriptional response, whereas a cell with fewer transcripts (i.e. transcriptcell < transcriptcutoff) has not.Assume that a given value of FCthresh, quantify the number of false positives and false negatives. False positives were defined as a cell for which FCcell ≥ FCthresh, but transcriptcell < transcriptcutoff. Thus this cell had a greater than threshold response, as indicated by change in nuclear NF-κB, but was not transcriptionally activated based on assumption in (1). False negatives were defined as a cell for which FCcell < FCthresh, and transcriptcell ≥ transcriptcutoff. Thus this cell had a sub-threshold response based on change of nuclear NF-κB, yet was transcriptionally activated as per (1).For each value of FCthresh is the total number of false positives and false negatives divided by the total number of cells. The total error was calculated across a range of FCthresh values in increments of 0.01.The total error for a value of To estimate the error in determining whether a cell has had a transcriptionally significant response to TNF based on the fold-change of nuclear NF-κB in a cell , we consFCthresh) where total error is minimized.Finally, the nuclear EGFP-RelA fold-change threshold for cellular response to TNF was defined as the fold-change threshold , 2) no-slip boundary conditions on the channel walls and 3) steady-state conditions were reached. The hydrostatic pressures at each inlets were calculated from its height relative that of the outlet reservoir.Three-dimensional simulations were carried out using COMSOL Multiphysics 3.5 , modelling the actual shape and dimensions of the device. Simulations solved Navier–Stokes equations with the different boundaries conditions and the following assumptions: 1) fluids similar to water , kf1 and kr1 are the association (1.833 × 107 M−1s−1) and dissociation rate (3.5 × 10−4 s−1) constants for TNF-TNFR1 and kf2 and kr2 are the association (2.5 × 107 M−1s−1) and dissociation rate (0.011 s−1) constants for TNF-TNFR2 .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


In addition, TNF increases the TRPV1 protein level and number of TRPV1-expressing neurons. Both effects could be abolished by the administration of RTX. These results suggest that the involvement of TRPV1 in TNF-induced pain offers new TRPV1-based experimental therapeutic approaches and demonstrates the analgesic potential of RTX in inflammatory pain diseases.Transient receptor potential vanilloid-1 (TRPV1) is a nonselective cation channel, predominantly expressed in sensory neurons. TRPV1 is known to play an important role in the pathogenesis of inflammatory and neuropathic pain states. Previous studies suggest interactions between tumor necrosis factor- (TNF-) alpha and TRPV1, resulting in a modulation of ion channel function and protein expression in sensory neurons. We examined the effect of intrathecal administration of the ultrapotent TRPV1 agonist resiniferatoxin (RTX) on TNF-induced pain-associated behavior of rats using von Frey and hot plate behavioral testing. Intrathecal injection of TNF induces mechanical allodynia (2 and 20 ng/kg) and thermal hyperalgesia (200 ng) 24 h after administration. The additional intrathecal administration of RTX (1.9  TNF is expressed by microglial cells and astrocytes in the central nervous system and by Schwann cells in the peripheral nervous system, especially after nerve injury [Tumor necrosis factor- (TNF-) e injury . In the e injury . Both TNe injury , 4. While injury  and can e injury –8. In ade injury –11, TNF e injury –11.Euphorbia resinifera and Euphorbia poissonii which are cactus-like plants. RTX irreversibly binds TRPV1 and induces a sustained calcium influx [TRPV1 is a nonselective cation channel that is predominantly expressed in sensory neurons, but can also be detected in other tissues such as human epidermal keratinocytes, the submandibular gland, and human hair follicles , 13. Itsm influx , 16. Addm influx , 18.Previous studies suggest interactions between TNF and TRPV1, resulting in a modulation of ion channel function and protein expression in the pathophysiology of inflammatory and neuropathic pain , 19, 20.RTX was shown to enhance the frequency of spontaneous excitatory postsynaptic currents (EPSCs) between DRG neurons and dorsal horn neurons of the spinal cord. This results in the reduction of pain sensitization, because of TRPV1 overactivation, leading to receptor inhibition . A decreThese data suggest a critical role for TRPV1 in TNF-induced neuropathic pain and an involvement of TNF in the potential analgesic effect of RTX.In this study, we examine the interactions of TRPV1 and TNF for pain processing after intrathecal administration of RTX by using behavioral tests and detection of protein levels in DRG neurons of rats.For behavioral experiments, male Sprague-Dawley (CD) rats were used . All experiments were performed in accordance with the guidelines of the Animal Care and Use Committees of the University of Duisburg-Essen, Germany, and the German regulatory authorities (G939/07). All animals were kept on a 14/10 h light/dark cycle with water and standard food pellets available ad libitum.TNF  was dissolved in a mixture of bovine serum albumin  and 0.9% sodium chloride (NaCl). TNF was used at concentrations of 2 ng/kg, 20 ng/kg, and 200 ng/kg. RTX  was dissolved in ethanol.μl) as described previously [5 and L6, perpendicular to the spine. When the needle entered the subarachnoid space, a lateral tail movement could be observed, as an indicator of successful puncture. TNF was applied in concentrations of 2 ng/kg, 20 ng/kg, and 200 ng/kg per injection . RTX wated from ). All prBaseline data were recorded daily for 3 days. TNF, NaCl, RTX, or TNF/RTX was administered on day 4. Behavioral tests were performed 1, 3, 6, and 24 h after drug administration.Thermal hyperalgesia was assessed with an algesimeter , as described previously . Three cn = 7 for each condition; total of 56 animals).Mechanical sensitivity was assessed using von Frey hairs and the up-down method, as described elsewhere . The 50%μm) of snap-frozen whole ganglia were placed onto microscopic slides. Three independent experiments were performed. DRGs of two animals per condition were pooled and randomly chosen for slice preparing. Five slices per condition were analyzed .DRG tissues were obtained as described previously . AnimalsTissue sections were fixed in 4% paraformaldehyde for 15 min and washed three times in phosphate-buffered saline (PBS). Cell membranes were permeabilized in PBS + 0.5% Triton X-100 for 15 min at room temperature. Unspecific binding sites were blocked with 5% milk powder in PBS for 1 h at room temperature with subsequent washing three times for 5 min at room temperature.To investigate the relative protein level of TRPV1, slices were incubated with rabbit primary antibodies specific for TRPV1 receptor protein .Mouse primary antibody for NeuN  was used as a neuronal marker. Incubation in the primary antibody was performed for at 4°C overnight. After washing, the slices were incubated with secondary antibodies  for 1 h in the dark at room temperature.μM) by using wet blotting technique. Membranes were blocked in Tris-buffered saline Tween-20 (TBST) containing 5% nonfat milk powder (Sigma-Aldrich) for 1 h at room temperature with gentle agitation. Membranes were incubated overnight at 4°C with rabbit polyclonal TRPV1 , rabbit polyclonal, and mouse polyclonal beta-III-tubulin antibodies . Afterwards, membranes were incubated with anti-rabbit IgG and anti-mouse IgG horseradish peroxidase  for 2 h at room temperature. Immunoreactivity was detected by using enhanced chemiluminescence substrate (Thermo Fisher Scientific). For Western blot analysis, three independent approaches were performed with one animal per condition for each experiment .For Western blot analysis, animals were treated as described above. DRG were removed 24 h after injection. The tissue was homogenized in RIPA buffer (Thermo Fisher Scientific) including protease inhibitor cocktail (Roche). Homogenates were subjected to 4–12% Bolt-Tris gels (Thermo Fisher Scientific). Proteins were transferred to nitrocellulose membranes . All data are reported as means ± SEM.Data were analyzed using ANOVA and Tukey's post hoc test. Values have been checked for normal distribution using a Shapiro-Wilk normality test. A result was considered to be significant when For examining alterations in TRPV1 protein level, DRG slices were analyzed with Zoe Fluorescent Cell Imager . Each image was taken with the same gain, brightness, and contrast values from all slices. TRPV1 protein levels were quantified using ImageJ software (NHI). Cells were selected using the Free Hand tool. The integrated density of TRPV1 in each NeuN-positive neuron was measured and normalized against the background signal in each image. Data were calculated from three independent approaches per condition. For each approach, a minimum of 100 cells from two rats were measured. Additional the number of TRPV1-positive neurons was counted and compared between each condition. Sensory neurons within the DRG were identified as TRPV1-positive when somata were clearly stained against NeuN (red) and TRPV1 (green), while TRPV1-negative neurons showed just a slight or no signal in the green channel.Analysis of Western blot signals were performed by using ImageJ software (NIH). Intensity of TRPV1 were normalized to intensity of beta-III-tubulin as a neuronal marker.In von Frey testing, a concentration of 2 ng/kg or 200 ng/kg TNF led to a significant reduction of paw withdrawal threshold after 24 h compared to untreated control animals, resulting in an induction of mechanical allodynia. When TNF was administered in a concentration of 20 ng, no significant change in withdrawal threshold was observed in comparison to control animals, suggesting an inversed bell-shaped dose dependency.p > 0.05). When RTX was administrated solely, no change in behavior was observed . Twenty-four hours after TNF administration, paw withdrawal threshold was significantly decreased to 8.9 ± 1.1 g (p < 0.01). Additional administration of RTX resulted in a significant change of paw withdrawal threshold after 1 h , while after 3 (16.9 ± 2.3 g) or 6 h (16.1 ± 1.2 g), no significant change in paw withdrawal threshold was observed (p > 0.05), compared to pretests. Twenty-four hours after administration, paw withdrawal threshold was not significantly altered (12.1 ± 2.3 g) compared to the pretest (p < 0.05). There was no difference between TNF and TNF + RTX administration after 24 h (p > 0.05), but the additional administration of RTX increased of paw withdrawal threshold significantly 3 and 6 h after, compared to TNF administration , 3 (13.7 ± 1.2 g), or 6 h (11.8 ± 1.1 g) compared to pretesting . Additional administration of RTX did not change the paw withdrawal threshold over the time range, compared to pretesting . When comparing data of TNF and TNF + RTX, a significant difference was observed after 24 h. Paw withdrawal threshold was increased after additional RTX administration, compared to sole TNF administration (p < 0.05)  .For examining heat hyperalgesia after intrathecal TNF administration, hot plate testing was performed. Only TNF in a concentration of 200 ng/kg led to a significant reduction of paw withdrawal threshold after 24 h compared to pretesting of untreated control animals. Administration of 2 or 20 ng/kg of TNF did not lead to significant change in paw withdrawal threshold over the experimental time.p > 0.05). When RTX was administered to control animals, no change in behavior was observed . Additional administration of RTX resulted in changes in paw withdrawal threshold over the experimental time compared to pretesting. When TNF + RTX data were compared to TNF, a significant increase of paw withdrawal threshold could be observed 3 h , 6 h , and 24 h  after administration , 3 h (11.5 ± 0.5 g), 6 h (11.6 ± 0.7 g), or 24 h (10.9 ± 0.6 g) compared to pretesting (stration .p > 0.05). Additional administration of RTX resulted in an increased paw withdrawal threshold of 23.8 ± 2.8 g after 24 h compared to pretesting (p < 0.05). When administration of TNF + RTX was compared to TNF administration alone, an increase of withdrawal threshold was observed after 1 h , 3 h , 6 h , and 24 h   .p > 0.05). Additional administration of RTX led to a significant increase of paw withdrawal threshold after 24 h (23.7 ± 2.6 g), compared to pretesting (p < 0.05). Combined administration with RTX abolished the TNF-induced reduction of paw withdrawal threshold after 24 h  , 3 h (11.5 ± 0.6 g), or 6 h (11.6 ± 0.4 g), no changes in withdrawal thresholds were observed ( < 0.05) .μg/kg), or a combination of TNF and RTX and sacrificed after 24 h. Protein levels of TRPV1 were determined in DRG slices by immunohistochemical staining (p < 0.05), compared to control conditions. In vivo administration of RTX led to a significant decrease of the TRPV1 protein level after 24 h in comparison to untreated control DRGs . When RTX was administrated additionally to TNF, the TNF-induced increase of theTRPV1 protein level was abolished. Here, the protein level of TRPV1 was significantly decreased , compared to TNF administration additionally , RTX . This effect was reduced by the additional administration of RTX   administration was observed  .p < 0.05). In contrast, RTX administration significantly decreased the number of TRPV1-positive cells within the DRG to 27.3 ± 0.5% (p < 0.05), compared to control DRGs. When TNF and RTX were administrated simultaneously, RTX abolished the TNF-mediated increase of TRPV1-positive neurons   .This study demonstrates the mechanism-based analgesic potential of intrathecal RTX administration on symptoms of TNF-induced pain, presenting as mechanical allodynia and thermal hyperalgesia in an inflammatory pain model. Additional intrathecal administration of the TRPV1 agonist RTX abolished TNF-induced thermal hyperalgesia and mechanical allodynia. These effects could be mediated by TNF-mediated increase and RTX-mediated decrease of TRPV1-expressing neurons within the DRG, underlining the critical involvement of TRPV1 in inflammatory and neuropathic pain.μg/kg). In contrast, carrageenan-induced mechanical allodynia was not affected by RTX administration [TNF induces mechanical allodynia and thermal hyperalgesia 24 h after administration. This result is in concordance with recent studies showing similar results in rat and mice models of neuropathic pain –30. The stration . Capsaicstration , 32. Thistration –36. Furtstration . In compstration . This maTNF neutralizing antibodies were given intraoperatively after 4 days, in two different models of neuropathic pain, chronic constriction injury (CCI), and partial sciatic nerve transection (PST) . Those fAdditionally, electrophysiological studies suggest a higher analgesic potency of TRPV1 agonist in the presence of TNF. In this study, we provide evidence by observing the strongest induction of neuropathic symptoms and an analgesic effect of RTX 24 h after TNF or TNF + RTX administration during our behavioral experiments. This finding could be explained by TNF-induced overexpression of TRPV1 or by enhanced inhibition of VGCC .Our results support a critical role of TRPV1 for typical signs of TNF-induced neuropathic pain, including thermal hyperalgesia and mechanical allodynia. Furthermore, we demonstrate an analgesic effect of TRPV1 agonist RTX, suggesting intrathecal TRPV1-acting drugs as a potential therapeutic approach for the treatment of neuropathic pain.


Inflammation plays a crucial role in kidney damage after crush syndrome (CS). Several researchers report that high mobility group box-1 protein (HMGB1) may be the vital trigger in kidney damage, and tumor necrosis factor-α (TNF-α) and c-Jun N-terminal kinase (JNK) are involve in this pathophysiological process, but their biological roles are unclear. This study aimed to explore the relationship between HMGB1, JNK, and TNF-α in kidney damage.The crush injury model was established using weight compression. The reliability of the crush injury model was determined by hematoxylin-eosin (HE) staining. Western blot was used to detect the expression of HMGB1, JNK, and TNF-α, and TUNEL was used to mark apoptotic cells in the renal cortex.The results showed that the highest expression of HMGB1 in muscle was 12 h after CS. JNK and TNF-α increased and peaked at 1 day after CS in kidneys. Western blot analysis revealed that anti-HMGB1 antibody could downregulate the expression of JNK and TNF-α. Anti-TNF-α could downregulate activation of JNK, and SP600125 could downregulate expression of TNF-α in the kidneys. In addition, anti-HMGB1 antibody, anti-TNF-α antibody, and SP600125 could reduce cellular apoptosis in the renal cortex.It is possible that JNK and TNF-α commonly contribute to kidney damage by assembling a positive feedback cycle after CS, leading to increased apoptosis in the renal cortex. HMGB1 from the muscle may be the trigger. Crush syndrome (CS) is a serious medical condition that can develop after traumatic events, such as earthquakes, landslides, and vehicle accidents . Most stRecently, numerous studies have focused on high-mobility group box 1 protein (HMGB1), since HMGB1 is reported to have a crucial role in the pathogenesis of systemic acute inflammation , 10. In Previous studies have demonstrated that HMGB1 could increase the production of TNF-α indirectly  and alsoHowever, the trigger effect of HMGB1 and the relationship between JNK and TNF-α expression is uncertain. In addition, it is not clear whether administration of anti-TNF-α, anti-HMGB1 antibodies, and SP600125, a JNK inhibitor, exerts a protective effect against TNF-α induced kidney damage. We investigated whether these administrations affected the apoptosis in kidneys.In total, 90 C57BL/6 male mice weighing 20–25 g were purchased from the Laboratory Animal Center of Xi’an Jiaotong University. Animals were housed and fed in a temperature- and humidity-controlled environment with standardized light/dark cycle (12 h day/night) for 1 week. All animal procedures were in accordance with the ARRIVE guidelines and the National Institutes of Health guide for the care and use of Laboratory animals. The study was approved by the Ethics Committee of Xi’an Jiaotong University.n = 6), and CS group (n = 24). Animals in the CS model were anesthetized with an intraperitoneal injection of 10% chloralhydrate (0.35 mL/kg) and were placed in a prone position, with both hind limbs (2 cm from the ankles up) compressed by 20 kg weights [n = 6 in each group) after compression. Mice in the normal group were euthanized on day 2. One kidney and muscle from one hind limb in each mouse was taken for HE staining, and the remaining muscle and kidney were obtained for western blotting.In the first phase, 30 mice were randomly assigned to 2 groups: normal group , CS + vehicle (n = 12), CS + anti-HMGB1 antibody (n = 12), CS + anti-TNF-α antibody (n = 12), and CS + SP600125 (n = 12). The CS model was established as described above. Intraperitoneal injections were performed 1 h after compression was released, at a total volume of 200 μl. Dimethyl sulfoxide (DMSO) was used as a solubilizer. The dosage of anti-TNF-α antibody (5 μg/200 μl), SP600125 (10 μg/200 μl), and anti-HMGB1 antibody (2.5 μg/200 μl) [In the second phase, 60 mice were randomly assigned to 5 groups: CS  were separated on a sodium dodecyl sulfate-polyacrylamide gel 12% gel) and electrotransferred onto polyvinylidene fluoride membranes. After 2 h incubation in blocking solution , the membranes were blotted with primary antibodies against HMGB1 , TNF-α , p-JNK , JNK , or β-actin  overnight at 4 °C. After extensive rinsing with TBST buffer, the blots were incubated with HRP-conjugated anti-rat/mouse secondary antibodies . Specific bands were detected using X-ray film. The density of the bands was analyzed by Quantity one software version 4.62 .2O2 in methanol to inactivate endogenous peroxidase. After washing with PBS, specimens were incubated in the labeling reaction mixture containing terminal deoxynucleotidyl transferase. After incubation, the sections were incubated with horseradish peroxidase. The sections were then treated with DAB solution and were counterstained with hematoxylin. Finally, the sections were mounted to a glass slide with a cover slip.We used a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) reaction by means of TUNEL Kit  to detect apoptotic cells. The tissue sections were deparaffinized with xylene, and were treated with HP < 0.05 was considered statistically significant.SPSS 18.0  was used for statistical analyses. All data were presented as mean ± SD. Comparisons among multiple groups were performed using ANOVA. Comparisons between the two groups were performed using the LSD test. In total, 25 mice died after the operation and administration of drugs during the experiments. There was no death in the normal, while the number of deaths was 1, 3, 2, and 2 in the 0 h, 12 days, 1 day, and 2 days groups, respectively. The death in 0 h was during the process of compression. In the second phase, 5, 5, 2, 3, and 2 deaths were observed in the CS, CS + vehicle, CS + anti-TNF-α antibody, CS + SP600125, and CS + anti-HMGB1 antibody groups, respectively. The 1 and 2 deaths in CS and CS + vehicle were during the process of compression. After CS, physical activities of mice reduced obviously, with both hind limbs prolapsing. Dead mice were excluded from further analysis. In all the living mice, injured skeletal muscles were found to be hemorrhagic and with edema.Pathological changes in muscles and kidneys were detected via HE staining, to assess the reliability of our CS model. As shown in muscle pathological sections, there was bleeding, degeneration, swelling, and necrosis apparent in part of the muscle fibers and lymphocytic infiltration in the CS groups Fig. , compareWestern blot analysis revealed a significant upregulation of HMGB1 in muscle, and p-JNK, and TNF-α in kidneys following CS, compared with the normal groups. It showed a gradual increase of HMGB1 expression until reaching a peak 12 h after crush injury in muscle tissue Fig. . In kidnSince the expressions of JNK and TNF-α increased to a peak 1 day post-CS, we chose this time point to sacrifice mice for the second phase experiments. When anti-HMGB1 antibody was administered to block the activation of p-JNK and production of TNF-α, western blot analysis revealed that anti-HMGB1 induced a downregulation of p-JNK and TNF-α expression, compared with the high levels detected in the CS and vehicle group Fig. , b.Fig. When anti-TNF-α antibody was administered, western blot analysis showed that p-JNK/t-JNK was downregulated compared to the CS group Fig. . When SPThere were few TUNEL-positive cells in the normal renal cortex region Fig. , but theThis study focused on the trigger role of HMGB1 from the muscle, and the detailed relationship between JNK and TNF-α in the kidneys. Our results indicate the following main findings. Firstly, HMGB1 and pro-inflammatory cytokines are activated rapidly in the skeletal muscle and released from the nucleus to the extracellular matrix. JNK is then activated gradually in the kidneys, while TNF-α shows a similar tendency to JNK. Secondly, JNK and TNF-α in the kidney can be downregulated by blocking the action of HMGB1 (anti-HMGB1 antibody). Three types of TNF-α in kidney can be downregulated by blocking the action of JNK (SP600125). Thirdly, administration of anti-HMGB1, anti-TNF-α, and SP600125 can decrease apoptosis in kidneys, respectively.Currently, the animal models of CS are mainly rats , mice 2, or rabbBased on our results, HMGB1, JNK, and TNF-α seem to assemble a positive feedback cycle. HMGB1 from injured muscle activates JNK in the kidneys and JNK enhances production of TNF-α, which in turn enhances the production of JNK, resulting in soaring TNF-α level, which contributes to kidney damage. The level of HMGB1 in serum would be a meaning marker to evaluate the severity of CS injury. SP600125 and anti-HMGB1 antibody may be the possible drugs to alleviate the inflammatory response and damage.Anti-HMGB1 antibody is reported to have a neutralizing effect on renal ischemia-reperfusion injury, and it also suppresses the number of apoptotic cells . The resThere are some limitations in this study. Firstly, apoptosis has been reported to be involved in kidney damage at the cellular level after experimental CS , 30. In In conclusion, it is possible that JNK and TNF-α commonly contribute to kidney damage, by assembling a positive feedback cycle after CS, leading to increased apoptosis in the renal cortex. HMGB1 from the muscle may be the trigger.


Toxoplasma gondii is an intra-cellular protozoan parasite that can infect almost all nucleated cells, eliciting host immune responses against infection. Host tissue damage is mainly caused by cellular lysis when T. gondii egresses from infected cells. However, the effects of cytokines released by host immune cells on egression of T. gondii remain elusive. This study aimed to investigate the role of tumor necrosis factor-alpha (TNF-α) on the egress of T. gondii from infected human foreskin fibroblast (HFF) cells and to elucidate the underlying mechanisms that regulate TNF-α-induced egress. Using flow cytometry to count tachyzoites of T. gondii released into cell culture medium, we found that egress of T. gondii from infected HFF cells could be induced by 10 ng/mL TNF-α in a time-dependent manner. Pre-treatment of infected HFF cells with BAPTA-AM to chelate intra-parasitic calcium could greatly inhibit TNF-α-induced egress. Similar results were obtained when using cytochalasin D to block parasite motility before the TNF-α-induced egress assay. In addition, blocking host apoptosis by Z-VAD-FMK could decrease TNF-α induced egress, while blocking necroptosis by necrostatin-1 has little impact on TNF-α-induced egress. The egressed tachyzoites displayed a normal growth rate and lost no virulence. Our results suggest that host cytokines could influence the cellular lytic processes of T. gondii, providing new insights into the relationship between host TNF-α and T. gondii pathogenesis. Toxoplasma gondii is an obligate intra-cellular parasite that can infect almost all kinds of nucleated cells in warm-blooded animals, leading to toxoplasmosis [T. gondii commonly causes a self-limiting, usually asymptomatic infection in immunocompetent hosts, it can cause serious disease in the central nervous system in individuals with immunocompromized immune systems, such as AIDS patients, or immature immune systems, such as in newborns [lasmosis . Althougnewborns .T. gondii goes through three distinct processes: active invasion, intra-cellular replication and egress. Host tissue or organ damage is mainly caused at the egress stage when parasites exit from infected cells. The egressed motile parasites further invade neighboring cells to expand their distribution in hosts. Like invasion, parasitic motility plays a critical role in T. gondii egress [Toxoplasma perforin-like protein 1 (TgPLP1) displays structural features necessary for pore formation, which facilitates the natural egress of tachyzoites from infected cells [Toxoplasma calcium-dependent protein kinase 1 (TgCDPK1) and TgCDPK3 [T. gondii, both of which function in a calcium (Ca2+)-dependent manner. To investigate the molecular mechanisms involved in parasitic egress, researchers have used various chemicals to induce early egress of T. gondii. Dithiothreitol (DTT) can activate parasitic nucleoside triphosphate hydrolase (NTPase), leading to depletion of host cell ATP levels and release of tachyzoites from infected cells [2+ flux induced by DTT may be plausibly linked to NTPase activation and parasitic egress [Staphylococcus aureus α-toxin-induced egress assay and a nigericin-induced egress assay [T. gondii also detect potassium (K+) efflux from host cells before moving out of infected cells.To complete its cellular lytic life cycle, i egress . Moreoveed cells . Other p TgCDPK3 ,13, haveed cells , while ac egress . In a Stss assay ,16, it wT. gondii infection are crucial for the establishment of a balanced host-parasite relationship. The typical host immune responses against T. gondii infection are production of IL-12, which further activates NK cells to produce IFN-γ, driving the proliferation of type I CD4+ and CD8+ T cells to produce more IFN-γ. IFN-γ further activates T. gondii-infected macrophages to produce nitric oxide (NO) or tumor necrosis factor (TNF), which control replication of the intra-cellular parasite. A number of studies have since provided evidence indicating that certain immune factors, such as IFN-γ [T. gondii from infected cells. T lymphocytes acting on T. gondii infected target cells via death ligand- or perforin/granzyme-dependent cytotoxicity also trigger the egress of infectious parasites with the ability to infect surrounding cells, including the effector cells [T. gondii from infected target macrophages. This suggests that other immune factors could serve as a bridge linking host immune responses and parasitic egress [Host immune responses induced by as IFN-γ ,18 and Nas IFN-γ ,23, may c egress .T. gondii succumbed within the first 2 week of the infection, with high transcription levels of TNF-α [T. gondii-infected macrophages, and neutralization of TNF-α inhibited the effect of CD154 on macrophage anti-T. gondii activity. A high TNF-α level contributes to the inflammatory response and to damage of the choroid and retina in ocular toxoplasmosis patients [T. gondii infection, TNF-α may induce egress of tachyzoites from infected cells, which helps parasites invade other cells for mass replication. However, in the late stage of T. gondii infection, activated macrophages can eliminate TNF-α-induced egressed T. gondii. In this study, we investigated the effects of TNF-α on egress of T. gondii from infected human foreskin fibroblast cells (HFFs). TNF-α could trigger early egress of tachyzoites in a time-dependent manner. Intra-parasitic calcium, parasitic motility and the host apoptosis pathway were crucial for TNF-α-induced egress. There were no significant changes in growth rate and virulence of egressed parasites.TNF-α is considered a pro-inflammatory cytokine involved in innate immune response. IL-10 knock-out mice infected with of TNF-α . CD154 ipatients . HoweverToxoplasma tachyzoites  expressing yellow fluorescent protein (YFP) in parasitic cytosol were propagated by serial passage in monolayers of human fibroblasts (HFFs). Briefly, HFFs  were grown in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum (FBS) . Parasites were harvested after 3 to 5 days in culture, when the fibroblast host cell monolayer reached 80 to 90% lysis. Recombinant human tumor necrosis factor-alpha (TNF-α) was purchased from Sangon Biotech . Other reagents used in this study were Cytochalasin D (Cyto-D), calcium chelator BAPTA-AM, necroptosis inhibitor Necrostatin-1 (Nec-1), and apoptosis inhibitor Z-VAD-FMK .T. gondii and TNF-α-induced egress, HFFs infected with tachyzoites at various times  were treated with 10 ng/mL TNF-α for 6 h. The egressed tachyzoites were collected by centrifuging at 1000 rpm for 10 min. Then, tachyzoites were re-suspended with 1 mL cell culture medium and counted using flow cytometry.HFFs cultured in 6-well tissue culture plates were used as host cells in this study. Near confluent HFF monolayers were infected with tachyzoites at a multiplicity of infection (MOI) of 0.5 to 1 in a 37 °C incubator for 30 minutes (min) in invasion medium, followed by removal of extracellular parasites through washing with pre-warmed phosphate-buffered saline (PBS). 24 to 36 hours post-infection (hpi), infected cells were treated with 10 ng/mL TNF-α for 3 hours (h) or 6 h. To determine the relationship between intra-cellular development of In some experimental sets, infected HFFs (about 36 hpi) were pre-treated for 10 min with 1 µM or 5 µM cytochalasin D  to block parasitic motility. Intra-parasitic calcium was chelated by BAPTA-AM  before TNF-α treatment.T. gondii for 24∼36 h were pre-treated with 20 µM necrostatin-1 (necroptosis inhibitor) or 10 µM Z-VAD-FMK (apoptosis inhibitor) for 20 min, followed by TNF-α treatment .To determine whether TNF-α-induced tachyzoite egress is dependent on the host apoptosis or necroptosis pathway, HFFs infected with 2 tissue culture flasks and then treated with 10 ng/mL TNF-α for 6 h; egressed parasites were collected at 1000 rpm for 10 min. For the in vitro growth assay, egressed tachyzoites were added to HFF cells at an MOI of 0.5-1.0, the number of PV containing different numbers of parasites was determined by microscopic examination 24 hpi. The numbers of rosettes  were also counted. Ten fields were directly (without fixing) counted using an Olympus IX 51 fluorescent microscope. Meanwhile, egressed tachyzoites were used to infect C57BL/6 mice intra-peritoneally , and the survival rates after challenge were confirmed. Natural egressed parasites (about 72 hpi) were used in the control group. All animals received humane care, and all testing methods were performed in accordance with the Institutional Guidelines of Anhui Medical University for the Care and Use of Laboratory Animals; the study methods were carried out under the protocol approved by the Institutional Animal Care and Use Committee of Anhui Medical University.RH-YFP tachyzoites were allowed to grow in HFF cells for 36 h in 25 cmt-test or one-way analysis of variance (ANOVA). Differences were considered statistically significant at a p-value < 0.05.All statistical analyses were processed by SPSS 20.0 Data Editor software , and all data expressed as means ± SD values. Data analysis methods were T. gondii, HFFs infected with tachyzoites for 36 h were treated with 10 ng/mL TNF-α for various time periods. As shown in vs 1608.2 ± 274.68, p < 0.05). Next, we determined whether TNF-α-induced parasite egress was dependent on the intra-cellular development of T. gondii. We infected HFFs with tachyzoites for various time periods, and then treated infected cells with TNF-α. As we can see in vs TNF-α, 1850.6 ± 508.15 vs 30273.6 ± 7957.53, p < 0.05), indicating that TNF-α-induced egress may depend on the developmental stages of T. gondii in host cells.To test whether exogenous TNF-α could trigger early egress of T. gondii, we performed experiments to determine the essentiality of calcium in TNF-α-induced egress of T. gondii. As shown in vs 6568.2 ± 1507.51, p < 0.05). Pre-treatment with Cyto-D to block parasitic motility yielded similar results (p < 0.05). Thus, TNF-α-induced egress of T. gondii relied not only on intra-parasitic calcium but also on parasitic motility.As previous studies showed that intra-parasitic calcium plays an important role in egress of  results B, althouT. gondii through activation of the host apoptosis pathway [T. gondii, we pre-treated infected HFFs with apoptosis inhibitor Z-VAD-FMK and found that, compared with the control group . Meanwhile, there was no significant difference of virulence between egressed parasites and control parasites.To detect whether the tachyzoites were infectious after TNF-α-induced egress, we collected egressed parasites to infect HFF cells or inoculate C57BL/6 mice intra-peritoneally. As shown in T. gondii can infect all nucleated cells in a diverse array of species, causing tissue or organ damage by lysis of infected host cells through egress. Currently, many researchers have shifted their focus to study the molecular mechanism of how T. gondii egress from their host cells [T. gondii from infected cells [in vivo egress of T. gondii.st cells ,4,20. Pred cells ,20. HoweT. gondii infection elicits host immune responses, mainly CD8 T cell-mediated immune responses, accompanied by the production of cytokines, such as IL-12 and IFN-γ. It has been reported that IFN-γ could induce early egress of tachyzoites from astrocytes and HFFs [T. gondii from HFFs. However, unlike the results of studies showing that IFN-γ-induced egress of tachyzoites from astrocytes and calcium ionophore-induced parasite egress from LLC-MK2 cells did not depend on intra-cellular development of T. gondii [Immunologically, and HFFs ,17. In t. gondii , TNF-α-iT. gondii was mediated through the release of intra-cellular calcium as a consequence of caspase activation early in the apoptotic cascade [T. gondii, which was partly similar to a previous report showing that IFN-γ-stimulated cell death, which could induce parasitic egress without replication, was not inhibited by necrostatin-1 [T. gondii tended to induce apoptosis of infected cells before egress [T. gondii egress need further in-depth investigation.Consistent with previous reports demonstrating that death receptor ligation or exposure to perforin triggering rapid egress of  cascade , we founstatin-1 . As a pre egress , combineT. gondii shortly after invasion into LLC-MK2 cells (2 hpi), but these egressed parasites could not further multiply inside host cells, indicating that such parasites lost their infectivity [Calcium ionophore induced egress of ectivity ,23. It iectivity . AlthougT. gondii from infected fibroblasts, suggesting a novel role of host inflammatory factors in intra-cellular pathogen infections. Further investigations are needed to confirm whether this inflammatory factor-induced egress of T. gondii is found in vivo. The effector protein of T. gondii interacting with molecules of the host TNF-α signal pathway should be identified, which may provide new directions for anti-toxoplasmosis drug development.In conclusion, our study found that host cytokine TNF-α could serve as an inducer to trigger egress of The authors declare that they have no conflicts of interest in relation to this article.


TNF-α) is a multifunctional cytokine that regulates different cellular activities related to spermatogenesis. Tumor necrosis factor-alpha receptor 1 (TNFR1) mediates TNF-α activity and polymorphism in TNFR1 could lead to gene dysfunction and male infertility.Tumor necrosis factor-alpha  method was used to investigate the polymorphism in both case and control groups. PCR fragments were digested by Mspa1I enzyme and products were appeared by gel electrophoresis. The abundance of A→G was calculated in the azoospermic and healthy men.This case-control study included 108 azoospermic and 119 fertile men. This research investigated the frequency of According to the present study, GG and AG genotypes frequency in the azoospermic men group were higher than the control group (OR= 2.298 (1.248-4.229), p=0.007), . Our findings also showed that G allele frequency in azoospermic men had significant difference compared to the control group (OR=2.302 (1.580-3.355), p<0.001).It seems that the GG genotype and G allele have an association with increased risk of non-obstructive azoospermia. Infertility is defined as an inability of a couple to conceive after one year of unprotected sexual intercourse , 2. SeveStudies on specific genes in humans and experimental models have shown the impact of genetic factors on infertility . Tumor nTNFR1 gene is located on chromosome 12p13.2  were enrolled as the case group and 119 normal fertile men as the control group. Cases diagnosed by urologists after semen analysis and clinical examination in Research and Clinical Center for Infertility according to criteria of World Health Organization . Cases hSamplingTNFR1 gene fragment, and then electrophoresis was done on 2% agarose gel that 183-bp band was observed. The enzymatic digestion was carried out by the MSPA1I enzyme, then the samples were electrophoresed on 3% agarose gel.Five ml of peripheral blood sample was collected in tubes containing anticoagulant EDTA (ethylenediaminetetraacetic acid) from each participant. Salting out method was used for DNA extraction. Extracted DNA quality was examined using Nanodrop device and the samples within the standard range were selected. The target gene was amplified using specific primers and polymerase chain reaction (PCR) techniques. PCR products were transferred onto agarose gel after affecting the restriction enzyme. The results of electrophoresis were analyzed. Digestion by MSPA1I enzymeAfter PCR reactions, the final analysis was performed by PCR-RFLP method with the help of restriction MSPA1I enzyme for all participants. After optimizing the enzymatic digestion conditions, samples were cut and then were taken on 3% agarose gel by enzyme MSPA1I (New England Biolabs (Neb) Co. Catalog no. R0577S). The length of amplified products containing this polymorphism was 183 base pairs. In the present study, three different patterns were expected to be observed on gel electrophoresis, including homozygous individuals (AA): a band in 183-bp region, heterozygous individuals (AG): three bands in 183-bp, 108-bp and 75-bp regions, as well as homozygous individuals (GG): two bands in 108-bp and 75-bp regions. Ethical committeeEthical committee in Yazd Reproductive Sciences Institute approved the methods of this study and informed consent form was signed by all participants before starting this study.Statistical analysisAfter obtaining the relative frequency of alleles and genotypes in both studied groups (infertile and control), significant levels of frequencies, the difference between the two groups and their relationships with azoospermia were assessed by chi-square test using Statistical Package for the Social Sciences, version 20, SPSS Inc, Chicago, Illinois, USA (SPSS). P-value, Odds Ratio and OR Confidence Interval were calculated for the frequencies and parameters. In the present study, 108 non-obstructive azoospermic men and 119 controls were participated. The Mean age of cases and controls were 33.67 and 33.25 years respectively (p=0.104). Minimum and maximum duration of infertility in patients were between 2 and 20 years.Three different patterns were observed for bands appeared on gel electrophoresis, including AA genotype (183 bp), AG genotype  and GG genotype (108bp and 75bp). Present results demonstrated that the frequency of GG genotype in cases and controls was significantly different (p=0.007).The frequency of GG genotype was higher in cases than controls. The frequency of A and G alleles of TNF R1 36 A/G polymorphism was significantly different between cases and controls, and the risk of non-obstructive azoospermia was greater in men with GG genotype . Also, the difference in A and G alleles frequency was significant between groups (p<0.001) and the risk of azoospermia in people with G allele was higher  .This study was conducted to assess this polymorphism and its prevalence among patients with idiopathic azoospermia. Results of the present study showed G allele and GG genotype of TNFR1 36 A/G polymorphism is significantly higher in azoospermic infertile men. There are numerous reasons for infertility, which may be related to abnormalities in male or female or both . The infIt is estimated that about 40-50% of men have qualitative and quantitative abnormalities in their sperms . SpermatHosono et al examined the expression levels of TNF-α and its receptors in patients with Colorectal Adenoma. They determined that serum levels of TNFR1 were significantly higher in these patients compared with the control group, as well as found no significant correlation between serum levels of TNF-α and TNFR2 between patients and control group . The serTronchan et al in France population examined the relationship between 36 A/G polymorphism of TNFR1 with male infertility and sperm abnormalities using PCR-RFLP and ASPCR methods. The results demonstrated that allele A of TNF-α is associated with sperm motility and count, which increases the TNF-α expression . TNF-α aIn addition, TNF-α decreases germ cell apoptosis through suppressing Fas ligand system. On the other hand, increase of TNF-α level is correlated with sperm DNA chromatin abnormalities and reduction of testosterone synthesis. Increased expression of TNF-α is associated with impaired sperm motility and count. For this reason and due to the role of TNFR1 as the TNF-α receptors, it is expected to be observed the effects similar to the effect of promoted TNF-α by increasing expression of this receptor. Bialas and colleagues in 2009 investigated the role of IL-6, IL-10, TNF-α-, TNFR1, and TNFR2 in the processes of normal and abnormal spermatogenesis as well as testicular tumors using Real-Time PCR technique. Their study showed a significant relationship between promoted expression of IL-6 and testicular tumors, but no association about IL-10. In addition, it was observed that TNFR1 expression levels increased significantly in patients with impaired sperm maturation compared with patients with testicular tumors. However, no significant correlation was found in the TNFR2 . In compThe findings of the present study revealed a significant increase in G allele among patients with azoospermia compared with normospermic male. It can be concluded that the elevation in G allele might be effective in increasing male azoospermia.


To investigate whether GILZ could antagonize TNF-α-induced arthritic inflammation and protect bone in mice, we generated a TNF-α-GILZ double transgenic mouse line (TNF-GILZ Tg) by crossbreeding a TNF-α Tg mouse, which ubiquitously expresses human TNF-α, with a GILZ Tg mouse, which expresses mouse GILZ under the control of a 3.6kb rat type I collagen promoter fragment. Results showed that overexpression of GILZ in bone marrow mesenchymal stem/progenitor cells protected mice from TNF-α-induced inflammatory bone loss and improved bone integrity . However, mesenchymal cell lineage restricted GILZ expression had limited effects on TNF-α-induced arthritic inflammation as indicated by clinical scores and serum levels of inflammatory cytokines and chemokines.TNF-α plays a key role in the development of rheumatoid arthritis (RA) and inflammatory bone loss. Unfortunately, treatment of RA with anti-inflammatory glucocorticoids (GCs) also causes bone loss resulting in osteoporosis. Our previous studies showed that overexpression of glucocorticoid-induced leucine zipper (GILZ), a mediator of GC’s anti-inflammatory effect, can enhance osteogenic differentiation  Chronic inflammatory conditions such as rheumatoid arthritis (RA) and inflammatory bowel disease are known to cause bone loss , 2. TumoHigh doses of GCs have been used for decades in the treatment of autoimmune disorders such as RA , 27. HowAll chemicals were purchased from Thermo Fisher Scientific  or Sigma-Aldrich  except where specified.2 overdose followed by thoracotomy according to AU-IACUC approved animal protocols.TNF-α transgenic (TNF-α Tg) mice overexpressing human the TNF-α gene were provided by Dr. George Kollias . The GILAll mice were evaluated weekly for signs of arthritis at the age of 7 weeks in a blinded fashion. A clinical score was assigned to each ankle joint using a semi-quantitative scoring system : 0 for nTen to twelve mice from each experimental group  were assessed for bone density and architecture. The left femur of each mouse was dissected free of soft tissues and scanned by DXA densitometry  for bone mineral density (BMD) and bone mineral content (BMC) measurement. Bone structural parameters of the right femur and tibia of each mouse were analyzed by μ-CT scan  as previously described .After DXA scan, the left femur and ankle of each mouse were decalcified in EDTA, embedded in paraffin and sectioned at 4–6 μm for histomorphometry. Sections were stained with hematoxylin and eosin for calculation of osteoblast numbers. A Bioquant Osteo image analysis system  was used to count the number of osteoblasts and measure the osteoblast covered areas. A defined region of interest was established ~0.5mm proximal to the distal growth plate and extended a further 0.5mm, all within the endocortical edges at 40x magnification.2O2 in PBS. The sections were then washed in distilled water and heated at 95°C in antigen retrieval buffer . Nonspecific staining was blocked with 5% normal goat serum in PBS for 1 h. Endogenous biotin was inhibited with an avidin/biotin blocking kit . The sections were then incubated with an anti-osteocalcin polyclonal antibody  diluted at 1:1000 in 2% normal goat serum overnight at 4°C. Next, the sections were incubated with biotinylated goat anti-mouse/rabbit antibody   for 30 min. Sections were then washed and incubated with StreptABComplex/HRP (Dako) for 45 min. Antibody binding was revealed using a DAB kit . Negative controls were obtained by omission of the primary antibody. Counterstaining was performed with Mayer's hematoxylin (Sigma).For immunohistochemistry, paraffin sections of femurs were deparaffinized in xylene and rehydrated by passing the slides through graded alcohol solutions. Endogenous peroxidase was quenched with 3% HSynovial fibroblasts were isolated from the paws of both GILZ and TNF-α Tg mice according to the protocol of Armaka et al. . Briefly5′-GCTGCACAATTTCTCCACCT-3′ (forward) and 5′-GCTCACGAATCTGCTCCTTT-3′ (reverse); β-actin (NM_007393): 5′-CTGGCACCACACCTTCTACA-3′ (forward) and 5′-GGTACGACCAGAGGCATACA-3′ (reverse); IL-6 (NM_031168.2): 5’-GCCAGAGTCCTTCAGAGAGATACA-3’ (forward) and 5’-CTTGGTCCTTAGCCACTCCTTC-3’ (reverse).Total cellular RNA was isolated using TRIzol reagent following manufacturer's instructions . Equal amounts of total RNA (2 μg) were reverse transcribed using iScript cDNA synthesis kit  and the mRNA levels of the indicated genes were analyzed in triplicate using SYBR Green Master Mix and a Chromo-4 real-time RT-PCR instrument. The mRNA levels were normalized to β-actin , and gene expression was presented as fold change. The primer sequences used in the PCR reactions were: GILZ (AF024519): Three to four mice were used in each group and the experiment was repeated twice. Both tibiae and femur were dissected free of soft tissues and bone marrow cells flushed with RPMI 1640 medium supplemented with 10% FBS. Blood was collected by cardiac puncture. Cells were first incubated with antibodies against cell surface markers CD11b, and then fixed and permeabilized using Fixation/Permeabilization Concentrate  before incubation with antibodies for intracellular labeling of human TNF-α . After one wash, cells were run through a 4-color flow cytometer , and data were collected using CellQuest software  as previously described . As a gaBlood samples were centrifuged at 2,000 rpm for 10 min at 4°C and the serum collected and stored at −80°C in aliquots. Receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) levels were measured using Quantikine ELISA Kits . Serum concentrations of selected cytokines and chemokines were measured using a fluorescent bead-based Multiplex immunoassay on a Luminex 200 machine . Serum collected from TNF Tg and TNF-GILZ Tg mice  were analyzed. All samples were assayed in duplicate.t tests, using Prism 5.0 software. A p value less than 0.05 is considered significant.The results are expressed as means ± S.D. The data were analyzed using either analysis of variance with Bonferroni post hoc testing or unpaired To test whether GILZ can offset or reduce the degree of TNF-α-induced arthritic inflammation, we created TNF-GILZ double transgenic (TNF-GILZ Tg) mice by crossbreeding GILZ Tg mice with TNF-α Tg mice. The TNF-α Tg mouse carries a human TNF-α gene and is expressed ubiquitously . This moWe then examined whether GILZ can protect TNF-α-induced inflammatory bone loss. DXA analysis showed that both the bone mineral density (BMD) and bone mineral content (BMC) exhibit a trend of increased mineralization in TNF-GILZ mice when compared with TNF-α mice, although these increases were not statistically significant . HoweverTo determine if the inflammatory profile was altered in TNF-GILZ double Tg mice, we measured serum levels of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL), two factors that play important roles in bone metabolism, and several inflammatory cytokines and chemokines that are involved in arthritic inflammation and bone loss , 43–47. Finally, we examined the local effects of GILZ in bones and joints, as well as in fibroblast-like synoviocytes (FLS) since these cells are considered bona fide MSCs . High leRheumatoid arthritis (RA) is a chronic inflammatory disease affecting not only joints but also the skeleton leading to systemic bone loss and increased risk of fractures. TNF-α is one of the key factors responsible for inflammation and bone loss in RA . Studiesin vitro and it promotes bone acquisition in mice [in vitro [Glucocorticoids (GCs) are among the most potent anti-inflammatory drugs for treatment of RA, but their clinical usage is limited due to their severe side effects on bone (GC-induced osteoporosis) , 53, 54. in mice , 37, Fur in mice  and antain vitro . These sin vitro , 37, butInterestingly, we noticed that local expression of IL-6 in bone and joint, and in FLS is significantly reduced in TNF-GILZ double Tg mice compared to that in TNF-αTg mice. This seemed unlikely since the FLS cells reside in joint which is outside of bone marrow cavity and should not overexpress GILZ in TNF-GILZ mice. However, there are evidence showing that the onset of RA is associated with a massive influx of mesenchymal cells into the joint , 57. TheS1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.


RTN3 both evades cooling-induced translational elongation repression and is also bound by RBM3, which drives the increased expression of RTN3. In mice, knockdown of RTN3 expression eliminated cooling-induced neuroprotection. However, lentivirally mediated RTN3 overexpression prevented synaptic loss and cognitive deficits in a mouse model of neurodegeneration, downstream and independently of RBM3. We conclude that RTN3 expression is a mediator of RBM3-induced neuroprotection, controlled by novel mechanisms of escape from translational inhibition on cooling.Cooling and hypothermia are profoundly neuroprotective, mediated, at least in part, by the cold shock protein, RBM3. However, the neuroprotective effector proteins induced by RBM3 and the mechanisms by which mRNAs encoding cold shock proteins escape cooling-induced translational repression are unknown. Here, we show that cooling induces reprogramming of the translatome, including the upregulation of a new cold shock protein, RTN3, a reticulon protein implicated in synapse formation. We report that this has two mechanistic components. Thus,   •RTN3Cooling-induced reprogramming of the translatome increases synthesis of •RTN3 mRNA and drives its expressionThe neuroprotective protein RBM3 binds •RTN3 overexpression prevents synaptic loss in mice with prion disease•RTN3 expression is a mediator of RBM3-induced neuroprotection Therapeutic hypothermia is neuroprotective, and the cold shock protein RMB3 plays a critical role in mediating synaptic repair processes that accompany cooling. Bastide and Peretti et al. show that cooling selectively reprograms the translatome and identify RTN3 as a cold-induced protein that acts downstream of RBM3 in the neuroprotection pathway. Therapeutic hypothermia is a powerful neuroprotectant, acting through multiple mechanisms, although the underlying pathways are not fully understood , 2. We rUpon cooling, the changes to the protein synthesis machinery are similar to those observed with other stress inducers  , 11 and RBM3 and RTN3 evade translational repression and that RBM3 binds RTN3 mRNA and plays a major role in driving cooling-induced upregulation of RTN3 expression. Finally, we show that RTN3 expression, downstream of RBM3 induction, mediates cooling-induced neuroprotection in mice with neurodegenerative disease and importantly is neuroprotective even in the absence of cooling.To examine the relationship between cold stress, RBM3 induction, and the modulation of mRNA translation for the synthesis of putative neuroprotective proteins, we investigated the post-transcriptional response to hypothermia in vitro and validated the data in vivo in a mouse model of neurodegeneration, in which we know cooling is protective, mediated by RBM3. We show that, following cold shock/cooling, the global decrease in protein synthesis rates is associated with selective reprogramming of the translatome. We find enhanced synthesis of specific proteins: not only the cold shock protein RBM3 (as predicted) but also of a number of proteins with a role in development and function of the nervous system, including reticulon 3 (RTN3). RTN3 has a role in synaptic plasticity and synapse formation , 19 and Both transcriptional and post-transcriptional control mechanisms are required for the overall response to cell stress . In ordeSucrose density gradient analysis was carried out on cooled samples to separate polysomes and the associated transcripts C, which RTN3 and Noggin, contained codons that require less abundant tRNAs in the 5′ end of the transcripts . For induction of cold stress, cells were incubated at 32°C or at 25°C for 24 hr before harvesting. Small interfering RNAs (siRNAs) were transfected using DharmaFECT 1 and plasmids with Lipofectamine 2000 (Invitrogen).Western blots were performed as described previously  with mod35S-methionine into trichloroacetic acid (TCA)-insoluble material as described previously [The rate of protein synthesis was measured by the incorporation of eviously . FurtherSucrose density gradient analysis was carried out as described . Full deTotal RNA extraction was performed using Trizol reagent (Invitrogen) according to the manufacturer’s instructions.The human cDNA microarrays were manufactured in Nottingham University. This custom cDNA microarray consists of 29,593  oligonucleotide probes derived from MWG Human 30K slides A, B, and C. RNA from sucrose density gradient fractionation was pooled into subpolysomal or polysomal fractions, labeled, and hybridized to the arrays as described previously . MicroarGenePix Pro 6.0 was used to quantify fluorescence intensities for individual spots on the microarray. All statistical analysis was performed in the statistical environment R, version 2.6.1, and the Limma package .Northern analysis was performed as described previously . VisualiPost-nuclear extracts were incubated with either anti-RBM3 antibody or immunoglobulin G (IgG)-coated protein G magnetic beads and processed as described in the Reverse transcription was carried out using random primers and Superscript III Reverse Transcriptase (Invitrogen) according to manufacturer instructions. qPCR was carried out using SensiFAST SYBR Lo-ROX Kit (Bioline) according to manufacturer instructions. Primers used are in the As described previously , hemizygFVB wild-type (WT) or hemizygous Tg37 mice weighing more than 20 g were cooled using 5′ AMP as described [GenTarget generated lentiviral plasmids. Viruses were injected stereotaxically into the CA1 region of the hippocampus as described ; additioBurrowing was performed as described . BrieflyParaffin-embedded brains and pancreases were sectioned at 5 μm and stained with H&E as described , 39. NeuTranslation elongation rates on human mRNAs were estimated using a published computational model . The modA.B., D.P., J.R.P.K., S.G., X.P., J.R., A.R., and D.V. conducted the experiments. R.V.S. and T.S. analyzed the data. T.v.d.H. generated the computational model and analyzed decoding speeds. A.E.W., M.B., G.R.M., and C.M.S. designed the experiments and wrote the paper.


Most drug therapies distribute the agents throughout the entire body, even though the drugs are typically only needed at specific tissues. This often limits dosage and causes discomfort and harmful side‐effects. Significant research has examined nanoparticles (NPs) for use as targeted delivery vehicles for therapeutic cargo, however, major clinical success has been limited. Current work focuses mainly on liposomal and polymer‐based NPs, but emerging research is exploring the engineering of viral capsids as noninfectious protein‐based NPs—termed virus‐like particles (VLPs). This review covers the research that has been performed thus far and outlines the potential for these VLPs to become highly effective delivery vehicles that overcome the many challenges encountered for targeted delivery of therapeutic cargo. CCMVcowpea chlorotic mottle virusCPMVcowpea mosaic virusEDC1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide hydrochlorideEGFepidermal growth factorEPRenhanced permeability and retentionHBVchepatitis B virus coreMS2enterobacteria phage MS2NHSn‐hydroxysuccinimideNPnanoparticleP22Salmonella typhimurium P22PEGpolyethylene glycolQβenterobacteria phage QβVLPvirus‐like particle1Currently, numerous diseases lack adequate treatment, most notably cancer and various genetic disorders. In 2016, the National Cancer Institute estimates that 1,685,210 new cases of cancer will be diagnosed in the United States and 595,690 people will die from the disease—a 35% mortality rate.In addition to cancer, the development of gene therapies for treatment of genetic disorders, such as mitochondrial disorders and Parkinson's disease has been a major focus of research. By 2012, over 1800 gene therapy clinical trials had been conducted or approved.Targeted delivery has long been one of the most promising, but also most challenging, opportunities for improving the treatment of these diseases. Targeted delivery offers three key advantages that systemic delivery lacks: (a) the therapeutic will act primarily at the desired site‐of‐action, limiting off‐target effects such as the harmful side‐effects involved with chemotherapy; (b) the delivery vehicle can provide much higher local concentrations of the therapeutic within the diseased tissues, allowing a more effective treatment; and (c) the delivery vehicle can carry the therapeutic to sites it would not normally be able to reach, improving the efficiency of gene therapies. The first attempts at developing delivery vehicles were antibody‐drug conjugates. These carriers have been extensively developed with two FDA‐approved examples [Trastuzumab emtansine (Kadcyla) and Brentuximab vedotin (Adcetris)], and many more are in clinical trials.2Despite many attempts, only a few liposomal and protein‐based NPs have been approved for cancer drug delivery, including Doxil and Abraxane—and these are all passive‐targeting delivery agents relying on the enhanced permeability and retention (EPR) effect for tumor localization.An alternate type of drug delivery NP that is showing promise is the VLP.3As mentioned previously, targeted drug delivery by NPs must overcome multiple challenges Table .31 The a4This review will focus on six of the most actively developed VLPs from: one animal virus, three bacteriophages, and two plant viruses Figure .32, 33, 4.1T = 4 icosahedral geometry.Escherichia coli cytosolic accumulation and cell‐free protein synthesis.The hepatitis B virus is comprised of an internal protein capsid and a lipid envelope containing other proteins. Two different VLPs can be produced from the virus, using either the core antigen that forms the internal capsid or the surface antigen that spontaneously combines with lipids to form NPs. We will focus on the VLP derived from the hepaitis B core (HBVc) antigen, which is formed from 240 copies of a single protein.4.2Salmonella typhimurium P22, all infect enterobacteria, most notably E. coli. Although all three are composed of only a nucleic acid‐filled viral capsid, P22 differs greatly from MS2 and Qβ. MS2 and Qβ are composed of 90 homodimers and require a specific stem‐loop hairpin secondary structure in their RNA genome to trigger VLP self‐assembly by binding to the coat proteins.E. coli, but Qβ can also be produced in yeast and both Qβ and MS2 can be produced using cell‐free protein synthesis.The three bacteriophages, MS2, Qβ, and 4.3E. coli or yeast.2 or by purifying hexahistidine tagged dimers directly.E. coli or yeast; insect cells or plants must be used.E. coli or obtain purified coat proteins adds another challenge for targeted drug delivery; however, CPMV has been actively evaluated for therapeutic use due to the ability to easily display ligands on its surface and load cargo through association with its genome.The final two commonly‐used VLPs to be discussed are derived from plant viruses that infect the cowpea leaf: cowpea chlorotic mottle virus (CCMV) and cowpea mosaic virus (CPMV). Neither virus has a lipid envelope. Both VLPs assemble with icosahedral geometry.5Because of their precise and repeated structures and relatively large cargo capacities, VLPs have many advantages over other types of NPs. Since they are expressed biologically and formed from multiple copies of the same protein, the VLPs are highly uniform and are easily expressed in bacteria . Also, they have evolved in nature to encapsidate their viral genomes, which could be advantageous for loading therapeutic cargo. Particularly in the case of MS2 and Qβ, specific stem‐loop RNA secondary structures that are required for assembly can carry other molecules into the VLP during assembly.5.1As discussed, a delivery vehicle must provide several different functionalities. For several of these attributes, the VLP surface must be extensively modified with various biomolecules. These ligands can provide specific cellular targeting, reduce immune responses, and potentially facilitate extravasation. Most approaches require covalent attachment, however, P22 can display ligands through noncovalent interactions.5.1.1Arguably the most commonly used reactive amino acid residue, cysteine, can be presented either naturally or by mutation on the VLP surface. Because of its free sulfhydryl group, cysteine will readily and spontaneously form disulfide bonds with other sulfhydryl‐containing ligands under oxidative conditions. However, the disulfide bond is also easily reduced and may not be ideal for surface attachments. Alternatively, a series of compounds based on maleimide readily and irreversibly form thioether linkages with cysteine residues at a pH between 6.5 and 7.5. This attachment strategy has been used to display cell‐penetrating peptides, fluorescent probes, and heterologous peptides and proteins on the surfaces of MS2, P22, CCMV, and CPMV VLPs.5.1.2n‐hydroxysuccinimide (NHS) ester reactions (because NHS is released as part of the reaction), amide bonds are formed at surface‐exposed lysine residues. The reaction occurs spontaneously between pH 7.2 and 9. This attachment chemistry has been used to display transferrin on MS2, which may allow the VLP to transcytose the blood‐brain barrier, a development that could open up a new library of therapies for neurological disorders.Another common amino acid residue that is easily modified is lysine because of its primary amine. Using reactions termed 5.1.3Although not as commonly used, the last class of reactive natural amino acid residues includes the carboxylic acids aspartate and glutamate. Unlike strategies involving cysteine and lysine, coupling to these residues requires multiple steps. First, the carboxylic acid must be activated using 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide hydrochloride (EDC). Once activated, it will react with NHS to form an NHS ester. Now that the carboxylic acid side‐chain has essentially become an NHS ester, previously described with regard to lysine modifications, we can use a ligand with an exposed primary amine to form a stable amide bond. This strategy has been used primarily with CCMV to display peptides or fluorescent probes.5.1.4p‐amino‐phenylalanine (pAF). These amino acids are incorporated into proteins in two ways: global methionine replacement and amber stop codon suppression. Because AHA is very similar to methionine, AHA will be incorporated at each AUG codon if the methionine supply is rate‐limiting; this is termed global methionine replacement.Beyond the 20 natural amino acids, many nonnatural amino acids have been used for site‐specific protein conjugation reactions. The two nonnatural amino acids most frequently incorporated into VLP coat proteins are azidohomoalanine (AHA) and Despite the drawbacks, these methods have been used to incorporate nonnatural amino acids with uniquely reactive side‐chains. AHA, displaying an azide, will participate in copper(I)‐catalyzed azide‐alkyne cycloaddition (“click” reaction) and form covalent triazole rings with alkyne‐containing ligands.5.1.5The final covalent attachment method we will discuss is genetic modification, in which the gene for the desired surface ligand is fused to the gene for the coat protein of the VLP. While the added peptide can inhibit protein folding as well as VLP assembly, this approach has been shown to work for most VLPs, but not CCMV.5.1.6P22 is unique compared to the other VLPs because of the existence of the decoration (or “dec”) protein. This protein has high affinity for the surface of P22.5.2VLPs have been used to load a range of molecules, including small molecules , nucleic acids, peptides, proteins, and even other NPs.5.2.1Unfortunately, there has been no published work describing the loading of small molecules within the HBVc VLP. The bacteriophages, on the other hand, have been used extensively to load small molecules. MS2 has been shown to encapsidate chemotherapeutics (taxol and doxorubicin), fluorescent probes (Alexa Fluor® 488 and fluorescein), and other small molecules (porphyrin and DOTA chelators) through conjugations to interior cysteines , interior tyrosines (diazonium coupling), and the stem‐loop RNA hairpin required for VLP assembly.The plant virus‐based VLPs are unique compared to the others because they allow noncovalent loading of small molecules. CCMV has been shown to load polystyrene sulfonate through noncovalent electrostatic interactions between the cargo and the coat proteins.Although these molecules may not all be therapeutically relevant, the results indicate that the methods can successfully load small molecules into many of the VLPs and the approaches can easily be extended to load other small molecule drugs. However, the fact that the only noncovalent loading of small molecules uses a polymerized cargo (that is bigger than most chemotherapeutics) or adsorbs the molecule within nucleic acids shows how difficult it is to load and retain these small cargoes. This is due to the presence of pores throughout the VLP structures, as seen in Figure 5.2.2MS2 VLPs are particularly suited to loading RNA. They require a short stem‐loop RNA hairpin, which is typically part of their genomic RNA, to assemble into capsids.5.2.3There are four main ways to load peptides or proteins into VLPs: (a) fusing the peptide or protein sequence to the amino acid sequence of the coat protein; (b) conjugating the peptide or protein to the genome; (c) engineering electrostatic interactions between the cargo and the coat protein; and (d) passive encapsidation. The first method loads hundreds of peptides or proteins per VLP. Both the first and second methods are facile, but have two major drawbacks. First, the peptide or protein must be amenable to genetic fusion or nucleic acid conjugation and still fold into an active form while also allowing the VLP subunits or nucleic acid to fold properly. Second, the peptide or protein must be able to exert its effect while fused to the coat proteins or conjugated to the nucleic acid. The third and fourth methods are less effective, though they allow loading of free peptides and proteins. Loading via electrostatic interactions is more effective than passive loading, but only works for peptides or proteins that have (or can be engineered to have) an affinity for the internal surface of the VLPs. HBVc VLPs have been shown to load proteins through genetic fusions either to the C‐terminus or within the protein sequence as well via passive encapsidation.5.2.4A significant body of work has studied the use of VLPs for the development of improved contrast agents. By loading the standard NP‐based contrast agents within VLPs, the new NPs gain improved relaxivities which then give higher resolution images. Additionally, if the VLPs are further modified to target specific cells, the signal‐to‐noise ratio is increased even further giving clear images of, for example, tumors. To that end, HBVc and CPMV VLPs have been loaded with iron oxide NPs through coordination to the coat proteins or through passive encapsidation.5.3Unlike the metal‐based, liposomal, and polymer‐based NPs, VLPs are highly uniform. VLPs, produced with an exact number of coat proteins and arranged in a consistent geometry, will have significantly lower lot‐to‐lot variability and identical cargo release profiles. Additionally, once inside the targeted cells, the VLPs should degrade and release all of the therapeutic cargo at once—unlike polymer NPs which slowly degrade and release the cargo over time.At the same time, protein‐based design means that the VLPs are not as stable as polymer NPs. Fortunately, this drawback is known and has been studied in the hopes of making better VLPs. These studies focused on HBVc, MS2, and Qβ VLPs. The HBVc VLP forms intradimer disulfide bonds that stabilize the 120 dimers, and Qβ forms disulfide bonds that link the pentameric and hexameric subunits at the 5‐ and 3‐fold axes of symmetry.5.4Although there have not been in vivo biodistribution studies for HBVc and P22 VLPs to our knowledge, in‐depth studies have been performed for MS2, Qβ, CCMV, and CPMV. We focused on studies using intravenous administration into mice or rats as model systems, which are the systems likely to be studied for initial evaluation of VLP‐based targeted therapeutics.The distribution of MS2 VLPs, labeled internally with The biodistribution of the plant virus‐based VLPs, CCMV, and CPMV, intravenously injected in mice at various times, are mostly similar. They primarily accumulate in the liver, spleen, kidney, and GI tract.Because developing VLP‐based targeted therapies for cancer is a primary application, biodistribution studies in mice possessing tumor xenografts were also conducted. MS2 or PEGylated CPMV VLPs were injected intravenously and partially accumulated in the tumors after 24 hr. This was hypothesized to be because of the EPR effect.5.5While many different cell targeting ligands have been evaluated, ranging from glycans to specific receptor‐ligands such as folate and transferrin, the most common targeting ligand is the antibody fragment, although recently RNA and DNA aptamers have been used more frequently.It has been shown that single‐domain antibody fragments could be displayed on the surface of HBVc VLPs; however, no cell targeting results have been reported.Notably, the M.G. Finn group functionalized Qβ with human transferrin and observed cellular uptake and internalization of the VLPs through clathrin‐mediated endocytosis in BSC1 cells.Although there has not been a specific targeting study using CCMV, CCMV VLPs containing EYFP RNA were transfected into mammalian BHK cells.In some cases, VLPs have been engineered to display cell‐penetrating peptides as well—either to aid in the initial cell targeting and entry or, when used in lower concentration, to aid in escaping the endosome. The use of such agents for endosomal escape may be needed to enhance the delivery of functional therapeutic cargo. After VLPs containing macromolecular cargo are endocytosed by the targeted cells, they must escape the endosome before reaching the end of the endosomal pathway: the lysosome. The lysosome will degrade the VLPs and any nucleic acid, peptide, or protein cargo they contain. Conversely, many small molecule cargoes should remain functional after VLP degradation. Previous work has displayed three different cell‐penetrating peptides on three different VLPs: MS2 has been functionalized with the HIV‐Tat peptide and a histidine‐rich H5WYG peptide, P22 has also been functionalized with the HIV‐Tat peptide, and CPMV has been functionalized with arginine‐rich R5 peptides.6can be produced using cell‐free protein synthesiscan load small molecules, nucleic acids, and proteinscan be stabilized with disulfide bondscan incorporate nonnatural amino acids for ease of surface functionalization through the “click” reactioncan be functionalized to display antibody fragments for specific cellular targetingnot shown for HBVc VLPs)can be functionalized to display PEG to avoid the immune system (not shown for MS2 VLPs)will disassemble in the reducing conditions of the cytosol to release their cargo (Although VLP‐based targeted drug delivery remains a nascent technology that requires further studies to prove its clinical efficacy, significant progress has been made. Many of the initial disadvantages of using VLPs have been remedied, as shown in Table Although it has not been experimentally proven, the disulfide bonded mutant of MS2 should behave similarly to Qβ and the disulfide bonded mutant of HBVc and disassemble in cytosolic conditions.It is also suggested that additional work focus on fully overcoming the challenges listed in Table


Two major conclusions have been drawn from these analyses: first, charred plant matter has been identified as a main component of these prehistoric black pigments; and second, angiosperm and conifer charcoal was a primary raw material for pigment production, identified by means of the archaeobotanical study of plant cells. For the second goal, black charcoal pigments were replicated in the laboratory by using different raw materials and binders and by reproducing two main chaînes opératoires. The comparative study of the structure and preservation of plant tissues of both prehistoric and experimental pigments by means of SEM-EDX underlines both a complex preparation process and the use of likely pigment recipes, mixing raw material with fatty or oily binders. Finally, the formal and stylistic analysis of the motifs portrayed at Les Dogues allowed us to explore the relationship between identified stylistic phases and black charcoal pigment use, raising new archaeological questions concerning the acquisition of know-how and the transfer of traditionally learned chaînes opératoires in Spanish Levantine rock art.We present a new multi-analytical approach to the characterization of black pigments in Spanish Levantine rock art. This new protocol seeks to identify the raw materials that were used, as well as reconstruct the different technical gestures and decision-making processes involved in the obtaining of these black pigments. For the first of these goals, the pictorial matter of the black figurative motifs documented at the Les Dogues rock art shelter  was characterized through the combination of physicochemical and archeobotanical analyses. During the first stage of our research protocol,  Spanish Levantine rock art is a unique form of pictorial expression in European prehistory. Located in rock shelters in the inland regions of the Iberian Mediterranean basin, this prehistoric art is of particular interest due to both the naturalism of figures and the narrative component of its scenes, which portray specific economic and social activities. The remarkable variations in form, technique, themes, and composition over time and throughout the territory that exist within this graphic horizon underline the interaction between complex social dynamics and a highly structured territory, as manifested through interaction and exchange networks that likely experienced several phases of expansion and contraction through time. This implies that the analysis of Levantine rock art is particularly useful for gaining insight into the prehistoric societies responsible for their elaboration.th millennium cal. BC), and rather relate it to the fully established Neolithic societies .The cultural and chronological framework of Levantine rock art is currently imprecise, resulting mainly from the fact that the primarily mineral-based pigments that are used are impossible to date using conventional radiocarbon methods. Nevertheless, the latest chrono-cultural hypotheses support an affiliation between Neolithic societies and these graphic representations –2. Recenchaînes opératoires.The pigment palette of Spanish Levantine rock art consists of just three colours: red, black and white, with a clear prevalence of the first throughout the entire relative chronological sequence as well as in most of the Levantine regions. To date little is known regarding the composition, preparation and use of these prehistoric pigments, and subsequently even less is known regarding chronological and geographical variations in these hypothetical Regarding colour selection, we have yet to identify the motivations behind colour choice, whether they correspond to specific social rules or rather are a function of limited access to mineral or organic raw materials. Some regional patterns can be established via differential use of certain colours, yet this argument cannot be pushed further as to date a geographical analysis of spatial distributions of differential colour use and boundary variation through time has not been completed. Regarding raw materials, no geological survey allowing for the geographic reconstruction of possible colouring material sources in the areas surrounding Levantine rock art sites has been conducted.From a more technical standpoint, data available concerning components used in the preparation of pigments is scarce, and we are far from being able to establish social patterns via the identification of recurring pigment mixtures or “recipes”. Although the physicochemical characterization of Spanish Levantine pigments has been a primary research focus in recent years, the theoretical scope, methods used, and experimental techniques explored are extremely heterogeneous, thereby hampering any sort of interregional comparative study of results produced –6. Inveschaînes opératoires. These chaînes opératoires–modalities through which raw material is acquired, transformed, and used–correspond to social rules that were learned, shared, and accepted by a community, and therefore should be considered as identity-revealing . These formal attributes are distinctive of the last stylistic phases–the Linear horizon–of the regional sequence –51. The Bearing these formal differences in mind, we can attribute the use of charcoal pigments in the Gassulla area to two different stylistic phases. Furthermore, it is worth noting that the Linear horizon also shows a wide range of formal variation and an extensive geographic distribution in all Levantine regions, all of which points to a long period of use of this type of figures. Even if we cannot define the specific time lapse characterized by the use of charcoal pigments, due to the lack of radiocarbon dates, the fact that they were used in two different stylistic phases raises new questions with regard to the mechanisms responsible for the transfer of knowledge and know-how of pigment production within Spanish Levantine rock art traditions. Additionally, Raman spectra did not highlight any differences in the samples taken from the two motifs belonging to these two different styles.Another aspect yet to be explored is the geographical distribution of Levantine charcoal pigment use. To date, outside the Gassulla area, amorphous carbon has only been detected in black pigments of the Ciervos Negros rock art shelter  , about 314C dates, with reasonable standard deviations, is damaging to the paintings. This means that for the time being, the application of radiocarbon dating methods to the Levantine charcoal pigments is unfeasible.It is also worth noting that the identification of organic matter in the pigments would make radiocarbon dating possible, potentially enabling us to refine the chronological and cultural context of Levantine rock art. The significant quantity of charcoal currently needed to obtain calibrated chaîne opératoire. Such an approach has allowed for the accurate physicochemical characterization of pigment components, in addition to the holistic comprehension of the technical gestures and decision-making processes embedded in the specific techniques of pigment production. Moreover, the botanical identification of plants used in the preparation of these black pigments constitutes a new line of research that will contribute to our understanding of plant resource exploitation by Neolithic communities.The study of pigments from Les Dogues has successfully demonstrated the benefits of a multi-analytical approach integrated within the theoretical and methodological framework of In this study, we have demonstrated the use of binders by comparing plant tissues from both experimentally produced and prehistoric pigments. The next logical step of this research axis will be to investigate binder use in Levantine rock art and to characterize them via the novel application of analytical techniques in this field of study. This will allow for the identification of other organic compounds, which would be a major advance in our comprehension of the elaboration of Levantine pigments.chaîne opératoire, into a larger and more complex archaeological discussion regarding the characterization of different Levantine art styles, their areas of influence, and their interaction and exchange networks. All these aspects are integral to the comprehension of the Neolithic expansion into the Iberian Peninsula, whose resulting cultural mosaic requires precise definitions should we hope to apprehend the historical and societal processes in play.Moving forward we aim to integrate this new data regarding Levantine pigments, obtained via the experimental reconstruction of their