Bilateral simultaneous acute optic neuritis is a rare occurrence in adults, which has been reported mostly in the setting of untreated systemic autoimmune disorders. Such clinical presentations are encompassed in the spectrum of ‘atypical’ optic neuritis with resultant poor visual outcome and mainly associated with neuromyelitis optica spectrum disorders. We report an unusual presentation, that is, severe atypical optic neuritis in a patient of pemphigus foliaceous on immunosuppressive therapy with significant visual recovery after treatment. Optic neuritis is a condition that is characterized by inflammation of the optic nerve typically presenting unilaterally in young adults with a preponderance in females and frequently associated with multiple sclerosis. Bilateral simultaneous acute optic neuritis is a rare occurrence in adults, which has been reported mostly in the setting of systemic autoimmune disorders and is encompassed in the spectrum of ‘atypical’ optic neuritis. In atypical optic neuritis, the inflammation may be triggered by an inflammatory or immune-related disease like sarcoidosis, neuromyelitis optica (NMO), or by a different process such as infection  or vitamin B12 deficiency.Autoimmune optic neuropathy has been described in the presence of serological evidence of an autoimmune disorder and must be considered when a patient with optic neuritis has atypical features. Pemphigus foliaceus is an acquired autoimmune disorder characterized by blistering and intra-epidermal deposition of IgG auto-antibodies against intracellular adhesion glycoprotein desmoglein-1. An isolated case of NMO in a patient with pemphigus foliaceus has been reported till date.We report a case of bilateral severe atypical optic neuritis with profound visual loss at presentation in a patient of pemphigus foliaceus on immunosuppressive therapy, where NMO was ruled out and a favorable outcome was observed following high-dose systemic corticosteroid therapy.A 37-year-old male presented to our outpatient department with complaints of rapid, progressive profound vision loss over last three days. He was a known case of pemphigus foliaceus on immunomodulators for last three years, but was irregular with follow-up and non-compliant to medications. On ocular evaluation, his best-corrected visual acuity was absence of light perception in both eyes with bilateral afferent pathway defect, amaurotic pupil, and painful ocular movements at extremes of gazes. Fundus evaluation revealed bilateral disc edema, inferior disc hemorrhages, and peripapillary retinal nerve fiber layer edema suggestive of severe papillitis Figure , 1B. An Atypical optic neuritis is usually seen as an early manifestation of NMO or NMO spectrum disorder (NMOSD) ,2. AtypiOur present case is a middle-aged male, a known case of pemphigus foliaceus on irregular immunosuppressive therapy who developed atypical optic neuritis after a few years of treatment. The most probable cause of such presentation, NMO or NMOSD, was ruled out with relevant investigations. The patient also had multiple atypical findings like male gender, bilateral non-perception of light, severe papillitis, and optic disc hemorrhages. But, it did not correlate with any common cause of atypical optic neuritis as suggested by the investigations for immunological and/or infective disorders as well as CSF analysis for cells, protein, or sugar levels as well as immunoglobulins. Atypical optic neuritis can have devastating effects on the vision of the patient if not treated timely, and even then, the results are less than satisfactory in a significant number of cases.A single case of NMO-associated optic neuritis in a patient with pemphigus foliaceus has been reported till date . Velez eTherefore, it is critical to recognize and monitor patients with autoimmune disorders for possible development of atypical optic neuritis and institute prompt management, so as to achieve better visual outcomes and reduce ocular morbidity.


The renin–angiotensin system and the AT1 receptor are the targets of several classes of therapeutics  used as first-line treatments in cardiovascular diseases. The importance of AT1 in the regulation of the cerebrovascular system is also acknowledged. However, despite numerous beneficial effects in preclinical experiments, ARBs do not induce satisfactory curative results in clinical stroke studies. A better understanding of AT1 signaling and the development of biased AT1 agonists, able to selectively activate the β-arrestin transduction pathway rather than the Gq pathway, have led to new therapeutic strategies to target detrimental effects of AT1 activation. In this paper, we review the involvement of AT1 in cerebrovascular diseases as well as recent advances in the understanding of its molecular dynamics and biased or non-biased signaling. We also describe why these alternative signaling pathways induced by β-arrestin biased AT1 agonists could be considered as new therapeutic avenues for cerebrovascular diseases.The physiological and pathophysiological relevance of the angiotensin II type 1 (AT I. I1 S-ni1 structure, it is noteworthy that C2897.40 is one of the residues involved in the “long-range allosteric network” , whereas it points outward from the receptor in the inactive  and canonical active (in green) states. This suggests that any modification on this particular residue could alter the process involved in conformational changes and may preclude or stabilize one or more conformations of AT1. This hypothesis is strengthened by several reports focusing on single nucleotide polymorphism of AT1 in humans. Among the few naturally occurring variants of AT1, one is related to C2897.40 where the cysteine is replaced by a tryptophan residue (C289W variant). This variant has not been associated with a pathology, but seems to be less expressed at the plasma membrane and decreases the potency of Ang II on phosphatidylinositol hydrolysis consecutive to Gq stimulation [1/2 [7.40, such as S-nitrosation, may bias AT1 signaling, but further investigations are needed.When looking at ATork” see . As showmulation . At leasmulation ,114. How1 is prone to biased agonism towards the β-arrestin pathway, we will now review the potential interests of promoting this AT1/β-arrestin signaling. AT1 recruits both β-arrestins equally and internalizes as a stable complex, inducing a slow recycling to the plasma membrane [Having established that ATmembrane ,116.1 which reduce Gq signaling, the activation of the β-arrestin pathway induces several signaling cascades involved in cellular processes, such as cell proliferation and growth, nutrient uptake and migration  [1 independently of G protein or Ang II [Hence, TRV023 is another ATrrestins a. In micpathway) . TRV023 pathway) . These rr Ang II . Moreover Ang II . Finallyr Ang II .1 ligands appear promising, although not yet fully understood. These biased agonists also seem to bind to AT2, which could contribute to counteracting the cardiovascular harmful effects of AT1. Although AT2 does not couple to heterotrimeric G proteins and does not recruit β-arrestins [1/AT2 heterodimer signaling. However, to date no comprehensive study has been conducted to support this hypothesis.Thus, the beneficial actions of these β-arrestin biased ATrrestins ,140, thi1 on cerebrovascular diseases. The literature, however, supports the idea that these drugs might represent new hope for these clinical situations in which alternative treatments are lacking.To the best of our knowledge, no study has focused on the impact of specific β-arrestin biased agonists of AT1 [1, together with its AT2 agonistic properties might contribute to the cerebrovascular beneficial effects of Ang-(1–7) [1/AT2 or AT1/MasR seem to inhibit AT1 signaling by Ang II, β-arrestin recruitment and receptor internalization [Interestingly, Ang-(1–7), a major component of the Ang-(1–7)/MasR/ACE2 protective axis of the renin–angiotensin system, has also been described as a β-arrestin biased ligand of AT1 ,141. Num1 ,143,144 1 ,145,146.1 . Howeverng-(1–7) ,148,149.lization ,152,153.Unlike reports at the cardiac level, both β-arrestin1 and β-arrestin2 seem to have neuroprotective effects on cerebrovascular diseases. The MCAo ischemic stroke model in mouse revealed an increase in protein expression of the two β-arrestins, suggesting their involvement in the pathophysiological process . This stStudies exploring the alterations of β-arrestin2 expression in strokes and neurovascular disease are scarce and the role of β-arrestin2 in neurological disorders is not yet clear. One study showed that β-arrestin2 plays a pivotal role in the immunodeficiency syndrome induced by strokes . In part1 in cerebrovascular diseases has been increasingly highlighted in recent years; however, therapeutic advances in this area have not followed. The development of new therapeutic approaches requires a better understanding of AT1 signaling pathways and its possible interactions with other components. Since β-arrestin-biased AT1 agonists have showed promising results in the cardiovascular disease context, it would be interesting to study them in models of cerebrovascular diseases such as strokes.The pathophysiological role of AT1 signaling, whether through β-arrestin signaling or characterization of the potential beneficial effects of β-arrestin biased agonists.There is still much to learn about the regulation of AT


This study aimed to investigate the effect of different classes of obesity on the energetics and mechanics of walking and to explore the relationships between body mass, NetCw/kg and gait mechanics by using principal component analysis (PCA). NetCw/kg and gait mechanics were computed in severely obese , moderately obese  and normal-weight  adults during five walking trials  on an instrumented treadmill. NetCw/kg was significantly higher in SOG compared to NWG (p = 0.019), with no significant difference between SOG and MOG (p = 0.14), nor between MOG and NWG (p = 0.27). Recovery was significantly higher in SOG than in NWG (p = 0.028), with no significant difference between SOG and MOG (p = 0.13), nor between MOG and NWG (p = 0.35). PCA models explained between 17.0% and 44.2% of the data variance. This study showed that: (1) obesity class influences the gait energetics and mechanics; (2) PCA was able to identify two components, showing that the obesity class is associated with lower walking efficiency and better pendulum-like characteristics.Higher mass-normalized net energy cost of walking (NetC The World Health Organization (WHO) categorizes obesity and the associated health risks into class I (30.0 ≤ BMI ≤ 34.9 kg·m−2), II (35.0 ≤ BMI ≤ 39.9 kg·m−2), and III (BMI ≥ 40.0 kg·m−2) [In recent decades, the prevalence of obesity has continued to increase across the world , represePhysical activity can be an effective strategy to prevent or minimize weight gain in adults  and obes−1; NetCw) and relative  values at predetermined walking speeds [w/kg (+80%) [k) and the potential energy (Ep) of the center of mass (COM) are transferred (some Ek is converted into Ep and vice versa), reducing the external mechanical work performed  and the associated energy expenditure during walking [Several investigators have found that net energy cost of walking is higher in individuals with obesity compared with their leaner counterparts in absolute   than tha walking .w/kg  than class I (+8.5%), because individuals with class I obesity showed only a tendency toward a greater NetCw/kg when compared with their lean counterparts [w/kg in individuals with class III obesity was still lower than theoretically expected (+80%) [ext  at all experimental walking speeds (0.56–1.67 m·s−1) [ext/kg, but only at fast speeds (>1.1 m·s−1) [w and Recovery in individuals with class III obesity seems to suggest that the obesity level may be involved in the increased NetCw/kg, as well as in pendular exchange optimization. However, it seems that this profitable mechanism runs up to a functional body mass threshold beyond which the increase in NetCw cannot be completely—or at least partially—be compensated by pendular transduction [w/kg and with a decrease in Wext/kg, along with a more skillful Recovery. However, for loads beyond 30% of their body mass, the results showed an increase in NetCw/kg and a tendency to maintain a constant Wext/kg, even though Recovery remained improved [w and Wext/Recovery may be due to (i) a decrease in muscular efficiency, (ii) an increase in the isometric contractions required to support the load and maintain posture, or (iii) the inadequate/inappropriate muscle strength necessary to carry these loads beyond this amount [added mass walking [w cannot completely be mitigated by a more skillful pendular recovery. Altogether, these results highlight a relationship between the level of obesity and the energetics and mechanics of walking. However, to the best of our knowledge, thus far, no studies have investigated this relationship.Recently, Fernandez-Mendez et al.  reportedterparts . The hig7 m·s−1) , whereas1 m·s−1) . The higsduction , as demoimproved . This “ds amount . If we cw has many predictors which are time-dependent and often correlated with each other [Moreover, the NetCch other . For thech other , and prich other . Howeverw/kg would be higher in SOG than MOG and NWG and this extra cost would be associated with a lower Wext/kg along with greater Recovery in SOG than NWG and MOG. On the other hand, NetCw/kg would be non-significantly higher in MOG than NWG and it would be associated with a lower Wext/kg along with greater Recovery only at fast walking speeds in the former compared with the latter group.Therefore, the first aim of the study was to investigate the effect of different obesity levels on the energetics and mechanics of walking in individuals with class II–III  and class I  obesity, and normal-weight adults  while walking at different speeds. It was hypothesized that NetCw and gait mechanics at different walking speeds by using PCA in order to identify any specific data patterns.The second aim was to explore the relationships between body mass, NetC−2; n = 18, 3 men and 7 women: class II; 1 man and 7 women: class III) and MOG , respectively. An NWG  was also included. All subjects were in good health and were free of musculoskeletal disorders or other conditions that could affect the gait pattern. This study used pooled data of two previously published studies of our research group [Thirty-five sedentary  adults with obesity and thirteen normal-weight adults took part in this study. The median BMI of the participants with obesity (median = 35.1) was used to divide them into SOG  on an instrumented single-belt treadmill . The order of the speeds was determined randomly. A minimum of a 5 min resting period between each walking trial was carried out. Metabolic and mechanical data (10 strides) were collected for each walking speed.Prior to testing, anthropometrical characteristics and body composition were assessed in individuals with obesity through a dual energy X-ray absorptiometric scan . Subsequently, all study participants visited the laboratory once to perform (1) a 10 min treadmill familiarization session  and (2) Standing height was measured using a Harpenden Stadiometer. Body mass was measured to the nearest 0.1 kg using a precision digital scale with the subject wearing shorts and a T-shirt. Total and regional masses for each body segment  as well as body composition  were assessed in obese subjects through a dual-energy X-ray absorptiometric scan.®, Italy [2 output  was calThe two metabolic cards used provide gas exchange measurements that are similar, reliable, and valid relative to each other .−1) data from the last minute of each walking trial and SMR were averaged. The energy equivalent of 1 L of O2 was then used to compute the gross metabolic rate (W) [w (J·m−1); the relative was obtained by dividing this latter by the body mass .The gas exchange was measured breath-by-breath during walking trials. Steady states for rate (W) . The netFor each walking trial, step length  and frequency  were assessed during twenty consecutive steps in the last minute of each walking trial using the instrumented treadmill (sampling rate: 1000 Hz).v), forward (Ff) and lateral (Fl) components of the ground reaction forces (GRFs) for the 20 steps selected. These components, along with the body mass of each subject, were used to calculate the 3D accelerations of the COM following the method described previously by Massaad et al. [f was equal to zero [v, Vf and Vl, respectively). A second mathematical integration of Vv and Vl was applied to assess the COMv and COMl. To guarantee step consistency, only steps were selected in which the sum of the increments of the three components did not differ by more than 25% from the sum of decrements [kv, Ekf, and Ekl) of the COM were then calculated and used to assess the instantaneous Ek of the COM (Equation (1)).m is the body mass.The mechanical energy fluctuations of the COM in the 3 axes were computed from the vertical , m and gravity constant (g = 9.81 m·s−2), instantaneous Ep of the COM was calculated (Equation (2)).Using the vertical position of COM (tot) of the COM was computed as the sum of the instantaneous Ek and Ep (Equation (3)).Subsequently, the instantaneous total mechanical energy  and relative  terms.The amount of the external mechanical work done per step was defined as the sum of the positive increments in Ek and Ep curves, respectively.Then, the amount of mechanical energy saved via the pendular energy transduction was assessed by Recovery (Equation (4)).k and Ep curves was computed by determining α , as previously described by Cavagna et al. [pk+ indicates the difference between the period at which Ek and Ep increase simultaneously . Given this definition of α, if Ek and Ep curves are 180° out of phase, α would be equal to 0° [k:Wp−1 ratio.The phase shift between the Ea et al.   was computed as the ratio of the Wng speed .t-tests were performed to test the differences in a few experimental variables between SOG and MOG.Descriptive statistics (mean ± SD) were performed to characterize the sample. Chi-squared tests  were used for testing sex differences among groups. A one-way ANOVA with Tukey correction was applied to test the difference between groups concerning a first set of the participant’s characteristics . Data normality and homogeneity of variances were assessed by using Shapiro–Wilk’s and Leven’s tests, respectively. If the normality assumption was violated, non-parametric tests were employed. Independent-sample −1). A random subject effect was introduced in the models  to account for repeated measures for each subject. Fixed effects were added . Holm correction was applied to identify where statistical differences  occurred. The main effects of speed are omitted in the presentation of results, because it is well accepted that it affects metabolic and mechanics observations [Linear mixed models (LMMs) were used to determine differences in energetics and mechanics between groups while walking at different speeds  were not able to complete the 5 min walking trial at 1.67 m·s−1. The analysis process comprised various steps . Kaiser–Meyer–Olkin (KMO) and Barlett’s tests were performed to confirm the suitability for PCA. Only PCs that explained ≥70% of the data variance were retained [p ≤ 0.05.Based on sample size (between 5 and 10 subjects for each item)  and releretained . Loadingretained ; the othThe participant’s characteristics are presented in p < 0.22). Age was significantly higher in SOG compared to NWG and MOG (p < 0.001 for both), with no significant difference between MOG and NWG (p = 0.15). Height was significantly lower in SOG compared to NWG and MOG (p < 0.001 for both), with no significant difference between MOG and NWG (p = 0.49). BMI and body mass were significantly higher in SOG compared to NWG and MOG (p < 0.001 for both) and in MOG compared to NWG (p < 0.001). Lean and fat body masses (kg and %) were significantly higher in SOG compared to MOG . Head and trunk, upper limb, and lower limb masses were significantly higher in SOG than in MOG . Lower limb lean and fat masses (kg and %) were not significantly different between these groups .Sex was not significantly different among groups , with no significant difference between MOG and NWG (p = 1.0).The SMR was significantly higher in SOG compared to NWG and MOG (/kg was significantly lower in SOG compared to NWG (p < 0.001) and between MOG and NWG (p < 0.001), with no significant difference between SOG and MOG (p = 0.83).The SMRw was significantly higher in SOG than in MOG and NWG (p < 0.001 for both) and in MOG than NWG (p < 0.001). There was a significant interaction effect (p < 0.001). The NetCw was significantly higher in SOG compared to MOG at all walking speeds, except at 0.83 m·s−1 (p = 1.0) (The NetCp = 1.0) A.w/kg was significantly higher in SOG compared to NWG (p = 0.019), with no significant difference between the former and MOG (p = 0.14), nor between MOG and NWG (p = 0.27). There was a significant interaction effect (p = 0.006). The NetCw/kg was significantly higher in SOG than in NWG at the fastest speed (1.67 m·s−1) (p = 0.019) (The NetC= 0.019) B.v and COMl at the experimental walking speeds are presented in The spatiotemporal parameters, COMp = 0.002), with no significant difference between the former and NWG (p = 0.06), nor between MOG and NWG (p = 0.28). When normalized by height, step length was not significantly different among the three groups (p = 0.61).Step length was significantly lower in SOG compared to MOG , with no significant difference between MOG and NWG (p = 0.85).Step frequency was significantly higher in SOG compared to NWG and MOG (v (p = 1.0).There was no significant group main effect on COMl was significantly higher in SOG compared to NWG (p = 0.052), with no significant difference in the former compared with MOG (p = 0.30) and in MOG compared with NWG (p = 0.30).COMp ≥ 0.07).For all these analyses, no significant interaction was found  and in MOG than NWG . There was a significant interaction effect for these three variables (p ≤ 0.005) (The W≤ 0.005) A,C,E.ext/kg was significantly lower in MOG than in NWG (p = 0.022), with no significant difference between SOG and NWG (p = 0.16), nor between the former and MOG (p = 0.26). There was a significant interaction effect (p < 0.001). Wext/kg was significantly lower in MOG than in NWG at 1.11 and at 1.67 m·s−1 (p = 0.003) (The W= 0.003) B.p/kg was significantly higher in SOG compared to NWG and MOG  with no significant difference between MOG and NWG (p = 0.53). There was no significant interaction effect (p = 0.73) (The W = 0.73) D.k/kg (p = 0.13) nor a significant interaction effect (p = 0.22) (There was neither a significant group main effect on W = 0.22) F.p = 0.028), with no significant difference between the former and MOG (p = 0.13), nor between the latter and NWG (p = 0.35). There was a significant interaction effect (p < 0.001). Recovery was significantly lower in MOG than in NWG at the slowest speed (0.56 m·s−1) (p = 0.050) (Recovery was significantly higher in SOG than in NWG (= 0.050) A.p = 0.039) and in MOG than in NWG (p = 0.039) with no significant difference between SOG and MOG (p = 0.94). There was a significant interaction effect (p = 0.002). The phase shift was significantly higher in MOG than NWG at the slowest speed (0.56 m·s−1) (p = 0.002) (The phase shift was significantly higher in SOG than in NWG (= 0.002) B.k·Wp−1 (p = 0.14), nor a significant interaction effect (p = 0.23) (There was neither a significant group main effect on W = 0.23) C.p = 0.014), with no significant difference between the former and MOG (p = 0.34), nor between the latter and NWG (p = 0.09). There was a significant interaction effect (p < 0.001). The NetE was significantly lower in SOG than in NWG at the fastest speeds  (p < 0.001), and in MOG than in NWG only at 1.67 m·s−1 (p = 0.017) (The NetE was significantly lower in SOG than in NWG (= 0.017) D.1, respectively, indicating an acceptable sample adequacy for PCA. Barlett’s test of sphericity was statistically significant for each walking speed (p < 0.001). PCA models explained between 17.0% and 44.2% of the data variance. Body mass, Wext, and NetCw were loaded highest on PC1  at all walking speeds, whereas Recovery and COMl were loaded highest on PC2 (labeled pendulum-like characteristics) at 0.83, 1.11, and 1.39 m·s−1  and in MOG than in NWG (p < 0.001). PC2 and PC3 scores were not significantly different between groups  (At 0.56 m·sctively) .−1, PC1 scores were significantly higher in SOG than in NWG and MOG  and in MOG than in NWG (p = 0.001). PC2 and PC3 scores were not significantly different between groups  (At 0.83 m·sctively) .−1, PC1 scores were significantly higher in SOG compared to NWG and MOG (p < 0.001 for both) and in MOG than in NWG (p < 0.001). PC2 scores were also significantly higher in SOG than in NWG (p = 0.016) and in MOG than in NWG (p = 0.019), with no significant difference between SOG and MOG (p = 0.99). PC3 scores were not significantly different between groups (p = 0.13) (At 1.11 m·s = 0.13) .−1, PC1 scores were significantly higher in SOG than in NWG (p < 0.001) and MOG (p = 0.026) and in MOG than in NWG (p = 0.001). PC2 scores were significantly higher in SOG compared to NWG (p = 0.024) with no significant different between the former and MOG (p = 0.59) nor between MOG and NWG (p = 0.18) (At 1.39 m·s = 0.18) .w/kg and higher Recovery than normal-weight adults, regardless of the lower Wext/kg performed which, in turn, was significantly lower in subject with moderate obesity than in their normal-weight counterparts, despite similar Recovery and NetCw/kg. PCA was able to identify two components: walking efficiency and pendulum-like characteristics. PC scores were sensitive to distinguish individuals with obesity with lower walking efficiency from their normal-weight counterparts and to differentiate adults with severe obesity with better pendulum-like characteristics from other groups, especially at the fastest walking speeds.The main findings of the present study were that individuals with severe obesity exhibited both a greater NetCw/kg in SOG was 15% greater  [l  than in NWG across all walking speeds tested  . This wad (+80%) , who repd (+80%) . Our fins tested , along wd to NWG . These do NWG  compared to their normal-weight counterparts  in the former (int was not assessed in our study). However, previous results from Fernandez-Mendez et al. [int and greater NetCw/kg compared to their lean counterparts, which suggests that swinging of the limbs is not responsible for the extra energy cost in this population. For this reason, we can assume that the NetE (Wext·NetCw−1) assessed in our study can well approximate the overall mechanical efficiency (Wtot·NetCw−1). The lower NetE found in SOG compared to NWG may be explained by a more knee-flexed lower limb at the heel strike [w [functional body mass threshold. Indeed, below this threshold, adults with class I obesity (MOG) presented a lower increase in NetCw/kg, which might be better  cushioned, by a lower Wext/kg. Moreover, the lower the level of obesity, the higher the critical speed at which mechanical efficiency is penalized , especially in sedentary people with obesity who are in the so-called “unregulated zone”, in which appetite and food intake are not affected by TEE; thus, food intake drives body weight gain [w/kg could be useful to shift individuals with obesity into the “regulated zone”, in which appetite and food intake are affected by physical activity [w/kg in adults with obesity may be beneficial to increase non-exercise activity thermogenesis (NEAT) [ control . A greatght gain . Althougactivity , the rolactivity ,45,46. Ns (NEAT) , and thus (NEAT)  and has s (NEAT) .v and mechanical work during walking [ext [Some methodological limitations need to be addressed. First, our groups were not matched for age, sex, or height. However, all subjects in our study were no older than ∼65 years, which is considered the critical age when the energetics and mechanics of walking change substantially ,50. Sex  walking . This coing [ext . Howevering [ext . Third, ing [ext . Therefow/kg compared to their normal-weight counterparts. This extra cost is partially mitigated only in adults with class II–III obesity (SOG) by a more skillful pendular mechanism, consequent to the optimal exploitation of lateral movements that enhance the phase shift between the Ep and Ek curves at the expense of amplitude optimization. This energy mismatch seems to explain the nonsignificant lower Wext/kg that may relate to the attainment and/or the exceeding of a functional body mass threshold. Indeed, obesity level may be paradoxically associated with lower mechanical efficiency and with better pendulum-like characteristics during walking.In conclusion, this study shows that obesity class appears to have an effect on the energetic and mechanics of walking. Individuals with obesity  have a greater NetC


Amundson’s letter  in respoNonetheless, we do not claim that innovation or adoption will be easy, nor is this transition inevitable. Our goal was to introduce technologies  and call for changes in policy and investment to broaden deployment and make the transition a reality. We do not propose that any of the transitional states should be the goal of the sector. As Amundson  notes, aWe chose a broad brush and a key metric—greenhouse gas emissions—for optimization of agricultural systems, as it fits current policy, incentives, and emerging markets. Amundson  highlighTo alter the aphorism from George Box, “all predictions are wrong, some are useful,” the purpose of our manuscript  was to aAmundson  notes th


A variety of techniques were utilized to evaluate detailed functional roles of miRNA-221-3p during odontogenesis, including in vitro tooth cultivation, renal capsule transplantation, in situ hybridization, real-time PCR, and immunohistochemistry. Two-day in vitro tooth cultivation at E13 identified altered cellular events, including cellular proliferation, apoptosis, adhesion, and cytoskeletal arrangement, with the loss and gain of miRNA-221-3p. qPCR analysis revealed alterations in gene expression of tooth-related signaling molecules, including β-catenin, Bmp2, Bmp4, Fgf4, Ptch1, and Shh, when inhibited with miRNA-221-3p and mimic. Also, the inhibition of miRNA-221-3p demonstrated increased mesenchymal localizations of pSMAD1/5/8, alongside decreased expression patterns of Shh and Fgf4 within inner enamel epithelium (IEE) in E13 + 2 days in vitro cultivated teeth. Moreover, 1-week renal transplantation of in vitro cultivated teeth had smaller tooth size with reduced enamel and dentin matrices, along with increased cellular proliferation and Shh expression along the Hertwig epithelial root sheath (HERS), within the inhibitor group. Similarly, in 3-week renal calcified teeth, the overexpression of miRNA-221-3p did not affect tooth phenotype, while the loss of function resulted in long and slender teeth with short mesiodistal length. This study provides evidence that a suitable level of miRNA-221-3p is required for the modulation of major signaling pathways, including Wnt, Bmp, and Shh, during tooth morphogenesis.miRNAs are conserved short non-coding RNAs that play a role in the modulation of various biological pathways during tissue and organ morphogenesis. In this study, the function of  Shh, Wnt, and Fgfs throughout the various stages of tooth development including initiation, bud, bell, cap, and root morphogenesis  morphogenesis . This prompted the investigation into the role of miRNA-221-3p in signaling modulation during tooth development, which is important for understanding the regulatory pathways involved in tooth development.miRNAs are conserved short non-coding RNAs, an average of 22 nucleotides in length. Mature miRNA are up to several hundred nucleotides in length, called pri-miRNA, before being processed by the enzymes Drosha and Dicer . The bioignaling , miR-31,incisors , and miRntiation . More thntiation , and it miRNA-221-3p, one of the conserved miRNA, was investigated via an in vitro organ cultivation system, and a loss and gain of function assay using miRNA-221-3p specific inhibitors and its mimics. miRNA-221-3p is has been well studied in cancer  was assigned to the day in which a vaginal plug was confirmed. All experiments described in this study were independently performed in triplicate as a minimum.All experiments involving animals were performed according to the guidelines of the Kyungpook National University, School of Dentistry, Intramural Animal Use and Care Committee (KNU-2020-0107). Mice were kept in an optimal environment, maintaining relative humidity (55%) and temperature (25°C) with access to food and water in situ hybridizations were performed as described previously . The miRCURY LNATM miRNA custom detection probe for 221-3P (/5DigN/AAACCCAGCAGACAATGTAGCT/3Dig_N/) was purchased from Qiagen . For section in situ hybridizations, digoxigenin (DIG)-labeled antisense RNA probes were hybridized overnight to examine the detailed expression patterns of genes as described previously  or  of the miRNA-221-3p as previously described  with 10% fetal bovine serum  and antibiotics, and cultivated using a modified Trowell’s culture method for 2 days, as previously described . To checescribed . After tescribed . Differiin vivo and in vitro tooth. Primary antibodies were directed against Ki67 ; ROCK1 ; E-cadherin ; β-catenin ; pSMAD ; AMELX  and NESTIN . The secondary antibodies used in this study were biotinylated goat anti-rabbit or anti-mouse IgG. Immunocomplexes were visualized using a diaminobenzidine tetrahydrochloride (DAB) reagent kit  and goat Anti-Rabbit Alexa Fluor@488 ; goat Anti-Mouse Alexa Fluor@647 . The DAB stained slides were mounted with xylene based mounting medium Malinol and immunofluorescent slides were mounted with VECTASHIELD® Antifade Mounting Medium containing DAPI  and cover slipped.Histology and immunostaining were carried out as described previously . Hematox1 was used to measure the fluorescence of the images as described previously (n = 3) for each group. Data were evaluated for significance using unpaired, two-tailed t-test. A p-value of <0.05 indicates significance.Histological sections were photographed using a Leica DM2500 with and without fluorescent setting. ImageJeviously . BrieflyFor colocalization analysis the correlation index (Pearson’s correlation coefficient) of colocalized pixels between the green (β-Catenin) and blue (DAPI for nucleus) colors were measured in ImageJ and plotted as graph in GraphPad Prism as described previously . Briefly2 in mesenchyme adjacent to the enamel knot. Whereas number of Ki67 positive cells from fluorescent labeled sections were presented as % of Ki67 positive cells (green) in total numbers of nuclei (blue) in the given field. GraphPad Prism (version 8) was used to calculate the test statistics and plot the graph. Briefly, after splitting the channels to blue (DAPI for nuclei) and green (Ki67) in ImageJ, the individual channels were processed through thresholding, binary processing  and finally particle measurement  was performed as previously described . RNA wasmiRNA-221-3p was restricted along the invaginated epithelium  and 1-week renal calcified teeth was similar to the in vivo post-natal 3 (PN3) mice. The double-layered HERS grew apically and meets OEE and IEE leaving no interposing layers of stellate reticulum (SR) and stratum intermedium (SI)  . The ameium (SI) . Consistoth size . Therefooth size . Meanwhioth size . In addioth size . This leoth size . As HERSormation . The secnd mimic . To confnd mimic . The parnd mimic . Howevernd mimic .in vitro cultivation and loss and gain of function of signaling molecules, demonstrate their precise roles during tissue and organ morphogenesis (miRNA-221-3p, a conserved miRNA within the animal kingdom (miRNA-221-3p has been identified to overlap with the expression of known transcription and paracrine factors, such as Bmp2, Bmp4, Bmp7, Wnt3, Wnt10a, Wnt10b, Fgf4, and Shh (The complex and dynamic molecular interactions involved in tooth development are well understood and have highlighted the importance of spatiotemporal functions of signaling molecules during odontogenesis . Experimogenesis . In this kingdom , was eva and Shh , during  and Shh . This demiRNA-221-3p during the bud stage of tooth development, highlighted its role in signal modulation during odontogenesis. Normally, the activation of Wnt/β-catenin signaling occurs in the dental epithelial and adjacent mesenchymal cells. However, upon inhibition, it resulted in the developmental arrest at the bud stage and short root phenotype (miRNA-221-3p during bud stage showed downregulation of β-catenin along the dental epithelium. Therefore, it can be suggested that miRNA-221-3p plays a modulating role in Wnt/β-catenin signaling during crosstalk between epithelium and mesenchyme, due to the optimal level of Wnt/β-catenin signaling being essential for the developmental progression of the tooth (miRNA-221-3p resulted in increased Bmp signaling and intense mesenchymal localization of pSMAD1/5/8 in the inhibitor-treated specimen. This altered Bmp signaling resulted in decreased cellular proliferation and apoptosis in the dental epithelium and adjacent mesenchyme, as in previous reports (miRNA-221-3p. Among some of the notable targets are cyclin-dependent kinase inhibitors, Bcl2 like 11, Bmpr1a, and Lrp10 suggesting that change in tooth size resulted from change in combination of different signaling pathways related to cell cycle arrest, apoptosis, BMP and WNT signaling. Future works are needed for the validation of putative role of miRNA 221-3p on modulation of these molecules.The epithelial–mesenchymal interactions up to, and beyond the bud stage plays a significant role in normal tooth progression and development . The althenotype . In our he tooth . Similarhe tooth . In our  reports . This wo reports . A miRNA reports  predictsmiRNA-221-3p decreased the expression of Fgf4, which in general, promotes proliferation, determines tooth cusps (Fgf4 is completely lost in Lef1 knockout teeth which is arrested at bud stage and recombinant Fgf4 rescued this phenotype. Consistent with this, we expected smaller tooth size with decreased level of Fgf4 in inhibitor treated specimen. Pharmacological inhibition of Fgf signaling by SU5402 blocked the epithelial morphogenesis and tooth mineralization in zebrafish (Fgf4 is important signaling for proper tooth crown morphogenesis. It also contributed to decreased tooth size through decreased cell proliferation and increased apoptosis. At the same time, Shh induced proliferation in a differential manner and determines tooth shape and affects ameloblast polarization through odontoblast differentiation and loss of Shh did not change the expression of other signaling molecules like Fgf4, Bmp2, and Wnt10b (Shh altered the expression patterns in developing dental tissues suggesting that the loss of Shh results in a change of cell fate, from proliferative to a less proliferative enamel knot-like population (Shh signaling is crucial for crown morphogenesis and evident in inhibitor-treated specimen. Consistent with our results, inhibition of Shh by 5E1 and Forskolin during tooth organ culture resulted decreased proliferation, increased apoptosis leading to altered tooth phenotype (Ptch1 suppress the Smoothened which in turn controls the Gli transcriptional effectors in the repressive forms. In the presence of SHH ligand, the Ptch1 suppression of Smoothened is relieved and the signal is transduced through activation of Gli transcription factors (miRNA-221-3p inhibitor, Ptch1 is elevated which may interfere the regulation of Shh signaling through extended Smoothened suppression. On the other hand, mimicking the miRNA-221-3p lowers the Ptch1 in the system and Smoothened may no longer be suppressed which may bypass the early step of Ptch1-Shh binding which ultimately affects the Shh signaling cascade resulting altered Shh expression in the cultured tissues. Also, these altered expression area would be explained by spatiotemporal specific expression of genes which are required for harmonious organogenesis rather than just changing in expression levels during development. These results demonstrate the tight regulation of signaling molecules during the early stages of tooth development, which lay the foundation for the complete morphogenesis of crown and root. In addition, mesenchymal expressed Bmp signaling is also important for HERS development and tooth root formation (miRNA-221-3p will guide the tooth root formation possibly through modulation of Wnt/β-catenin and Bmp signaling. Previous studies have demonstrated the role of ROCK1 in ameloblast differentiation (miRNA-221-3p in our study (miRNA-221-3p modulates ROCK1 and E-cadherin-mediated maintenance of actin cytoskeleton and cellular adhesion that ultimately regulates the differentiation stage of tooth and crown morphogenesis.Inhibition of th cusps , and preth cusps . Kratochebrafish  which ald Wnt10b . Howeverpulation  which rehenotype . In quie factors  which isormation , furtherntiation  and the ntiation . Howeverur study , suggestmiRNA-221-3p showed increased proliferative cells along the HERS and its adjacent mesenchymal cells (Shh expression, and the reciprocal interactions of Shh signaling along the HERS epithelium and adjacent mesenchymal cells, would play a significant role in tooth root formation (Shh along the HERS in the inhibitor group compared to control and mimic (Shh in the rootless K14-Cre;Smad4fl/fl (mes mutants affecting the tooth root formation (miRNA-221-3p inhibitor-treated teeth are a result of increased proliferation and intense Shh expression. The inhibition of miRNA-221-3p at the bud and cap stage resulted in a decreased expression of Shh after 2 days (Shh along the HERS in the inhibitor group compared to control and mimic (miRNA-221-3p may also be regained and upregulate the Shh expression in the HERS after 1-week.During mice molar development, at around PN2, crown formation is almost completed and the root formation initiates with the emergence of HERS . The coral cells . The epiormation . Howevernd mimic . This maad4fl/fl . In addiormation  further r 2 days , howevernd mimic . As the nd mimic , it can miRNA-221-3p modulates the Shh and Wnt signaling during cap and bell stages of tooth development, and the reciprocal interaction between the mesenchyme and epithelium, in turn, regulates other signaling molecules, such as Fgf and Bmp. These changes in signaling eventually determine the fate of tooth crown and root morphogenesis through hard tissue formation. Therefore, we conclude that the modulation of miRNA-221-3p signaling may explain some of the processes involved in the integrative and fine-tuning networks of signaling molecules involved in tooth development. In addition, it is required to examine the detailed signaling regulations of SHH signaling through miRNA-221-3p using pharmacological modulators of miRNA inhibited tooth culture and, rescue of β-catenin and Fgf signaling. Furthermore it also would be necessary to reveal the consequence signaling including p-ERK, Etv4, Etv5, and so on using genome wide screening methods to define the detailed signaling modulations in tooth morphogenesis.In conclusion, The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by KNU-2020-0107.YA, SN, and J-YK contributed to conception, design, and data interpretation, critically revised the manuscript. T-YK, E-SL, C-HA, J-YK, HY, SL, YL, and W-JS contributed to data analysis, interpretation, and critically revised the manuscript. All authors gave final approval and agreed to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Phenolic compounds from fruits and vegetables have shown antioxidant, anticancer, anti-inflammatory, among other beneficial properties for human health. All these benefits have motivated multiple studies about preserving, extracting, and even increasing the concentration of these compounds in foods. A diverse group of vegetable products treated with High Hydrostatic Pressure (HHP) at different pressure and time have shown higher phenolic content than their untreated counterparts. The increments have been associated with an improvement in their extraction from cellular tissues and even with the activation of the biosynthetic pathway for their production. The application of HHP from 500 to 600 MPa, has been shown to cause cell wall disruption facilitating the release of phenolic compounds from cell compartments. HPP treatments ranging from 15 to 100 MPa during 10–20 min at room temperature have produced changes in phenolic biosynthesis with increments up to 155%. This review analyzes the use of HHP as a method to increase the phenolic content in vegetable systems. Phenolic content changes are associated with either an immediate stress response, with a consequent improvement in their extraction from cellular tissues, or a late stress response that activates the biosynthetic pathways of phenolics in plants. Phenolics are a group of specialized metabolites with antioxidant, antimicrobial, anticancer, anti-inflammatory activity, among other biological properties for human health ,2. FruitThermal treatments are frequently used during fruit and vegetable processing to inactivate pathogenic and spoilage microorganisms and enzymes to ensure food safety and quality . HoweverPressure is a physical parameter that affects the state of physical, chemical, and biological systems. Pressure can modify the chemical configuration of some food molecules changing the rate of chemical and enzymatic reactions . HHP is HHP to inactivate microorganisms was first proposed in the late 1980s. Since then, the use of this technology as a preservation method has increased in the food industry. The ability of HHP to inactivate microorganisms and enzymes related to food oxidation are some examples of the benefits involved . HHP-treIn addition to food preservation, HHP has been proposed as a method to enhance bioactive compound content in foods and to improve their extraction. Some pressurized foods have shown higher contents of functional compounds such as phenolics compared with untreated products. The increments have been associated with the release of these compounds from cellular compartments, resulting in increased extractability due to the mechanical stress occurring during the pressurization, which compacts the cellular morphology, cell wall, and organelles . AnotherThis review analyzes the mechanisms for phenolic increment in vegetables and fruits immediately after HHP treatment and during storage, differentiating between increment due to cellular disruption (improvement of extraction yield) or activation of the metabolic pathways for compound biosynthesis.The biosynthesis of phenolic in plants is achieved by the malonate and the shikimate pathways . The malAfter the production of chorismate, the phenic acid is formed by the enol-pyruvate transferase action, the phenic acid goes under a decarboxylation, and the substitution of the oxygen for an amino group results in the formation of L-phenylalanine . The mosThe production of specialized metabolites in plants is affected by biotic and abiotic factors, such as environmental conditions, microorganisms, insect attacks, among others . Biotic Phenolic compounds are implicated in the biotic and abiotic stresses by reinforcing cell walls and scavenging of ROS (Reactive Oxygen Species) . Biotic Mechanical force is another important abiotic stress factor that has shown a positive effect on the production of phenolics in plants. HHP at pressure levels below 100 MPa acts as a mechanical stressor resulting in an increment of phenolic levels ,45. PresAlthough the effects of HHP on the biosynthesis of phenolics have been evaluated in fruits and vegetables such as mangoes, carrots, strawberries, and suspension cultures of grapes and potato, there is not yet enough research on the mechanism(s) implied in the increment for the biosynthesis of phenolics by HHP. Several studies using pressures from 10 to 100 MPa at treatment times from 10 to 20 min at room temperature have been tested immediately after processing or during the storage at different temperatures and relative humidities; and the results have revealed a change in phenolics biosynthesis with increments up to 155%. Some studies conclude biosynthesis of phenolics due to the low-pressure levels used and the increment in their content, nevertheless, some of them do not show studies related to increment in PAL activity, ROS production, or gene expression, which are important to conclude this .Phenolic biosynthesis due to HHP does not seem to increase proportionally with the pressure. The increments primarily depend on the type of fruit treated and the ripening stage, as well as, the storage conditions such as temperature, relative humidity, and storage time ,47. In a2O2, which later acts, controls, and initiates enzymatic responses to repair the damaged cell wall via stress-responsive gene, oxidative burst linked with cell wall reinforcement, biosynthesis of phenolics, among others [2O2 is carried out in minutes, acting as the elicitor in the biosynthesis. The production of H2O2 at the cellular level acts as Ca+2 signaling, activates kinases, hormonal signaling, and regulates gene expression [2O2 as a signaling molecule activating metabolic pathways [Ortega et al.  suggesteg others ,48,54. Ig others . The relpression . H2O2 aspathways , leads tpathways ,31,32.2O2, while the late response involves the biosynthesis of secondary metabolites during storage [Similar to mango, for HHP-treated carrots after one day of storage, the total phenolic content increased 69.1% at 60 MPa and 154.9% at 100 MPa . After d storage ,19,47.Solanum tuberosum) suspension culture treated at 100–200 MPa for 10 min, phenolic content increased immediately after processing by 54, 81, 267, 456, and 453% at pressures of 100, 125, 150, 175, and 200 MPa, respectively; nevertheless, these increments appear not to be associated with biosynthesis, but rather with the loss of compartmentation and subsequent release of the content of the vacuoles into the cytoplasm [The synthesis of phenolics in cell cultures has also been evaluated. Cai et al.  studied ytoplasm .Bioactive compounds, such as phenolics, are contained in specific organelles in the cell, which can vary depending on each type of product and variety . The inc2) [Several methods have been applied for phenolic extraction, such as conventional solvent extraction (CSE), and novel technologies like ultrasonic-assisted extraction (UAE), microwave-assisted extraction (MAE), and supercritical fluid extraction (SFE-CO2) ,60,61. THHP can also favor the release of bioactive compounds from cellular compartments, enhancing their extractability . When frThe effects on plant tissues, organelles, cell walls, and membranes, depend on the pressure level . For exaIn most cases presented in Silvetia compressa, where 400 MPa (15 min) and 600 MPa (5 min) decreased phenolics by 30 and 41%, respectively [The levels of improvement during HHP processing depend on a variety of factors such as treatment conditions, type of compound, food physical characteristics, and composition. While, after HHP processing, the method used for phenolic extraction  could influence the yield obtained among different studies from different authors. In addition, the storage conditions and handling after and during processing could influence results among different studies. As mentioned before, during processing, HHP could influence enzymes increasing their activity due to factors such as the release of membrane-bound enzymes or configuration changes, this last mechanism is also related to decreasing activity . Accordiectively . In caseectively , wild beectively , and eveectively .Granny Smith) treated at 500 MPa, it was showing a higher absorption of minerals produced by high solubility in the intestine [It has been suggested that the increment in bioactive compounds by HHP could result in better bioavailability. Bioavailability determines the number of bioactive compounds that are digested, absorbed, and metabolized and therefore it determines their action in the human body ,79,80. Fntestine , while fntestine ,4.2O2 activates metabolic routes that increase the phenolic content. Despite very few cases, the use of HHP showed to be a very innovative and promising technology to improve the phenolic compounds in plants. The results presented in this review are highly relevant for the future use of HHP technology for both biosynthesis and extraction of phenolics or even other secondary metabolites with functional activities. This technology could help to generate food with better nutritional and functional value, including enhanced antioxidant activity, in addition to a better extraction could even influence the bioavailability of these compounds in the human body, increasing their beneficial effects on health.This work describes two possible mechanisms for the increment of phenolics in foods after HHP treatment and during storage. The overview presented suggested that the increments could be related to phenolic biosynthesis and improvement of their extractability in food. The enhancement of the extraction of phenolic through HHP has been related to an immediate response to stress, where the HHP disrupts cellular compartments enhancing mass transfer and extractability or due to the disruption of non-covalent interactions between phenolics and cell wall. Despite a few cases, HHP has shown to be more effective at higher pressure levels, (>200 MPa); however, the level of yield achieved highly depends on the type of food and intensity of the other variables  as well as the extraction/analysis methods used. The use of HHP at lower values of pressure (<100 MPa) has been shown to activate biosynthesis of phenolic in a late response where the higher production of ROS such as H


Erythropoietin (EPO) is a glycoprotein cytokine known for its pleiotropic effects on various types of cells and tissues. EPO and its receptor EPOR trigger signaling cascades JAK2/STAT5, MAPK, and PI3K/AKT that are interconnected and irreplaceable for cell survival. In this article, we describe the role of the MAPK and PI3K/AKT signaling pathways during red blood cell formation as well as in non-hematopoietic tissues and tumor cells. Although the central framework of these pathways is similar for most of cell types, there are some stage-specific, tissue, and cell-lineage differences. We summarize the current state of research in this field, highlight the novel members of EPO-induced PI3K and MAPK signaling, and in this respect also the differences between erythroid and non-erythroid cells. Erythropoiesis is the process of creating mature red blood cells from pluripotent hematopoetic progenitor cells, resulting in the production of approximately two million erythrocytes per second . ErythroProduction of mature red blood cells is a sophisticated multistep process located in erythroblastic islands, a specialized niche within the bone marrow with a population of erythroid cells in different stages of differentiation and the central macrophage  that is As we have described the role of the STAT5 protein as a key molecule of EPO signaling in our recent paper , we will3, E, and A in addition to the upregulation of specific markers of immature erythroid progenitors c-KIT and E-cadherin [The EPOR/PI3K signaling cascade is crucial in mediating signals for survival, proliferation, and terminal differentiation of erythroid progenitors ,13. PI3Kcadherin . Interescadherin  proved tDuring erythroid maturation, MAPK as activated by EPO plays an important role in myeloid gene suppression, while the PI3K pathway is critical for erythroid gene induction . RecentlMAPKs are a family of evolutionarily conserved Ser/Thr kinases that play crucial roles in proliferation, migration, differentiation, senescence, and apoptosis. They are also frequently connected and/or involved in the signaling pathways of oncogenesis, tumor progression, and drug resistance. In mammals, MAPK proteins can be divided into several subgroups including extracellular signal-regulated kinases 1 & 2 (ERK1/2), p38 MAP kinases, and c-Jun amino-terminal kinases (JNK), and the family of ERK5 proteins and atypical ERK3, 4, 7, and 8  Figure . The actDuring erythroblast differentiation, an upstream signaling molecule of EPOR spleen tyrosine kinase (SYK) is constitutively associated with EPOR in the plasma membrane and is required for STAT5 and ERK activation induced by EPO . MaintenJAK2 kinase is crucial for successful EPO/EPOR activation of the PI3K and MAPK pathways. The pair of JAK2 molecules is constitutively associated with cytoplasmic box 1 on the intracytoplasmic site of EPOR . After E2 also mediates both AKT and MAPK signaling by activating protein kinase C (PKC) that mediates the signal to RAF [2 signals to another. PREX1, an important guanine nucleotide exchange factor, is also an activator of both AKT and MAPK signaling during tumorigenesis [PI3K/AKT and MAPK pathways have numerous overlaps and cross-talks between each other to support mechanisms of cell survival. Indeed, they act as reciprocal inhibitors . It is wl to RAF . Moreoveigenesis . Interesigenesis .EPO signaling cascades are activated in many non-hematopoietic tissues in order to prevent against tissue injury and damage . Either The EPOR/PI3K pathway operating in the nucleus is well described in erythroid progenitors, while non-hematopoietic tissues also use mitochondrial engagement. It is well known that the protection of the mitochondrial membrane integrity and the stopping of cytochrome c release from the mitochondria is the main mechanism by which AKT prevents cell death . MoreoveCarelli et al.  discoverDiverse cross-talks between EPO-mediated MAPK and PI3K/AKT signaling exist in the nervous system and their complex interaction depends on the respective cellular process and the cell type. The synergistic effect of ERK1/2 and AKT activation by EPO was required for neuroprotection  and for It is known that endogenous EPO regulates the bone environment on diverse levels. In bone, EPOR receptors are present on osteoblasts, osteoclasts, and on bone marrow stem cells (BMSCs) that can differentiate into osteoblasts, chondrocytes, and bone marrow adipocytes . During Regulation of the bone marrow microenvironment is associated with the EPO/MAPK pathway activity, inhibiting the adipogenic differentiation of bone marrow mesenchymal stem cells . InteresEPO’s protective effect was described in a wide variety of processes in cardiovascular pathophysiology, particularly in ischaemia, cell proliferation, apoptosis, and platelet activation. EPO exhibited myocardial protection through the MAPK/ERK induced GATA-4 stability, reduction of caspase-3 activity, upregulation of Bcl-2 , and decEPO and its derivatives such as helix B surface peptide (HBSP) revealed protective effects in transplant-related renal injuries such as ischemia-reperfusion injury (IRI) and immunosuppressant nephrotoxicity . RenoproThe importance of EPO in the proliferation, migration, and invasion via p38 MAPK and ERK1/2 signaling was confirmed on vascular smooth muscle cells . FurtherThe expression of EPO and EPOR in retinal tissues indicates its autocrine or paracrine action. EPO can protect retinal tissues including microvascular endothelium, retinal epithelium, Müller cells, and retinal neurons. This effect of EPO is partly dependent on the activation of PI3K and MAPK pathways. During diabetic retinopathy, inner blood-retinal barrier (BRB) breakdown occurs while EPO prevents this damage via the inhibition of microglial activation and phagocytosis mediated by the SRC/AKT/COFFILIN signaling pathway . FurtherResearch progress in the area of erythropoiesis with extensive proteomics, transcriptomic, and epigenetic studies on EPO signaling is currently absent regarding malignant cells. Nevertheless, we can conclude that EPO signaling has several different features in cancer cells compared to erythroid tissue. Firstly, cancer cells have a lower number of receptors for EPO compared to erythroid progenitors . Indeed,In addition, microenvironments of bone marrow and tumors differ from each other, therefore EPO signaling may be influenced by distinct mechanisms and interactions of signaling molecules. In tumor tissues, autocrine or paracrine secretion of EPO is typical compared to endocrine distribution of EPO required for erythropoiesis. Moreover, the hypoxic microenvironment of cancer tissues stimulates the local EPO production, supporting viability and independent tumor growth. While EPO signaling during erythropoiesis plays a main role in differentiation and proliferation, in cancer cells the signaling refers more to anti-apoptotic cell survival and migration activity. Importantly, the main framework of EPO signaling remains unchanged across all types of cells.EPOR and its downstream signaling pathways STAT5, PI3K/AKT, and MAPK were found as constitutively active and independent of EPO stimulation in non-small cell lung, renal, and ovarian cancer cells but the mechanism underlying this phenomenon is not sufficiently described ,115,116.EPO activates the PI3K/AKT pathway in human melanoma cells  and bothCell migration is considered an initial step in metastasis and may be regulated by a variety of signaling pathways. Interestingly, the migration of human breast cancer cells with stable overexpression of EPOR was promoted by increased activation of the ERK1/2 pathway but not through the JAK2-STAT5 axis . On the The interaction of EPO and EPOR triggers the activation of several signaling pathways including PI3K/AKT and MAPK. They are known to be important regulators of the differentiation, proliferation, and cell survival of erythroid cells. More information is emerging about their role in the protection of non-hematopoietic tissues and cancer cells. In this article, we have attempted to summarize recent findings on the EPO-induced PI3K and MAPK pathways, and described their role in numerous cellular events. Currently, while the entire process of erythroid differentiation is influenced by many different interventions, more investigations are required to uncover the diverse effects of EPO on the erythropoiesis in addition to its well-characterized proliferative and pro-survival effects. The future challenge in this area is to intersect detailed interactions of signaling molecules and determine their contribution to and beyond erythropoiesis. Advances in this area of research may be useful for improving the efficacy of EPO therapy in clinical use and to gain a better understanding the overall processes of cellular signaling.


Background: Aging is associated with decline in mitochondrial function and reduced exercise capacity. Urolithin A (UA) is a natural gut metabolite shown to stimulate mitophagy and improve muscle function in aged animals, and induce mitochondrial gene expression in elderly. Purpose: Investigate if oral administration of UA improved walking distance (6MWT), muscle fatigue resistance in hand (FDI) and leg (TA) muscles, and had an impact on plasma biomarkers. Method: We conducted a randomized, double-blind, placebo-controlled study (NCT03283462) in elderly subjects (65-90 yrs.) supplemented daily with 1000mg UA or placebo for 4 months. 128 subjects were screened and 66 randomized. 6MWT and ATPmax via MRS were assessed at baseline and at 4 months. Muscle fatigue tests and plasma analysis of biomarkers were assessed at baseline, 2 and 4 months. Results: UA significantly improved muscle endurance  in two different muscles  compared with placebo at 2-months. Plasma levels of several acylcarnitines, ceramides and C-reactive-protein were decreased by UA at the end-of study. 6MWT distance  and ATPmax increased in both groups from baseline  with UA supplemented group exhibiting greater improvements, although these were not statistically different between groups. Conclusion: UA supplementation improved muscle endurance, metabolic and inflammatory plasma biomarkers after 2-months, suggesting that UA can have a positive impact on muscle and cellular health in the elderly.


The HIV co-receptors, CCR5 and CXCR4, are necessary for HIV entry into target cells, interacting with the HIV envelope protein, gp120, to initiate several signaling cascades thought to be important to the entry process. Co-receptor signaling may also promote the development of neuroHIV by contributing to both persistent neuroinflammation and indirect neurotoxicity. But despite the critical importance of CXCR4 and CCR5 signaling to HIV pathogenesis, there is only one therapeutic (the CCR5 inhibitor Maraviroc) that targets these receptors. Moreover, our understanding of co-receptor signaling in the specific context of neuroHIV is relatively poor. Research into co-receptor signaling has largely stalled in the past decade, possibly owing to the complexity of the signaling cascades and functions mediated by these receptors. Examining the many signaling pathways triggered by co-receptor activation has been challenging due to the lack of specific molecular tools targeting many of the proteins involved in these pathways and the wide array of model systems used across these experiments. Studies examining the impact of co-receptor signaling on HIV neuropathogenesis often show activation of multiple overlapping pathways by similar stimuli, leading to contradictory data on the effects of co-receptor activation. To address this, we will broadly review HIV infection and neuropathogenesis, examine different co-receptor mediated signaling pathways and functions, then discuss the HIV mediated signaling and the differences between activation induced by HIV and cognate ligands. We will assess the specific effects of co-receptor activation on neuropathogenesis, focusing on neuroinflammation. We will also explore how the use of substances of abuse, which are highly prevalent in people living with HIV, can exacerbate the neuropathogenic effects of co-receptor signaling. Finally, we will discuss the current state of therapeutics targeting co-receptors, highlighting challenges the field has faced and areas in which research into co-receptor signaling would yield the most therapeutic benefit in the context of HIV infection. This discussion will provide a comprehensive overview of what is known and what remains to be explored in regard to co-receptor signaling and HIV infection, and will emphasize the potential value of HIV co-receptors as a target for future therapeutic development. Infection with human immunodeficiency virus (HIV), the cause of acquired immunodeficiency syndrome (AIDS), has been a major public health issue since the emergence of the virus in the early 1980s and more than 38 million people are currently infected with HIV . Indeed,Infection with HIV is almost always mediated by interactions between the HIV envelope protein, gp120, the CD4 receptor and a co-receptor, generally the chemokine receptors CCR5 or CXCR4. These chemokine receptors , were fiαi [αq and through non-Gα protein pathways, including those initiated by Gβγ and β-arrestins [Much of the current research on CCR5 and CXCR4 focuses on their role as HIV co-receptors. However, both CXCR4 and CCR5 are also chemokine receptors, and their activation mediates complex signaling cascades that initiate a variety of other functions under both pathological and homeostatic conditions –30. As mαi  but therrrestins , 34–36. rrestins –41. Howerrestins , 43, furrrestins , 44, 45.Despite these challenges, there is a critical need for novel drugs and strategies that target co-receptor signaling. This is particularly true in the context of neuroHIV, where the interaction of HIV virions with CCR5 and CXCR4 expressed on both myeloid and neuronal cell populations seems to play a role in persistent neuroinflammation and neuronal dysfunction –51. UnfoWhile the number of new HIV infections per year has declined since 2000, and people are now living longer with HIV, nearly 2 million new infections and 800,000 HIV-associated deaths still occurred in 2019 , 53. ThiThese comorbidities result from HIV infection and associated inflammation in organs throughout the body. HIV primarily infects CD4 + T-cells and myeloid lineage cells including monocytes and tissue specific macrophages such as microglia and alveolar macrophages , 67, 68.Following entry, the viral capsid undergoes remodeling by host proteins in a process called uncoating, and the viral reverse transcriptase enzyme converts the HIV RNA into proviral DNA, although the precise order of these operations is still not completely clear . There i+/CD16+ monocytes, and possibly infected CD4 + T-cells, which transmigrate across the blood–brain barrier (BBB) and release virus into the CNS [Neurological symptoms associated with HIV infection have existed since the start of the epidemic , 88, and the CNS , 96–100. the CNS , 102. Th the CNS –103. The the CNS , 104–108 the CNS –111. It  the CNS –115. Alt the CNS , 116 andPrior to cART, HIV-associated neuropathology was characterized by the formation of multinucleated giant cells, presence of microglial nodules, infiltration of lymphocytes, activated CNS macrophages, and neuronal loss , 117–120Current data indicate chronic neuroinflammation is a key factor in neuropathogenesis in cART-treated individuals , 143–145Studies indicate that CNS viral infection is incompletely suppressed by cART, suggesting that neuronal injury and inflammation associated with co-receptor signaling in cART-treated individuals may be due to intact virions. HIV RNA is detectable in the CSF and CNS of cART treated populations, indicating the persistence of viral replication –60, alth3C receptors [Both CCR5 and CXCR4 are G-protein coupled, 7-transmembrane receptors (GPCR) that belong to a family of 20 proteins known as chemokine receptors. These receptors are expressed on a wide range of cell types and are classified into four subgroups depending on which subfamilies of chemokines they bind: CXC, CC, XC, and CXeceptors , 30, 183eceptors , 184. Theceptors , 186. Wheceptors , 188, CCeceptors , 190, maα, Gβ, and Gγ subunits [α subunit by exchanging a bound guanine diphosphate (GDP) for a guanine triphosphate (GTP). This causes the Gα subunit to dissociate from the receptor to initiate distinct downstream signaling cascades [α proteins: Gαs, Gαi, Gαq, and Gα12/13, each associated with distinct signaling cascades, though distinct G-proteins can activate the same downstream effectors or have overlapping functions [α proteins are more frequently studied, but Gβ and Gγ subunits, which form an obligate dimer (Gβγ), can also activate several signaling mechanisms [αi signaling [Like other GPCRs, chemokine receptors transmit signals resulting from ligand binding via an associated complex of three distinct G-proteins; Gsubunits . In the cascades . There aunctions . The sigchanisms –195. Thechanisms , 197. Thchanisms , 198–200chanisms , 201, whignaling , and canignaling –205 as wignaling , 206.Chemokine receptor signal transduction is also regulated by blocking receptor signaling. This involves GPCR kinases (GRKs) phosphorylating several different residues on the active carboxyl terminus of the GPCR, recruiting β-arrestins , 208. Foαi [αi can also activate Src and Lyn kinases [βγ subunits can also stimulate cell movement [2) to diacylglycerol (DAG) and inositol triphosphate (IP3), initiating intracellular calcium (Ca2+) release from the endoplasmic reticulum as well as activation of protein kinase C (PKC). Several of these cascades, including activation of PI3K and AKT [The primary function of chemokine receptors is to mediate cell migration, and much of the signaling originating from these receptors promotes cell movement. Classically, these receptors couple to Gαi , 213–215αi , 215–217 kinases , 219, wh kinases –222 and  kinases –228. Relmovement , activatmovement , 230; thmovement ; or phosmovement , 233, wh and AKT , 206, or and AKT –205, can and AKT , 198–200These are just some of the vast network of signaling pathways associated with chemokine receptor activation, and both CCR5 and CXCR4 have a number of important pathways specifically associated with their activity. The complexity of these and other signaling networks initiated by co-receptor binding is often amplified by the use of different types of cell systems. The use of different types of cells has enabled more precise interrogation of specific signaling processes, but it has also led to confusing and contradictory data, as co-receptors in distinct cell types often show different responses to similar stimuli. This is particularly problematic when comparing transfected cells and cell lines to primary cells and in vivo systems, as it can create confusion about the physiologically relevant actions of co-receptors during disease. These signaling processes, their downstream effects and the caveats associated with their study are discussed below, and those involved in HIV pathogenesis are described in detail in the subsequent section. Further discussion on the functions and pathways activated by chemokine receptor signaling can be found in additional reviews , 234–236The CCR5 receptor was first identified as a human monocyte chemokine receptor in 1996, following the discovery of its primary ligands CCL3 (MIP1α), CCL4 (MIP1β), and CCL5 (RANTES) . Only CCUnder homeostatic conditions, chemokine signaling through CCR5 is associated with numerous physiological processes including leukocyte migration , 242–244Due to its broad influence, CCR5 signaling is tightly regulated by several processes, generally triggered by phosphorylation of serine residues on its C-terminus and the Asp-Arg-Tyr (DRY) motif leading to GRK/arrestin mediated receptor desensitization and internalization , 252. Thαi inhibits the activation of adenylate cyclase, regulating the production of cAMP [αi can be influenced by the formation of oligomers, changing the responses to select ligands [αi inhibitor pertussis toxin (PTX) block CCL5-induced Ca2+ release in cells expressing only CCR5, but not in cells co-expressing CCR5 and CCR2 and treated with CCL2 and CCL5 [The CCR5 receptor can signal through several distinct G-protein mediated pathways. Signaling through G of cAMP , 251 and of cAMP , 264, an of cAMP , 265–267 ligands . For exaand CCL5 . Homodimand CCL5 , 270, al2+ release and PLCβ activation, which can be mediated by Gβγ [αi-independent effects via its B-oligomer [βγ, rather than Gαi is essential for chemotaxis mediated by Gαi-coupled receptors [βγ may be more critical in mediating the effects of CCR5 than Gαi.However, several CCR5 signaling pathways are insensitive to pertussis toxin, indicating the involvement of alternative G-proteins and/or G-protein independent signaling mechanisms –273. Mucd by Gβγ , 274. Mooligomer , 275, oreceptors , 229. Boeceptors  and T-lyeceptors , 276. Sieceptors , 267, wheceptors  and chemeceptors , 279. Aceceptors , 280, aneceptors , 267. Thαq, which can also initiate IP3-mediated intracellular Ca2+ release [2+ flux in HEK.CCR5 cells, and this was inhibited by the Gαq inhibitor YM-254,890 [αq-specific CCR5 signaling is unclear, due to the extensive overlap between Gβγ and Gαq signaling. However, the centrality of Gβγ-mediated PI3K signaling to chemotaxis suggests CCR5 mainly acts through Gαi and Gβγ in response to physiological ligands [αq signaling, as siRNA against Gαq, but not Gαi, prevented CCR5-mediated changes in viral fusion [αi independent signaling effects [CCR5 may also couple to G release , 281. A -254,890 . The phy ligands , 229. In effects , 282–285αi, Gαq, and Gβγ signaling, several signaling processes may be independent of G-protein activity. Stimulation of CCR5 by either CCL2, CCL3, or CCL5 leads to activation of janus kinases 1 and 2 (JAKs) [In addition to G2 (JAKs) , 272, an2 (JAKs) .αi-mediated activation of both the MEK/ERK and PI3K/AKT signaling cascades, reducing apoptosis in mouse macrophages [2+ mobilization and the activation of JAK1/STAT5, triggering cell polarization and migration. However, this did not occur with AOP-RANTES, a synthetic CCL5 derivative that can also bind to CCR5 but does not induce chemotaxis [αi-association and subsequent release of Gβγ [The responses to CCR5 binding are mediated by a number of effectors, including but not limited to MAPKs , 286, sirophages . Stimulaemotaxis , 290, due of Gβγ . Many ofe of Gβγ . This dee of Gβγ , 291. Thnd messenger induction [αi-associated GTPγS binding, but not Ca2+ flux, suggesting stabilization of specific conformations of CCR5 is associated with distinct signaling cascades [In addition to complications due to overlapping pathways and distinct effects in different cell types , 272, annduction , 297. Innduction , 298. Mocascades .Other studies show differences in the β-arrestin mediated internalization of specific CCR5 populations, suggesting subpopulations of CCR5 conformations may be resistant to chemokine-induced internalization in macrophages, but not T-cells . This isThe CXCR4 receptor was first identified on peripheral blood lymphocytes in 1994  and is hnd and 3rd extracellular loop [In adults, CXCL12-CXCR4 binding promotes the survival and trafficking of immune cells , 308. Dular loop .Like CCR5 and other GPCRs, CXCR4 signaling is regulated by desensitization (homologous and heterologous), internalization, and degradation. Direct activation of PKC by phorbol esters , 323, T αi mediated inhibition in cAMP production. Recombinant overexpression systems, such as HEK293T and Sf9 cells show that CXCR4 can activate different Gαi proteins, including Gαi1, Gαi2, Gαi3, and Gαo in response to CXCL12 stimulation [αi1 and Gαi2 rather than Gαi3 and Gαo [αi triggers activation of MAPK and PI3K pathways [α13 [αq [αq in dendritic cells and granulocytes but not T and B cells [α13 may have particular relevance in cancer, where Gα13 is overexpressed [αq can induce IP3 mediated Ca2+ release through PLCβ [βγ subunit [α13 in Jurkat T cells mediates cell migration via activation of Rho [Like CCR5 and other chemokine receptors, CXCR4 signals primarily through G-protein dependent pathways, primarily Gmulation , althoug and Gαo , 334. Acpathways , mediatipathways –338 as wpathways , 339. CXays [α13  or Gαq [[α13 [αq , 342, al B cells , and couxpressed , 345. Asugh PLCβ , 342; th subunit , 346. Acn of Rho , and alsn of Rho .2+ from intracellular stores, and after its nuclear translocation, the transcription of several target genes [αi with CXCR4 is dependent on JAK, further supporting a co-dependent mechanism between members of the JAK/STAT pathway and G-protein coupled signaling [Like CCR5, CXCR4 can also signal through G protein independent mechanisms, such as β-arrestin-mediated signaling. Both β-arrestin-1 and -2 enhance CXCR4-mediated ERK activation , and β-aet genes . Studiesignaling   and this was dependent on the presence of microglia . Increasoverload , alteratoverload , 154, anoverload , all of overload –106. ThuSubstance use disorders (SUD) have been comorbid with HIV infection since the start of the epidemic. Rates of SUD vary widely by region, substance, and specific sub-population, but are found in roughly in 9–48% of HIV-infected individuals globally, compared to 0.7–8.6% of the population as a whole –495. TheStimulants include methamphetamine (Meth), cocaine, and prescription drugs used to treat conditions such as ADHD, all of which act by directly increasing CNS dopamine –510. DopThese affects may be further amplified through the direct interaction of stimulants with CCR5. Cocaine can increase mesolimbic CCR5 expression, and the conditioned place preference behaviors associated with cocaine are reduced by the use of maraviroc, a CCR5 antagonist . Meth haOpioids are also commonly abused in HIV-infected populations and can exacerbate the symptoms of HAND , 533–535Co-receptors are essential for HIV entry and therefore make an attractive target for anti-retroviral therapeutics. Blocking cell entry prevents viral replication, and when combined with other drugs this can achieve sustained suppression, halting disease progression. Further, the role of co-receptor signaling in modulating neuroinflammation suggests that targeting the downstream signaling events may reduce the inflammatory effects of HIV, particularly in combination with other antiretrovirals. However, despite more than 30 years of antiretroviral development and numerous claims that co-receptor targeting was an emerging, promising therapy –549, theThere are a number of reasons co-receptors are difficult to target, including the ability of HIV to utilize multiple different conformations , 300 andEarly in the HIV epidemic, it was observed that individuals homozygous for the CCR5- defects , 559. Thd of HIV . Anotherd of HIV , and CCRd of HIV , necessid of HIV . Unfortud of HIV –462, 563d of HIV –294, 300d of HIV , 564.This may be due to the capacity for gp120 to efficiently interact with CCR5 in multiple structural conformations, whereas natural ligands and inhibitors such as Maraviroc can only target specific conformations , 301. MuThis has led to an interest in developing biased ligands or small molecules that promote the internalization of CCR5 or bias signaling pathways to prevent HIV entry without stimulating downstream signaling , 566. SeWhile blocking CCR5 has been considered a more promising strategy for inhibition of viral entry and replication, an increasing number of patients are developing X4 tropic or dual-tropic viral strains that can bind to CXCR4 . This suThere are two primary challenges associated with developing CXCR4-targeting therapies, accommodating receptor oligomerization and minimizing undesirable side effects due to the normal, homeostatic functions of CXCR4. The ability of receptors to affect one another through oligomerization makes it essential to consider the in vivo state of CXCR4, particularly because CXCR4 may homodimerize in both the absence  or preseThe biggest challenge in the development of CXCR4-targeted HIV entry inhibitors is overcoming undesirable side effects that result from inhibition of CXCR4, as this receptor has essential roles in numerous homeostatic functions in the periphery and CNS. Studies on two of the CXCR4 antagonists brought into clinical testing (AMD3100 and AMD11070) were terminated early due to toxicity . Thus, iWhile strategies to target CCR5 and CXCR4 via small molecules, antibodies, and chemokine analogues are promising, none of these holds promise in blocking both receptors concurrently. Dual co-receptor antagonists have been described , but theCCR5 and CXCR4 have a number of common pathways, with several specific effectors such Pyk2 and p38 MAPK that are involved in a number of HIV-associated pathologies. These effectors are also involved in other diseases, including cancer , 574, soA huge body of work indicates co-receptor signaling pathways activated by HIV are not only critical to the viral entry and replication process, but also to the broader neuroinflammatory and neurotoxic impact of HIV in the CNS. There have been numerous attempts to develop therapeutics targeting CXCR4 and CCR5, but most of these candidates did not or have not yet advanced out of trials, and many failed due either lack of efficacy or serious side effects , 583–587It is important to note that the majority of studies discussed here have been performed using highly artificial conditions. Thus, while they suggest a critical role for co-receptors, they preclude drawing a definitive conclusion regarding the impact of co-receptor signaling on HIV neuropathogenesis. This further highlights the need for future studies to make use of physiologically relevant cell types, and take advantage of new technologies, such as 3D culture systems and iPSC-derived primary cells, as well as animal models that more closely parallel the human immune system. Many of the neuroinflammatory effects of HIV-co-receptor interaction involve multiple cell types and show distinct activity in co-cultures relative to monocultures. Therefore, the development and use of mixed culture systems and organoids using combinations of neurons, glia and other myeloid cells will be critical to better mimic the interactions in the CNS. Combining data from these systems with more reductionist, single cell models that capitalize on newer technologies such as CRISPR and single cell genomic analyses such as single cell RNA sequencing (scRNAseq) and single cell Assay for Transposase Accessible Chromatin (scATACseq), will enable targeted analyses of changes in the expression, transcription and or function of specific effectors within different signaling pathways. Moreover, pairing these assays with conformational studies will enable precise assessment of the effects of specific conformational changes on HIV binding and signaling though CXCR4 and CCR5. These new systems should be combined with experimental consistency and physiological accuracy in regard to the use of HIV concentration and treatment paradigms. This is particularly true in the use of monomeric gp120 versus intact HIV virions, given the lack of a demonstrable role of gp120 in driving neuroHIV in the cART era. The use of monomeric gp120 has been valuable in a number of areas, including studies highlighting the complexity of signaling pathways associated with HIV activation of co-receptors. However, the field needs to better address signaling in response to HIV virions, as this may reveal distinct differences in co-receptor engagement and point towards more physiologically relevant signaling effectors that can be targeted.A better understanding of the dynamic interaction between HIV and the CXCR4 / CCR5 co-receptors should enable the efficient development of ligands that precisely target only the signaling processes involved in entry. More broadly, both CXCR4 and CCR5 affect a wide array of diseases and homeostatic processes, from cancer to cardiac disease, indicating that effectively targeting co-receptors and manipulating their signaling could be a useful tool in the treatment of a number of diseases. Thus, these same studies, experimental pipelines and drug candidates will be used to develop therapeutics that not only inhibit the effects of co-receptors on HIV neuropathogenesis but could potentially be used to treat a number of other important human diseases.