The involvement of the testis by metastatic medullary thyroid carcinoma has never been described before. We describe the first case of metastatic medullary thyroid carcinoma affecting testis and inguinal lymph nodes.A 73-year-old Caucasian man was referred to undergo urologic surgery due to a painless nodule in the right testis and an homolateral inguinal lymphoadenomegaly. The patient had a history of medullary thyroid carcinoma with relapsing disease to the spine and lung nodules. Serum calcitonin and CEA levels were 175 pg/ml and 22 ng/ml, respectively. With suspected testicular cancer, the patient underwent radical right orchiectomy with the excision biopsy of the right inguinal lymph node. Histopathology and immunohistochemistry revealed that both the lesions were due to metastases from medullary thyroid carcinoma.Metastases to the testis and inguinal lymph nodes may be due to various solid and hematological tumors. This case, despite its rarity, suggests that testis and inguinal lymph nodes should be considered as potential secondary sites of medullary thyroid carcinoma as well. RET) have a pathogenic role in the onset of familial MTC (FMTC) or MEN2, the two clinical forms of hereditary MTC [RET mutations predict prognosis and guide the choice of treatment timing and follow-up. However, genetic testing for germline RET mutations is recommended not only for screening children and adults in known kindreds with inherited forms of MTC, but also for all newly diagnosed patients with clinically apparent sporadic MTC [Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor that arises from the thyroid parafollicolar calcitonin producing C cells and accounts for about 5% of thyroid carcinomas . MTC maitary MTC . In thesadic MTC , 2.Sporadic MTC usually manifests in the fifth-sixth decade, while familial forms have an earlier onset . MTC usuCompared with papillary/follicular thyroid cancer, MTC is more aggressive having a higher rate of recurrence and increased mortality . Older aSurgery is the main treatment for primary MTC and for local and distant metastases, whenever feasible. External-beam radiation to the neck/upper mediastinum is useful in patients with extrathyroidal disease or extensive nodal metastases not undergoing curative resection and for palliative purposes . CytotoxHerein, we report the case of a patient with MTC that metastasized to the right testis and to an homolateral inguinal lymph node, many years later to the detection of skeletal and lung metastases.In 2002, a 63 year old Caucasian man presenting a large solitary nodule in the upper right lobe of thyroid was cytologically diagnosed with medullary thyroid carcinoma (MTC). No family history of MTC or multiple endocrine neoplasia (MEN) was reported. Appropriate endocrine work up excluded MEN2. Preoperative serum calcitonin was 122 pg/ml (reference range: 0-9.6 pg/ml) with normal carcinoembryonic antigen (CEA) serum levels. The patient underwent total thyroidectomy and bilateral neck lymph node dissection. Histology confirmed a 52 mm in diameter MTC confined to thyroid without metastases in the 54 resected lymph nodes . The genetic testing identified the non-conservative functional c.2071G > A polymorphism in codon 691 of the RET proto-oncogene that was not mutated.18FDG-PET) and 68Ga-DOTATOC-PET confirmed the fifth lumbar vertebra as the only metastatic site. In 2011, the disease progressed (at bone scan the known uptake area enlarged and a new pathologic area appeared in the right hemisacrum). Bone lesions were osteoblastic on X-ray evaluation. Lung metastases remained stable in the absence of new lesions at CT-scan. Serum calcitonin and CEA levels were 141 pg/ml and 21 ng/ml, respectively. The patient was still asymptomatic.During the follow-up calcitonin  and CEA were in the normal range until September 2008, when basal calcitonin started to slightly increase (up to 64 pg/ml). Neck ultrasound was negative, but contrast-enhanced computed tomography scan (CT-scan) showed diffused lung micronodules. Then, the 99mTc-MDP bone scan showed an uptake area at the pedicle and right lamina of the fifth lumbar vertebra (November 2009) and 18-Fluorodeoxyglucose Positron Emission Tomography  where tumor cells infiltrated the Post-surgery calcitonin and CEA levels decreased, but did not normalize. Recently, progressive disease was documented in the lung, bone and inguinal lymph nodes. The patient is still asymptomatic, but is going to start vandetanib.Testicular metastases are extremely rare, except in patients affected by leukemia or lymphoma . Metastaet al. demonstrated connections between spermatic and renal capsular veins and between spermatic and ipsilateral colonic veins. These findings may sustain the retrograde venous spread from both kidney and colon cancer [The mechanisms of metastatic spread to the testis is still speculative. It has been suggested that metastases from solid tumors might join testis via retrograde arterial, venous or lymphatic embolization . Specifin cancer . Moreoven cancer . Metastan cancer , 23.In patients presenting a solitary testicular mass, the differential diagnosis must include metastatic carcinoma. A personal history of cancer should first be investigated. Older age is suggestive of a secondary testicular tumor. Measurement of serum α-fetoprotein and β-HCG, if negative, does not exclude a testicular primary germ cell tumor. However, post-surgery/-biopsy histopathology evaluation is almost invariably required to address the differential diagnosis of a testicular mass .Histology features of testicular metastases are quite different from primary tumors of the testis. The former present as a nodule of small nests of tumor cells more commonly located in the epididymis and para-epididymal area, with a widespread involvement in the interstitium and the relative sparing of the seminiferous tubules . InterstEven though, the rarity of metastatic involvement of testis remains unexplained, it may be due to blood-testis and blood-epididymis barrier , 35. TheInguinal lymph nodes are not a known site of metastatic spread from MTC. Metastases to inguinal lymph nodes are more commonly involved by cells from lymphoma and other malignant tumors located in the skin of lower extremities, in the scrotum, penis, vulva, clitoris, lower third of the vagina, anal canal, and infraumbilical region of the anterior abdominal wall. Melanoma or squamous cell carcinoma arising on the skin of the trunk may also metastasize to the inguinal lymph nodes. Rarely prostate, testicular or colon primary malignancies metastasize to inguinal lymph nodes , 39. Wherete testis. However, it is impossible to establish whether metastatic spread occurred vice versa or simultaneously.It is presumable that MTC metastases reached the testis first and then the inguinal lymph node via the infiltration of the Another interesting aspect of the present case, lies in the unexpectedly low levels of serum calcitonin and CEA in relation to the widespread dissemination of the disease. Calcitonin is a specific serum marker of MTC . PreoperTo the best of our knowledge, this is the first case report on testicular metastasis of MTC. Also inguinal lymph node involvement as a site of relapsing MTC has never been described before. In this patient, MTC metastases to the testis and inguinal lymph node were associated with disseminated disease, which is commonly observed in testicular metastases from solid malignancies. The present report, beyond its rarity, emphasizes that physicians need to consider testis and inguinal lymph nodes as an unusual, but possible site of metastases from MTC, even in the presence of relatively low serum markers of malignancy.Written informed consent was obtained from the patient for publication of this Case report and any accompanying images. A copy of the written consent is available for review.


Background. Rotavirus vaccination reduces childhood hospitalization in Africa, but cost-effectiveness has not been determined using real-world effectiveness and costing data. We sought to determine monovalent rotavirus vaccine cost-effectiveness in Malawi, one of Africa's poorest countries and the first Gavi-eligible country to report disease reduction following introduction in 2012.Methods. This was a prospective cohort study of children with acute gastroenteritis at a rural primary health center, a rural first referral–level hospital and an urban regional referral hospital in Malawi. For each participant we itemized household costs of illness and direct medical expenditures incurred. We also collected Ministry of Health vaccine implementation costs. Using a standard tool (TRIVAC), we derived cost-effectiveness.Results. Between 1 January 2013 and 21 November 2014, we recruited 530 children aged <5 years with gastroenteritis. Costs did not differ by rotavirus test result, but were significantly higher for admitted children and those with increased severity on Vesikari scale. Adding rotavirus vaccine to the national schedule costs Malawi $0.42 per dose in system costs. Vaccine copayment is an additional $0.20. Over 20 years, the vaccine program will avert 1 026 000 cases of rotavirus gastroenteritis, 78 000 inpatient admissions, 4300 deaths, and 136 000 disability-adjusted-life-years (DALYs). For this year's birth cohort, it will avert 54 000 cases of rotavirus and 281 deaths in children aged <5 years. The program will cost $10.5 million and save $8.0 million in averted healthcare costs. Societal cost per DALY averted was $10, and the cost per rotavirus case averted was $1.Conclusions. Gastroenteritis causes substantial economic burden to Malawi. The rotavirus vaccine program is highly cost-effective. Together with the demonstrated impact of rotavirus vaccine in reducing population hospitalization burden, its cost-effectiveness makes a strong argument for widespread utilization in other low-income, high-burden settings. Rotavirus gastroenteritis is a leading cause of illness and death in African children, accounting for >197 000 deaths annually, just over half of the global rotavirus mortality burden . Since 2Malawi, a low-income country in southern Africa with under-5 mortality of 71 per 1000 live births and GDP per capita of $253 . PreviouBetween 1 January 2013 and 21 November 2014, we conducted a prospective cohort study of children <5 years of age resident within the study site catchment areas presenting with acute gastroenteritis in northern and southern Malawi. The northern rural site was located in Chilumba Rural Hospital campus, Karonga, on which site are colocated an “under-5” primary health center providing childhood outpatient services and, adjacently housed, a first-referral-level inpatient facility. Both service the population within a demographic surveillance site in Chilumba that has been described previously , 11. TheUsing a standard case report form, we undertook detailed itemized individual patient–level determination of actual expenditures related to the current illness. From a household perspective, we collected by parental interview self-reported recall of all illness-related expenditures from symptom onset to convalescence at home after discharge, including transport costs to and from health facilities, loss of income and other opportunity costs, costs of formal and informal healthcare seeking , and costs related to accommodation and food for visiting family members during admission of the index child and any direct medical costs. We did not include long-run costs arising from any disability related to the acute illness episode.From a government healthcare provider perspective, we collected actual costs incurred in our cohort Table . From thOngoing rotavirus vaccine program cost projections were provided by the 2012–2016 comprehensive Expanded Programme on Immunization (EPI) multiyear plan , with thDisease burden estimates were based on our observed incidence in Malawi and other published burden estimates from Africa , 15; we Sample size was specifically calculated for this costing study. Taking a healthcare provider perspective, using predefined precision about a continuous cost estimate, a sample size of 88 provided a diarrheal illness cost estimate with a margin of error of ≤10%, assuming a coefficient of variation of 0.5 and at least 1000 children with diarrhea presenting to our study site annually –23. LargWe conducted univariate sensitivity analysis on variables that impacted our TRIVAC model results most dramatically Table . These vWe recruited a total of 530 children with gastroenteritis, comprising 118 outpatient and 282 children admitted at Queen Elizabeth Central Hospital (urban setting), and 108 outpatient and 22 children admitted at Chilumba Rural Hospital  . We did not take into account costs of possible secondary household cases linked to our index patients, nor did we take into account indirect benefits of vaccination in averting secondary cases. If such indirect effects will occur in Malawi as they have elsewhere , this woAlthough this study included primary healthcare, first-referral-level, and tertiary-level facilities in urban and rural settings and included inpatients and outpatients, caution is warranted in extrapolating our findings to other settings, particularly those in which vaccine effectiveness has not been evaluated, or whose healthcare systems differ substantially from Malawi. In addition, the very presence of our longstanding research activities at our recruitment sites may reduce applicability to other government clinics where research and, by implication, an emphasis on good quality clinical care are enhanced. Malawi's population utilization of primary and subsequent referral-level care was uncertain; thus, we assumed proportions based on healthcare utilization reported by our cohort. We ran our models with differing but plausible distributions, and this did not dramatically affect cost-effectiveness estimates. We therefore believe it is likely that in many resource-limited settings our findings have applicability, but clearly local health planners should be mindful of their own settings when considering the relevance of our findings. Model-based expectations of cost-effectiveness have been reported from Southeast Asia, where the vaccine is yet to be introduced .The Malawian economy underwent a period of substantial instability in the wake of the devaluation of the kwacha in 2012. This ushered in inflation and marked increases in costs of goods and services. Inflation had stabilized by the time we embarked on the bulk of our recruitment, but the uncertain economic climate persists. Such turbulence is difficult to adjust for in analysis, but is all too common in low-income countries. Indeed, such instabilities are often associated with increased illness, thereby strengthening further the argument for introducing and maintaining vaccine programs that are effective and cost-effective even in the short run.In conclusion, gastroenteritis episodes represent a substantial economic burden to government and to families. We found monovalent rotavirus vaccine to be highly cost-effective in Malawi, even under challenging possible future scenarios. Together with the demonstrated impact of monovalent rotavirus vaccine in reducing population hospitalization burden, the additional cost savings afforded by this vaccine make a strong argument for the widespread introduction in other low-income, high-burden settings.


Diaphorina citri, Hemiptera: Liviidae), is the principal vector of the phloem-limited bacteria strongly associated with huanglongbing (HLB), the world’s most serious disease of citrus. Host plant resistance may provide an environmentally safe and sustainable method of controlling ACP and/or HLB. Two xCitroncirus accessions (hybrids of Poncirus trifoliata and Citrus spp.), that are relatively resistant (UN-3881) or relatively susceptible (Troyer-1459) to ACP adults with regard to adult longevity, were compared in relation to ACP feeding behavior and some structural features of the leaf midrib. The settling (putative feeding/probing) sites of ACP adults on various parts of the leaf were not influenced primarily by plant accession. However, fewer ACP stylet sheaths were found in the midrib and fewer stylet sheath termini reached the vascular bundle (phloem and/or xylem) in UN-3881 compared to Troyer-1459 plants. Furthermore, in midribs of UN-3881 leaves the fibrous ring (sclerenchyma) around the phloem was significantly wider (thicker) compared to that in midribs of Troyer-1459 leaves. Our data indicate that feeding and/or probing by ACP adults into the vascular bundle is less frequent in the more resistant (UN-3881) than in the more susceptible (Troyer-1459) accessions. Our results also suggest that the thickness of the fibrous ring may be a barrier to stylet penetration into the vascular bundle, which is important for successful ACP feeding on the phloem and for transmitting HLB-associated bacteria. These results may help in the development of citrus plants resistant to ACP, which in turn could halt or slow the spread of the HLB-associated bacteria by this vector.The Asian citrus psyllid (ACP,  Diaphorina citri Kuwayama (Hemiptera: Liviidae), is an invasive insect species that transmits the phloem-limited bacteria (Candidatus Liberibacter spp.) strongly implicated as the causative agents of huanglongbing , the most serious disease of citrus worldwide Candidatus Liberibacter asiaticus (LAS) is known to be transmitted by ACP in a persistent, probably propagative, manner, and thus was found in many tissues of this vector including the gut, salivary glands and hemolymph The Asian citrus psyllid (ACP), Following the discovery of HLB in Florida in 2005, a three-component management program against HLB was advocated initially and implemented by some citrus growers: intensive chemical control of ACP, removal of HLB-infected trees, and planting disease-free nursery stock Poncirus trifoliata L. P. trifoliata in a field survey P. trifoliata is a trifoliate species that is used as rootstock in many citrus-growing regions. It is cross-compatible with Citrus species and thus has been an important parent in the development of new rootstocks and scions through intergeneric hybridization with CitrusP. trifoliata, and many accessions of xCitroncirus spp. (hybrids of P. trifoliata and other parent species), were found to reduce oviposition and lifespan of ACP in no choice tests P. trifoliata appears to have antixenosis (deters or prevents colonization) and/or antibiosis (toxicity) in relation to ACP Host plant resistance to ACP, i.e. prevention of colonization or multiplication of ACP on a plant, ultimately may provide the most effective, economical, environmentally safe, and sustainable method of controlling ACP, especially if the plant also is resistant to HLB or other important pests of citrus Citrus sinensis (L.) Osbeck var. ‘Ridge Pineapple’ or ‘Valencia’] (susceptible to ACP and LAS), we previously demonstrated that the majority (80–90%) of the stylet sheaths termini produced by ACP adults or nymphs that reached a vascular bundle were associated with the phloem whereas only 10–20% were associated with xylem vessels ACP and other phytophagous hemipteran insects produce salivary secretions during their feeding or probing. Some of these salivary secretions solidify around the stylets and are termed ‘salivary sheaths’, ‘stylet sheaths’ or ‘stylet tracks’ because, after an insect withdraws its mouthparts, these sheaths remain in plant tissues showing exactly where the insect had been probing and/or feeding Citroncirus accessions: one is relatively resistant (UN-3881) and the other is relatively susceptible (Troyer-1459) to ACP adults with regard to adult longevity. Mean life span of ACP adults maintained on UN-3881 plants was shown previously to be significantly shorter (nearly one third) that of ACP adults maintained on Troyer-1459 although egg laying was similarly reduced in both of these accessions compared to control plants (Citrus macrophylla Wester) Citroncirus accessions as far as ACP adult longevity/life span is concerned, but not to other aspects of resistance .In the present work, we tested the hypothesis that the fibrous ring is thicker, and that ACP stylet sheath occurrence and termini are different, in leaves of two xMurraya paniculata (L.) Jack] or citrus trees (Citrus macrophylla Wester) in a greenhouse as described by Hall et al. Citroncirus were obtained from the USDA-ARS National Clonal Germplasm Repository for Citrus and Dates located at the University of California at Riverside  plants were also used. The numbers associated with these accessions are those given by the Citrus Research Center (CRC). Seeds were planted and grown in a greenhouse (with daily averages of 25–29°C) as previously described Young adults of ACP, within one week of eclosion from the last nymphal instar, were taken from a healthy (non-HLB infected) laboratory colony established during the year 2000 that has been maintained on healthy orange jasmine [To study ACP settling behavior (putative feeding/probing sites), five younger trifoliate leaves and five older trifoliate leaves  were detached from plants of the two accessions: UN-3881 and Troyer-1459. The cut-end petioles were placed in small (0.5 ml) microfuge tubes filled with water, and capped with a piece of Parafilm membrane. Each leaf was then placed in a tightly-covered plastic Petri dish along with 5 ACP adults. The dishes were positioned vertically, so that ACP adults could reach both sides of the leaf as previously described Comparisons were made between Troyer-1459 and UN-3881 plants with respect to leaf midrib structure and ACP stylet sheath occurrence using clip cages to confine ACP adults on young leaves of each plant. The clip cages used  were madAfter removing adult ACP from each clip cage, the midrib from the entire leaf area that was inside the cage (exposed to ACP feeding) was cut out longitudinally on both sides with a sharp razor blade. In experiment 2a, the midrib was then cut into four pieces nearly equal in length. Each piece was immediately transferred to a drop of phosphate buffered saline  on a clean glass slide, and sectioned by hand using a new clean razor blade to the thinnest possible sections under a stereomicroscope (at 20x or higher). These sections  were quickly transferred to a fixative solution  for 1–3 days, before washing 3 times with PBS-T (PBS with 0.1% TritonX100). Sections were then stained for 5 min in 3 nM solution of the fluorescent nucleic acid stain propidium iodide, before washing again in PBS-T. In experiment 2b, however, a simpler method was used, in which the midrib pieces were fixed and washed before sectioning, and the sections were not stained. This latter method was adequate when sections were examined only by epifluorescence microscopy, whereas staining in propidium iodide was necessary when using confocal laser scanning microscopy (CLSM) [14,17). In both cases, all the sections obtained from the whole midrib area under the clip cage were processed, mounted on microscope slides using Fluoro-Gel , and examined. Mounted sections were kept in the dark at 4°C until examination with UV light using an epifluorescence inverted microscope  fitted with a camera system. These sections were examined using autofluorescence (with no filter cubes). Some sections (stained with propidium iodide) were also examined with CLSM  with excitation wavelength of 543, sometimes simultaneously with differential interference contrast (DIC) in order to show cell boundaries.http://rsb.info.nih.gov/ij/index.html”.Morphometrics of the leaf midrib, including width of the phloem and the fibrous ring (sclerenchyma), as well as diameters of the vascular bundle and midrib, were determined from photographed images of these sections using the computer program ‘ImageJ’ “We used a model selection framework using likelihood ratio to evaluate differences in settling (putative feeding or probing) sites of ACP adults on various locations of the leaf in the relatively resistant and susceptible accessions . This stAnalysis of variance (ANOVA) and Tukey’s studentized HSD test  were used to test differences in the width/thickness of the fibrous ring and other structures in the midrib. Additionally, Chi-square analysis was used to test differences in the occurrence of stylet sheath and their termini between resistant and susceptible plants.P<0.001). ACP were more likely to feed on adaxial small veins and abaxial mid veins (midribs) in old leaves than in young leaves, and less likely to feed on abaxial small veins in old leaves than young leaves (P<0.001). ACP settled more often on adaxial mid veins in young Troyer leaves than old UN-3881 leaves, adaxial small veins in young or old UN-3881 leaves than in young or old Troyer leaves, and abaxial mid veins in young Troyer leaves than old leaves of Troyer and UN-3881 (Tables1 and 3). There was no difference in ACP mortality during the five day period of each experiment between UN-3881 (0–5%) and Troyer-1459 (2–5%) accessions.The settling (putative feeding/probing) sites of ACP adults on various parts of the leaf were not influenced primarily by plant accession , 2, 3. Hg leaves  and 2. ICitroncirus plants, that are relatively resistant (UN-3881) or relatively susceptible (Troyer-1459) to ACP adults (with regard to adult longevity), showed that their midrib structure is essentially similar to that described earlier for ACP susceptible Citrus plants Epifluorescence microscopy of cross sections in the leaf midrib of xstant UN-881 or re Troyer-159 to ACPSignificant differences in the morphometrics of some structures in the leaf midrib were found between the relatively resistant (UN-3881) and the relatively susceptible (Troyer-1459) plants . The widCitroncirus accessions with regard to the occurrence of ACP stylet sheaths in the leaf midrib, and in the proportion of stylet sheath termini found in the vascular bundle (phloem and/or xylem) . Additionally, the percentage of stylet sheath termini found in the vascular bundle (as opposed to those that were found in the mesophyll) were significantly higher in Troyer-1459 (85.7%) than in UN-3881 (62.1%) . In both accessions, however, the percentage of sheath termini that were found associated with xylem elements (11.1–16.7%) was much lower than that of those found in the phloem (83.3–88.9%), with no significant differences between Troyer-1459 and UN-3881 in this regard. Stylet sheaths were observed to extend or branch from the phloem to the xylem elements apparently either from the inner periphery of the phloem closest to the xylem (Significant differences were found between the two tested xr xylem) . Stylet he xylem , 2D or bhe xylem .Generally, in both accessions, the stylet sheaths entering the midrib from the upper  leaf side were mainly associated with the upper-side phloem , 2D, wheCitroncirus accessions studied, the stylet sheaths starting either from the upper  or the lower  sides of the leaf midrib normally took a slightly tenuous (apparently intercellular) course through the palisade or ground/spongy parenchyma layers before entering the vascular bundle , partially surrounding the phloem in the leaf midrib is composed of 1–5 layers of thick-walled fibers with little or no space between fiber cell walls , 3A, 4B.Citroncirus) and resistant (P. trifoliata) plants  appearance around a central canal that was previously occupied by the stylet bundle , 2, 3, 4) plants . Using H) plants .Citroncirus accessions compared to control plants (Citrus macrophylla Wester) C. macrophylla) were not used here because their leaf shape and size are vastly different from the trifoliate leaves of xCitroncirus spp., which would make morphometric and structural comparisons between the two types of leaves very difficult and unreliable. Thus, it should be noted that our investigation is only related to resistance of the two tested xCitroncirus accessions as far as ACP adult longevity is concerned, but not to other aspects of resistance .Host plant resistance is an essential component of integrated pest management programs for ACP, HLB and other pest/pathogen systems Megoura viciae (Buckton) (Hemiptera: Aphididae) was reported to make a stylet ‘detour’ when encountering arches of tough sclerenchyma cells along the phloem in their host plant Vicia fabae L. Using Raman microscopy analysis, Richter et al. CitrusM. viciae was almost exclusively secreted in sieve elements, whereas the nonsoluble (stylet sheath) fraction was preferentially secreted at cell wall conditions In the present work, among ACP stylet sheaths that reached the vascular bundle in both accessions, the great majority of sheath termini reached the phloem while very few were associated with xylem vessels. This is consistent with our previous report on ACP stylet sheaths in Blissus insularis Barber (Hemiptera: Blissidae), the salivary sheaths in St. Augustinegrass were more abundant on the outermost leaf sheath of axillary shoots of resistant cultivars compared with susceptible ones, suggesting that the thick-walled sclerenchyma cells play a role in chinch bug resistance possibly by reducing stylet penetration into the vascular tissue Cacopsylla pyri L.) do not ingest from the phloem in resistant selections of pear for any prolonged period, which suggests that resistance factors are located in the phloem sap rather than factors outside the phloem Aphis glycines Matsumura where EPG studies showed the following: a. soybean aphids feeding on susceptible genotypes had a significantly greater duration of sieve element phase than when feeding on resistant genotypes, b. the time taken to reach the first sieve element phase in resistant genotypes was significantly greater than in susceptible ones, and c. most of the aphids reached sieve element phase in susceptible genotypes but only a few reached this phase in resistant genotypes Cicadulina mbila (Naude) Trialeurodes vaporariorum (Westwood), resistance factors were associated with phloem ingestion in resistant tomato genotypes, but with other factors (not related to the phloem) in sweet pepper genotypes Using electrical penetration graphs (EPG), Bonani et al. Rhopalosiphum padi L., vector of the phloem limited barley yellow dwarf virus (BYDV), indicated differences in the phloem salivation and phloem feeding phases between aphids feeding on barley lines resistant or susceptible to BYDV, and suggested that the phloem salivation time was too short for successful infection by BYDV in the resistant lines Differences in the feeding behavior of a pathogen vector may affect its ability to acquire and/or inoculate this pathogen from or to its host plant. An EPG study on the aphid


The free radical theory of aging proposes that reactive oxygen species (ROS)-induced accumulation of damage to cellular macromolecules is a primary driving force of aging and a major determinant of lifespan. Although this theory is one of the most popular explanations for the cause of aging, several experimental rodent models of antioxidant manipulation have failed to affect lifespan. Moreover, antioxidant supplementation clinical trials have been largely disappointing. The mitochondrial theory of aging specifies more particularly that mitochondria are both the primary sources of ROS and the primary targets of ROS damage. In addition to effects on lifespan and aging, mitochondrial ROS have been shown to play a central role in healthspan of many vital organ systems. In this article we review the evidence supporting the role of mitochondrial oxidative stress, mitochondrial damage and dysfunction in aging and healthspan, including cardiac aging, age-dependent cardiovascular diseases, skeletal muscle aging, neurodegenerative diseases, insulin resistance and diabetes as well as age-related cancers. The crosstalk of mitochondrial ROS, redox, and other cellular signaling is briefly presented. Potential therapeutic strategies to improve mitochondrial function in aging and healthspan are reviewed, with a focus on mitochondrial protective drugs, such as the mitochondrial antioxidants MitoQ, SkQ1, and the mitochondrial protective peptide SS-31. Under normal conditions, ROS are maintained at the physiological levels by several endogenous antioxidant systems, including superoxide dismutatase (SOD), catalase, glutathione peroxidases, and glutathione reductase (GR). Other antioxidant systems involving thiol-disulphide oxidoreductase systems include the cytosolic proteins thioredoxin (TRX) and glutaredoxin (GRX). These antioxidant systems are complex, located in different cellular compartments and are often redundant or complementary in various conditions. Physiological levels of ROS interact with redox state and play a role in mediating cell signaling, while pathological levels of ROS can result in oxidative damage to cellular components and activate several cell death pathways  increased lifespan [Drosophila melanogaster early results were confounded by uncontrolled genetic background effects. Later analyses suggested that over-expression of catalase, SOD1, or SOD2 using the genes’ native promoters does not increase life span [Based on the free radical theory of aging, several scientists have attempted to increase lifespan by genetic manipulation of antioxidant system components, however, the results have generally been conflicting. In lifespan . In Drosife span ,7, but tife span . It has ife span . In tranife span -11. Whilife span ).Several clinical trials using antioxidant supplementation in various study populations have been performed during the last three decades and the results are often equivocal or conflicting. Meta-analyses of large numbers of individual reports are often required to reach conclusions, however these too vary. A large scale analysis of 68 randomized trials including 232,606 participants from general population or patients with heterogeneous diseases have reported no effect of antioxidant supplements on overall mortality, or even a significant increase in mortality in subjects receiving beta carotene, vitamin A, and vitamin E . A recenThe lack of anti-aging effect with antioxidant supplements led Harman to modify his original theory to specify mitochondria as both the primary sources of ROS and the primary targets of ROS damage . One of The revised mitochondrial free radical theory suggests that failures of antioxidants to extend murine lifespan or failure of antioxidant supplements in clinical trials might be explained by poor distribution of antioxidants to mitochondria, and proposes that antioxidants targeted to mitochondria might be beneficial for lifespan extension. Several lines of evidence have supported the mitochondrial theory of aging. One of the most direct experimental evidence for the role of mitochondrial ROS in longevity was shown in mice overexpressing catalase targeted to mitochondria (mCAT), which resulted in a significant median and maximal lifespan extension in two independent lines of C57Bl6 mice . InteresShc gene. These mice display reduced ROS generation and increased resistance to ROS-mediated apoptosis and thereby have a prolonged lifespan [Shc is a mitochondrial redox enzyme located in the mitochondrial intermembrane space, which produces H2O2 from electron leakage during oxidative phosphorylation [Shc was later shown to accumulate within mitochondria to activate mitochondrial Ca2+ response, and subsequently induce apoptosis [D257A/D257A , abbreviated Polgm/m). These mice were susceptible to accumulation of mtDNA point mutations and deletions with age [m/m mouse heart was attenuated by mCAT [Additional evidence for the involvement of mitochondrial ROS in aging comes from observations of mice with a targeted mutation of the p66lifespan . Furtherrylation . Phosphopoptosis . Furtherwith age ,23. Theywith age . Intereswith age . MoreoveIn spite of these ‘attractive’ aspects of the mitochondrial free radical theory of aging, there remain many unsolved questions. Damage to mitochondrial DNA has been shown to increase with age, even more so than nuclear DNA ; howeverThe mitochondrial free radical theory has led to a focus on development and refinement of drugs to specifically target ROS specifically in the mitochondria of cells. The most common approach is based on delivery of known redox agents to the mitochondrial matrix by conjugation to delocalized cations (such as the triphenylphosphonium ion (TPP+)), including MitoQ and SkQ1 ,31. The C. elegans demonstrating that inhibition of respiration increases mitochondrial ROS production and significantly increases lifespan via mitochondrial ROS mediated activation of HIF-1 [Drosophila melanogaster[Caenorabditis elegans[C. elegans extends lifespan by inducing mitochondrial respiration and increasing oxidative stress, and this AMPK-dependent lifespan extension is abolished by pre-treatment of antioxidant N-acetyl cysteine, suggesting that oxidative stress is required for lifespan extension of dietary restriction [Apart from generating detrimental oxidative damage, ROS have numerous crucial biological roles in signaling and stress response (reviewed in -35). Emeof HIF-1 . Low dosanogaster and Caens elegans. For instriction . Althoug2O2 concentrations that may be involved in signaling or hormesis [The theory of mitohormesis could have important translational implications as an ideal antioxidant therapy might be one that prevents oxidative damage induced under pathological conditions without interfering with ROS needed for hormesis and cellular signaling. We speculate that the targeted expression of catalase in mitochondria (mCAT) might be such an example, as there are beneficial effects of mCAT in aging and several disease models with negligible adverse effects Table . Key to hormesis ,42.Increasing evidence suggests that abnormal mitochondrial ROS (mtROS) production and detoxification contributes to mitochondrial dysfunction and cardiomyopathy in old age . The Framingham Heart Study and the Baltimore Longitudinal Study on Aging (BLSA) demonstrate that aging is associated with increased prevalence of left ventricular hypertrophy and decline in diastolic function (measured by the ratio of early to late ventricular filling (E/A) by Doppler echocardiography) in otherwise healthy individuals. Left ventricular (LV) wall thickness increases and maximal exercise capacity decreases with age in both sexes, indicative of LV hypertrophy, while systolic function is relatively preserved at rest . CarData from our laboratory demonstrated that mCAT greatly attenuated many of these cardiac aging phenotypes Figure A-D. The m/m) have substantial increases in mtDNA mutations and deletions with age [m/m mice display cardiac hypertrophy  injury often occurs during acute myocardial infarction, either due to spontaneous recanalization of the occluded artery or as a result of a reperfusion therapy. ROS are well known to be primary mediators in IR injury. ROS begin to accumulate during ischemia , causing release   in type IIB muscle fibers leads to an increase in mitochondrial oxidative stress and dysfunction in fast-twitch mouse muscles. Mitochondria from fast-twitch muscles in these mice had significantly reduced aconitase and succinate dehydrogenase (complex II of the electron transport chain) activities and increased capacity for superoxide production resulting in elevated Frostanes . Both farostanes , while vrostanes . However-/- knockout mice accumulated mitochondria around the neuromuscular junction, showed a loss of motor units and disruption of neuromuscular junctions. However, muscle specific knockout of CuZnSOD did not result in muscle atrophy, increased oxidative stress, nor mitochondrial dysfunction [-/- mice leads to significant increases in force production over that achieved by nerve stimulation [-/- mice suggest an important role of increased oxidative stress in age-related muscle atrophy, although it remains unclear whether oxidative stress originating in the myofibers or the motorneurons are the primary drivers of this process. This conclusion is supported by recent work demonstrating that treatment with SS-31 during hind limb unloading ameliorated muscle atrophy and mitochondrial dysfunction [The current evidence strongly supports an important role of mitochondrial oxidative stress in the decline in skeletal muscle function with age, while its role in skeletal muscle atrophy with age is still controversial. The strongest evidence in support of a role for oxidative stress in age-related muscle atrophy comes from mice lacking CuZnSOD (SOD1). CuZnSOD is found in both the cytosol and the inner membrane space of the mitochondria. The absence of CuZnSOD leads to an accumulation of peroxinitrite and oxidative damage , increasfunction , but didmulation . This refunction .1F0 ATPase [There are multiple ways in which an increase in oxidative stress can affect skeletal mitochondrial and contractile function. Work to date has primarily focused on the accumulation of oxidative damage to proteins, lipids, and DNA. Most studies find a clear accumulation of oxidatively damaged macromolecules with age in skeletal muscle and most other tissues. However, oxidation of the mitochondrial and cellular redox environment can also exert control over cellular function through post-translational modification of proteins Figure . Glutath0 ATPase  and comp0 ATPase , and of 0 ATPase  and α-ke0 ATPase . In addi0 ATPase  resultinRedox modification of proteins can also affect contractile function. The skeletal muscle ryanodine receptor 1 (RyR1) in aged rats is oxidized by cysteine nitrosylation . This leOld age is associated with progressive decline in the functional performance of the nervous system. This intrinsic nervous system aging includes slowed reaction times, degeneration of sensory and motor function, and a decline in cognitive performance. In addition to intrinsic nervous system aging, several neurodegenerative diseases demonstrate strong age-related onset including the highly prevalent Alzheimer’s disease (AD) and Parkinson’s disease (PD), among others.Age-related sensorineural hearing loss or presbycusis is gradual loss of hearing with aging. The prevalence in the elderly is estimated to be 30% to 35% of people aged 65 to 75 years and 40% to 50% of people aged older than 75 years . The senAD is the most prevalent neurodegenerative disease, affecting approximately 5 million Americans. The clinical presentation of AD is primarily memory impairment and dementia. The early memory deficit in AD is often described as ‘recent memory impairment’  . Deficit1-42. Presenilins are integral membrane proteins that function as the proteolytic components of γ-secretase. Mutations of presenilins result in increased production of Aβ1-42.There are two principle pathologic lesions in AD: neurofibrillary tangle (NFT) and the amyloid plaque . The NFT2+ homeostasis [Several lines of evidence have shown the central roles of mitochondria in AD (see reviews ). Both Aeostasis , increaseostasis . Furthereostasis -150. It eostasis . IncreasMore direct evidence of the role of mitochondrial oxidative stress in AD is demonstrated by studies using mCAT mice or mitochondrial targeted antioxidants. Mao et al.  showed t2a cells with Aβ led to reduced neurite outgrowth, lower cell viability, mitochondrial dysfunction, and fragmentation and loss of ATP, all of which were partially protected by simultaneous incubation with either SS-31 or MitoQ [2O2 production, reduced cytochrome oxidase activity, and decreased ATP levels [AD is associated with neuronal cell death, loss of synapses, as well as mitochondrial abnormalities. Incubation of Nor MitoQ . PrimaryP levels . There wP levels .PD, the second most common neurodegenerative disorder, is characterized by defects in motor functions, manifested as resting tremor, bradykinesia, rigidity, and postural instability. The hallmark pathology of PD is a gradual loss of pigmented dopaminergic neurons in the substantia nigra pars compacta, and accumulation of Lewy bodies in catecholaminergic neurons of the brainstem in the substantia nigra and locus ceruleus. Lewy bodies are abnormal aggregates of proteins composed predominantly of α-synuclein and ubiquitin.There is substantial support for the central role of mitochondria in the pathogenesis of PD. A few genetic loci have been mapped in rare familial PD cases, and are sequentially named PARK1 to PARK11 (review in ). SeveraSeveral rodent models have been used to recapitulate pathology and pathophysiology of PD, including mice with genetic manipulation of many of the genes mentioned above and rodents treated with environmental toxins (see review ). Of not+-induced inhibition of oxygen consumption, ATP production, and mitochondrial swelling in isolated mitochondria. MitoQ was also reported to be protective against MPTP toxicity [Direct evidence of the role of mitochondrial ROS in PD was shown by the protective effect of mCAT mouse brains against MPTP induced mitochondrial ROS production and subsequent dopaminergic neuron degeneration in substantia nigra pars compacta . In conttoxicity .Growing evidence has implicated the involvement of oxidative stress in insulin resistance and the pathogenesis of diabetes. Hyperglycemia is associated with increased ROS production from glucose autoxidation, advanced glycosylation end-products (AGEs) formation, polyol pathway, and ROS-producing enzymes including NADPH oxidase . Nishika2O2 emission and showed preserved insulin sensitivity in skeletal muscle, further support of the mitochondrial ROS in muscle insulin resistance [The involvement of mitochondrial oxidative stress in muscle insulin resistance has been demonstrated by the protection of mCAT mice in age-related reductions in mitochondrial function and lipid-induced insulin resistance in skeletal muscle . This prsistance . Thus miDiabetes is linked with multiple cardiovascular complications including accelerated atherosclerosis, augmented ischemic injury post-myocardial infarction, diabetic retinopathy, and nephropathy. In a mouse model of diabetes induced by streptozotocin injection, retina of diabetic mice had two-fold increase in superoxide levels, 40% reduction in GSH levels and 20% reduction in complex III activity, and increased mitochondrial membrane permeability. All these changes were attenuated in mice overexpressing MnSOD, which also experience reduced vascular histopathology, indicating the role of mitochondrial oxidative stress in retinopathy . CardiacDespite the evidence supporting the role of mitochondrial oxidative stress in experimental models of diabetes, there are mixed results on the protective role of antioxidant treatments in diabetes or its complications from clinical trials . For insIt is possible that increased mitochondrial ROS may play a larger role in the development of insulin resistance before the onset of chronic hyperglycemia. A recent study using the streptozotocin-induced mouse model of type 1 diabetes actually found reduced mitochondrial function and superoxide production in diabetic kidneys and suggested that this may be due to reduced mitochondrial biogenesis caused by lower PGC1α expression . The invIt was recently reported that SS-31 significantly reduced diabetic retinopathy . Daily t2O2-induced cell death, which may confer the protective effects in mCAT mice [Mitochondrial ROS leads to oxidative damage in nucleic acids and proteins and has been implicated in carcinogenesis. A recent study demonstrates that loss of mitochondrial cytochrome oxidase is associated with the development of colonic dysplasia (precancerous state) in patients with ulcerative colitis . Direct CAT mice .-/-) develop thymic lymphomas, despite very mild neurodegenerative phenotypes. Reducing mitochondrial ROS by mCAT in ATM-/- mice reduced propensity to develop thymic lymphoma, improved bone marrow hematopoiesis, and macrophage differentiation in vitro, and partially rescued memory T-cell development [Ataxia telangiectasia mutated (ATM) kinase plays a central role in the DNA-damage response and redox sensing by the phosphorylation of many key proteins that initiate activation of the DNA damage checkpoint, leading to cell cycle arrest, DNA repair, or apoptosis. In addition to severe ataxia due to cerebellar degeneration, ataxia telangiectasia patients also have increased risk of lymphomas and leukemias, as well as immune defect . ATM nulelopment .+) to take advantage of the potential gradient across the inner mitochondrial membrane. The second major category is aromatic-cationic tetrapeptides that selectively target the inner mitochondrial membrane without relying on mitochondrial potential.Meta-analyses of several clinical trials using antioxidant supplement have shown largely disappointing results . With st+ has been conjugated to coenzyme Q (MitoQ) and plastoquinone (SkQ1) [+-conjugated antioxidants are more potent than their lipophilic counterparts in reducing intracellular ROS, preserving reduced thiols, and reducing oxidative cell death [The mitochondrial inner membrane has a negative potential gradient -150-180 mV) that is generated as a result of the release of protons from the mitochondrial matrix to the intermembrane space. The negative potential serves as a basis for the use of lipophilic cations to deliver redox agents into the mitochondrial matrix. This method can potentially result in 100- to 1,000-fold accumulation of drugs within the mitochondrial matrix . TPP+ hae (SkQ1) ,31. By p mV that ll death , This lill death ).MitoQ had been shown to improve pathology associated with antioxidant deficiency and prolong lifespan of SOD-deficient flies, however, it failed to show lifespan extension in normal WT flies . Indeed,+-conjugated antioxidants can reduce mitochondrial ROS, they may also reduce oxidative phosphorylation and ATP production.However, recent reports suggest that MitoQ can actually increase superoxide production at Complex I ,190 and MitoQ has been evaluated in two clinical trials. A small trial of MitoQ in 30 patients with hepatitis C revealed a significant reduction in alanine aminotransferase after 28 days of treatment . However2) was originally thought to exert its beneficial effect solely by the free radical scavenging activity of dimethyl tyrosine [2O2 hydroxyl radical and peroxynitrite in vitro in a dose-dependent manner [The Szeto-Schiller (SS) compounds are tetrapeptides with an alternating aromatic-cationic amino acids motif, which was serendipitously found to preferentially concentrate in the inner mitochondrial membrane greater than 1,000-fold compared with the cytosolic concentration ,190,195.tyrosine . SS-31 it manner .A recent study revealed that in addition to this ROS scavenging capacity, SS-31 selectively binds to cardiolipin on the inner mitochondrial membrane via both electrostatic and hydrophobic interactions . CardiolWe recently showed that the binding of SS-31 to cardiolipin alters the interaction of cardiolipin with cytochrome c, and favors its electron carrier function while inhibiting peroxidase activity by protecting the Met80-heme ligand ,206. By These unique properties of SS-31 are particularly effective in minimizing ischemia-reperfusion injury. After prolonged ischemia, the hydrophobic interaction between cardiolipin and cytochrome c is enhanced by low ATP concentration ,208 and SS-31 has also been shown to reduce cardiac ischemia reperfusion injury and reperfusion arrhythmia and better preserve myocardial function in various infarct models ,64,196. In addition to ischemia-reperfusion injury, SS-31 has shown impressive effects in preclinical models of heart failure. SS-31 ameliorated Angiotensin-II induced cardiac hypertrophy and diastolic dysfunction, as well as Gαq overexpression-induced heart failure, despite the absence of a blood pressure lowering effect . SS-31 aSS-31 has also been shown to be beneficial in many other models of age-associated diseases, including PD , AD 152152, skelGiven the very promising preclinical efficacy data, SS-31 entered into clinical trials using a clinical formulation named Bendavia . SeveralThese clinical studies are generally designed to address the efficacy of SS-31 in the treatment of age-associated cardiorenal diseases. It will eventually be important to also establish whether these mitochondria-targeted antioxidants can delay aging and other age-related degenerative diseases.Substantial evidence supports the central role of mitochondrial oxidative stress in aging and healthspan. Despite the disappointing outcomes of non-targeted antioxidants in clinical trials, there is growing evidence for the beneficial effects of mitochondrial-targeted antioxidants in aging and age-related diseases. Genetic and pharmacological approaches reducing mitochondrial oxidative stress  attenuate the phenotypes of cardiac aging, age-related cardiovascular diseases, skeletal muscle aging, neurodegenerative diseases, diabetes, and cancer various animal models (summarized in Table HH Szeto is the inventor of SS-31 and the Cornell Research Foundation (CRF) holds several patents covering the SS peptides and a patent application has been filed for the findings described in this article, with HH Szeto, PS Rabinovitch and DF Dai as inventors. CRF has licensed the SS peptide technology for further research and development to a commercial enterprise in which CRF and HH Szeto have financial interests.DFD and YAC drafted and revised the manuscript. DM helped to draft and made critical revision to the manuscript. HHS and PSR made critical revision to the manuscript. All authors read and approved the final manuscript.


ALK) positive NSCLC patients to diagnose ALK-EML4 gene rearrangement and to guide therapeutic management of these patients. The ability to detect genetic abnormalities such ALK rearrangement in CTCs shows that these cells could offer new perspectives both for the diagnosis and the monitoring of ALK-positive patients eligible for treatment with ALK inhibitors.The advent of rationally targeted therapies such as small-molecule tyrosine kinase inhibitors (TKIs) has considerably transformed the therapeutic management of a subset of patients with non-small-cell lung cancer (NSCLC) harboring defined molecular abnormalities. When such genetic molecular alterations are detected the use of specific TKI has demonstrated better results  compared to systemic therapy. However, the detection of such molecular abnormalities is complicated by the difficulty in obtaining sufficient tumor material, in terms of quantity and quality, from a biopsy. Here, we described how circulating tumor cells (CTCs) can have a clinical utility in anaplastic lymphoma kinase ( EGFR) gene  are present in 10–30% of patients with NSCLC and confer sensitivity to gefitinib, erlotinib, and afatinib. As first-line treatment, EGFR inhibitors can produce overall response rates (ORR) of 75% in selected NSCLC patients  has considerably shifted with the emergence of rationally targeted therapies for a subset of molecularly defined lung cancers. NSCLCs and in particular adenocarcinoma, the most frequent histologic subtype, have been segmented into clinically relevant molecular subsets according to a classification based on multiple so-called oncogenic “driver” alterations Figure A 1). Th. Th1). TFR) gene –4.CommonEGFR mutations, the ALK-gene rearrangement defines a unique molecular subset in 3–7% of NSCLC patients (ALK-gene rearrangement involves the anaplastic lymphoma kinase (ALK) gene and, most often, the echinoderm microtubule-associated protein-like 4 (EML4) loci which both map to the short arm of the human chromosome 2 in opposite directions  of 8–9 months in ALK-positive NSCLC patients receiving crizotinib , immunohistochemistry (IHC), or reverse transcriptase-polymerase chain reaction (RT-PCR) on these tumor samples. Wang et al. recently reported that FISH and IHC results were concordant in 98% of cases while RT-PCR results were less concordant with FISH (89%). FISH is highly specific but is costly and requires technical expertise. The IHC assay developed by Ventana (Ventana ALK assay) enables to detect ALK rearrangement with a high sensitivity and concordance when compared to FISH results, and has the advantage to be automated, faster, and less expensive than FISH (ALK break apart FISH probe kit (Abott molecular). Finding alternatives to a tumor biopsy and more effective means to diagnose an ALK rearrangement is a critical issue in order to identify NSCLC patients who may benefit from an ALK inhibitor treatment.Prospective molecular profiling of NSCLC is now performed in routine clinical practice to identify oncogenic “driver” abnormalities and to screen eligible patients for targeted TKI therapies. Tumor tissue may be difficult to obtain in advanced/metastatic NSCLC patients for whom surgery is rarely a component of the treatment strategy. The molecular characteristics or biomarkers are most commonly identified on tumor biopsy samples although tissue adequacy, both in terms of quantity and quality, is often insufficient for patients with advanced/metastatic NSCLC. The detection of an han FISH . Treatme6 or 107 leukocytes. Most methods of CTC detection rely on the combination of two successive steps, an initial enrichment process followed by CTC detection so as to increase the sensitivity of the assay  that was optimized for high cell recovery. By combining blood filtration and FA-FISH, we demonstrated that ALK-rearranged CTCs could be detected in a cohort of 18 ALK-positive NSCLC patients  may inform on the status of predictive biomarkers for drug sensitivity and therapy selection. CTCs are, however, very rare events occurring at rates, as low as one cell per 10he assay . Numerouhe assay . In spitve NSCLC , 17. IliALK rearrangement can be reliably detected in the CTCs of ALK-positive NSCLC patients, these two studies provide new perspectives for the diagnosis of ALK-positive patients eligible for treatment with ALK inhibitors. These non-invasive molecular analyses performed on CTCs could be easily repeated at different time-points during treatment and could help to guide therapeutic decisions in a patient’s treatment course. Single cell analyses of CTCs captured on filters at the time of disease progression are also anticipated to help characterize resistant subclones and mechanisms of resistance to ALK inhibitors.By demonstrating that 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.


Altered energy metabolism is a biochemical fingerprint of cancer cells that represents one of the “hallmarks of cancer”. This metabolic phenotype is characterized by preferential dependence on glycolysis (the process of conversion of glucose into pyruvate followed by lactate production) for energy production in an oxygen-independent manner. Although glycolysis is less efficient than oxidative phosphorylation in the net yield of adenosine triphosphate (ATP), cancer cells adapt to this mathematical disadvantage by increased glucose up-take, which in turn facilitates a higher rate of glycolysis. Apart from providing cellular energy, the metabolic intermediates of glycolysis also play a pivotal role in macromolecular biosynthesis, thus conferring selective advantage to cancer cells under diminished nutrient supply. Accumulating data also indicate that intracellular ATP is a critical determinant of chemoresistance. Under hypoxic conditions where glycolysis remains the predominant energy producing pathway sensitizing cancer cells would require intracellular depletion of ATP by inhibition of glycolysis. Together, the oncogenic regulation of glycolysis and multifaceted roles of glycolytic components underscore the biological significance of tumor glycolysis. Thus targeting glycolysis remains attractive for therapeutic intervention. Several preclinical investigations have indeed demonstrated the effectiveness of this therapeutic approach thereby supporting its scientific rationale. Recent reviews have provided a wealth of information on the biochemical targets of glycolysis and their inhibitors. The objective of this review is to present the most recent research on the cancer-specific role of glycolytic enzymes including their non-glycolytic functions in order to explore the potential for therapeutic opportunities. Further, we discuss the translational potential of emerging drug candidates in light of technical advances in treatment modalities such as image-guided targeted delivery of cancer therapeutics. PET imaging, combined with computed tomography (CT), plays an indispensable role in modern diagnostic oncology [Glucose metabolism in cancer cells is primarily characterized by two major biochemical events: (i) increased glucose uptake and (ii) aerobic glycolysis, the process of conversion of glucose into pyruvate eventually resulting in the production of lactate (fermentation). The former has already been exploited clinically to diagnose cancer and assess tumor response through the utilization of radiolabeled glucose analog, oncology ,3. Yet, oncology . As a reoncology .It is increasingly evident that oncogenes and tumor suppressors regulate altered energy metabolism. Oncogenic mutations culminate in the up-regulation of glucose transporters  ,7 thus f+ channel axis of cancer cells is suppressed, a mere restoration of mitochondrial-K+ channel function is sufficient to promote apoptosis. This report supports two major hypotheses, (i) reversal of the glycolytic phenotype to oxidative phosphorylation can promote cancer cell death and (ii) glycolysis can facilitate tumor growth despite a suppressed mitochondria-K+ channel axis.The fact that cancer cells express the glycolytic phenotype has long been known . HoweveUnderstandably, the metabolic switch from mitochondrial respiration to glycolysis during hypoxia (where oxidative phosphorylation will be inactive) as well as mitochondrial dysfunction ,22 are cOn the other hand, cancer cells subjected to mtDNA gene mutations or deletions show reduced colony formation, growth rate and diminished tumorigenicity . Based oRecent investigations have shed light on the understanding of the benefits and selective advantages of aerobic glycolysis. Although glycolysis yields a lower amount of ATP compared to mitochondrial OXPHOS, several key benefits inherent in aerobic glycolysis drive cancer cells to favor glycolysis over mitochondrial oxidation . First, The PPP plays a pivotal role in macromolecular biosynthesis. Recent evidence indicates that it also contributes to therapeutic resistance as an antioxidant system to chemo- and radiation therapies . Among sMany enzymes of the glycolytic pathway also play significant roles in several non-glycolytic processes that enable the cancer cells to meet other cellular demands. As shown in Figure For example, the mitochondrial membrane-bound HKII antagonizes the proapoptotic machinery there by provides survival advantage to cancer cells ,44. In aAs with glycolytic enzymes, some of the metabolic intermediates of glycolysis have also been associated with non-glycolytic pathways. For example, Fructose-1, 6 bisphosphate plays an anti-apoptotic role in cancer cells by maintaining the cytochrome C in a reduced, non-active state . SimilarIncreasing evidences demonstrate that glycolytic enzymes translocate to different subcellular compartments where they can interact with subcellular structures to an appreciable degree resulting in significant differences in their primary and secondary functions Figure . The enzWhereas the necessity and significance of the association between glycolytic enzymes and cytoskeletal structures remains obscure, the nuclear functions of glycolytic enzymes are sufficiently documented. Indeed, one of the isoforms of phosphofructo kinase (PFK), the PFKFB3 impacts cancer cell proliferation by its nuclear translocation . SimilarA higher lactate level significantly correlates with tumor recurrence and the metastatic potential of tumors resulting in poor patient outcomes . As lactRecently, using untransformed primary breast cells (HMEC) as controls, Hussien and Brooks  demonstrin vitro models, where spheroid-formation resulted in the promotion of a central hypoxic area eventually leading to an increase in the glycolytic flux [Multiple lines of evidences have established that a higher expression levels of GLUTs and of certain enzymes such as HKII, GAPDH, LDH and PFK-B is linked to malignant growth -75. As dtic flux . Akt, thtic flux . Akt acttic flux .Coordinated networks involving signaling pathways enable cancer cells to detect and integrate the immediate environmental conditions to balance their anabolic and catabolic processes. The mammalian Target of Rapamycin (mTOR) represents such a pathway where the intracellular energy sensing molecule AMPK can impact the mTOR complex I (mTORC1) mechanism of activation to either delay or halt the energy consuming synthetic processes . Such anFigure There are several approaches to disrupting glycolysis. Since cancer cells depend on increased utilization of glucose as compared to normal healthy cells, glucose deprivation could be an effective anticancer approach and possibly used as a cancer-preventive strategy. Indeed, carbohydrate-restricted diets to treat cancer patients have been reported to have therapeutic benefits .An obvious direct approach would be to block the GLUTs, which would prevent glucose entry into the cancer cell and lead to total disruption of the glycolytic pathway. Several such compounds  demonstrated anticancer effects in preclinical models ,85. HoweAnother approach is to target the enzyme HKII that is responsible for the first step of glycolysis that converts glucose to glucose-6-phosphate. This enzyme plays a pivotal role in tumor glycolysis. First, it is a rate limiting step that provides direct feedback inhibition thereby preventing the consumption of cellular ATP in turn preserving precious energy within the cancer cell. Second, it has a low Km (high affinity) for glucose. This characteristic facilitates the initiation of glycolysis specifically in times of low serum glucose levels, and along with its subcellular localization,  plays a pivotal role in the energy metabolism of cancer cells ,87. LoniPFK catalyzes another rate-limiting step of glycolysis and is regulated by allosteric effectors and covalent modifications such as phosphorylation. It is activated by AMP and fructose 2,6-bisphosphate . An abundance of ATP inhibits the activity of PFK, presumably representing a regulatory mechanism. However, F-2, 6-BP has the capability to override the inhibitory effect of ATP, and to perpetuate uninterrupted glycolytic flux. Predictably, F-2, 6-BP is elevated in cancer cells . It is rAn alternative promising therapeutic approach to date in terms of inhibiting tumor glycolysis has been targeting the enzyme GAPDH. In many ways the GAPDH reaction is unique because GAPDH catalyzes the very first step in which energy in the form of NADH is produced, the so-called “pay-off phase” Figure . As suchin vitro in multiple cell lines and in vivo in numerous animal models of cancer [Since GAPDH represents an attractive target for therapeutic intervention several inhibitors have already been tested for their efficacy in cell cultures as well as animal models . One of f cancer ,102. By f cancer ,104. As PK catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate, and generates ATP in the process. Among various isoforms, the M2 isoform has gained much attention due to its elevated expression in tumor cells. PK-M2 exists in either active or inactive forms. The activity of this isoform depends on its conformation . PK-M2 is critical for aerobic glycolysis and tumor energy metabolism ,105. As LDH catalyzes the final step in the glycolytic pathway that converts pyruvate into lactate. The intracellular accumulation of lactate is extremely detrimental as the abundance of lactate drastically lowers intracellular pH. The export of lactate into the extracellular space is therefore necessary. It takes place through an active process involving specific transporters known as MCTs. Among the described isoforms, MCT1 and MCT4 have been the subject of intense investigation due to their role in the import and export of lactate. FX11, an inhibitor of LDH depletes intracellular ATP levels, which in turn significantly increase oxidative stress resulting in tumor cell death . SimilarMCTs are the final port of entry for the lactate shuttle. Depending upon the isoform of MCT (1 or 4) the lactate could be either exported or imported . A knownAs discussed earlier, the reliance of cancer cells on the glycolytic pathway for their energy needs has been known for decades and has even been successfully exploited diagnostically (FDG-PET imaging) for many years. Yet, targeting this pathway for therapy has not been translated to the clinic. One of the major impediments is to overcome undesirable effects such as systemic toxicity. Plausibly, this is due to the ubiquitous nature of the enzymes of glucose metabolism including glycolysis. But in all fairness, systemic toxicity is a major impediment to clinical progress of any anticancer agent, not just those targeting tumor glycolysis. The emergence of the concept of selective targeting with targeted delivery has provided an additional option to circumvent the problem of systemic toxicity. In the last few years, there has been growing interest in revisiting this approach. This strategy employs the use of image-guidance to deliver the drug where it is required, i.e. in the vicinity of the tumor. Recent advances in imaging technology allow for such precise targeting of tumors, be it directly intra-tumorally or intraarterially where the blood supply to the tumor can be exploited . Such thCertainly, the scientific rationale for targeting tumor glycolysis is clearly sound and logical. It is based on the fact that tumor glycolysis is a true signature of cancer cells. A number of drug candidates have been tested mostly pre-clinically with mixed success. But some have been extremely promising and are about to enter the clinical arena. One of the keys to clinical success will reside in our ability to develop glycolytic inhibitors with a very high specificity for the molecular target. Recently, Birsoy et al.  demonstrAntiglycolytic agents may provide an additional line of attack in combination therapy. Combining chemotherapeutic drugs and glycolytic inhibitors have already been demonstrated to be promising strategy to overcome drug resistance in cancer. Since tumor glycolysis also plays a significant role in chemoresistance of cancer cells glycolytic inhibitors therefore have the potential to sensitize tumor cells and to improve the outcome of conventional chemotherapy. Such combination therapies have yielded better results in preclinical models. For example, the use of chemotherapeutic agents (adriamycin or paclitaxel) or radiation therapy resulted in improved efficacy, when applied after sensitizing tumor cells with 2-DG, a HKII inhibitor ,95. SimiThe inhibition of glycolysis can also transform tumor cells into forms that are susceptible or sensitive to immunotherapy, thus opening a new window of opportunity for immunotherapy . In summ3-BrPA: 3-bromopyruvate; GLUT: Glucose transporters; HKII: Hexokinase II; GAPDH: Glycerladehyde-3-phosphate dehydrogenase; PFK: Phospho fructo kinase; PFKFB: 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatases; PK: Pyruvate kinase; LDH: Lactate dehydrogenase; PK-M2: Pyruvate kinase M2; MCT: Monocarboxylate transporters.Dr. Geschwind is the founder of Presciencelabs LLC, a biotech firm currently developing the pyruvate analog 3-bromopyruvate (3-BrPA) for clinical use in liver cancer.Both authors wrote and approved the manuscript.


ARDS has a wide variability of lung morphological characteristics. Both alveolar collapse and airway narrowing or closing are present, often heterogeneously. Despite advances in ARDS imaging, we have thus far been unable to distinguish regional airway opening from airway dilatation in PEEP-induced lung recruitment. We demonstrate the technique of functional respiratory imaging (FRI) to differentiate these two entities.2O; median value between 1 and 3; clinically acceptable minimal; and 0 cmH2O. FRI methods as described by De Backer et al. [Six patients with early-stage ARDS were included in this prospective single-centre cohort trial. The lower infliction point on a pressure-volume curve was considered as the clinically acceptable minimal PEEP value. Subsequently, four distinct PEEP levels were chosen to perform CT scans: at 20 cmHAirway stretching  could be quantified and distinguished from airway recruitment with this technique. Higher PEEP pressures not only recruit, but also expand the bronchi. The ratio of dilation/recruitment of bronchi was higher in the upper lobes than in the lower lobes, as illustrated in Figure The novel technique of FRI can be used to visualise the airway structures in ARDS and distinguish airway stretching from airway recruitment. This pilot study shows that, in ARDS, the upper lung regions are subject to airway dilation, whereas the lower (atelectatic) lung lobes have more airway opening with higher PEEP levels.


Human Immunodeficiency Virus (HIV) infection and use of protease inhibitors have been associated with accelerated atherosclerosis. Increased rates of coronary in-stent restenosis are reported in these patients. There is limited data available on peripheral vascular disease interventions on these patients. Herein we report an aggressive subclavian in-stent restenosis with an unexpected response to balloon angioplasty treatment with a large, mobile tissue flap formation and its treatment with another stent. The survival of patients with Human Immunodeficiency Virus (HIV) infection has dramatically increased with advent of highly active antiretroviral therapy (HAART). Serious metabolic problems with the use of antiretroviral drugs in addition to the HIV infection itself might be causative in accelerated atherosclerosis and unexpectedly higher rates of coronary in-stent restenosis rates in these patients , 2. AlthHerein, we report an HIV infected patient with an occluded symptomatic left subclavian artery which had been successfully treated with stenting who developed subsequent aggressive in-stent restenosis that was resistant to balloon angioplasty (BA) and responded to BA with the tear of the neointimal tissue causing a large mobile tissue flap requiring repeat stenting to prevent embolic complications.A 56-year-old Caucasian male with hypertension, hyperlipidemia with LDL of 78 on day of procedure, HIV infection being currently treated with HAART  with a CD4 count of 420, coronary artery disease status after implantation of a drug eluting stent in an obtuse marginal two years ago, asymptomatic bilateral carotid artery stenosis, left subclavian artery occlusion with subclavian steal status after percutaneous angioplasty, and stenting with a Visi-Pro 7.0 × 37 mm stent (Covidien) one year ago  and angioplasty of the lesion was performed with a Viatrac 7.0 × 15 mm balloon  inflated at 14 atmospheres pressure . After bIn-stent restenosis is an increasingly recognized problem in patients with HIV infection. It is caused by excessive smooth muscle proliferation and accumulation of extracellular matrix . IncreasStudies have shown that patients with higher levels of inflammatory markers, such as C-reactive protein, at the time of PCI have higher restenosis rates , 4. HIV Further, HIV, in itself, is an atherogenic state. HIV disease induces accelerated T-cell proliferation, heightened T-cell activation, and high levels of inflammatory marker expression and immunologic perturbations that persist even after the introduction of antiretroviral therapy. T lymphocytes and inflammatory cytokines play key roles in atherosclerosis. Thus, immunodeficiency and immune reconstitution may accelerate atherosclerosis and in-stent restenosis . NecropsOn the other hand, though highly active antiretroviral therapy (HAART) treatment has led to a dramatic decline of morbidity and mortality in HIV infected patients, it is also associated with significant metabolic adverse effects, that is, alteration of lipid profile, insulin resistance, and impaired endothelial function –8. AlterAll these above-mentioned mechanisms contribute to aggressive form of in-stent restenosis in HIV infected patients. In contrast to coronary arteries, the peripheral vasculature can harbor excessive amounts of smooth muscle proliferation and extracellular matrix at sites of in-stent restenosis. Conventionally, balloon angioplasty has been the treatment of choice for in-stent restenosis in peripheral vasculature. But, in this particular set of patients, balloon angioplasty may not always be adequate and can lead to complications like dissection of neointimal hyperplasia within in-stent restenosis that may require stenting to prevent distal embolization as illustrated in our case.Aggressive risk factor modification and lipid-lowering drug therapy including statins are required in this subset of patients to prevent future cardiovascular events. Future directions in treating these patients need to be explored. One possibility would be the use of drug eluting stents rather than bare metal stents in peripheral vasculature in these patients that would reduce the risk of aggressive in-stent restenosis as observed in this patient. This was reinforced by lack of any in-stent restenosis observed in drug eluting stent placed in obtuse marginal coronary artery 2 years ago in our patient.