P. yoelii. PAT and PanKs were shown to be dispensable for blood stage development, but they were essential for mosquito stages development. Yet, little is known about the cellular functions of the other enzymes of the CoA biosynthesis pathway in malaria parasite life cycle stages. All enzymes of this pathway were targeted for deletion or deletion/complementation analyses by knockout/knock-in plasmid constructs to reveal their essential roles in P. yoelii life cycle stages. The intermediate enzymes PPCS (Phosphopantothenylcysteine Synthase), PPCDC (Phosphopantothenylcysteine Decarboxylase) were shown to be dispensable for asexual and sexual blood stage development, but they were essential for oocyst development and the production of sporozoites. However, the last two enzymes of this pathway, PPAT (Phosphopantetheine Adenylyltransferase) and DPCK (Dephospho-CoA Kinase), were essential for blood stage development. These results indicate alternative first substrate requirement for the malaria parasite, other than the canonical pantothenate, for the synthesis of CoA in the blood but not inside the mosquito midgut. Collectively, our data shows that CoA de novo biosynthesis is essential for both blood and mosquito stages, and thus validates the enzymes of this pathway as potential antimalarial targets.Coenzyme A (CoA) is an essential universal cofactor for all prokaryotic and eukaryotic cells. In nearly all non-photosynthetic cells, CoA biosynthesis depends on the uptake and phosphorylation of vitamin B5 (pantothenic acid or pantothenate). Recently, putative pantothenate transporter (PAT) and pantothenate kinases (PanKs) were functionally characterized in  To create individual gene deletion of PyPPCS and PyPPCDC, a targeting vector, pAA20, containing the enhanced GFP (eGFP) reporter cassette and the human DHFR resistance selection marker cassette was used to clone 5′ and 3′ regions of each gene. Following transfection with the targeting vectors, drug selection and cloning of transgenic parasites, genomic PCR analyses were performed to confirm the genetic replacement events and the deletion of PyPPCS and PyPPCDC  in the development of parasite within host erythrocytes and in transmission to the mosquito, a genetic approach was employed to generate individual deletion of PPCS and PPCDC in PyPPCS and PyPPCDC deletion during the intraerythrocytic phase of P. yoelii development, we compared blood stage (BS) parasitemia of groups of four BALB/c mice per genotype intravenously (IV) infected with 5,000 BS of Pyppcs(−) clone Q4, Pyppcdc(−) clone R3, WT-like parasites Pyp230p(−) clone A5  and Pyppcdc(−) parasites in mouse erythrocytes made it possible to assess the function of both PyPPCS and PyPPCDC in gametocyte development and maturation, as well as in gametogenesis. Outbred Swiss Webster (SW) mice were intravenously (IV) infected with 1 × 106 BS of Pyppcs(−), Pyppcdc(−), or Pyp230p(−) parasites. Blood samples were collected and smears were made simultaneously at day three post infection to determine the percentages of male and female gametocytes in giemsa-stained thin blood smears . These experiments confirm that the lack of the second and third enzymes of CoA biosynthesis PPCS and PPCDC do not interfere with the growth and development of any asexual or sexual blood stage parasites.The viability of s Figure , and mals Figure . SimilarPPCS and PPCDC for the development of ookinetes, female Anopheles stephensi mosquitoes were fed on infected mice displaying the highest male gamete exflagellation rate from the previous experiment. After 20 h post mosquito-feeding (pmf), mosquito midguts from at least 20 blood-fed female A. stephensi were dissected, and mature ookinetes were counted for each parasite genotype , Pyppcdc(−), and Pyp230p(−) parasites were all designed to constitutively express eGFP in all life cycle stages to visualize the development of early oocyst. Midguts of mosquitoes fed on Pyppcs(−), Pyppcdc(−), and Pyp230p(−) parasites were dissected, and the number of oocysts on their midguts were counted using fluorescence microscopy at day 4 and 7 pmf, respectively  at day 4 pmf. More significant reduction at day 7 pmf was displayed by both knockout strains compared to the control strain at day 7 pmf, and to the respective knockout strains at day 4  or Pyppcdc(−) infected mice in four independent experiments  clone A5, Pyppcs(−), clone Q4 and Pyppcdc(−) clone R3 parasites, as previously described  mice and BALB/c mice (6–8 weeks old) were purchased from Envigo Research Models and Services, formerly known as Harlan Laboratories, . All Animal experiments were conducted according to the approved protocols of the Institutional Animal Care and Use Committee (IACUC) of Tulane University. All other experimental protocols and the use of recombinant DNA were conducted according to the approved protocols of the Institutional Biosafety Committee (IBC) of Tulane University. Detailed phenotypic analyses in mice and mosquitoes were performed, using P. yoelii 17X-NL strain using primer pairs 71–72 and 73–74 for PPCS, respectively, and primer pairs 81–82 and 83–84 for PPCDC, respectively, and primer pairs 91–92 and 93–94 for PPAT, respectively, and primer pairs 101–102 and 103–104 for DPCK, respectively. Primer sequences are listed in Supplementary Table SacII and BamHI, and KpnI and HindIII, respectively. For the knock-in constructs of PPAT and DPCK enzymes, the 5′ UTR region cloned in with SacII and BamHI and used for the homologous recombination in the knockout constructs were replaced by the coding sequence of PPAT and DPCK, amplified by primers 97–98 and 107–108, respectively. All the resulting targeting vectors were linearized with SacII and KpnI prior to transfection of P. yoelii 17X-NL parasites by electroporation with Lonza Nucleofector II. Selection of resistant parasites and cloning of transfectants were performed as previously described  values were used to represent error bars in all graph figures.One-way Analysis-of-Variance (ANOVA) was used to assess the mean values for statistically significant differences in all experiments. GraphPad InStat software was used for all statistical analyses. ASIA designed the experiments; RH and AA conducted the experiments; RH, AA, and ASIA analyzed the results; RH, AA, and ASIA wrote the manuscript.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.


Dopamine and serotonin neurotransmission relies mostly on the action of four factors: serotonin and dopamine transporters (SERT and DAT) and enzymes monoaminooxidase A (MAO-A) and catechol-O-mehtyltransferase (COMT). The goal of this research was to closely examine schizophrenia symptom domains in relation to the investigated polymorphisms.Study group was composed of 300 schizophrenic patients. Severity of schizophrenia was assessed by the Positive and negative syndrome scale (PANSS), depressive symptoms were assessed with Calgary depression scale for schizophrenia (CDSS). SERT (5-HTTLPR), DAT (VNTR), COMT  and MAO-A (VNTR) gene polymorphisms were analyzed. Schizophrenia symptom dimensions were determined with multivariate statistical methods, while logistic regression and ANOVA were used to investigate the influence of a genotype on a symptom domain.Factor analysis of PANSS scale retained all 30 items and identified 5 separate factors . Analysis of CDSS scale revealed 2 separate factors .Statistically significant PANSS variables were those of aggressive/impulsive and negative symptoms, while suicidality was the only significant CDSS variable.Our PANSS scale factor analysis established 5 distinct factors. Previous factor analyses provided from 3, up to 7 different factors, but mostly 5 distinct ones: negative symptoms, positive symptoms, depressive symptoms, excitement and disorganization. That factor distribution corresponds to our findings in terms of identified number of factors, but seems to differ in terms of item distribution within those factors.When testing the influence of investigated gene polymorphisms on the variable of total PANSS score and five distinct factors we did not establish significant findings regarding four variables: total PANSS score, positive, cognitive and affective/depressive symptoms. While that is in line with majority of other investigations, SERT promoter polymorphism and COMT Val158Met gene polymorphism have been previously associated with depressive and positive symptoms. SERT and MAO-A polymorphisms separately had a significant effect on the variable of aggressive/impulsive symptoms, which has not been reported earlier. Furthermore, significant influence of COMT gene polymorphism was established for the variable of negative symptoms, which is a confirmation of some earlier reports, although there have been contrary findings.Previous reports of CDSS scale factor structure are limited to data from its initial validation to the few recent findings of its three-factor structure . We identified 2 separate factors using factor analysis, “depression” (which included seven out of nine items) and “suicidality”. To the best of our knowledge this is the first investigation of the putative association between any of the four investigated polymorphisms and depressive symptoms of schizophrenia measured by the CDSS scale. Ultimately, we did not establish a significant association of investigated gene polymorphisms and total CDSS score, as well as the “depression” factor. However, there was a significant association between the “suicidality” factor and SERT and MAO-A gene polymorphisms, as well as their interaction. The fact that the significant association was established for only one of the two obtained CDSS factors suggests that the association is subtle and, at least partially, explains rarely reported associations between investigated gene polymorphisms and schizophrenia symptom domains, which is especially true for depressive symptomatology.


Cell size is amenable by genetic and environmental factors. The highly conserved nutrient-responsive Target of Rapamycin (TOR) signaling pathway regulates cellular metabolic status and growth in response to numerous inputs. Timing and duration of TOR pathway activity is pivotal for both cell mass built up as well as cell cycle progression and is controlled and fine-tuned by the abundance and quality of nutrients, hormonal signals, growth factors, stress, and oxygen. TOR kinases function within two functionally and structurally discrete multiprotein complexes, TORC1 and TORC2, that are implicated in temporal and spatial control of cell size and growth respectively; however, recent data indicate that such functional distinctions are much more complex. Here, we briefly review roles of the two complexes in cellular growth and cytoarchitecture in various experimental model systems. Tor genes together with the FKBP12 homolog fpr1 were first isolated in Saccharomyces cerevisiae  is an evolutionarily conserved Ser/Thr-protein kinase functioning as the heart of signaling networks toward nutrient and hormonal sensing. The role of these networks is to regulate anabolism and catabolism by coordinating numerous cellular metabolic processes, such as protein translation, formation of ribosome components, mRNA stability, autophagy, cell-cycle, transcription, and cellular architecture  has major contributions in temporal aspects of cell size control. For example, inhibition of TORC1 using rapamycin advances mitotic onset in fission yeast. The effect is similar to cell size reduction observed when cells sense and experience poor nitrogen sources. This is achieved through the stress MAPK pathway via the Pyp2 phosphatase  promotes cell growth by stimulating synthesis of the primary building blocks of macromolecules, by inhibiting autophagy and influencing cell cycle progression  has also been implicated in special aspects of cell growth in mammals and fish. Mammalian injured mature CNS axons do not regenerate. PTEN inhibition results in TORC1-dependent, rather than TORC2, CNS axon regeneration and growth. The effect is mediated through the S6-kinase  is reported to have opposing roles to TORC1 in fission yeast  results in reduced binding of MBF to its target promoters. Moreover, MBF target genes fail to be induced in the aforementioned mutants upon DNA replication stress. Finally, the protein levels of Cdt2 and Cig2 (both of them MBF targets) are reduced when TORC2 or Gad8 function is abolished. These data highlight pivotal roles of TORC2 within the cell nucleus and reveal a TORC2-dependent mechanism in DNA replication stress response through the control of MBF transcriptional targets  morphology and constriction (Baker et al., Trypanosoma brucei four TOR kinase orthologues TbTOR1, TbTOR2, TbTOR3, and TbTOR4 have been identified. TbTOR1 and TbTOR2 resemble the structure of all TOR kinases (Saldivia et al., Roles of TORC2 in temporal aspects of cell size and growth are emerging in systems as diverse as protozoa and human cancer cells. In the protozoan parasite Traditionally TORC1 is linked to temporal aspects of cell size and growth while TORC2 with spatial growth. Nevertheless, emerging data indicate more complex relationships (Figure CR conceptualized the review, decided on the content, drafted the work and prepared the figures. SG provided reference searching, proofreading and editing. Both authors approve the final version of the manuscript.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.


Although the field of oncology has developed and expanded dramatically, a single drug has not yet been discovered that can cure all patients, even those with similar cancer types. We now know that cancer is an extremely heterogeneous disease, which explains differences not only between cancer cells from different patients, but also between cancer cells within a single patient.4 Clearly, more effective strategies are critically needed to defeat the long-standing enemy known as cancer.Based on early evidence of fossilized bone tumors that were found in ancient Egyptian mummies, cancer is an ancient disease.5 Cancer is a major focus of the precision medicine initiative and developments in precise and effective treatments could benefit many other chronic diseases. Precision oncology or precision medicine of cancer focuses on matching the most accurate and effective treatment to each individual cancer patient based on the genetic profile of the cancer and the individual. Because every single cancer patient exhibits a different genetic profile and the profile can change over time, more patients will benefit if therapeutic options can be tailored to that individual, thus avoiding the idea that “one-size-fits-all” in terms of cancer treatment.The concept and practice of precision medicine is a methodical and systematic movement aimed at defeating diseases such as cancer.10 focusing on the concept of precision medicine have had limited success.12 Naturally, opponents of precision medicine have criticized the strategy based on the results of the small number of clinical studies.13 However, concluding that precision medicine will not work is premature because the precision medicine approach has not yet been fully tested in a sufficient number of trials. In addition, the suggestion has been made that having inadequate access to specific therapeutic agents and an insufficient number of tumor samples may have contributed to the limited success.13 Because cancer is a highly heterogeneous disease both between patients and within the same patient, the ability to cope with changes in the clinical trial setting is extremely challenging. Many creative and bold ideas of precision medicine have not yet made the transition from the lab bench to the clinic and need to be more fully evaluated in small clinical studies. For example, the microbiota is now recognized as a key player in health.14 The microbiota influences endocrinology and disease status and alters drug response and resistance, and this could hold true for cancer and the efficiency of cancer treatments. Sequencing the human microbiome and modulating the host-microbiota interactions in individual patients may be one approach to improve therapeutic outcomes.15Results of one randomized clinical trial and a small number of feasibility and/or tumor response studiesBcr-Abl gene fusion in chronic myeloid leukemia (CML). Uncovering this genetic driver of CML lead to the development of a selective inhibitor of BCR-ABL, imatinib, which exhibited broader treatment coverage because, unlike other gene mutations, the Bcr-Abl gene fusion occurs in almost all CML patients. This compound improved the overall survival rates of CML patients to 90% over 5 years and 88% over 8 years.16 Another example includes the effectiveness of drugs like trastuzumab, lapatinib, pertuzumab, or ado-trastuzumab emtansine against human epidermal growth factor receptor 2 (HER2)-positive breast cancer. Compared with chemotherapy alone, the addition of trastuzumab to chemotherapy significantly slowed the disease progression , increased the objective response rate , prolonged survival time , and reduced the risk of death by 20%.17 Lapatinib plus chemotherapy  achieved a longer median time to disease progression compared with chemotherapy alone .18 A combination of pertuzumab, trastuzumab, and chemotherapy  further improved the median overall survival time to 56.5 months compared with a combination of only trastuzumab and chemotherapy .19 Treatment with ado-trastuzumab emtansine, a conjugate of a HER2 monoclonal antibody and a cytotoxic drug, significantly improved the length of progression-free survival and overall survival with lower adverse effects when compared with lapatinib and chemotherapy .20 These two examples demonstrate how the identification of key mutations like the Bcr-Abl fusion or HER2 can clearly benefit a larger number of selected cancer patients.Questions that must be addressed include whether precision oncology is just a theory or whether it realistically assures a better future, and if truly promising how can the application of precision oncology be improved and effectively implemented into the clinic? Several lines of evidence strongly support the idea that precision oncology could likely benefit more patients compared with traditional chemotherapies. First, some patients’ lives have already been substantially improved by target-based therapies compared with conventional cytotoxic therapies. One of the most notable examples is the discovery of the 21 This strategy is based on the discovery that cell death is caused by a combination of deficiencies in the expression of two or more genes, whereas deficiency in only one of these genes can increase viability.22 In 2005, the BRCA and poly ADP ribose polymerase (PARP) genes were found to have a synthetic lethal relationship.24 In 2014, the FDA (Food and Drug Administration) authorized accelerated approval of olaparib, a PARP inhibitor, to treat BRCA-mutant ovarian cancer patients.28 Progression-free survival of BRCA-mutant ovarian cancer patients was significantly prolonged by olaparib compared with the placebo treatment .28 Likewise, several candidate proteins were identified to treat cancers that over-express Myc, a transcription factor for which small-molecule inhibitors are currently unavailable.33 Within just a 5-year timespan, researchers found that Myc-amplified tumors were sensitive to CDK1, aurora kinase B, and BRD4 inhibition.30 Although many of these inhibitors have yet to translate into clinical successes, Phase I trials of three BRD4 inhibitors are ongoing (CPI-0610 and TEN-010) or have just finished (OTX015).35 These findings suggest that changes in gene A in cancer does not necessarily mean that gene A is the best target, but instead targeting its synthetic lethal partner might be a more effective strategy.Second, many strategies are now available to identify important molecular targets for therapeutic intervention. Synthetic lethality is one unique strategy directed toward identifying cancer vulnerabilities.36 The cancer patients will be assigned to 1 of 25 different treatment arms based on their genetic mutation profile. The overall response rate will be the endpoint to measure success. However, no control arms will be included, which could dramatically affect the interpretation of the final results. Even though a number of questions have been raised, the investigators are very optimistic that the results will further the efforts to implement precision oncology treatments. The Molecular Profiling-based Assignment of Cancer Therapy (NCI-MPACT) is another innovative clinical trial to test the hypothesis that targeting an oncogenic driver mutation is more efficacious than not targeting it. NCI-MPACT will recruit advanced cancer patients who have been unresponsive to standard therapeutic options and possess mutations in one of three genetic pathways that include DNA repair, PI3-K/mTOR , and Ras/Raf/MEK (mitogen-activated protein kinase). Patients without a driver mutation will not be eligible for further treatment.37 Although this trial is similar to the NCI-MATCH in that patients will undergo tumor biopsies when enrolled, patients in the NCI-MPACT study will be assigned to one of two arms: treatment with drug(s) designed to target the mutation or treatment with drug(s) not prospectively identified to target the mutation. By evaluating gene targets across the histologic subtypes with NCI-MATCH and addressing the importance of driver mutations with NCI-MPACT trials, the efficacy of diagnosis and therapies could be significantly enhanced.Third, clinical trial design is constantly evolving to overtake tumor heterogeneity from patient to patient. The Molecular Analysis for Therapy Choice (NCI-MATCH) is a clinical trial selecting treatments based on genetic features of patients, not traditional tumor histology.38 Recently, a gene therapy trial to treat myeloma, melanoma, and sarcoma with CRISPR/Cas has been approved by the National Institute of Health and is awaiting approval from the FDA.39 In addition to CRISPR/Cas, cryo-EM is a promising tool for precision oncology. Cryo-EM is a type of transmission EM in which samples are examined at cryogenic temperatures.40 Because the samples  are frozen in their native states, researchers can study biological events accurately at the subatomic or atomic level. For an instance, a 2.3 Å resolution cryo-EM structure of p97 showed a large corkscrew-like hexameric form, revealed its interactions with an allosteric inhibitor, and displayed conformational changes induced by adenosine tri-phosphate.41 Visualization of intact proteins and anti-cancer drugs at subatomic or atomic levels will assist researchers in understanding the consequences of genetic alterations on drug response and resistance.Finally, the influence of new technologies such as the CRISPR/Cas system and cryo-electron microscopy (cryo-EM) will broaden and sharpen our ability to identify novel therapeutic targets for precision oncology. CRISPR/Cas technology enables controlled exchange, insertion and deletion of DNA sequences unlike spontaneous mutation, and can easily generate animal models that mimic mutations status of patients.In spite of some early setbacks, precision oncology still has a great deal of promise and should not be abandoned hastily. The challenge of tumor heterogeneity should not discourage or intimidate efforts to overcome cancer but should push the field forward. As practice makes perfect, precision mends patients.


Mycobacterium abscessus represents an important respiratory pathogen among the rapidly-growing non-tuberculous mycobacteria. Infections caused by M. abscessus are increasingly found in cystic fibrosis (CF) patients and are often refractory to antibiotic therapy. The underlying immunopathological mechanisms of pathogenesis remain largely unknown. A major reason for the poor advances in M. abscessus research has been a lack of adequate models to study the acute and chronic stages of the disease leading to delayed progress of evaluation of therapeutic efficacy of potentially active antibiotics. However, the recent development of cellular models led to new insights in the interplay between M. abscessus with host macrophages as well as with amoebae, proposed to represent the environmental host and reservoir for non-tuberculous mycobacteria. The zebrafish embryo has also appeared as a useful alternative to more traditional models as it recapitulates the vertebrate immune system and, due to its optical transparency, allows a spatio-temporal visualization of the infection process in a living animal. More sophisticated immunocompromised mice have also been exploited recently to dissect the immune and inflammatory responses to M. abscessus. Herein, we will discuss the limitations, advantages and potential offered by these various models to study the pathophysiology of M. abscessus infection and to assess the preclinical efficacy of compounds active against this emerging human pathogen. Mycobacterium abscessus (Mabs) is a rapidly-growing mycobacterial species, regarded as an important pathogen responsible for a wide array of clinical manifestations in humans, ranging from cutaneous infections to severe chronic pulmonary infections, usually encountered in immunocompromised and in cystic fibrosis (CF) patients  or a rough (R) colony morphotype that can result in different clinical outcomes. Epidemiological studies indicate that the presence of R variants are associated with the most severe cases of pulmonary infections which can persist for years  and Dectin-1 have been shown to participate in phagocytosis of Mabs  have been widely exploited during the last two decades to decipher the intricate interactions between pathogens and the host immune system  did not develop a sustained progressive infection. Conversely, when infected with a high-dose aerosol inoculum , C57BL/6 and Ob/Ob mice established an infection resulting in an early influx of IFN-γ+ CD4+ T cells in the lungs. This primary immune response preempted the clearance of Mabs in both C57BL/6 and Ob/Ob mice. On the other hand, IFN-γ knockout (GKO) mice infected with a LDA or HDA of Mabs resulted in a low amount of persistent Mabs lung infection inducing influx of T cells, macrophages and dendritic cells, which contributed to granuloma formation. Interestingly, a HDA Mabs infection of the GKO mice provoked CD4+ and CD8+ T cells capable of producing IL-4 and IL-10 in the pulmonary cavity.Early studies confirmed that most immunocompetent mouse strains result in clearance of Mabs infection, further research has led to establishment of mouse models with deficits in innate or acquired immunity, resulting in a high level of Mabs disease. Initial studies established that immunocompromised mice with defects in innate or acquired immunity infected intravenously with 1 × 106Mabs controlled the infection , iNOS−/−, Cybb−/− (devoid of super-oxide generating enzyme), TNFαR−/−, C3HeB/FeJ, GKO, and MyD88−/− mice. Through a 40 days chronic infection, Mabs was still present at low levels in the lungs of the C3HeB/FeJ, GKO and MyD88−/− mice. Furthermore, the GKO and MyD88−/− mice sustained diminished amounts of Mabs in the spleen and liver after 40 days, whereas SCID, nude and GM-CSF−/− mice infected intravenously with Mabs showed progressive Mabs burden  for modeling Mabs infection was the presence of foamy cells and necrotizing and non-necrotizing granulomas in the lungs after 40 days of infection, commonly observed in the histopathologic sections of human NTM lung disease. The challenge remains to overcome RGM avirulence and immune clearance in immunocompetent models for future research advances. Multiple CF models have also been developed for other human CF pathogens. Current studies are focused on developing these CF models for Mabs infection advancing into the development of pulmonary infection rather than using the intravenous route.Despite the aforementioned challenges associated with establishing a high level of Mabs to most commonly used antimicrobials, the discovery of new and active compounds is urgently needed. A major step in drug discovery relies on in vivo evaluations of the identified hits, requiring studies in adequate animal models. As already mentioned, immunocompetent mice are characterized by a transient infection with a rapid clearance of Mabs  further validated the zebrafish as a potent preclinical model (Dubée et al., in vivo activity of a new piperidinol-based compound, PIPD1, inhibiting mycolic acid transport in Mabs was evaluated in zebrafish (Dupont et al., cftr expression (Phennicie et al., cftr-deficient environment as it is currently not known whether a CFTR defect affects susceptibility to antibiotics. Of note, embryos are particularly conducive to high throughput screening, as shown for Mycobacterium marinum (Carvalho et al., Drosophila melanogaster has also been reported for the rapid evaluation of potential drug candidates against Mabs (Oh et al., However, despite the fact that immunocompromised mice present a significant advance in comparison to wild-type mice in preclinical assessments, they remain costly, time-consuming and may not reflect the predictive value required for compound testing. Zebrafish were also successfully developed to test the suitability and sensitivity of clarithromycin and imipenem, two clinically relevant drugs in Mabs, its environmental and human hosts. These biological systems have undoubtedly allowed to better elucidate the pathogenesis of Mabs disease and to highlight the distinctive intra- and extracellular traits characterizing the lifestyle of the R and S forms. However, despite considerable progress, our knowledge of the immunopathology of Mabs infection remains largely incomplete. As discussed here, these recently developed models represent innovative tools for better manipulating both the pathogen and the host immune response and should lead to an in-depth comprehension of the intricate interactions between the pathogen and its hosts, which may also unveil novel strategies to combat the infection and disease progression. Although several challenges still need to be overcome, these models hold also great promises for future development of novel and improved drug combinations to control one of the most difficult-to-treat mycobacterial species.Herein, we have summarized compelling models developed to better understand the interplay between All authors contributed to writing the manuscript. AB designed the figures.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.


Molecular disturbances of neurotransmitter systems have long been held to be a core feature of the pathophysiology of schizophrenia. Despite years of study of neurotransmitter associated protein expression at multiple levels of gene expression, reports of abnormal neurotransmitter receptor transcript, protein, and signaling complex expression in schizophrenia brain have often been conflicting. These inconsistencies led us to reconsider neurotransmitter-based hypotheses of schizophrenia not as a problem of receptor number, or as a defect of neurotransmitter systems, but rather as a dysregulation of central cellular processes regulating the intracellular distribution of signaling proteins. Our working hypothesis is that a fundamental dysregulation of intracellular processes exists in schizophrenia, resulting in abnormal assembly, trafficking, and intracellular targeting of many key proteins involved in neurotransmission and other critical cellular functions. Previous studies have shown the important roles posttranslational lipid and carbohydrate modifications play in targeting receptors, transporters, and other proteins between intracellular compartments and the synapse, and in the lateral translocation of such molecules between lipid microdomains at the distal end of forward trafficking pathways. Accordingly, we have predicted that abnormal posttranslational lipid and/or sugar modification of proteins by occurs in schizophrenia. We have previously reported changes in extent of N-linked glycosylation as well as of the lipid modifications palmitoylation and N-myristoylation on target proteins in schizophrenia brain. In an ongoing project to elucidate mechanisms of these changes, we have studied expression patterns of key enzymes associated with these posttranslational modifications.Using well characterized samples of postmortem brain from schizophrenia and matched comparison subjects, we assayed transcript expression of enzymes associated with posttranslational protein modifications by lipids and carbohydrates using microarrays and qPCR. Next, we assayed protein expression of a subset of enzymes using western blot analyses. To determine the brain cell specificity of protein changes, we used laser capture microdissection (LCM) of neuronal and glial cells to harvest specific cell populations from postmortem brains, and developed and validated a capillary electrophoresis system for ultra-low quantity protein concentration (the ProteinSimple WES system) to measure protein expression within LCM harvested cells.Using microarray and qPCR, multiple transcript changes were found in schizophrenia cortex. The most substantial number of altered transcripts were found for those encoding enzymes associated with multiple aspects of posttranslational carbohydrate modifications of proteins, including N-acetylglucosaminyltransferases (GlcNAcTs), glucosyltransferases, glucosidases, N-acetylgalactosaminyltransferases , galactosyltransferases, mannosidases, fucosyltransferases, fucosidases, sialyltransferases, and sialidases. Fewer changes were found for lipid modification enzymes.Subsequently, protein expression of candidate proteins associated with these posttranslational modifications were determined by western blot analyses. Changes in protein expression of mutlple enzymes associated with glycan modification of proteins were found in schizophrenia. These include significant decreases in expression of the N-acetylglucosaminyltransferases B3GNT8 and MGAT4A, and the fucosyltransferase FUT8. Increased protein expression was found for the fucosyltransferase POFUT2, the sialyltransferase ST8SIA2, the glucosyltransferase UGGT2, and the mannosidase EDEM2. Numerous protein changes were also found in enzymes associated with lipid and glycolipid modifications. These include decreased expression of the prenylation associate denzyme subunits farnesyl-protein transferase α-subunit (FNTA), geranylgeranyltransferase type I β-subunit (PGGT1B), and rab geranylgeranyltransferase β-subunit (RABGGTB). Glycophosphatidylinositol (GPI)-anchor attachment 1 protein (GPAA1) is increased in these subjects.To determine the cell-specific pattern of these protein changes, we have developed an LCM-capillary electrophoresis assay to isolate protein from LCM harvested cells to allow multiplex protein quantification in 500 ng of protein obtained from these cells. We have validated that we can reliably harvest cortical neuronal subtypes and astroglia, are able to measure 4 proteins simultaneously in samples from these cells lines, and are currently collecting cells to extend these findings into cell-specific studies to determine if the changes we have found in posttranslational modification proteins are widely specific or specific to given subpopulations of brain cells.These data support our earlier findings of altered patterns of the posttranslational modifications of both glycosylation and lipid modification of proteins in the cortex of schizophrenia. By identifying changes in both mRNA and protein expression of key enzymes associated with these posttranslational modifications, we have begun to elucidate potential mechanisms of these earlier observations.One of the challenges that has plagued schizophrenia research for decades is that many different neurotransmitter and neurochemical systems have been implicated and studied in this illness, and reconciling this large literature is challenging. These many changes in numerous different systems suggest, however, that rather than schizophrenia being a disorder of a given neurotransmitter system, it is rather a disturbance of core intracellular processes that underlie regulation of multiple neurochemical systems. The machinery associated with posttranslational modifications of proteins is a possible substrate that could reconcile prior abnormalities identified in myriad systems. We have proposed that a fundamental defect in the brains of those affected with this illness is abnormal assembly, trafficking and receptor dynamics of many different proteins in schizophrenia that is due mechanistically to abnormal posttranslational modifications that influence intracellular targeting and trafficking of proteins between subcellular compartments. Dysregulation of lipid and glycan modification of proteins are likely candidates for such a process, and these present data begin to elucidate the mechanisms from which these abnormalities occur.


Autophagy and macropinocytosis are processes that are vital for cellular homeostasis, and help cells respond to stress and take up large amounts of material, respectively. The limbal and corneal epithelia have the machinery necessary to carry out both processes; however, autophagy and macropinocytosis are relatively understudied in these two epithelia. In this Perspectives, we describe the basic principles behind macropinocytosis and autophagy, discuss how these two processes are regulated in the limbal and corneal epithelia, consider how these two processes impact on the physiology of limbal and corneal epithelia, and elaborate on areas of future research in autophagy and macropinocytosis as related to the limbal/corneal epithelia. Nonetheless, Christian de Duve coined the term “autophagic vacuoles” to describe the self-eating function of lysosomes,1 which still is the operative definition used today. Christian de Duve won the Nobel Prize in Physiology and Medicine in 1974 for the discovery of the lysosome, and the same prize was awarded in 2016 to Yoshinori Ohsumi for his work in yeast identifying genes essential for autophagy.3 Ohsumi's work has led to our current understanding that autophagy regulates numerous physiologic processes where cellular components must be degraded and recycled. Examples of such processes are the cell's response to starvation and other stresses,4 removal of bacteria and viruses,5 elimination of damaged proteins and organelles that occur during aging,6 and maintenance of stem cell homeostasis.7 Given the numerous ways that autophagy impacts cellular homeostasis, it is not remarkable that this cellular process has been implicated in ocular health and disease (see prior reviews9). Surprisingly, scant attention has been directed at dissecting autophagy in the corneal and limbal epithelia.The term “autophagy” is attributed to the venerable morphologist and cell biologist Christian de Duve.10 and enables cells to nonselectively engulf and take up large volumes of fluid and membrane via the closure of plasma membrane protrusions.11 In addition to enabling cells to “gulp” large amounts of fluids, macropinocytosis is now recognized as a means of pathogen egress into cells as well as having important roles in tumorigenesis and cancer therapeutics.12 Similar to autophagy, macropinocytosis has been markedly understudied in the corneal/limbal epithelia. We offer a view of how the regulation of autophagy and macropinocytosis differs between limbal and corneal epithelia, and discuss areas that are ripe for future investigation.Macropinocytosis was described first by Lewis in 193114 Autophagy can be divided into early and late stages  is segregated into double membrane vesicles (autophagosomes) that fuse with lysosomes for degradation.e stages . The eare stages . Nucleat11 and Maltese and Overmeyer,17 and references therein). Macropinocytosis is initiated as a response to growth factor stimulation, such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), or the tumor promotor tissue plasminogen activator (TPA).19 Such stimulation results in actin-mediated membrane ruffling (lamellipodia) at the plasma membrane . Consequently, there has been high interest in the use of macropinocytosis for targeted therapy development using macropinocytosing antibodies.23 Given that varying cell types can have wide ranges in macropinocytotic activity,17 determining how macropinocytosis is regulated in limbal and corneal epithelia seems warranted.Macropinocytosis is an endocytic process resulting in the formation of large (0.2–2 μm) macropinosomes . Such a diversity for two adjacent stratified squamous epithelia has made the corneal/limbal epithelia a valuable model for interrogating a variety of biologic processes. Perhaps the most consequential distinction is that the limbal epithelial basal cells are the preferential site of the corneal epithelial stem cells.2729 As such, the limbal epithelium has been recognized to have major roles in maintaining corneal epithelial homeostasis, serving as the source for epithelial cells to aid in tissue regeneration following wound healing, and acting as a barrier to the egress of the conjunctival epithelium. Thus, for the past three decades much research has been directed toward understanding the structural, biochemical, molecular, and cell biological aspects of the limbal versus corneal epithelial basal cells and their microenvironments (niches). Broad conclusions drawn from these studies are that limbal epithelial basal cells are less differentiated than corneal epithelial basal cells28; have a subpopulation of cells that are mitotically quiescent27; exhibit a greater proliferative capacity than corneal epithelial basal cells30; express distinct cell proteins, such as transporters,32 transcription factors,34 and keratins,35 to name a few; are regulated by microRNAs that are distinct from corneal epithelia basal cells3640; and are supported by a unique basement membrane/stromal interface.4143 Despite this impressive examination of limbal versus corneal epithelial basal cells, negligible attention has been directed to macropinocytosis and autophagy, cellular processes recently demonstrated to be coordinately regulated  expression profiles in limbal versus corneal epithelial basal cells, we reported that the miRs-103/107 family, which was preferentially expressed in the limbal epithelium,45 In this analysis, apoptosis, metabolic processes and response to stress were major biological events predicted to be affected by this miRNA family.37 Interestingly, we noted that HLEKs deficient in miRs-103/107 rapidly developed large vacuoles that originated, in part, from a dysregulation in macropinocytosis.44 miRs-103/107 regulate macropinocytosis, in part, at two stages. First, by targeting NEDD9 and SHC3, Src, and Ras activation are attenuated and the initiation of macropinocytosis is blocked. Secondly, by targeting ANKFY1 (Rabankyrin5), miRs-103/107 also interferes with the formation of the macropinosome, which contributes to the large vacuoles  and conducted an mRNA profiling study.vacuoles . It is i46 and Rab11 is a marker of autophagosome maturation.47 Both of these markers colocalized with Lysotracker  on the large vacuoles, strongly implicating an involvement with autophagy. Pharmacologic and genetic studies strongly suggested that large vacuole retention was due to a defect in the end stages of autophagy and that miRs-103/107 functioned to positively regulate this aspect of autophagy. This provided us with an opportunity to unravel the mechanisms underlying the end-stage defect. In a series of experiments, we focused on the dynamin/AP2/clathrin complex since this combination has been implicated in lysosome reformation and clearance.4850 Dynamin 1 is a GTPase enzyme that functions in endocytosis, lysosomal reformation, and vacuole clearance. When dynamin is phosphorylated, phospholipid binding is blocked, effectively attenuating endocytosis.50 Loss of miRs-103/107 inactivated (phosphorylate) dynamin, which resulted in vacuole retention. This occurred via two pathways: (1) miRs-103/107 target CDK5R1,51 which is an activator of CDK5. CDK5 is a kinase for dynamin52 and, thus, inactivates (phosphorylation) this protein. By targeting CDK5R1, miRs-103/107 enable dynamin to be activated. (2) miRs-103/107 target PLD1 and PLD2 which downregulate phosphatidic acid (PA) and diacylglycerol (DAG) synthesis and diminishes protein kinase C (PKC) activity.53 Such attenuation of PKC activity dephosphorylates (activates) dynamin enabling proper end-stage autophagy. Collectively, our findings reveal that miRs-103/107 coordinately suppress macropinocytosis and preserve end-stage autophagy, which is the first demonstration that these two processes can be linked ; however, many of these proteins are associated with lysosomal storage disease and/or in keratoconus corneas. Consequently, their involvement in limbal/corneal epithelial physiology is questionable. An exception is recent studies in cultured human corneal epithelial cells, demonstrating that lacritin, a tear-derived epithelial mitogen,55 acetylates FOXO3.56 Such acetylation results in a coupling with the autophagy-related protein 101 (ATG101) and the subsequent initiation of autophagy.56 Lacritin-induced stimulation of autophagy appears to be a relatively rapid and transient event56 having the potential to enable corneal epithelial cells to quickly respond to stress.Previous work on autophagy and the ocular surface revealed 15 autophagy-related proteins associated with ocular pathology  poor.6365 A recent study demonstrated that Cx43 is degraded by the autophagy machinery.66 Our observations that autophagy is enhanced in the basal limbal epithelial cells,44 plus the fact that miRs-103/107 are preferentially expressed in the limbal epithelium,37 is consistent with the idea that this miRNA family might regulate Cx43 expression by positively regulating autophagy and targeting the protein tyrosine phosphatase receptor type M (PTPRM), which negatively regulates connexin 43-based gap junctions.37Preferential expression of a miRNA family in the limbal epithelium that is involved with maintenance of autophagy implies that this digestive process may differ in the limbal versus corneal epithelium. Using mice that transgenically express a green fluorescent protein (GFP)-labeled LC3 to assess autophagy,44 Human limbal epithelial keratinocytes and hTCEpi seem to tolerate such large vesicles, as we did not detect evidence of cell death or necrosis in these cells. However, macropinocytotic-derived vacuoles in other cell types and cell lines have been associated with cell death.67 Whether limbal keratinocytes are more resistant to macropinocytotic-induced cell death is not apparent. Nonetheless, our work and that of others67 indicates that the induction of macropinocytosis also can negatively affect cell metabolism and cell survival when macropinosomes are involved in vacuole formation.Less clear is the biological significance of macropinocytosis in corneal/limbal physiology. Since the limbus is highly vascularized, we speculated that the epithelial cells may not need macropinocytosis as a means of obtaining nutrients and, thus, miRs-103/107 serve to prevent dysregulation of this process. Additionally, as stem cells are believed to be relatively quiescent, their energy needs might not be as extensive as the more differentiated corneal epithelial cells, hence a minimal requirement for rapid uptake of fluids. Our work indicated that dysregulation of miRs-103/107 in HLEKs or the limbal-derived corneal cell line (hTCEpi) leads to a rapid and massive induction of macropinocytotic-derived vacuoles concomitant with ineffective protein metabolism.69 which would argue against macropinocytosis in corneal basal cells. Another source of corneal epithelial nutrients is the tears, which supply oxygen, as well as immunologic and growth factors that are critical for epithelial homeostasis and repair. Conceivably, macropinocytosis could be a means by which superficial and wing cells “gulp” tear fluid for nutrition thereby bypassing the tight junctions, which serve as a barrier to the diffusion of molecules by sealing the intercellular space.70 Another role for macropinocytosis might be in response to corneal perturbations. For example, following wounding the corneal epithelium becomes compromised and one consequence is stromal swelling.71 In such a scenario, newly reepithelialized corneal epithelial cells might use macropinocytosis to uptake excess stromal fluid and thereby aid in stromal restitution.As macropinocytosis has largely been ignored in the anterior ocular segment, opportunities abound for fruitful research. We speculated above on why the highly vascularized limbal stroma might not require the ability of the epithelium to internalize large quantities of solutes. The question remains does the corneal epithelium participate in and/or require active macropinocytosis? The lack of miRs-103/107 in the corneal epithelium implies that regulation against massive or dysregulated macropinocytosis is absent. It is tempting to think that the avascular nature of the corneal stroma might require a macropinosomal mechanism for nutrient uptake by the corneal basal cells. However, the dogma is that the adult resting corneal stroma is relatively desiccated, particularly in the anterior portion,72 The EphA2 receptor is a member of the Eph receptor tyrosine kinase family and in many tissues upon interacting with its ligand, ephrin A1, has been implicated in regulating proliferation, differentiation, migration, and boundary formation.73 Specifically in the corneal epithelium, the Eph/ephrin complex affects migration.72 Importantly, in other tissues and cancer cell lines, the EphA2 receptor has been implicated as a positive inducer of macropinocytosis.74 For example, the malarial parasite Plasmodium's P36 protein activated the extracellular ligand-binding region of the hepatocyte EphA2 receptor, which was central in the formation of a protective vacuole made of hepatocyte membrane.74 Macropinocytosing antibodies targeting EphA2 have been shown to be effective in killing a panel of EphA2-positive tumor cell lines.23 These studies raise the question of whether corneal epithelial EphA2 may be involved in macropinocytotic events? One potential role could be in the Pseudomonas- or Acanthamobea-mediated cell death of corneal epithelial cells under certain conditions, such as tear gland insufficiency, where the ocular defense system  is compromised.75 While much work has been done to identify the binding and epithelial traversal of these organisms (see prior reports77 and references therein), our understanding of the binding to specific corneal epithelial receptors is incomplete. We suggest that during colonization and adherence to the corneal surface, these organisms may cause clustering of EphA2 receptors, which can activate macropinocytosis,79 and/or secrete proteins that can activate an EphA2-mediated macropinocytotic entry into corneal epithelial cells.74 In support of this idea, transmission electron microscopy (TEM) of corneal epithelium from eyes of mice infected with P. aeruginosa revealed the presence of bacteria within membrane-bound vacuoles; whether these vacuoles arose via macropinocytosis was not determined.80 Given the availability of EphA2 null mice, the idea that macropinocytosis has a role in aiding pathogen entry into the cornea can be easily tested.Another potential role for macropinocytosis revolves around interactions with pathogens on the corneal epithelial surface. We hypothesize a possible involvement of EphA2 with macropinocytotic activation leading to corneal infections. This idea is based, in part, on the observation of EphA2 expression on the surface of corneal epithelial cells.54 and references therein). Since corneal and limbal epithelia have distinct physiologies, are the degradation processes similar for both epithelia? As mentioned previously, based on LC3 expression, corneal epithelial basal cells appear to be less active than their limbal counterparts.44 In contrast, high expression of LC3 was noted in the corneal wing and superficial cell layers. These regions are in relatively close approximation to the surface and are the first areas to experience environmental stresses. Thus, it is not surprising that autophagy would be active in wing and superficial cells. Another consideration is the idea that autophagy might have a role as a mediator of early differentiation. In many tissues, autophagy is highly active during differentiation . Thus, we reason that the marked LC3 expression in superficial cells may, in part, reflect a role for autophagy-related proteins as initiators of early differentiation in these cells. When HaCaT cells (a keratinocyte cell line) were induced to differentiate, release of Beclin 1 and enhancement of ATG12 and LC3II were noted, suggestive that autophagy might have a role in the early stages of differentiation.82 Recently, the molecular machinery involved in the removal of nuclei (nucleophagy) during the end-stages of keratinization have been detailed and this form of autophagy was demonstrated to be induced when keratinocytes differentiate.84 Taken together these observations raise questions of how autophagy is regulated in the corneal epithelium and the role of autophagy and nucleophagy in corneal epithelial differentiation. We have preliminary evidence implicating a regulatory role for miR-184, which is the most highly expressed corneal epithelial miRNA.38 miR-184 was initially shown to function in the corneal epithelium by inhibiting miR-205, which targeted the tumor suppresser SHIP2.40 By preserving SHIP2 levels in the corneal epithelium, proper Akt signaling was assured, which maintained corneal epithelial survival.40 More recently, miR-184 was shown to have angiostatic properties and, thus, functioned in maintaining corneal avascularity.86 Other functions for miR-184 in the corneal epithelium have been lineage specification,39 controlling familial severe keratoconus as well as cataract formation.88 We now have evidence (unpublished observations) that miR-184 targets the Nogo-B receptor (NUS1). The Nobo-B receptor stabilizes Neimann-Pick Type C2 protein (NPC2),89 which is required for the proper clearance of autophagosomes and, thus, has a role in regulation of autophagy-lysosomal activity.91 We posit that miR-184 targeting of NUS1 leads to a failure of NPC2 to maintain proper autophagic flux, which is detected by a decrease in LC3 activity in corneal epithelial basal cells.44 In support of this idea is the reciprocal association of miR-184 and LC3 expression in the corneal epithelium. miR-184 is primarily detected in corneal epithelial basal cells, with little expression in wing and superficial cells.38 Conversely, LC3 pucta are low to absent in basal cells and high in wing and superficial cells. It is important to remember that all cells have autophagic capability, including corneal epithelial basal cells. A major gap in our knowledge is the mechanism(s) that corneal epithelial basal cells use to invoke autophagy during normal and pathologic conditions.With respect to digestion, while we are beginning to understand how this process is regulated in the limbal epithelium, there still are several areas that need attention. For example, knowledge of the early stages of autophagy has not been defined from either a regulatory or biochemical perspective. Does the degradation of intracellular and extracellular material follow the canonical autophagy pathway or the phagocytic pathway? There is emerging evidence for an autophagosome-independent role for autophagy proteins in lysosome fusion and turnover of extracellular substrates (see the report of Florey et al.54 The biological significance of this interrelationship requires a more in depth interrogation. It is apparent that aspects of each of these processes are regulated differently in the corneal and limbal epithelia. We also know that within either the limbal or corneal epithelium the activity of these processes may vary from basal to the outermost cells. Many of the observations have been made using submerged culture systems. While this has been a logical starting point, we live in a three-dimensional world and much work must be done using 3D-organotypic raft culture systems and/or mouse models. Ultimately, our concepts concerning autophagy and macropinocytosis must be investigated in the context of common pathologies of the corneal/limbal epithelia, such as pathogenic infections, dry eye, aberrant wound healing, and diabetic keratopathies. With the library of mouse models for human disease steadily increasing, we anticipate answers to many of these questions in the not too distant future.The corneal/limbal epithelial system has enhanced our understanding of the biology of epithelial stem cells and their transient amplifying cell progeny, due, in part, to the distinct morphogenetic characteristics of these adjacent tissues. We believe there is an equal opportunity for similar advances to be made in our fundamental knowledge of autophagy and macropinocytosis using the corneal/limbal epithelia as a model. For example, we showed that aspects of autophagy and macropinocytosis are regulated by miRs-103/107 and others have shown biochemical commonality.


MKRN3 gene in ‘idiopathic’ CPP Gonadotropin-releasing hormone agonists are the standard treatment for progressive CPP.Central precocious puberty (CPP) results from premature re-activation of the gonadotropic axis. CPP is much more common in girls than in boys and is idiopathic in most cases. In boys, precocious puberty is more likely to be linked to hypothalamic lesions (≈40%). Recent studies have implicated the inactivation of  The signs of puberty include breast development in girls and testicular enlargement in boys (testicular volume greater than 4 ml or testicular length greater than 25 mm).Precocious puberty (PP) is defined as the onset of clinical signs of puberty before age 8 years in girls and 9.5 years in boys. However, the onset of puberty may be subject to constitutional  environmental  and nutritional (body mass index) variations,1PP leads to the progressive development of secondary sexual characteristics, together with the development of pubic hair, and an acceleration of growth velocity and bone maturation, resulting in premature fusion of the growth plates, potentially responsible for adult height deficit. It may have consequences for growth and psychosocial development. PP may be caused by central or peripheral mechanisms.4 results from premature reactivation of the hypothalamo-pituitary-gonadal axis and pulsatile gonadotropin-releasing hormone (GnRH) secretion, with a hormonal pattern similar to that of normal puberty. CPP may be due to hypothalamic lesions but is idiopathic in most cases, particularly in girls1. Recent studies have implicated the activation of Kisspeptin and its receptor and the inactivation of Makorin ring finger 3 (MKRN3) genes in “idiopathic” CPP6. MKRN3 is an imprinted gene located on the long arm of chromosome 15, with a potentially inhibitory effect on GnRH secretion. MKRN3 gene defects have been identified as a cause of paternally transmitted familial CPP, but such defects do not underlie maternally transmitted CPP and are rarely involved in sporadic forms.Central PP (CPP), which is much more common in girls than in boys,8 Therapeutic abstention is appropriate in most cases, because puberty progresses slowly, with the menarche occurring, on average, 5.5 years after the onset of clinical signs of puberty and normal adult height relative to parental target height being reached. However, in some cases (about one-third of subjects), final stature prognosis may worsen during the progression of puberty, in parallel with the emergence of evident biological signs of oestrogenisation. Clinical assessments should therefore be systematically carried out in children for whom no treatment is justified at the initial assessment, at least until the age of 9 years, to identify girls subsequently requiring treatment to block precocious puberty.9Premature sexual maturation is a frequent cause for referral. Clinical evaluation is generally sufficient to reassure the patients and their families, but premature sexual maturation may reveal severe conditions, and thorough evaluation is therefore required to identify its cause and potential for progression so that appropriate treatment can be proposed. If a non-progressive form of PP is suspected, it is recommended to wait a few months and then to reassess the patient, to avoid unnecessary treatment. The heterogeneity of CPP, in terms of its clinical presentation and definition, can be accounted for by the gradual nature of the transition to puberty. Indeed, in many girls with idiopathic CPP, puberty progresses very slowly and may even be regressive, resulting in an unchanged predicted final stature and a normal adult height close to parental target height.10 Oestrogenic impregnation is assessed on pelvic ultrasound scans, which may show an oestrogenised appearance of the uterus (length ≥35 mm).11 Central magnetic resonance imaging focusing on the hypothalamic region is required in most cases of CPP12In both sexes, the central cause of precocious puberty is demonstrated by an increase in pituitary gonadotropin levels. Indeed, the mechanism of precocious puberty involves premature activation of the hypothalamic-pituitary-gonadal axis, with the initiation of pulsatile luteinising hormone (LH) secretion and an increase in the secretion of pituitary gonadotropins, both in basal conditions and after stimulation with LH-releasing hormone (LHRH). Before the onset of puberty, the follicle-stimulating hormone (FSH) peak is greater than the LH surge. During and after puberty, the LH surge predominates. In cases of central precocious puberty, basal serum LH concentration is usually ≥0.3 IU/l and LH concentration after stimulation is ≥5 IU/l.12 Such treatment results in the regression or stabilisation of pubertal symptoms and decreases in growth velocity and bone age advancement. The factors affecting height outcome include initial patient characteristics and treatment duration. After the cessation of GnRHa therapy, generally at an age of about 11 years, biological and clinical signs of puberty reappear within months, with most girls achieving menarche, with menstrual ovulation cycles, during the following year.14 PP associated with the presence of a hypothalamic lesion may progress to gonadotropin deficiency. The available data indicate that long-term GnRHa treatment does not seem to cause or aggravate obesity or have repercussions for body composition, bone mineral density, fertility and metabolic or cancer comorbidities. General health status is not different from that of women with normal puberty.15 However, data concerning psychosocial outcomes are scarce,16 and studies of this aspect are required.GnRH agonists (GnRHa) are the standard treatment for progressive CPP.


Serum lactate levels are traditionally interpreted as a marker of tissue hypoxia and often used clinically as an indicator of severity and outcome of sepsis/septic shock. Interestingly, recent studies involving the effects of tumor-derived lactate suggest that lactate itself may have an immunosuppressive effect in its local environment. This finding adds to the recent advances in immunometabolism that shed light on the importance of metabolism and metabolic intermediates in the regulation of innate immune and inflammatory responses in sepsis. In this article, we summarize recent studies, showing that the activation of immune cells requires aerobic glycolytic metabolism and that lactate produced by aerobic glycolysis may play an immunosuppressive role in sepsis. Sepsis is a clinical syndrome characterized by systemic inflammatory response to infection –3. In thThe measurement of serum lactate levels is often incorporated in the clinical management of critical illness, particularly in cases of severe sepsis and septic shock . In thisOver half a century ago, Dr. Otto Warburg observed that various cancer cells metabolize glucose directly to lactate despite the presence of abundant oxygen in the environment . Initialin vitro inhibition of glycolysis seems to reprogram innate immune cells to a more anti-inflammatory state, further highlighting the importance of metabolism in cell phenotype  are critical in the induction of innate immune and inflammatory responses. TLRs could bind with their ligands, including bacterial components and endogenous ligands, and initiate a cellular signaling cascade that ultimately results in transcriptional regulatory changes within the cell , 52. Forhenotype , 57.eff) cells and T-regulatory (Treg) cells. Michalek et al. recently reported that Teff cells exhibit greater expression of the glucose transporter 1 (GLUT1) and elevated levels of glycolysis, while Treg cells primarily rely on fatty acid oxidation  or PKM2 inhibitor also markedly improves survival outcome in polymicrobial sepsis, and reduces serum lactate levels and HMGB1 release  and markers  in a dosage-dependent manner  when compared with the control . MDSCs aIn vitro studies by this group demonstrated that sodium lactate inhibited CD4+ cell motility, whereas an acidic lactate was required to inhibit the motility of CD8+ T cells. In CD4+ T cells, the effect of lactate (at physiologic pH) seemed to be dependent on the interruption of glycolysis and required expression of the Na+/lactate cotransporter, Slc5a12 (+ T helper cells toward a TH17 subset, whereas unbuffered lactic acid inhibited the cytolytic function of CD8+ T cells (reg cells. It is possible that the proposed Treg cells are less dependent on aerobic glycolysis and primarily use oxidative phosphorylation for their energy production (In addition to influencing the function of the innate immune cells, lactate has also been reported to have effects on T-cell functioning . Haas et Slc5a12 . In cont Slc5a12 . These e Slc5a12 . However T cells . At presoduction . Thus, tWe have highlighted some of the current research surrounding the potential role of lactate as an immunosuppressive metabolite see Fig. . Althoug


Four different smooth muscle tissues  with a 5-fold difference in maximal shortening velocity were obtained from mice and classified according to expression of the inserted myosin heavy chain (SMHC-B). Western blotting and quantitative PCR analyses were used to determine 15 metabolic and 8 cell signaling key components in each tissue. The slow muscle type (aorta) with a 12 times lower SMHC-B had 6-fold lower expression of the phosphatase subunit MYPT1, a 7-fold higher expression of Rhokinase 1, and a 3-fold higher expression of the PKC target CPI17, compared to the faster (urinary bladder) smooth muscle. The slow muscle had higher expression of components involved in glucose uptake and glycolysis  and in gluconeogenesis , but lower expression of the metabolic sensing AMP-activated kinase, alpha 2 isoform (5 times). The slow type also had higher expression of enzymes involved in lipid metabolism . We present a refined division of smooth muscle into muscle types based on the analysis of contractile, metabolic, and signaling components. Slow compared to fast smooth muscle has a lower expression of the deactivating phosphatase and upregulated Ca Smooth muscle performs a wide range of important physiological tasks, including the rapid contractions of the urinary bladder to expel urine and the sustained tension maintenance in the wall of arteries to maintain blood pressure. Although they all share the common smooth structural appearance, the smooth muscle types are uniquely adapted to their requirements and exhibit a broad range of structural, mechanical, regulatory, and metabolic properties. Still, it has not been possible to clearly identify specific smooth muscle types. In early work by Emil Bozler , smooth It seems logical to divide muscles according to the properties of contractile system, and in the striated muscle the division into muscle types can be made based on the expression of the myosin heavy chain genes, which correlates with the contractile kinetics . In smoo2+ sensitivity of the contraction, mainly via the RhoA-Rhokinase and protein kinase C (PKC) pathways, e.g.,  and force was less inhibited by inorganic phosphate. However, the Vmax of slow muscle is highly sensitive to MgADP  and force are increased in depolarized muscle, whereas it increases in the portal vein [Although the oxygen consumption in slow (aorta) smooth muscle is lower than in fast  smooth muscle, the relative lactate production is higher in the slow muscle , reflectGlucose-6-phosphate dehydrogenase (G6PDH) is a key enzyme in the pentose phosphate pathway, important for generating substrates for synthesis of nucleotides and producing NADPH which is involved in, e.g., fatty acid synthesis and protection against oxidative stress. G6PDH was slightly lower in the fast urinary bladder smooth muscle. The functional correlate to this finding is not yet clear. However, it has been shown that G6PDH is upregulated in isolated smooth muscle cells during mechanical/oxidative stress and that downregulation of the enzyme leads to increased oxidative stress . In intaAdenosine monophosphate-activated kinase (AMPK) is a key metabolic sensor, activated by energy depletion and high AMP levels . It affeBoth LPL and HSL enzymes responsible for uptake and breakdown of triglycerides were higher in the slower tonic muscles compare to the faster. This would suggest that the slower muscles are more adapted to metabolism of lipids. Although the MCD, important for fatty acid synthesis, was similar between tissues, both ACC2 and FAS, two enzymes involved in fatty acid synthesis, were highly expressed in slower muscles. This even further points the fatty acids as an important substrate for the metabolism in slower muscles.2+-sensitizing component is highly expressed and in addition the sensitivity to PKC activation is enhanced. Although the energy turnover is low, the sustained contractions, while maintaining blood pressure, can be affected by varied levels of ADP resulting in “latch like” situations, which provides a link between contraction and a continuous input of metabolic energy from glucose and lipids.In summary, the main purpose of this study was neither to examine the exact role of all metabolic and signaling proteins nor to examine all steps in the different pathways. Rather, we aimed at obtaining a “finger print” of cellular components as a basis for characterizing the “fast” and “slow” smooth muscle types. Further work based on key parameters identified in the present study (as outlined in Table


The proneural factor Achaete-scute complex-like 1 (Ascl1/Mash1) acts as a pioneering transcription factor that initializes neuronal reprogramming. It drives neural progenitors and non-neuronal cells to exit the cell cycle, and promotes neuronal differentiation by activating neuronal target genes, even those that are normally repressed. Importantly, force-expression of Ascl1 was shown to drive proliferative reactive astroglia formed during stroke and glioblastoma stem cells towards neuronal differentiation, and this could potentially diminish CNS damage resulting from their proliferation. As a pro-neural factor, Ascl1 also has the general effect of enhancing neurite growth by damaged or surviving neurons. Here, a hypothesis that brain pathologies associated with traumatic/ischemic injury and malignancy could be targeted with pro-neural factors that drives neuronal differentiation is formulated and explored. Although a good number of caveats exist, exogenous over-expression of Ascl1, alone or in combination with other factors, may be worth further consideration as a therapeutic approach in brain injury and cancer. Central nervous system (CNS) pathologies due to traumatic or ischemic injury are often of devastating consequences, and the adult CNS cancer type glioblastoma has remained largely refractory to treatment despite intensive work on therapeutic interventions . AlthougEvolutionarily conserved pro-neural factors, including members of the basic helix-loop-helix (bHLH) class transcription factor and neurogenins, endow neural progenitor cells (NPCs) with a neuronal fate and drives neuronal differentiation ,5. AmongThe pivotal role of Ascl1 in neurogenesis has been known for more than twenty years now . KnockouA particularly interesting and important capacity of Ascl1 is its ability, either alone or in conjunction with other pro-neural factors, to directly reprogram various somatic cells into neurons . The GötThis direct neuronal reprogramming activity of Ascl1 thus allows for the following crude and simple-minded thought. The adult CNS consists largely of terminally differentiated neuronal and glia cells that no longer proliferate, which is a desirable state for the maintenance of its functional architecture. However, under pathological conditions, uncontrolled and undesirable cell divisions and proliferation occur, particularly during reactive glial responses in injury and in tumor growth, often with devastating consequences to neuronal survival and function. As terminally differentiated neurons would fully exit the cell cycle, a plausible way to curb uncontrolled and undesirable astroglia or tumor cell growth may be to simply turn them into neurons. In the following sections, evidence that this might be achievable with exogenous or forced Ascl1 expression, as well as the associated caveats of this approach, is discussed.In traumatic or ischemic brain injuries, astrocyte reactivation underlies glial scar formation. These processes of astrogliosis and glial scarring serve to limit the spread of the pro-inflammatory factors released from lysed cells, but also contribute to neuronal death and inhibition of regeneration. Interestingly, however, an increase in Ascl1-expressing proliferating progenitor was observed by Zhang and colleagues in a right middle cerebral occlusion stroke model, and these cells eventually gave rise to GABAergic neurons and mature oligodendrocytes . Frisén’The above findings are in line with other reports that Ascl1 can drive conversion of astrocytes to neurons in vivo , and attGlioblastomas are devastating brain cancers, as treatment of the primary tumor carries a high risk of endangering brain tissues, and these are often refractory to treatment because of the occurrence of cancer stem cells . Jiao anThe findings described in the sections above entice the following crude formulation of using Ascl1, in a form of gene therapeutic approach, to curb reactive astroglia proliferation and glioblastoma tumor growth. This forced conversion of unwanted proliferative brain cells into non-dividing neuron-like cells is a contrasting alternative to the more common approach of targeting proliferation drivers or oncogenic drivers, and may have a higher degree of specificity compared to other gene therapeutic approaches such as those that target, for example, more broadly acting micro-RNAs. To begin with, Ascl1 expression mediated by adenoviral-associated vectors or the like should be more widely tested in animal CNS injury and cancer xenograft models for their potential benefits. Other possible alternative modes of delivery include the use of functionalized Ascl1 protein, which could be delivered intracellularly , an apprThere are a number of immediately notable caveats associated with this strategy. The first, and perhaps most important as far as translational application is concerned, is the efficiency of conversion. Better understanding of the Ascl1 driven differentiation process in different cell types would be necessary to improve differentiation efficiency. As discussed above, reprogramming of reactive astrocytes may work better in certain parts of the CNS, and not in other parts, as the site and environment  could inAlthough astrogliosis and glial scar formation following injury were classically perceived to be detrimental for surviving cells and prevents axonal regeneration, it should be noted that these processes have beneficial aspects ,40. ReacIn the preceding paragraphs, the possibility of forced expression of Ascl1 as a potential therapeutic approach for brain injury and cancer is considered. While there are a number of caveats and the idea remains crude at the moment, its efficacy and safety is testable with well-established animal models of stroke  and glio


Autophagy and the DNA damage response (DDR) are biological processes essential for cellular and organismal homeostasis. Herein, we summarize and discuss emerging evidence linking DDR to autophagy. We highlight published data suggesting that autophagy is activated by DNA damage and is required for several functional outcomes of DDR signaling, including repair of DNA lesions, senescence, cell death, and cytokine secretion. Uncovering the mechanisms by which autophagy and DDR are intertwined provides novel insight into the pathobiology of conditions associated with accumulation of DNA damage, including cancer and aging, and novel concepts for the development of improved therapeutic strategies against these pathologies. The term ‘DNA damage response’ (DDR) refers to a network of intracellular pathways that sense and resolve damaged DNA. If unrepaired, DNA lesions may result in cell death  but can Figure 1) and the replication protein A (RPA) complex which responds predominantly to single strand DNA lesions and recruits ATR kinase. The binding of these kinases to damaged DNA triggers the recruitment of additional proteins, many of which become phosphorylated and activated to further transduce signals that orchestrate DNA replication, cell cycle control, transcription, repair of damage, and/or survival versus death.DNA damage response utilizes proteins involved in sensing, signaling, and repair of DNA damage. While the early activation events that follow DNA breaks are well elucidated, the primary signal which triggers DDR remains incompletely understood. It has, however, been proposed that when a DNA lesion occurs, it is accompanied by relaxation of chromatin through a series of post-translational histone modifications that include poly-(ADP-ribosylation) which is catalyzed by poly (ADP-ribose) polymerases (PARPs), phosphorylation and acetylation . These cThus, ATM bound to DSBs in conjunction with the MRN complex, undergoes autophosphorylation and activation. In turn, ATM activates various downstream effector proteins , includiFigure 1).In addition to the aforementioned phosphorylation targets, ATM forms a nuclear complex with PARP1 and NEMO . Within this complex, NEMO is subjected to a series of post-translational modifications including phosphorylation, SUMOylation and ubiquitination that lead to its nuclear export . Once in15 which reduces its affinity for the ubiquitin ligase HDM2 leading to p53 stabilization. However, ATM also impacts on p53 indirectly through activation of JNK which phosphorylates p53 at Thr81 enhancing its transcriptional activity . DNA damage has been reported to activate JNK also through the transcriptional repression of the JNK phosphatase MKP1 caused by transcription-blocking DNA lesions  which may damage both mitochondrial and nuclear DNA . MtDNA lAutophagy is a cellular ‘self-eating’ degradation process in which proteins or whole organelles are degraded in lysosomes and recycled to meet the anabolic and bioenergetic needs of the cell. As such, it plays a pivotal role in tissue homeostasis and various human pathologies, including cancer, neurodegeneration, autoimmunity and aging, have been associated with deregulated autophagy .Depending on the mechanism of delivery of the cargo to the lysosomes, three main types of autophagy have been so-far recognized: micro-autophagy that involves the direct delivery of cargo to lysosomes through lysosomal membrane invaginations; CMA which is typified by the lysosomal import of proteins through their interaction with specialized chaperones; and macro-autophagy which is the most widely studied mechanism of autophagy. In macro-autophagy (hereafter termed autophagy), the cargo is sequestered in double membrane vesicles known as autophagosomes, which are progressively formed by the finely interconnected activities of around 15 autophagy-related (ATG) proteins . Autopha63-ubiquitinated, p62-sequestered proteins by autophagy  to the microtubule-associated protein light chain 3 (LC3). PE-conjugated (“lipidated”) LC3, known as LC3-II, decorates mature autophagosomes which are targeted to lysosomes together with their cargo. The complete process of autophagy from nucleation to degradation is referred to as autophagic flux and reflects the actual ability of autophagosomes to degrade intracellular components. Autophagic flux can be monitored by determining the degradation of specific autophagic substrates such as SQSTM1  which interacts with LC3-II . SQSTM1/utophagy  and its utophagy .757 and disrupting the interaction between ULK1 and its activating kinase AMPK  . Released GATOR1 subsequently binds to and inactivates RAGA/B, resulting in TORC1 suppression  . Under sng TORC1 . Recent pression . Among vpression . Argininpression . TherefoHowever, it should be noted that in addition to the aforementioned “canonical” pathway of autophagy, alternative mechanisms of autophagosome formation that do not require the hierarchical participation of all ATG proteins have been described and represent an area of intense investigations . For exaL which are over-expressed in certain lymphomas and carcinomas , both of which result in Vps34 class III PI3K complex activation and autophagy initiation , nuclear dot protein 52 (NDP52) and SQSTM1/p62, which serve as a bridge for the assembly of the ULK1 complex and autophagosome formation . Among them, SIRT1 is of particular interest as beyond its role as deacetylase of proteins involved in DNA repair pathways  and rodent models of XPA possess dysfunctional mitochondria that contribute to the neurological and other pathologies manifested in these diseases  may target distinct Chk1 pools. Intriguingly, nuclear accumulation of Chk1 that ensues from defective CMA leads to destabilization of the MRN complex involved in the initial processing of DSBs prior to DNA repair by HR autophagy by ablation of ATG7 leads to Chk1 ubiquitination and proteasomal degradation which in turn impairs DNA damage repair by HR (but not NHEJ) and genomic integrity . Anotherted Chk1 . In contir by HR . Thus, lin vivo relevance of these observations is underscored by pharmacological studies in mice showing that inhibition of autophagy by the chemical spautin-1 promotes, whereas stimulation of autophagy by rapamycin reduces UVB-induced tumorigenesis , a p53 null human osteosarcoma cell line, which after prolonged expression of p21WAF1/Cip1 exhibit enhanced aggressiveness and chemoresistance by deregulating the replication licensing machinery causing replication stress and fuelling genomic instability . ACD is morphologically distinct from apoptosis and necrosis and is characterized by the sequestration of cytoplasmic materials in autophagosomes . TherefoThere is significant evidence supporting that DDR and immune response networks functionally interact  and thatBeyond SASP, autophagy may link DDR to the generation of local and systemic immune responses through the regulation of the so-called ‘immunogenic cell death,’ a form of cell death accompanied by the emission of immunostimulatory DAMPs. These include cell surface-exposed calreticulin and ATP and HMGB1 that are released by dying tumor cells following their exposure to certain genotoxic agents such as ionizing radiation (IR), doxorubicin and oxaliplatin . A studyIntriguingly, autophagy also operates in APCs in a cell-intrinsic manner to control secretion of IL-1β and other cytokines. Thus, basal autophagy inhibits IL-1β secretion by degrading both inflammasome proteins  and pro-Arabidopsis thaliana), amoebozoa (Dictyostelium discoideum), and metazoa  but not in prokaryotes  autophagy is activated upon exposure to diverse DNA damaging factors including radiation, chemicals, ROS and oncogenes; (2) the DDR-autophagy link operates in a variety of eukaryotic cell types representing different evolutionary and developmental stages; (3) autophagy is required for several fundamental processes associated with the cellular response to DNA damage such as repair of DNA lesions, senescence, cell death, and cytokine secretion; (4) reduced autophagic flux characterizes human pathologies that are associated with accumulation of DNA damage, including cancer and aging.Together, these observations raise the possibility that autophagy may have evolved as a quality control system that responds to a wide range of stress conditions including DNA damage, a major cellular stress factor. Unlike the more broadly conserved ubiquitin-proteasome system which regulates the turnover of short-lived proteins in both prokaryotes and eukaryotes, autophagy can target organelles or bulk cytoplasmic and nuclear material for lysosomal degradation. This is a property relevant to DDR in eukaryotic cells that enables them, for example, to deplete mitochondria bearing irreparable DNA damage and to control the number of mitochondria under stress conditions. Notably, the autophagic degradation of dysfunctional mitochondria (mitophagy) operates across the Eukaryota domain, including yeast, nematodes, flies, and mammals , contribThe exciting findings that continue to emerge in this field warrant further studies into the complex and often contrasting roles of autophagy in the onset, progression and therapy of various human diseases. This need is underscored by its dual role in malignancy: whereas autophagy prevents genomic instability, a hallmark of cancer, it may promote survival of tumor cells under stress conditions, including those induced by anticancer therapy . As the Figure 2.AE, SH, and VG contributed to the preparation, drafting and final review of the manuscript. SH provided the data depicted in 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.