Over the last few years, we have been evaluating a novel paradigm for immunization using viruses or virus-based vectors. Safety is provided not by attenuation or inactivation of vaccine viruses, but by the introduction into the viral genomes of genetic mechanisms that allow for stringent, deliberate spatial and temporal control of virus replication. The resulting replication-competent controlled viruses (RCCVs) can be activated to undergo one or, if desired, several rounds of efficient replication at the inoculation site, but are nonreplicating in the absence of activation. Extrapolating from observations that attenuated replicating viruses are better immunogens than replication-defective or inactivated viruses, it was hypothesized that RCCVs that replicate with wild-type-like efficiency when activated will be even better immunogens. The vigorous replication of the RCCVs should also render heterologous antigens expressed from them highly immunogenic. RCCVs for administration to skin sites or mucosal membranes were constructed using a virulent wild-type HSV-1 strain as the backbone. The recombinants are activated by a localized heat treatment to the inoculation site in the presence of a small-molecule regulator (SMR). Derivatives expressing influenza virus antigens were also prepared. Immunization/challenge experiments in mouse models revealed that the activated RCCVs induced far better protective immune responses against themselves as well as against the heterologous antigens they express than unactivated RCCVs or a replication-defective HSV-1 strain. Neutralizing antibody and proliferation responses mirrored these findings. We believe that the data obtained so far warrant further research to explore the possibility of developing effective RCCV-based vaccines directed to herpetic diseases and/or diseases caused by other pathogens. For a number of important diseases there exist only vaccines that are at best moderately effective and/or require frequent updating and re-vaccination. For some of diseases, no vaccines are available today, and this is not due to insufficient efforts by the academic and industrial communities. Herpetic diseases mediated by HSV-1 and HSV-2 belong to the latter category of diseases for which no vaccine has been successfully developed. This state of affairs should encourage searches for approaches that differ qualitatively from what has been explored before.Immunization with an enriched or pure protein of a pathogen tends to favor a humoral immune response over a cell-mediated immune response, i.e., a Th2-biased response instead of a more balanced Th1 and Th2 response. To mitigate this deficiency, vaccines containing split, subunit, or recombinant antigens are typically adjuvanted, which introduces the possibility of adjuvant-associated health problems that may surface over the life of a vaccinated individual [Historically, inactivated and attenuated pathogens were developed as vaccines. Not surprisingly, debates ensued regarding the relative efficacies of vaccines containing inactivated pathogens and vaccines containing attenuated pathogens. The best-remembered debate of this kind concerned the inactivated poliomyelitis vaccine of Salk and the live attenuated vaccine of Sabin ,3. The hThe key difference between inactivated and live attenuated viral vaccines is that the live attenuated vaccines have retained some capacity for replication. However, the two types of vaccines also differ in other respects. The process of inactivation may alter the antigens presented by an inactivated vaccine, whereas the surface of live attenuated vaccines is unadulterated. Furthermore, inactivated and live attenuated vaccines against a particular pathogen may be administered by different routes, as is the case for the poliomyelitis vaccines. Hence, the superiority of live attenuated vaccines over inactivated vaccines cannot be unambiguously ascribed to their residual ability to replicate. However, a number of studies have addressed the issue more directly, comparing immune responses to live attenuated viruses that retained some ability to replicate and corresponding replication-deficient viruses or to heterologous antigens expressed by them ,8,9,10. We hypothesized that if a replication-attenuated virus produces a more potent and/or more balanced immune response than a non-replicating virus, this immune response might be further enhanced by immunization with a virus that is capable of replicating, for a limited time, with a wild-type-like efficiency . This hyAlphaherpesviruses such as HSV-1 or HSV-2 appeared to be well-suited to serve as backbones for the construction of RCCVs for a number of reasons. They are double-stranded DNA viruses that employ the cellular transcription machinery for expression of their genes ,13,14. HAt the outset, there were several reasons for doubting that the above-described vaccination strategy could be successfully developed. One such concern was that it might not be possible to build RCCVs whose replication can be controlled with the appropriate stringency. The choice of suitable gene switches appeared to be rather limited. Subjecting replication of an RCCV to a drug-activated mechanism was considered to be unlikely to produce the desired sharp regulation. Before the concentration of a systemically administered drug had fallen below an effective level, the RCCV might have undergone multiple rounds of replication. Additionally, the rate of clearance of a drug may vary sufficiently between individuals to render the elaboration of a standard vaccination protocol difficult or unfeasible. A somewhat better but probably still inadequate control could be achieved by topical administration of the drug in a timed-release formulation. Only a physical cue or stimulus whose application can be precisely timed and confined to the RCCV administration site would provide for a sufficiently stringent temporal and spatial regulation of RCCV replication, provided that the response to the trigger subsides before infectious progeny of the RCCV are generated.A physical cue or stimulus that is applied to the virus administration site might impair the activity of cells of the innate immune system, including resident antigen-presenting cells, dampening any immune response that might be induced by the RCCV.Another question that could not be answered a priori was whether limited replication of an RCCV confined to the site of its administration could in fact produce a significant enhancement of the immune response to the virus. No information pertinent to this question was available at the time. Attenuated replicating vaccines (or candidate vaccines), including single-cycle vaccines, replicate systemically.Regarding the quality and potency of inflammatory and immune responses induced by an activated  RCCV, it could not be predicted with any confidence how much better they would be compared to responses induced by more conventional vaccines. On the one hand, efficient but limited replication of an RCVV that is concentrated in the administration region will result in localized lysis of many infected or superinfected cells and spillage of virus progeny and cell contents. This will result in very high local levels of PAMPs and DAMPs that can be expected to prompt a strong inflammatory response and stimulation of the innate immune system. Primarily infected apoptotic antigen-presenting cells such as dendritic cells may efficiently cross-present viral antigens to uninfected dendritic cells . PresentOn the other hand, viral immunomodulatory proteins that will be abundantly expressed in primarily infected cells may severely inhibit cytokine and interferon production , thereby dampening the  inflammatory and immune responses. Pattern recognition receptor and/or adapter function is inhibited by viral proteins UL37, ICP0, UL36USP, US3, and ICP34.5; interferon-regulatory factors (IRF) are interfered with by US3, US11, ICP34.5, VP24, ICP27, and ICP0; and NF-kB functionality is inhibited by UL36USP, US3, and ICP0. Furthermore, JAK/STAT signaling is blunted by the action of viral proteins VR3, UL13, UL41, and ICP27   indicate that RCCVs can be constructed whose replication is under sufficiently stringent deliberate control to provide the requisite safety as well as support our hypothesis that such recombinant viruses are capable of inducing superior immune responses without any apparent adverse effects. We submit that these results lay the foundation for the further development of RCCVs as candidate vaccines or vaccine vectors.RCCV-based vaccination may be accompanied by several additional important advantages:There is a recognized risk of run-away replication associated with vaccinating immunocompromised individuals with attenuated replicating viruses . This daFor the same reason, RCCVs should be resistant to reactivation from quiescence in infected nerve cells.RCCVs are activated in a chosen administration site. They should not replicate in a disseminated fashion. Hence, they are not expected to exhibit residual neurotoxicity, which has been observed even with single-cycle vaccines . As activated RCCVs will only replicate locally and for a limited time , there will be little opportunity for recombination with another virus. In the absence of activation, RCCVs are expected to be only present at low copy numbers in infected cells.HSP70B (HSPA7) gene has a very low basal activity in cultured cells and a heat-induced activity that is almost 1000-fold higher [The host mechanism that controls the expression of heat shock genes appeared to be ideally suited for controlling RCCV replication. The inducible transcription of heat shock genes, a group that includes the classical HSP genes as well as many additional genes, is under the control of heat shock transcription factor 1 (HSF1) . This ubd higher ,29,30. Id higher . SignifiHSP70B gene in this region. Activation of HSF1 is a proportional response to proteotoxic stress. Hence, the degree of activation is a function of heat dose, not temperature alone. Consequently, heating time could be decreased by increasing exposure temperature. In animal experiments employing high-intensity focused ultrasound, activation of the human HSP70B promoter could be achieved in discrete tissue regions by a 3 min exposure [Because alphaherpesviruses infect and replicate well in epithelial cells, an HSV-1-based RCCV may be administered preferably to a site in the skin or, possibly, a mucosal tissue. Heat delivery can be readily directed, in particular to skin sites. For example, disposable/recyclable or reusable heating pads may be employed. A pad that contains a supercooled solution of the essentially nontoxic salt sodium thiosulfate pentahydrate has been tested in human subjects . After cexposure . Activatexposure ,34,35.HSP70B promoter -activated transactivator GLP65 to mediate the expression of one or more replication-essential viral genes. Expression of the transactivator was placed under the control of a promoter assembly containing an promoter . In cellpromoter ,38. Thispromoter . An ideasyn+ [HSP70B/GAL4 promoter assembly that is functionally linked to a GLP65 gene. The native promoters of the regulated replication-essential genes were replaced by GAL4-responsive promoters. The regulated genes are the immediate early gene ICP4 in HSV-GS1 and HSV-GS3, the early gene ICP8 in HSV-GS3, and the late gene UL38/VP19c in HSV-GS7. Note that in HSV-GS3, two replication-essential genes were subjected to regulation, eliminating the possibility that a single mutation could give rise to an unregulated virus. The structures of RCCVs HSV-GS1, HSV-GS3, and HSV-GS7 are depicted schematically in syn+ ,40,41. Asyn+. Viral replication was not observed in untreated infected cells or in infected cells that were only heat-treated or only incubated in the presence of an antiprogestin. As an illustration, results from a single-step growth experiment in HSV-GS3-infected cells of a permissive human cell line are represented in Single-cycle growth experiments demonstrated that the RCCVs replicated efficiently in cells that had received an activating heat treatment and were exposed to an antiprogestin ,40. CompMost of the results discussed under this heading were originally disclosed in References ,40,41.syn+ administered to the rear feet. All animals developed signs of illness. Seventy-five percent of the mice immunized with activated HSV-GS7 and seventy percent of the mice immunized with activated HSV-GS3 recovered fully . Viral protein ICP47 is known to contribute importantly to the viral defense in humans, an effect that is not recapitulated in mouse models ,18. ICP4Regarding other initial concerns, our findings imply that a single round of spatially confined virus replication is indeed capable of inducing a greatly enhanced protective immune response. Furthermore, even if the RCCV activation procedure had any negative effect on the immune system, this effect was clearly not dominant. To summarize, our experiments provided strong evidence that an activated RCCV that undergoes efficient but limited replication is capable of inducing a superior functional immune response. Our findings motivate further studies employing animal models of specific herpetic diseases, e.g., models of ocular or genital herpes. Such studies may also address the important question of whether the enhanced immunity resulting from immunization with an activated RCCV will suffice to prevent or at least substantially mitigate reactivation of wild-type virus from latency.If the efficient but limited replication of an RCCV results in a substantially enhanced immunity against the type of herpesvirus from which the RCCV was derived when compared to the immunity induced by a non-replicating herpesvirus, one might expect a correspondingly strengthened immune response against a heterologous antigen if it is expressed from a replicating RCCV. Hence, RCCVs should be potent carriers of heterologous antigens. To test this hypothesis, expressible genes for the hemagglutinin (HA) or the nucleoprotein (NP) of equine influenza virus Prague/56 (EIV Prague/56) were introduced into the UL37/38 intergenic region of RCCV HSV-GS3 (see More recent experiments (unpublished) evaluated the functional immune responses to EIV antigens expressed from activated RCCV vectors using a mouse model that had been described previously . In thesBased on results obtained to date, we submit that RCCVs are powerful vectors of heterologous antigens. Regarding influenza, RCCVs expressing surface or internal influenza virus antigens may be considered as candidates for effective anti-influenza vaccines that may be well suited for the vaccination of the immunocompromised and senior populations. Whether such vaccines will also be beneficial to the general population may depend in part on the breath of protection they are capable of providing, i.e., whether they could be employable as non-seasonal influenza vaccines. Current work is aimed at defining the boundaries of the cross-protective activities of influenza virus antigen-expressing RCCVs. At this time, heat appears to be the only physical cue that can be employed for the stringent spatial and temporal control of RCCV replication. This is due to two properties of the heat shock system, i.e., the existence of HSP promoters that essentially behave as on–off switches and the transience of the activated state of HSF1, the transcription factor that transactivates HSP promoters.While it may be possible to build/employ other safeguards against inadvertent stress activation, we chose to co-control RCCV activation using a drug-mediated mechanism. One or more replication-essential viral genes are placed under the control of an SMR-activated transactivator that is expressed from a gene that is driven by an HSP promoter. The chosen transactivator comprises a GAL4 DNA-binding domain and a truncated progesterone receptor ligand-binding domain, and is activated by a narrow class of antiprogestins that include mifepristone and ulipristal. Replication-essential genes to be controlled by this transactivator have their native promoters replaced by minimal GAL4-responsive promoters, i.e., promoters that are bound by the GAL4 DNA-binding domain of the transactivator but not by transcription factors of the host cells. For the reasons discussed above, we believe that using a transactivator that is activated by certain antiprogestins for the co-control of RCCV replication represents a rational choice. If an RCCV of this type were administered to a subject, there would be essentially no possibility of run-away virus replication. Activation of the RCCV requires both triggers, i.e., an appropriate heat dose and an effective dose of an antiprogestin. Hence, even if the subject were exposed to a proteotoxic stress shortly after RCCV administration, e.g., experienced a severe fever, suffered from an exposure to a toxicant, engaged in particularly strenuous exercise, etc., virus replication would cease as soon as the antiprogestin concentration had fallen below the effective level. An accelerated decline of the antiprogestin concentration to an ineffective systemic level could be achieved by co-administering the drug with the RCCV rather than administering it orally. This should further minimize the possibility of inadvertent systemic replication of the virus. Replication in quiescently infected nerve cells should not be capable of occurring in the absence of antiprogestin (discussed in more detail below). Theoretically, some replication may take place in a stressed subject that ingests an effective dose of an antiprogestin . As long as such antiprogestins are essentially only used in single doses for emergency contraception, this possible complication would be de minimis.The appropriateness of employing an antiprogestin-activated transactivator in a RCCV-based vaccine may need to be re-evaluated in light of more recent developments. Pivotal clinical trials demonstrated that ulipristal is capable of reducing the size of uterine fibroids, resulting in marketing approvals of the ulipristal-containing drug Esmya  in Europe and Canada ,44,45. AShould some of the above-mentioned antiprogestins continue to be used for the treatment of uterine fibroids or for other indications, further control features could be added in the current heat- and antiprogestin-activated RCCVs. For example, an additional replication-essential gene could be subjected to the control of an appropriate tissue-specific promoter to prevent virus replication in untargeted tissues, particularly in nerve tissue. Having this additional feature present should effectively insulate the RCCVs against any untoward effects of therapeutic uses of antiprogestins.7-fold [Transactivators have been described that respond to doxycycline, erythromycin, pristinamycin, cumate, phloretin, ecdysteroids, diacylhydrazines  such as RG-102277 (RSL-1), LG335 (synthetic retinoic X receptor (RXR) α ligand), 4-hydroxytamoxifen (antiestrogen), 4,4′-dihydroxybenzil (DHB), parabens such as ethyl-4-hydroxybenzoate or propyl-4-hydroxybenzoate (RXRβ ligands with low estrogenic activity), non-immunosuppressive rapalogs and FK506 derivatives (reviewed in Reference ).The onl7-fold . DHB-dep7-fold . These tHeat- and antiprogestin-co-controlled RCCVs should not be susceptible to reactivation from quiescence in infected nerve cells. The promoters of one or two replication-essential genes were replaced with minimal GAL4-responsive promoters in these RCCVs. To the best of our knowledge, there are no cellular or viral transcription factors that can mediate transcription from these promoters. A GAL4 promoter drives the gene for the major single-strand DNA-binding protein ICP8 in HSV-GS3. In HSV-GS7, the gene for capsid protein VP19c is under GAL4 promoter control. ICP8 and VP19c are essential for lytic virus replication and should be equally indispensable for replication of a reactivated virus. In HSV-GS1 and HSV-GS3, the genes for ICP4 are controlled by GAL4 promoters. To the extent that this is known, early viral regulators that include ICP4 play a critical role in the late stages of reactivation, in particular during reactivation induced by fever/hyperthermia ,59,60,61Finally, it may be worth reflecting about the practicality of RCCV-based vaccination and the manner in which it could be practiced. For the reasons discussed above, it may be advantageous to administer the antiprogestin directly to the site of vaccine inoculation rather than systemically. Hence, an appropriate antiprogestin formulation may be included in the vaccine composition. In our experiments, heat treatment to the inoculation site was applied several hours after virus administration. If this interval were respected, two visits to a physician’s office might be required, one for vaccine administration and a second for the heat treatment. However, a second visit with the physician could be avoided by providing inoculated subjects with a simple heating device such as a small pad that is heated by crystallization of a supercooled, nontoxic salt solution (as described in more detail previously ) as well


Celiac disease (CD) is an autoimmune enteropathy caused by an intolerance to gluten proteins. It has been hypothesized that probiotic bacteria may exert beneficial effects by modulating inflammatory processes and by sustaining peptide hydrolysis at the intestinal level. This study aims at evaluating the capacity of a probiotic mixture (two different strains of lactobacilli and three of bifidobacteria) to hydrolyze gluten peptides following simulated gastrointestinal digestion of gliadin (PT-gliadin). The capacity of bacterial hydrolysates to counteract the toxic effects of gliadin-derived peptides in Caco-2 cells was also assessed. The protein and peptide mixtures, untreated or proteolyzed with the probiotic preparation, were analyzed before and after each proteolytic step with different techniques . These experiments demonstrated that PT-gliadin can be further digested by bacteria into lower molecular weight peptides. PT-gliadin, untreated or digested with the probiotics, was then used to evaluate oxidative stress, IL-6 cytokine production and expression of tight junctions’ proteins—such as occludin and zonulin—in Caco-2 cells. PT-gliadin induced IL-6 production and modulation and redistribution of zonulin and occludin, while digestion with the probiotic strains reversed these effects. Our data indicate that this probiotic mixture may exert a protective role in CD. Triticum genus. Gliadin is responsible for the intestinal damage in celiac disease (CD), an autoimmune disease mediated by T-cell activation in the gastrointestinal mucosa  of 33-mer peptide, i.e. 4.40 min. The 33-mer peptide was analyzed by this MS platform after probiotic treatment. The same peptide mixtures were also analyzed by UPLC//MS/DAD platform, applying analytical conditions optimized for 33-mer standard peptide. Analyses of PT-gliadin allow to identify, among others, a peptide with an a et al. , but no m/z signal corresponding to [MH+4] of 33-mer peptide after probiotic digestion.Since the main function of the intestinal epithelium is that of a selective barrier regulating the passage of various substances into the subepithelial compartment, here we examined the integrity of the tight junctions by analyzing the localization of zonulin (ZO-1) and occludin, the best studied components of the junctions. To this aim, we utilized the intestinal epithelial cell line Caco-2, that is widely used as a model for the in vitro study of human intestinal epithelium functions. We performed immunofluorescence analyses to establish whether the ZO-1 release and occludin rearrangement following PT-gliadin treatment would be prevented by further digestion with bacteria.Our results show that untreated Caco-2 monolayers had the typical ZO-1 and occludin localization at the cell periphery , whereasCaco-2 cells were also assessed for the production of IL-6 when stimulated with non-cytotoxic concentration of gliadin peptides. The PT-gliadin was able to cause a significant increase in IL-6 production compared to control. Conversely, when the cells were stimulated in the presence of PT-gliadin+bact no increase in IL-6 could be observed, instead a significant reduction in IL-6 production compared to control cells was observed A.In order to verify whether digestion with probiotic bacteria could modulate the pro-oxidant effects of PT-gliadin, Caco-2 cells were treated with PT-gliadin or PT-gliadin+bact and then assessed for intracellular hydroperoxides B. In ourThe intestinal microbiota is a complex ecosystem in which thousands of bacterial species are present and play a key role in the host’s health. Intestinal microbiota is involved, among other functions, in the maturation, maintenance and behavior of the mucosal immune system and contribute to the digestive process of food in a symbiotic manner . BacteriThe potential use of probiotics in CD management is supported by the intestinal dysbiosis generally associated with CD ,9,10,11  during the intestinal digestion of gliadin led to the generation of different fragment sequences in vitro, with lower molecular mass than those generated in non-treated samples [Flavobacterium meningosepticum [Some previous in vitro studies have demonstrated that the proteolytic activity of bacteria may modify gliadin fragments ,22,23. T samples , as obsesepticum . It has septicum . A recensepticum . A consosepticum ,27,28.L. paracasei, L. plantarum) and three strains of bifidobacteria  that probably exert complementary effects on the hydrolysis of gliadin peptides. The biological effect of proteolysis of gliadin by probiotic bacteria was analyzed in relation to intestinal permeability, inflammation or oxidative stress on the intestinal cells. Our results clearly show that the PT fragments from gliadin are able to induce tight junction destabilization and an inflammatory state on intestinal Caco-2 cells. On the other hand, the low MW fragments generated after bacterial treatment seem to prevent the disruption of tight junction proteins, antioxidant and anti-inflammatory activities, overcoming the negative effect induced by untreated PT-gliadin. The reversion of IL-6 increased production exerted by gliadin that was induced by probiotic digestion of PT fragments appears of great interest. Indeed, in CD patients, gliadins induce the production of pro-inflammatory cytokines, among others IL-6, which stimulates growth of T cells and differentiation of cytotoxic T cells and has been suggested as a marker of both CD activity and response to treatment [Noteworthy, the results observed in our study were obtained with a multi-strain probiotic preparation containing two strains of lactobacilli  reduced the production of TNF-α induced in cultured intestinal cells by gliadin peptides generated during gastrointestinal digestion [Other probiotic strains have shown the capacity to reverse the negative effect exerted by gliadin on the barrier function of intestinal cells and inflammation. pression , and Lac gliadin . Some Biigestion .It is well known that oxidative stress and inflammation are closely related phenomena and that gliadin-derived peptides are able to induce oxidative stress in Caco-2 cell cultures . Our resPromising results could come also by chymotryptic proteolysis experiment, because our preliminary evidences indicate that the probiotic product is able to modulate the amount of the 33-mer peptide generated by chymotrypsin digestion. This could be reasonable, considering the presence of different kind of proteases in the intestinal tract, each according to its specificity, to proteolyze the proteins in smaller fragments. Our results are in line with previous publications which demonstrate that probiotic lactobacilli and bifidobacteria may digest gluten peptides, thus reducing their immunogenic potential ,26,34. MB. longum CECT 7347 (previously named IATA-ES1) has been shown to reduce the production of inflammatory cytokines and the CD4+ T-cell mediated immune response in this animal model [In conclusion, our results suggest that the probiotic strains tested are able to reduce the toxicity of gliadin that remains after peptic-tryptic digestion by degrading immunodominant gliadin peptides, thus inhibiting their noxious effects on intestinal epithelial cells. The results obtained from our in vitro studies could have a higher value if verified in animals. In fact, recent work has identified an animal model of gliadin-induced enteropathy which has made it possible to confirm the beneficial effects of probiotic bacteria strains observed in vitro. Indeed, the administration of al model , while Lal model .


MicroRNAs (miRNAs) are a class of non-coding RNAs that hybridize to mRNAs and induce either translation repression or mRNA cleavage. Recently, it has been reported that miRNAs could possibly play a critical role in cellular processes like regulation of cell growth, differentiation, and apoptosis, emphasizing their role in tumorigenesis. Likewise, several miRNA's are involved in lung cancer tumorigenesis. The present review puts forth a database of human miRNA's involved in lung cancer along with their target genes. It also provides sequences of miRNA's and their chromosomal locations retrieved from different databases like microCosm (218 microRNAs), PhenomiR (293 microRNAs), and mir2Disease (90 microRNAs) and target gene information such as the pathways like cell cycle regulation, angiogenesis, apoptosis etc. Though miRNA's are still to be explored, they hold a promise as therapeutic targets and diagnostic markers of cancer. MicroRNAs were discovered in 1993 by Victor Ambros and group. However, their importance as an abundant class of regulatory non-coding RNAs came into light only after their rediscovery by Reinhart. MicroRNAs are reported to cover 1% to 3% of the genomes and 30% of the genes are shown to be regulated by them (Lai). Thousands of miRNAs involved in various human diseases have been identified by Jin. The reason for their involvement is mainly due to a change in the copy numbers or due to mutations in the sequence of miRNA or their target sites. They were also shown to be deregulated in majority of cancers. Recent studies have demonstrated the critical role of miRNAs in cancer pathogenesis-14. The potential of restoring levels of aberrantly under expressed miRNAs with miRNA mimics or inactivating over-expressed miRNAs with miRNA inhibitors has been explored and shows promise as the next generation of therapeutic strategies-19.MicroRNAs (miRNA) are short, noncoding RNAs regulating gene expression. Many factors potentially contribute to lung cancer formation for e.g. tobacco smoke, ionizing radiations and viral infections. However, the mechanisms involved in lung carcinogenesis remain largely unknown. Lung cancer is initiated by activation of oncogenes or inactivation of tumor suppressor genes. Similar to other cancers, many pathways are involved in lung cancer development and progression.Lung cancer is one of the most common causes of cancer related deaths in the world and is responsible for 1.3 million deaths worldwide every year. Lung cancer can be divided into two groups according to pathology: non-small cell lung cancer (NSCLC) (80.4%) and small cell lung cancer (SCLC) (16.8%). The epidermal growth factor receptor (EGFR)/ErbB1 regulates cell proliferation, apoptosis, angiogenesis and tumor invasion in non-small cell lung carcinoma. Recent investigations have demonstrated that miRNAs are likely to be involved in the regulation of EGFR in lung cancer. An inhibitor of miR-128b resulted in up-regulated EGFR expression in an EGFR-expressing NSCLC cell line, and treatment with a miR-128b mimic resulted in a concomitant reduction of EGFR expression. Inhibition of miR-21 was recently shown to enhance the anti-apoptotic potential of an anti-EGFR tyrosine kinase inhibitor in an EGFR-mutant lung adenocarcinoma cell line. The proto-oncogene RAS is the central molecule of the SOS-RAS-Raf-MAPK cascade which is the downstream cytoplasmic effector of the growth factor receptors. Approximately 20% of tumors in general and 30% of lung tumors have activating mutations in one of the RAS genes. One of the downstream effectors of the RAS signaling pathway is the MYC oncogene. MYC amplification and over-expression have been detected in different histologic subtypes of lung cancer. Both RAS and MYC are targets of the let-7/miR-98 family of miRNAs. Johnson  showed that members of the let-7 family of miRNAs bind to the 3′UTR of RAS, down-regulating RAS expression in human cells. It is seen that let-7 is poorly expressed in lung cancer compared to normal lung tissue, and that the expression of let-7 is inversely correlated with the expression of RAS in lung tumor samples. miR-29 is down-regulated in a number of human cancers including lung cancers, and its ability to repress Mcl-1 coupled with its role in regulating epigenetic DNA methylation means that enhancing expression of this regulatory element could be an effective therapeutic strategy-32. Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis during tumor development, and this molecule and its receptor (VEGFR) have been primary targets of therapies designed to target pathological angiogenic signaling. Liu investigated the involvement of miR-126 in the regulation of angiogenic processes in a lung cancer model. They found decreased expression of miR-126 and increased expression of VEGF-A in various lung cancer cell lines and showed that introduction of miR-126 using a lentiviral vector could down-regulate the expression of VEGF-A and inhibit growth.MicroRNAs are involved in many pathways related to lung cancer. ErbB2 (HER2/Neu), ErbB3 and ErbB4 belong to the family of receptor tyrosine kinases. These kinases initiate signal cascades leading to DNA synthesis and cell proliferation, and their over-expression is often critically involved in tumorigenesis and cancer cell proliferationp53 tumor suppressor gene, located on chromosome 17p, is affected in 60%-75% of lung cancer including both NSCLC and SCLC while Rb is more likely inactivated in SCLC. In lung cancer cells, induction of miR-34 results in apoptosis, 37 and miRNA profiling shows that the expression of miR-34a, miR-34b, and miR-34c are directly correlated with expression of the p53 tumor suppressor, suggesting that miR-34 is involved in regulating apoptosis as a regulatory target of p53. Evidence is emerging that tumor suppressors are likely to be regulated by miRNA activity. FUS1/TUSC2 is a tumor suppressor gene located on 3p21.3 that has been shown to be negatively regulated by the activities of miR-197, miR- 93, miR-98, and miR-378, 39. Reduced or complete loss of Fus1 expression was found in 82% and 100% of non-small cell and small cell lung cancer cell lines, and elevated levels of miR-93 and miR-197 have been shown to correlate with reduced Fus1 expression in NSCLC tumor specimens. Another important tumor suppressor gene is LKB1, whose loss-of-function mutation/deletion is observed in 30% lung adenocarcinomas and 20% of squamous cell carcinomas, 42. An investigation of miR-126 in the regulation of invasive potential in lung cancer revealed an inverse relationship between Crk and miR-126 expression in squamous cell tumors. miR-21 also targets PDCD4, a pro-apoptotic gene that inhibits tumorigenesis and whose down-regulation has been linked to poor survival in colon and lung cancer patients-46. The let-7 family has been shown to inhibit the expression of several oncogenes including RAS, MYC, and HMGA2, 47-49. Several studies have implicated the miR-17-92 cluster of miRNAs as an actual oncogene with important regulatory effects on pathologic tumor cell proliferation in various tissues including breast and lung-52.Inactivation of tumor suppressor genes plays important role in lung carcinogenesis. The . Identifying these miRNAs and their target genes provides a platform to work towards the understanding of lung cancer pathogenesis. Computational prediction of target genes for miRNAs in animals is a challenging task for both experimental and computational group, due to the complexity of miRNA target recognition.All these genetic pathways play a critical role in lung cancer progression. MicroRNAs play a very important role in the regulation of these pathways. Finding the targets of miRNA is equally important as finding the miRNA itself, human microRNA disease database, miRNAMap, and miRGen. miRBase serves as a central database for experimentally supported mature miRNA sequences for each supported miRNA. It also provides the genomic coordinates of the predicted precursor sequences. TargetScan, PicTar, TargetMiner, miRDB are the databases for miRNA target prediction and functional annotations in animals. microRNA.org target and expression 2005, are the databases used for microRNA target predictions based on the complementary perfect base-pairing in the seed region of the targets. TarBase is a resource of experimentally validated microRNA targets.Numerous databases are available which facilitate easy and efficient mining of microRNAs and their target genes involved in a specific cancer. Databases based on sequence annotation and microRNA genomics are miRBaseHowever, no database exists which provides a complete overview of microRNAs controlling lung cancer pathogenesis and progression. This review puts forth a database including almost all the microRNAs involved in lung carcinogenesis. The miRNAs involved in lung cancer are pooled up from databases, MicroCosm (218 microRNAs), PhenomiR (293 microRNAs) and mir2Disease (90 microRNAs). A supplementary excel document is provided with this review which gives a detailed description of the MiRNA's involved in lung cancer. It also includes information about the accession numbers, chromosomal positions and validated targets of the respective microRNA's., MiR2Disease database. After manually curating the miRNAs from different databases accession numbers and sequences of microRNAs were collected from the miRBase database. MicroRNAs chromosome positions were obtained from the HUGO gene nomenclature committee (HGNC). PhenomiR database contains the microRNA expression levels in the different lung cancer samples and cell lines, those were collected for specific microRNAs which are present in PhenomiR database and related to lung cancer.MicroRNAs involved in lung cancer were derived from miRBase, PhenomiRExperimentally validated targets for these lung cancer microRNAs were collected from the mir2disease and TarBase databases. These target genes were submitted to gene Go analysis software. Gene Go is the pathway analysis software which gives the information about interaction between the different biological molecules. Many target genes were found to be involved in lung cancer development and progression and these proteins are present in extracellular, membrane, cytoplasm and nucleus. All these target genes are positively or negatively regulated by the each other. Receptor ligand, generic kinase, transcription factors, lipid phosphatase, generic phosphatase, generic binding proteins and RAS super family proteins have been found to be involved in the lung cancer development. Generic binding proteins like cyclin D1, P27KIP1, and BCL2 are very important molecules in the cytoplasmic region and regulate many other targets. C-Myc is one of the important transcription factor which is present in the nucleus and regulates many proteins in the cytoplasmic region. Interactions between the extracellular, membranes, cytoplasmic and nuclear proteins are shown in the 1-S cell-cycle transition and is over expressed in many human tumors. A thorough analysis of the pathway circuit produced by genego software depicts that C-myc directly or indirectly regulates most of the proteins which involve in various other pathways. Thus c-Myc is a potent protein which needs to be studied.The major pathways associated in lung cancer based on genego analysis are pathways involving cell proliferation, cell cycle regulation, angiogenesis, signal transduction and apoptosis. The significant protein found to regulate most of the other proteins is c-Myc and the proteins regulated by c-Myc are cyclins, cyclin dependant kinases (CDKs), DNA methyl transferases, and CDK inhibitors. C-Myc is a basic-helix-loop-helix/leucine zipper (bHLH/LZ) transcription factor that controls the G1. But an altered expression of p27KIP1 dysregulates the expression of proteins involved in cell cycle regulation and thereby leads to cell proliferation. Interestingly, the pathway circuit obtained from genego software reveals that c-Myc indirectly regulates p27KIP1 through Bcl2 and H-Ras. Hence c-Myc becomes further more a vital protein to be focused on, apart from p27KIP1.It is also seen that p27KIP1 too regulates quite a few proteins. p27KIP1 is a cell-cycle regulatory protein that interacts with cyclin-CDK2 and -CDK4, inhibiting cell cycle progression at G. Exogenous delivery of a synthetic let-7 mimic has been used to mediate remission of established NSCLC tumors in mice. Future work will focus on both chemical modifications and delivery vehicles in order to maximize the therapeutic benefits and minimize the side effects of strategies designed to alter intracellular levels of specific miRNAs (MicroRNA regulation of lung cancer-related pathways provides numerous opportunities for therapeutic intervention. Competitive inhibition of miRNAs or the delivery of exogenously produced miRNAs could provide substantial therapeutic benefit by reducing the activity of prooncogenic miRNAs. These approaches are still in the preclinical stages, although intriguing evidence is emerging that supports their use in clinical applications. Expression of miR-29 family members has been shown to reduce tumorigenic potential in a lung cancer modelc miRNAs .The study of microRNAs has evolved rapidly since the discovery of this important class of regulatory molecules. The proposed lung cancer microRNA database would prove to be helpful to lung cancer biologists in one or the other way. Further studies on the miRNA's listed in the database would explore and explain the relation between miRNAs and the initiation and progression of malignant neoplasias and the basic biological roles that they play in tumor cell survival, invasion, metastasis, and angiogenesis. However, many issues are still to be addressed before the miRNAs can be used as therapeutic tools in the field of oncology. miRNA dysregulation in particular tumor subtypes need to be further investigated, which may aid the development of new therapies that specifically target tumor cells with particular molecular and regulatory profiles. Overall, miRNAs are ubiquitous regulatory molecules that hold great promise for increasing our understanding of lung cancer and for improving upon current diagnostic and therapeutic methods. Further evaluation of their value in cancer diagnostics and of the functional consequences of their manipulation will provide researchers and oncologists with a wealth of new tools to characterize and treat this prevalent and diverse disease.


Traumatic brain injury (TBI) is among the most prevalent causes of cerebrovascular and neurological damage worldwide. To this end, tobacco smoke (TS) has been shown to promote vascular inflammation, neurovascular impairments, and risk of cerebrovascular and neurological disorders through oxidative stress (OS) stimuli targeting the blood-brain barrier (BBB) endothelium among others. It has been recently suggested that premorbid conditions such as TS may exacerbate post-TBI brain damage and impact recovery.Our study investigated the mechanisms underlying the exacerbation of TBI injury by TS using a weight drop model. For this purpose, male C57BL/6J mice, age range 6–8 weeks, were chronically exposed to premorbid TS for 3 weeks. Test animals were then subjected to TBI by guided vertical head weight drop using a 30 g metal weight free felling from an 80 cm distance before reaching the target. We analyzed the physical activity and body weight of the mice before TBI and 1 h, 24 h, and 72 h post-injury. Finally, mice were sacrificed to collect blood and brain samples for subsequent biochemical and molecular analysis. Western blotting was applied to assess the expression of Nrf2  as well as tight junction proteins associated with BBB integrity including ZO-1, Occludin, and Claudin-5 from brain tissues homogenates. Levels of NF-kB (a pro-inflammatory transcript factor which antagonizes Nrf2 activity) and pro-inflammatory cytokines IL-6, IL-10, and TNF-α were assessed in blood samples.Our data revealed that premorbid TS promoted significantly increased inflammation and loss of BBB integrity in TBI when compared to TS-Free test mice. Additionally, mice chronically exposed to TS before TBI experienced a more significant weight loss, behavioral and motor activity deficiency, and slower post-TBI recovery when compared to TS-free TBI mice.The effects of premorbid TS appear consequential to the abrogation of physiological antioxidative and anti-inflammatory response to TBI leading to worsening impairments of the BBB, OS damage, and inflammation. These factors are also likely responsible for the retardation of post-traumatic recovery observed in these animals. Traumatic brain injury (TBI) has long been among the most common type of trauma and the leading cause of death and disability in the young-aged population (≤ 45 years of age) in the USA, thus becoming a serious public health concern in modern society , 2. AccoThe pathophysiology of TBI can be divided into primary and secondary injury mechanisms. The primary mechanical injury is due to the physical harm and may result in intracranial or extracranial hemorrhage following damage to the blood vessels, brain tissue, and the blood-brain barrier (BBB) . The secMoreover, disrupted tight junctional proteins (including occludin and claudins) reduce BBB integrity, thus increasing paracellular permeability. ROS is an active pro-inflammatory promoter which impacts endothelial viability and vascular biological responses. These latter include upregulation cell adhesion molecules, inflammatory cytokines, and, eventually, the influx of inflammatory cells into the brain parenchyma. These factors ultimately determine the progression of injury, including excitotoxicity and neuronal loss.ROS constituents and other reactive compounds profoundly enrich tobacco smoke (TS). Chronic smoking has been clearly shown to promote dysfunction of the BBB through activation of oxidative, inflammatory, and immune responses that support the onset and progression of cerebrovascular and neurodegenerative disorders, including TBI –26. TherThe well-known association between smoking and vascular endothelial dysfunction, as well as the increased risk of neurological disorders , is given. Despite the epidemiological and translational studies strongly suggesting activation of pathophysiological pathways by TS that exacerbate TBI outcome and influence recovery, determination and characterization of shared key modulators in BBB impairment due to TS and TBI lies unexplored. Identification and then targeting of these putative key modulators could help in preventing the initiation of metabolic/cerebrovascular complications due to TBI in smokers. Thus, the goal of the present study was to characterize the pathogenic impact of chronic smoking on TBI and assess the post-traumatic exacerbation of TBI, by studying key established pathological parameters leading to loss of BBB function and integrity. For this purpose, we have used a well-established weight-drop TBI mice model, to test the impact of premorbid TS exposure on post TBI injury and recovery animals with or without chronic pre-exposure to TS. We believe that a better understanding of TS potential influence on TBI can facilitate the development of more targeted and effective interventions to improve post-traumatic outcomes as well as reducing the impact of chronic smoking itself. This latter is of paramount importance for patients that cannot quit smoking. Furthermore, subjects who have recently stopped smoking remain at high risk of cerebrovascular disorders and worse secondary brain injury outcomes (due to the lingering effect of chronic smoking) for a relatively long period.Reagents and chemicals were purchased from Sigma-Aldrich  or Bio-Rad Laboratories . All Quantikine ELISA kits were purchased from R & D systems. The antibodies used in this study were obtained from various sources: rabbit anti-ZO-1 (#402200), mouse anti-Occludin (#331500), and anti-Claudin-5 (#4C3C2) from Life Technologies; mouse anti-PECAM-1 (#sc-376764), mouse anti-VCAM-1 (#sc-13160), rabbit anti-Nrf2 (#sc-722), mouse anti-NQO-1 (#sc-376023), mouse anti-HO1 (#sc-390991), and mouse anti-NFκB-p65 (#sc-(F-6)-8008) from Santa Cruz Biotechnology. Donkey anti-rabbit (#NA934) and sheep anti-mouse (#NA931) HRP-linked secondary antibodies were obtained from GE Healthcare .The experiment protocol in this study was abided by the Institutional Animal Care and Use Committee, TTUHSC, Lubbock, Texas . C57BL/6We used a weight-drop model of TBI , 32. ThiIn brief, mice were first anesthetized by inhaling isoflurane vapor for 3 to 5 min and then placed on a spongy platform positioned under the weight-drop device. Head movements were allowed parallel to the injury plane at the time of the induction to mimic a mild to moderate head injury. During the induction phase, mice were positioned to direct the trauma from the left anterior frontal area at the same distance between the eye and the ear. A hollow tube  was used as the guiding system for a metal weight (30 g) released in a free fall from the dropping point positioned 80 cm above the target  was collected by centrifugation at 14,000 g for 30 min. Samples were then aliquoted and stored at − 80 °C for subsequent protein expression analysis by Western blotting. Protein quantification was conducted using Pierce BCA Protein Assay Kit . Samples (60–90 μg for tissue lysates) were prepared as previously described by us , 37. BriBlood samples collected from mice were analyzed by Quantikine ELISA kits  for the quantitative determination of thrombomodulin and cytokines TNF-α, IL-6, and IL-10 according to the procedure per the manufacturer’s protocol.Tissue lysate was analyzed by Quantification Kit for Oxidized and Reduced Glutathione  according to the manufacturer’s guidelines. For the quantitative determination, samples were prepared by lysis of total cell protein in T-PER lysis buffer followed by dilution of 1:50 for glutathione (GSH) analysis. In brief, a serial dilution of reduced (GSH) and oxidized glutathione (GSSG) stock standards were prepared along with assay mixtures for detection of GSH and total GSH using 100 X Thiol green stock solutions, assay buffer, and GSSG probe. A one-step fluorometric reaction of the sample with the respective assay buffer was incubated for 30 min. Fluorescence intensity was assessed at Ex/Em of 490/520 nm. GSSG was determined by subtracting GSH from total GSH. Finally, GSH was plotted against GSSG to obtain the GSH/GSSG ratio.P values < 0.05 were considered statistically significant.All collected data were expressed as mean, standard ± deviation (SD). The sample size was chosen based on previous work by us and others to generate 80% power and a type 1 error rate = 0.05. The blind analysis was performed by one-way ANOVA using GraphPad Prism 8 Software Inc. . Post multiple comparison tests were performed as with Tukey’s or Dunnett’s test as recommended by the software. 2 (2 L/min) at atmospheric pressure (1 bar). Mice were housed in the smoking chambers (four mice/chamber), receiving an uninterrupted supply of normal oxygenated air in between puffs. At the end of each smoking cycle, mice were transferred immediately back to their regular housings with standard food and water supply.As shown in Fig. Weight analysis was regularly performed to assess whether smoke and/or TBI had any negative impact on body weight. Animals were divided into four groups at day 0 Fig. a represeOur primary hypothesis refers to the alteration in pro-inflammatory markers as one of the mechanisms that underlie the damage in the brains of TBI-induced mice. Hence, we evaluated the expression levels of inflammatory marker NF-kB, inflammatory adhesion molecules VCAM-1, and PECAM using Western blotting and enzyme-linked immunoassay (ELISA). As shown in Fig. Of relevant interest, TS exposure decreased the expression level of the anticoagulant factor thrombomodulin Fig. a, b. ThiThe effect of TS exposure and TBI on the expression of the antioxidative response nuclear factor erythroid-2-related factor 2 (Nrf2) was also evaluated by Western blot analysis of whole-brain homogenate, as shown in Fig. Our results show that except for the smoke + TBI condition, the overall total glutathione levels are not significantly different from controls Fig. a. PremorAdditional experiments were performed to assess the effect of TS exposure and TBI on BBB integrity. We evaluated the expression level of the BBB accessory protein zonula occludens-1 (ZO-1) and tight junction (TJ) protein expression encompassing the primary regulator of BBB paracellular permeability Occludin and Claudin-5.As shown in Fig. By contrast, TBI only has a significant impact on occludin . At the same time, the effect on claudin-5 was marginal, and no effect was observed regarding the expression of ZO-1. However, when the stimuli are combined, we see a synergistic downregulation of occludin, whereas that of claudin-5 and ZO-1 is attributable uniquely to TS exposure. The critical point of these results is that in addition to inflammation, premorbid TS exposure promotes the onset of a pre-existing state of weakened BBB integrity, which can be more readily impacted by TBI.Exploratory behavior and general activity of the test mice were regularly recorded to evaluate the impact of smoke and TBI based on their motor activity before and after induction of a mild traumatic injury.As shown in Fig. Moreover, as shown in Fig. TBI is a global health issue and one of the major leading causes of death and disability for individuals under the age of 50. According to the statistics, approximately half the global population will experience one or more TBI events over their lifetime . OxidatiIn the present study, we evaluated the potential influence of chronic tobacco smoking on pathophysiological mechanisms underlying the exacerbation of TBI and retardation of post TBI recovery using a weight-drop mice model. In this model, a fixed weight is released for a free fall based on a defined path and height. The weight and the height from which the weight is dropped determine the severity of the injury, which can range from mild to severe. This model was chosen because of its ability to simulate traumatic head injuries comparable to those observed in road accidents or falls . Based oMoreover, systemic and microenvironmental effects of neuroinflammation induced by TBI can affect neurotransmission and especially the dopaminergic pathways. This latter plays a significant role in secondary injury as well as behavioral and cognitive dysfunction following TBI –44. PrevNrf2, a basic region-leucine zipper (bZip) redox-sensitive transcription factor, is the master regulator of multiple cytoprotective responses which controls the redox state of cells in harmful stresses , 50, 51.In line with these findings, we assessed the impact of premorbid TS exposure and TBI on Nrf2 expression levels, as well as its downstream effector molecules NQO-1 and HO-1, which are known for exerting acute detoxification and cytoprotective functions. Our in vivo data show that the Nrf2-ARE system is activated in response to TBI see Fig. . This efThe oxidative stress impact of premorbid chronic smoking and TBI  was also confirmed by measurements of GSH as well as GSSG see Fig. . Our datThe downstream impact of Nrf2 downregulation includes further impairment of the BBB , which strictly maintains the brain homeostasis and controls the passage of substances in and out of the brain environment. Among the various control functions of the BBB, the inter-endothelial TJs rigidly control the paracellular pathways blocking the passage of polar molecules (including ions) from moving between adjacent endothelial cells . The mosAdditional pieces of evidence also indicate that inflammation is an essential contributor to the TBI pathophysiology exacerbating neuronal damage during post-traumatic brain injury. Therefore, sustained and excessive inflammation through the secretion of proinflammatory mediators can increase subsequent neurological impairment –66. This−/− mice have greater NF-κB activation and generation of pro-inflammatory cytokines in the brain and spinal cord injury compared to their wild-type Nrf2+/+counterparts [Moreover, in a recent study, it has been reported that Nrf2terparts . RelevanAn additional risk factor for TBI patients that is associated with chronic smoking may be derived from the impact of smoking on blood hemostasis. Thrombomodulin is a critical component of the anticoagulant protein C pathway, which ensures control over the process of blood coagulation. This anticoagulant system acts by blocking the activity of the prothrombinase complex and promoting fibrinolysis. This control system is activated following activation of the coagulation cascade in response to vessel injury and/or inflammation . InflammA shortcoming of this study is the exclusive use of male mice rather than a mixed-gender population of animals. Given the “early stage” nature of this study assessing the impact of TS on TBI damage and post-TBI recovery, we felt that it was necessary to minimize the variability of physiological responses  caused by gender-related hormonal differences. However, recent previous studies by our group have highlighted possible gender-specific cellular responses to chronic TS exposure  affectinAlthough outside the scope of this work, future experiments will focus on further detailing the concurrent underlying mechanisms through which TS impacts TBI. We will also focus on assessing the impact of gender as an additional risk factor in the TS-TBI interplay and expand further to evaluate the effects of vaping on TBI. Targeting Nrf2 to prevent TBI exacerbation by TS and other vascular comorbidities could provide a possible path toward the development of more effective treatments. This latter is of paramount importance for patients that either cannot quit smoking or those that recently stopped but will remain at high risk of developing CNS disorders for a significant amount of time (years) before full renormalization. Crucial consideration must be extended to several other factors that may hinder this renormalization process, including number and type of cigarette smoked per day, years spent smoking, and age of the subject when entirely quit. It is also possible that gender may play a significant role as well.Although the therapeutic window of BBB regulation after TBI remains unknown, further understanding of the dynamics regulating BBB dysfunction post-TBI would provide essential data to support the development of novel therapeutic approaches, including more selective therapeutic agents, and timing of treatment.Chronic cigarette smoking is a modifiable health risk, representing the third leading cause of preventable mortality in the USA . In this


However, 56% of the samples with a bidirectional shunt had a pre- and postductal saturation gradient of < 3%. Conclusions: The presence of a bidirectional ductal shunt is associated with hypoxemia and low pulmonary blood flow. The absence of a pre- and postductal saturation difference is frequently observed with bidirectional right-to-left shunting through the PDA, and does not exclude a diagnosis of PPHN in this model.Background: The aim was to evaluate the relationship between the direction of the patent ductus arteriosus (PDA) shunt and the pre- and postductal gradient for arterial blood gas (ABG) parameters in a lamb model of meconium aspiration syndrome (MAS) with persistent pulmonary hypertension of the newborn (PPHN). Methods: PPHN was induced by intermittent umbilical cord occlusion and the aspiration of meconium through the tracheal tube. After delivery, 13 lambs were ventilated and simultaneous 129 pairs of pre- and postductal ABG were drawn  while recording the PDA and the carotid and pulmonary blood flow. Results: Meconium aspiration resulted in hypoxemia. The bidirectional ductal shunt had a lower postductal partial arterial oxygen tension ([PaO The pre- and postductal gradient is thought to be secondary to right-to-left or bidirectional shunting across the PDA [Persistent pulmonary hypertension of the newborn (PPHN) is a disorder of unsuccessful circulatory transition at birth . It is c2) gradient of > 3% [As part of the screening for critical congenital heart disease (CCHD), an oxygen saturation by pulse oximetry  and saturation of arterial oxygen (SaO2) concentrations.We evaluated the relationship between the direction of ductal shunt and blood gas parameters in simultaneous preductal (right carotid) and postductal  samples in a lamb model of meconium aspiration syndrome (MAS) with PPHN. We hypothesized that the presence of a bidirectional or right-to-left shunt at the PDA will be associated with a clinically significant gradient between the pre- and postductal partial arterial oxygen tension  at the University of California Davis (UCD) and has been described in detail previously ,9,10. Th2) monitor. Following a laparotomy, the fetal lamb was partially exteriorized and intubated with a 4.5-mm cuffed endotracheal tube (ETT). The fetal lung fluid in the ETT was passively drained by lowering the head and, thereafter, the ETT was occluded to prevent gas exchange during gasping, following asphyxiation by cord occlusion. Under maternal anesthesia, and after infiltrating the site with subcutaneous bupivacaine, an incision was made to place a catheter in the lamb’s right carotid artery for the measurement of blood pressures and the collection of blood samples. The right jugular vein was catheterized for fluid and medication administration. A 3-mm flow probe  was placed around the left carotid to measure the blood flow. A left thoracotomy was performed for placement of flow probes to measure blood flow in the left pulmonary artery  and the ductus arteriosus . Finally, both the thoracotomy and neck incisions were surgically closed. The baseline hemodynamic measurements and arterial blood gases were recorded.Time-dated near-term  pregnant ewes were bred by Van Laningham Farm, Arbuckle, CA, USA. Following an overnight fast, the ewe was sedated with intravenous propofol or diazepam and ketamine. The ewe was then intubated with a 9.5-mm cuffed endotracheal tube (ETT), provided general anesthesia with 2–4% inhaled isoflurane, and continuously monitored with a pulse oximeter and an end-tidal carbon dioxide  was instilled into the ETT. Intravenous analgesic support was started prior to cord clamping. Acute prenatal asphyxiation was induced by occluding the umbilical cord for five minutes or until the heart rate decreased below 40 beats per minute. During this time period, the meconium solution was “spontaneously” aspirated during gasping and distributed into the lungs. The umbilical cord compression was relieved for two minutes to allow for hemodynamic recovery, followed by a second five-minute cord occlusion interval. Following meconium aspiration, the umbilical cord was tied and cut, and the lambs were delivered.2, and partial arterial carbon dioxide (PaCO2). A catheter was then placed in the umbilical artery to collect postductal blood samples. A pulse oximeter was placed on the right forelimb for continuous saturation monitoring (SpO2). A second pulse oximeter was placed around a hind limb for postductal SpO2 measurements. The inspired oxygen concentration was adjusted to achieve a preductal SpO2 as per the Neonatal Resuscitation Program guidelines for the first 15 min. Subsequent management was based on the consensus statement from the European Pediatric Pulmonary Vascular Disease Network (EPPVDN) [2 between 91% and 95%, a PaO2 between 50 and 70 mmHg, and a PaCO2 between 45 and 60 mmHg. Lambs were monitored for up to six hours, whereupon the blood gases and hemodynamic data were analyzed at 15 min intervals. At the completion of the study period, the lambs were euthanized.The lambs were transferred to a radiant warmer and mechanically ventilated. The peak inspiratory pressure (PIP) was adjusted based on the exhaled tidal volume, ETCO(EPPVDN) . We targ2, and flows are expressed as means with standard deviations (SDs). By convention, the right-to-left ductal flow was labeled negative and the left-to-right flow was labeled positive on the acquisition device. The ductal flow was recorded as the minimum (diastolic flow), mean, and maximum (systolic flow). Throughout the analysis, the right-to-left or bidirectional ductal shunting was defined by values with negative (right-to-left) flow either during the diastole, systole, or both phases of the cardiac cycle. The left-to-right ductal shunting was defined by positive flow throughout the cardiac cycle. We also evaluated the hemodynamic and gas exchange implications of a pre- and postductal SaO2 gradient of ≥ 3% and < 3%. The pre- and postductal comparisons were analyzed by a two-tailed, paired Student’s t-test. Comparisons between the right-to-left/bidirectional and left-to-right ductal shunt groups were analyzed using a two-tailed, Student’s t-test with unequal variances. Statistical significance was defined as p < 0.01.Blood gases were analyzed using a blood gas analyzer , and hemodynamic variables were continuously recorded using computer acquisition and analysis software . The ductal flow  was recorded simultaneously at the time of the pre- and postductal blood gas sampling. Blood gas variables, specifically SaOA total of 129 hemodynamic and blood gas time-points were compiled and sorted into right-to-left/bidirectional and left-to-right ductal shunt groups based on the ductal blood flow direction .2 gradient , and mean airway pressures were not significantly different between these two groups.The blood gas and hemodynamic parameters were also classified based on the pre- to postductal SaOgradient . The hem2 values and significantly higher preductal and postductal PaCO2 values compared to the samples drawn in the presence of left-to-right shunting  ductal shunting (as defined in the methodology) had significantly lower postductal PaOshunting . Only twtal PaO2 .p < 0.01). The left QCA was significantly lower in the bidirectional shunt group .Mean systemic blood pressure was significantly lower in the bidirectional group compared to the left-to-right ductal shunt group  and SaO2 were significantly lower than their preductal counterparts during bidirectional shunting . Oxygen consumption by the brain was calculated using the following equation QCA × (CaO2 − CvO2)/100, where CaO2 is the carotid arterial oxygen content and CvO2 is the jugular venous oxygen content. The brain oxygen delivery and oxygen consumption by the brain were lower in the bidirectional shunt group  and preductal arterial blood (with SaO2 of 90.9% ± 6.5%). Using the shunt equation, the right-to-left effective ductal shunt only contributes to 6.6% of the descending aortic flow in lambs with a bidirectional ductal flow. The presence of a saturation difference between the preductal and postductal samples is a reliable indicator of PPHN and a bidirectional or right-to-left shunt (89% of instances—In neonates with PPHN the absence of a pre- and postductal oxygenation gradient is thought to be due to the closure of the PDA or an exclusively left-to-right shunt at the PDA . The curIn lambs with parenchymal lung disease and secondary PPHN, the presence of a pre- and postductal oxygenation gradient was indicative of a right-to-left or bidirectional shunt. However, the lack of a pre- and postductal oxygenation gradient does not rule out bidirectional shunt. In clinical situations with parenchymal lung disease, the clinical absence of a pre- and postductal oxygenation gradient should not be considered to suggest the absence of PPHN, and higher emphasis should be placed on obtaining echocardiography to confirm the diagnosis.


N-methyl-d-aspartate receptors, transient potassium and dendritic sodium channels in regulating information transfer. Our results demonstrate that trial-to-trial variability, tuning-curve shape and biological heterogeneities critically regulate the relationship between the spatial tuning curve and spatial information transfer in hippocampal place cells.The relationship between the feature-tuning curve and information transfer profile of individual neurons provides vital insights about neural encoding. However, the relationship between the spatial tuning curve and spatial information transfer of hippocampal place cells remains unexplored. Here, employing a stochastic search procedure spanning thousands of models, we arrived at 127 conductance-based place-cell models that exhibited signature electrophysiological characteristics and sharp spatial tuning, with parametric values that exhibited neither clustering nor strong pairwise correlations. We introduced trial-to-trial variability in responses and computed model tuning curves and information transfer profiles, using stimulus-specific (SSI) and mutual (MI) information metrics, across locations within the place field. We found spatial information transfer to be heterogeneous across models, but to reduce consistently with increasing levels of variability. Importantly, whereas reliable low-variability responses implied that maximal information transfer occurred at high-slope regions of the tuning curve, increase in variability resulted in maximal transfer occurring at the peak-firing location in a subset of models. Moreover, experience-dependent asymmetry in place-field firing introduced asymmetries in the information transfer computed through MI, but not SSI, and the impact of activity-dependent variability on information transfer was minimal compared to activity-independent variability. We unveiled ion-channel degeneracy in the regulation of spatial information transfer, and demonstrated critical roles for  They receive, encode and process information about their surroundings in eliciting robust responses to challenges posed by the external environment. From an ethological perspective, it is essential that sensory information is efficiently encoded by neural circuits to ensure effective responses to environmental challenges. A dominant theme of neural circuit organization is the ability of individual neurons to encode specific features associated with the external environment, with different neurons responding maximally to distinct feature values. For instance, neurons in the primary visual cortex respond maximally to a specific visual orientation , neuronswithin the place field of hippocampal place cells has not been quantitatively assessed. Neurons in the hippocampus receive spatial information about a given arena and a substantial fraction of them respond to different spatial locations in the same arena , orientation-selective tuning curves and visual information transfer questions are posed with reference to the synaptic inputs received by the neuron  and a specific output characteristic   .within the place field. As our principal goal in this study is to assess the relationship between spatial tuning curves and spatial information transfer, it is essential that the information transfer measure also is specific to particular spatial locations. An ideal information metric that fulfills this requirement is the stimulus-specific information (SSI), a measure that was specifically defined to convey the amount of information that the responses of a neuron convey about a particular stimulus. SSI is defined as the average specific information across all the neural firing rates that are elicited when the animal traverses a particular spatial location, with specific information referring to the information that a particular firing rate response provides about which spatial location was being traversed  to distinct functions. Thus, we constructed a population of heterogeneous models, which were morphologically realistic and conductance-based, in arriving at our interpretations and conclusions. This heterogeneous conductance-based modeling framework allowed us to effectively address the second question on the impact of biological heterogeneities on spatial information transfer, and to assess the impact of individual ion channels and receptors on spatial information transfer.The answer to the second question on the impact of heterogeneities on neural function is critical from physiological and pathological perspectives. Experimental analyses typically interpret outcomes from summary statistics, and computational models often employ a perilous  because The relationship between tuning curves and information transfer depends on trial-to-trial variability in other neural systems . SpecifiHippocampal place cells manifest experience-dependent asymmetry in their place-field firing , 2000. WIn assessing the relationship between spatial information transfer and spatial tuning curves, it was essential to account for three characteristics in our experimental design: Our analyses show that each of these three characteristics — biophysical and physiological heterogeneities, the type and level of trial-to-trial variability, and behavior-dependent alterations to the tuning curve — critically regulated the relationship between the spatial tuning curve and spatial information transfer. We demonstrate that when hippocampal neurons exhibit low trial-to-trial response variability, they transfer peak spatial information at the high-slope locations (and not at peak firing location) of the spatial tuning curve within their place field. Importantly, we show that our model population manifested parametric degeneracy in the expression of similar tuning curves and similar information transfer metrics. As a consequence of the expression of degeneracy, we found heterogeneities in spatial information transfer and in the impact of knocking out individual ion channels on spatial information metrics, together pointing to a many-to-one relationship between different ion channel subtypes and spatial information transfer. Finally, our analyses also unveil a potent reduction in information transfer consequent to the elimination of transient potassium channels, NMDA receptors or dendritic sodium channels, thereby providing direct experimentally testable predictions.2aR), specific membrane resistivity (mR) and specific membrane capacitance (mC). In the base model, aR was set to 120 Ωcm and the specific membrane capacitance was set to 1 μF/cm2 for the entire neuron (x) , M-type potassium (KM), small-conductance calcium activated potassium (SK), T-type calcium (CaT), N-type calcium (CaN), R-type calcium (CaR), L-type calcium (CaL) and hyperpolarisation activated cyclic nucleotide gated (HCN or h). The current through these channels due to Na+, K+ ions were modeled in an ohmic formulation with the reversal potentials of Na+, K+ and h channels being 55, −90 and −30 mV respectively. The current due to calcium ions was modeled as per the Goldman–Hodgkin–Katz (GHK) conventions with the internal calcium concentration as 50 nM and external calcium concentration as 2 mM. The equations underlying the kinetics of these channels were obtained from prior electrophysiological recordings: NaF, KDR and KA , delayed rectifier potassium (KDR), R and KA , HCN gKAgh of a somatodendritic compartment, a hyperpolarizing current step of 100 pA was injected for 500 ms into the compartment. The local change in the membrane potential as a result of the step current was measured and the ratio of the local voltage deflection to the step current amplitude was taken to be the input resistance (To measure input resistance (sistance . For measistance .fR), maximum impedance amplitude (|Z|max), strength of resonance (Q) and total inductive phase (ΦL). To measure these a chirp stimulus, defined as a current stimulus with constant amplitude (peak to peak 100 pA) and linearly increasing frequency with time (0–15 Hz in 15 s), was injected in the compartment where the measurement was required. The local voltage response was recorded. To compute the impedance as a function of frequency, the Fourier spectrum of voltage response was divided with the Fourier spectrum of the current giving us the impedance profile as a complex quantity. The magnitude of impedance as a function of frequency was calculated using the following equation, To quantify the frequency dependence of neuronal responses, we used impedance based physiological measurements across the somatodendritic arbor Basak and Narayanan , 2020, NZ (f)) is the real part of the impedance profile and Im (Z (f)) is the imaginary part of the impedance profile and |Z (f) | is the magnitude of impedance. The maximum impedance amplitude was measured and the frequency at which it occurred was taken to be the resonance frequency. The strength of resonance was measured by taking ratio of the maximum impedance amplitude to the impedance amplitude at 0.5 Hz. For the phase related measures, the impedance phase profile was computed: In ϕ (f) is the phase as a function of frequency. The total inductive phase was measured by calculating the area under the positive portion of phase profile: In 2.2active synapses when the animal traverses within the place field of the postsynaptic neuron. The number of synapses was based on sensitivity analyses spanning different synapse numbers INMDCa =P 10.6, Na =P 1 and PK = 1. The ionic concentrations were set as, [Na]i = 18 mM, [Na]o = 140 mM, [K]i = 140 mM, [K]o = 5 mM, [Ca]i = 100 nM and [Ca]o = 2 mM. The magnesium dependence of the NMDAR current was calculated as follows MgBMg]o = 2 mM. The kinetics of the NMDAR current was determined by s (t): with [a is a normalization constant such that 0 ≤ s (t) ≤ 1, dτ is the decay constant, rτ is the rise time, with r =τ 5 ms and default d =τ 50 ms  was modeled in a manner similar to NMDAR with r =τ 2 ms and d =τ 10 ms. To normalize the unitary EPSP values associated with each synapse, we ensured that the attenuation along the dendritic cable did not affect the unitary somatic EPSP amplitude. Hence, the AMPAR permeabilities at the somato-apical trunk was tuned such that it produced a unitary somatic response of ~ 0.2 mV irrespective of the synaptic location  defined the center of the place field, f0 is the frequency of the cosine (8 Hz), σ is the standard deviation of the Gaussian (1 s). In our analyses, the virtual animal was assumed to traverse a linear arena at constant velocity, implying the equivalence of time and space as the independent variable in In In introducing experience-dependent asymmetry in place-field firing , 2000, wk (=25) governed the extent of asymmetry (In symmetry .dt = 25 μs), a random number was generated from a uniform random distribution spanning . An event corresponding to a presynaptic spike for this synapse was generated if this random number was less than dt × pre(tF) at a given time t. This process was independently repeated for each dt across each of the 80 synapses impinging on the postsynaptic neuron.Although each of the 80 synapses was driven by the Gaussian- or the Erlang-modulated cosinusoidal functions for the probabilistic generation of their respective pre-synaptic spike trains, they were independently generated, thereby ensuring that the input spikes are not temporally synchronous. Specifically, for a given synapse, at each integration time step  represents rectification to avoid negative firing rates, ξ(t) defined a GWN with zero mean and standard deviation σnoise. As the rectification governs the overall firing rate and not the noise term, this formulation allows for both negative and positive modulation of Fpre (t). The value of σnoise was increased to enhance the level of trial-to-trial variability, with Fpre (t) defined by a Gaussian- ) through convolution with a Gaussian kernel (σ = 200 ms). The maxima (Fmax) and the full-width at half maximum (FWHM) of the place-cell firing profile were employed as relative measures of place-field tuning sharpness. Specifically, high Fmax and low FWHM . Since the firing rate of the presynaptic neurons were modulated with a sinusoid of theta frequency , with space within the place field considered as the stimulus and the neuronal firing-rate considered the response. The aforementioned equivalence of time and space as the independent variable in Nloc) from the instantaneous firing-rate profile obtained for 30 different trials. To compute MI at these 20 locations, each location was subdivided into 4 bins, and the associated firing rate response was quantized into 20 bins. Mutual information between the spatial stimulus (S) and firing-rate response (F) was calculated at each Nloc as: To obtain location-dependent spatial information transfer, we computed mutual information in a piece-wise manner at 20 different locations  denoted mutual information between the response and the spatial stimulus at the ith location (i = 1… Nloc), and F defined the firing rate for S. The response entropy iH (F) was calculated as: where, ip  represented the conditional probability that the response was in the jth firing rate bin, given that the stimulus was in the kth spatial bin within the ith spatial location. pi (Sk) denoted the probability that the virtual animal was in the kth spatial bin within the ith spatial location, which was considered to follow a uniform distribution given the constant velocity assumption.In i (FH|S) in The noise entropy term iH (F|sk) represented the conditional noise entropy for the kth spatial bin within the ith spatial location, calculated as: where ip (j|SkF) denoted the conditional probability of the firing rate being in the thj bin given that the stimulus was in the Kth spatial bin within the ith location.where iI , the mutual information computed for the ith spatial location is different from I , the location-independent mutual information that could be computed for the entire place field . We employed the location-dependent formulation iI  to compare this with stimulus-specific information metrics.Together, this methodology of computing MI at several locations along the place field allowed us to assess spatial information transfer from all possible neural responses at that specific location. Note that 2.7The second set of metrics that we used to compute spatial information transfer was derived from stimulus-specific information (SSI), obtained for 30 different trials of the entire traversal spanning all spatial locations. SSI has been proposed as a measure of information in neuronal response about a particular stimulus, and conveys the average specific information spanning all responses to a particular stimulus. To calculate the SSI, the spatial stimulus and the firing rate response were segregated into 80 and 40 bins, respectively. The SSI was calculated using the expression given below : (26)SSIp (j|SiF) is the conditional probability of the firing rate being in the jth response bin given that the ith stimulus location was presented, and the specific information Isp (Fj) (where sp (Fj)  was compp (Fj) is the probability of the firing rate being in the thj response bin and p (i|FjS) defined the conditional probability for the stimulus in the ith bin given that the firing rate was in the jth response bin. The first term in H(S) and the second term represents the entropy of the stimulus distribution conditional on a particular firing rate response H(jS/F), providing Isp (jF = jH (S) − H(S/F) (Fj), and SSI constitutes the average reduction of uncertainty gained from all firing rate responses given a particular spatial location (Si). As Isp  would be defined as p Fj represents the probability of the firing rate lying in the jth response bin across the entire place field. As our focus in this study was on information metrics that were location-dependent (stimulus specific), we did not employ I , but have included the definition to illustrate the relationships and differences between Isp (jF), SSI (Si), Ii , and I .Here,  H(S/Fj) . Thus, s)I . Here, pIsp (jF) calculation was corrected using the Treves-Panzeri correction procedure  algorithm to arrive at a population of models that would satisfy the several biophysical heterogeneities , six signature intraneuronal functional maps (bAP), input resistance (Rin), resonance frequency (fR), maximum impedance amplitude (|Z|max), strength of resonance (Q) and total inductive phase (ΦL), each validated at three locations  and firing rate at the soma resulting from step current injections of 100 pA, 150 pA, 200 pA and 250 pA (4 measurements). Only the models that matched the bounds on these 24 measurements , and the p values are reported while presenting the results, or in the respective figure panels or associated captions. In qualitatively defining weak and strong correlations, we followed the nomenclature introduced by .We built a morphologically realistic, conductance-based model of a CA1 pyramidal cell, incorporating electrophysiologically characterized passive and active mechanisms . The mod3.1stratum radiatum where presynaptic afferent inputs impinged. These 80 synapses received independent presynaptic inputs governed by As a first step in evaluating the impact of heterogeneous ion channel combinations on sharp tuning of place-cell responses, we generated 12,000 random models by independent selection of parameter values from their respective uniform distributions . We randFmax and FWHM and asked if similar place-field tuning required similar parametric combinations  and the width of the firing rate profile (<2.5 s), and found 1024 of the 12,000 models (~8.5%) to satisfy these constraints . We pickinations –B. Consiinations , 2020, winations . Across Whereas place-field tuning constitutes one aspect of CA1 pyramidal neuron physiology, their well-characterized signature somatodendritic intrinsic properties form defining electrophysiological attributes. To match our model population with these signatures, we validated the 1024 sharply tuned models against 22 distinct electrophysiological measurements : each ofTogether, the unbiased stochastic search procedure provided us with a population of place-cell models that exhibited several signature electrophysiological properties, and manifested sharp place-field tuning in their firing rate profiles. We employed this population of place-cell models for assessing the impact of several biophysical and physiological characteristics on spatial information transfer within the place field.3.2e.g., The firing profile of a place cell within its place field represents a spatial tuning curve. For instance, in a symmetric firing profile e.g., , the spanoise (The introduction of input noise as additive Gaussian white noise (AGWN) manifested as trial-to-trial variability in the firing rate responses, enhanced the firing rate  and redunoise , resultenoise , and pronoise , theta pnoise  and the noise  of the pnoise  and mutunoise  for all We noted marked heterogeneity in spatial information, assessed with the SSI and MI profiles across models . Importanoise resulted in a marked reduction in spatial information across models, while still manifesting heterogeneity in spatial information transfer across the model population , SSIpeak/SSIslope was always lower than unity even with higher levels of trial-to-trial variability , this ratio was less than unity for low levels of trial-to-trial variability but transitioned to values higher than unity for higher levels of trial-to-trial variability  is exact in a single-compartmental model . HoweverAMPARP̄ and NMDARP̄) of the synapses according to the spatial location of the corresponding presynaptic CA3 neuron  in the frequency domain  that defined the 127 valid models and the 12 information transfer measurements , but the second peak was significantly larger than first peak for MI profiles  especially for low levels of trial-to-trial variability.We computed the asymmetric firing rate profiles for all valid models with low , medium noise; subpopulation with SSIdip > 0), but no such transition occurred in the MI profile  to be minimal on place cell properties  was the highest possible, as increases beyond that resulted in depolarization-induced block of action potential firing in several models. Experience-dependent asymmetry in firing profiles introduced asymmetry in the MI profiles, but not the SSI profile, even with MGWN-based trial-to-trial variability , and increased the FWHM values of both SSI . These observations offer a clear testable prediction that A-type potassium channels play a critical role in regulating spatial information transfer in hippocampal place cells. These results also establish a many-to-one mapping between the different ion channels and the efficacy of spatial information transfer, whereby different ion channels could contribute towards maintaining efficacious information transfer with heterogeneous contributions across neurons in the population. This many-to-one mapping provides a substrate for the expression of degeneracy where different combinations of ion channels could maintain similar functional outcomes in terms of spatial information transfer efficacy.In terms of information transfer, we found that the impact of knocking out individual channels was heterogeneous across the model population. There were models where the SSI  or MI F values is in SSI  and MI  sensors of animal location by spatially modulating their firing rate, transfer peak spatial information at the high-slope locations (and not at peak firing location) of the firing rate tuning curve within their place field. Importantly, we showed that there was significant heterogeneity across a population of models that received identical distributions of afferent synaptic patterns, owing to differences in ion channel composition of these models. The heterogeneity manifested quantitatively in terms of the amount of information transferred, and qualitatively in terms of how they responded to increases in the level of trial-to-trial variability. Specifically, with increases in trial-to-trial variability, whereas one subpopulation of models switched to transferring peak stimulus-specific spatial information at the peak-firing locations, another subpopulation continued to transfer peak information at the high-slope locations. These heterogeneities in spatial information transfer did not show strong relationships between heterogeneities in intrinsic or tuning properties of the models. We demonstrated the dependence of the spatial information transfer profile on the type of trial-to-trial variability, whereby activity-dependent variability had little impact on spatial information transfer compared to the significant reduction introduced by activity-independent variability.We demonstrated that hippocampal neurons, when they act as reliable , variability in neuronal responses under awake, behaving conditions is conceivably a mixture of both versions. Second, there are theoretical and electrophysiological lines of evidence for a critical role for asynchronous synaptic release, induced by active reverberation in recurrent circuits , on information transfer . Third, 4.3An important insight obtained from our study pertains to parametric degeneracy in effectuating spatial information transfer in place cells, with reference to ion channels and parameters that govern place cell biophysics and physiology ; Fig. 3.e.g., i.e., efficient rate coding) and concomitantly maintain intrinsic homeostasis, and ask if E–I–IE was essential to achieve these when the search space involves excitatory/inhibitory synaptic weights and ion channel conductances , such studies could also potentially assess the requirement of an intricate balance between mechanisms that promote and those that prevent dendritic spike initiation in maintaining efficient spatial information transfer.Degenerate systems show dominance of specific underlying parameters in regulating urements , 2019. Itransfer –13. Intetransfer , and forOur results proffer a testable prediction that experience-dependent asymmetry in place-field profiles do not markedly alter SSI. As experience-dependent asymmetry is considered to be predictive, reduction in spatial information transfer during the early parts of place-field firing would have rendered this predictive capability to be ineffectual. Our observations demonstrate that although the low values of slope during the early parts of firing profile reduces mutual information as a consequence of the asymmetry, stimulus specific information remains high. Further explorations could test this prediction on electro-physiologically obtained individual place cells transitioning with experience .relative perspective, with reference to fixed place-field widths (defined by preF(t)). Future analyses of spatial information transfer should relax these assumptions, and account for gradients in place-field width along the dorso-ventral axis (With specific reference to hippocampal place fields, future studies could explore the impact of systematic gradients in neuronal properties and ion channel expression along the dorsoventral, proximo-distal and superficial-deep axes of the hippocampus  on spatiral axis , along wral axis .Finally, the question on how spatial information transfer is regulated by activity-dependent plasticity and behavioral state-dependent neuromodulation of ion channels and receptors is critical in understanding the emergence of spatial information transfer in the context of novel place-field formation . Future From a broader perspective, our analyses here focused only on the relationship between spatial information transfer and spatially modulated neuronal firing rate. However, the hippocampal formation has been implicated in other functions, such as recognition, completion and separation of patterns, associative memory, and in engram formation . Future https://doi.org/10.1016/j.neunet.2021.07.026.Supplementary material related to this article can be found online at Supplementary Fig. S1 to Supplementary Fig. S22 & Supplementary Table S1