Influence of Connexin43 on the Bystander Effect Induced by Double Suicide Genes System in Vitro and in Vivo

Objective： To observe the influence of connexin 43 （Cx43） on the bystander effect induced by cytosine deaminase （CD） and herpes simplex virus thymidine kinase （HSV-tk） coexpression suicide genes system in human cholangiocarcinoma QBC939 cells and transplantation tumors in nude mice. Methods： In vitro, the CD＋tk＋ and CD＋tk＋Cx＋ cells were respectively treated with 5-fluorocytosine （5-Fc） and Ganciclovir （GCV）. The cytotoxic effect was evaluated by MTT method. In order to investigate the influence of Cx43 on the bystander effect, the size of transplantation tumors of the CD＋tk＋ and CD＋tk＋Cx＋ cells was measured before and after application of 5-Fc and GCV. Results： CD and tk genes were stably expressed in transfected QBC939 cells. The increased expression of Cx43 was determined by testing for the presence of Cx43 mRNA by RT-PCR and the presence of Cx43 protein by Western Blot in CD＋tk＋Cx＋ cells. The killing effect of 5-Fc and GCV on CD＋tk＋Cx＋ cells was more effective than that on CD＋tk＋ cells both in vitro and in vivo. Conclusion： Double suicide genes system CD/5-Fc＋tk/GCV could induce remarkable killing effect on cholangiocarcinoma cells in vitro and transplantation tumors in vivo. The cotransfection of Cx43 gene could enhance the bystander effect and hence the inhibition of carcinoma cells.

Radiation-induced Bystander Effect in Immune Response

Objective Since most reports on bystander effect have been only concerned with radiation-induced damage, the present paper aimed at disclosing whether low dose radiation could induce a stimulatory or beneficial bystander effect. Methods A co-culture system containing irradiated antigen presenting cells (J774A.1) and unirradiated T lymphocytes (EL-4) was established to observe the effect of J774A.1 cells exposed to both low and high doses of X-rays on the unirradiated EL-4 cells. Incorporation of 3H-TdR was used to assess the proliferation of the EL-4 cells, expression of CD80/86 and CD48 on J774A.1 cells was measured with immunohistochemistry and flow cytometry, respectively. NO release from J774A.1 cells was estimated with nitrate reduction method. Results Low dose-irradiated J774A.1 cells could stimulate the proliferation of the unirradiated EL-4 cells while the high dose-irradiated J774A.1 cells exerted an inhibitory effect on the proliferation of the unirradiated EL-4 cells. Preliminary mechanistic studies illustrated that the differential changes in CD48 expression and NO production by the irradiated J774A.1 cells after high and low dose radiation might be important factors underlying the differential bystander effect elicited by different doses of radiation. Conclusion Stimulatory bystander effect can be induced in immune cells by low dose radiation.

Bystander effect and abscopal effect in recurrent thymic carcinoma treated with carbon-ion radiation therapy:A case report

BACKGROUND Although the bystander effect and abscopal effect are familiar in medicine,they are relatively rare in clinical practice.Herein,we report the case of a patient who demonstrated an obvious bystander effect and abscopal effect response following carbon-ion irradiation for recurrent thymic carcinoma.CASE SUMMARY A 44-year-old female presented with shortness of breath.Eleven years prior,she was diagnosed with athymic tumor located in the anterosuperior mediastinum.She underwent extensive tumor resection,and the postoperative pathologic diagnosis was thymic carcinoma.She was administered 50 Gy/25 Fx of postoperative radiation.In 2019,she was diagnosed with a recurrence of thymic carcinoma,with multiple recurrent nodules and masses in the left thoracic chest and peritoneal cavity,the largest of which was in the diaphragm pleura proximal to the pericardium,with a size of 6.7 cm×5.3 cm×4.8 cm.She received carbonion radiotherapy.After carbon-ion radiotherapy treatment,the treated masses and the untreated masses were observed to have noticeably shrunk on the day of carbon-ion radiotherapy completion and on follow-up imaging.We followed the CARE Guidelines for consensus-based clinical case reporting guideline development and completed the CARE Checklist of information to report this case.CONCLUSION This report is the first of obvious abscopal and bystander effects following carbonion irradiation in a human patient,and further research is needed to better elucidate the mechanisms of bystander and abscopal effects.

Radiation Induced Bystander Effect: From in Vitro Studies to Clinical Application

In the past 20 years, the classic paradigm in radiobiology recognizing DNA as the main target for the action of radiation has changed. The new paradigm assumes that both targeted and non-targeted effects of radiation determine the final outcome of irradiation. Radiotherapy is one of the main modality treatments of neoplastic diseases with intent to cure, or sometimes to palliate only, thus radiation-induced non-targeted effect, commonly referred to as the radiation-induced bystander effect (RIBE) may have a share in cancer treatment. RIBE is mediated by molecular signaling from radiation targeted cells to their non-irradiated neighbors, and comprises such phenomena as bystander effect, genomic instability, adaptive response and abscopal effect. Whereas first three phenomena may appear both in vitro and in vivo, an abscopal effect is closely related to partial body irradiation and is a systemic effect mediated by immunologic system which synergizes with radiotherapy. From the clinical point of view abscopal effect is particularly interesting due to both its possible valuable contribution to the treatment of metastases, and the potential harmful effects as induction of genetic instability and carcinogenesis. This review summarized the main results of investigations of non-targeted effects coming from in vitro monolayer cultures, 3-dimentional models of tissues, preclinical studies on rodents and clinically observed beneficial abscopal effects with particular emphasis on participation of immunotherapy in the creation of abscopal effects.

Investigation of bystander effect in Molt-4 cells induced by ultraviolet ray

Objective： To find out whether ultraviolet ray, a kind of non-ionic ray, could cause the bystander effect, the UV exposed MOLT-4 cells had been investigated. Methods： Two experiment groups were carried out, in which cells were culture and treated at two concentrations： 2 × 10^5/mL and 5 × 10^5/mL. All other treatments were the same. Part of the cells was labeled with DID and exposed to UV ray for 40 s as effect cells; other cells was untreated as bystander cells. Then, the cells were co-cultured and harvested at 4 h interval over a period of 24 h. Annexin V-FITC/PI assay was used to evaluate the bystander effect in bystander cells co-cultured with effected cells. Laser confocal microscope method was used to observe the morphologic changes of the bystander cells. Results： The percentage of cells undergoing apoptosis in the bystander cells was increased over time compared with the control group. They were 6.84%, 8.09%, 9.88%, 17.64%, 17.43%, 30.99% and 37.93% respectively in 0 h, 4 h, 8 h, 12 h, 16 h, 20 h and 24 h. When observed by laser scanning confocal microscope, the bystander cells show some classic character of apoptosis such as chromosome condense, phosphatidylsedne transfer and formation of apoptotic bodies. Conclusion： Bystander effect is significant in un-irradiated bystander MOLT-4 cells when co-cultured with UV exposed cells.

Biophoton signaling in mediation of cell-to-cell communication and radiation-induced bystander effects

This paper presents a comprehensive overview of the historical trajectory and development in biophoton studies over the past 100 years,with a particular focus on the recent progress regarding the pivotal role of biophoton in mediating radiation-induced bystander effects(RIBE).The exploration of biophoton mystery starts from the initial observation of mitogenetic radiation and continues to develop to the contemporary science of biophotonics.The properties and underlying mechanisms of biophoton emission are described with illustrative examples from diverse biological systems such as plants,animals and humans.The conclusive evidence of cell-to-cell commu-nication facilitated by biophoton signaling is presented,followed by an elaborate interpretation of potential mechanisms through which biophoton mediates RIBE.The engagement of mitochondria and exosomes in this process is extensively clarified,by highlighting their significant roles in biophoton-mediated RIBE.The advances in biophoton research in respect of bystander response to ionizing radiation may offer profound insights into radiobiology and provide for possible future applications as well in radiation medicine and protection.

Pharmacokinetics and the bystander effect in CD：：UPRT/5-FC bi-gene therapy of glioma

Background Cytosine deaminase （CD） converts 5-fluorocytosine （5-FC） to 5-fluorouracil （5-FU） in CD/5-FC gene therapy, 5-FU will be mostly converted into nontoxic 13-alanine without uracil phosphoribosyltransferase （UPRT）. UPRT catalyzes the conversion of 5-FU to 5-fluorouridine monophosphate, which directly kills CD：：UPRT-expressing cells and surrounding cells via the bystander effect. But the pharmacokinetics and the bystander effect of CD：：UPRT/5-FC has not been verified in vivo and in vitro. Before the CD：：UPRT/5-FC bi-gene therapy system is used in clinical trial, it is essential to monitor the transgene expression and function in vivo. Thus, we developed a preclinical tumor model to investigate the feasibility of using ^19F-magnetic resonance spectroscopy （^19F-MRS） and optical imaging to measure non-invasive CD and UPRT expression and its bystander effect. Methods C6 and C6-CD：：UPRT cells were cultured with 5-FC. The medium, cells and their mixture were analyzed by ^19F-MRS. Rats with intracranial xenografted encephalic C6-CD：：UPRT glioma were injected intraperitoneally with 5-FC and their ^19F-MRS spectra recorded. Then the pharmacokinetics of 5-FC was proved. Mixtures of C6 and C6-CD：：UPRT cells at different ratios were cultured with 5-FC and the cytotoxic efficacy and survival rate of cells recorded. To determine the mechanism of the bystander effect, the culture media from cell comprising 25% and 75% C6-CD：：UPRT cells were examined by ^19F-MRS. A comparative study of mean was performed using analysis of variance （ANOVA）. Results ^19F-MRS on samples from C6-CD：：UPRT cells cultured with 5-FC showed three broad resonance signals corresponding to 5-FC, 5-FU and fluorinated nucleotides （F-Nuctd）. For the C6 mixture, only the 5-FC peak was detected. In vivo serial ^19F-MRS spectra showed a strong 5-FC peak and a weak 5-FU peak at 20 minutes after 5-FC injection. The 5-FU concentration reached a maximum at about 50 minutes. The F-Nuctd signal appeared after about 1 hour, reached a maximum at around 160 minutes, and was detectable for several hours. At a 10% ratio of C6-CD：：UPRT cells, the survival rate was （79.55±0.88）% （P 〈0.01）. As the C6-CD：：UPRT ratio increased, the survival rate of the cells decreased. ^19F-MRS showed that the signals for 5-FU and F-Nuctd in the culture medium increased as the ratio of C6-CD：：UPRT in the mixture increased. Conclusions ^19F-MRS studies indicated that C6-CD：：UPRT cells could effectively express CD and UPRT enzymes. The CD：：UPRT/5-FC system showed an obvious bystander effect. This study demonstrated that CD：：UPRT/5-FC gene therapy is suitable for 5-FC to F-Nuctd metabolism; and ^19F-MRS can monitor transferred CD：：UPRT gene expression and catalysis of substrates noninvasively, dynamically and quantitatively.

NO GOOD DEED GOES UNPUNISHED LOOPHOLES PERPETUATE ‘BYSTANDER EFFECT'

A public debate on the bystander effect,prompted by a viral video of a fatal traffic accident,has highlighted continued gaps in China’s'Good Samaritan'laws—legal protections for those who volunteer to help victims.The 94-second video from Zhumadian,Henan,recorded in April but circulated in mid-June,showed a

Emerging strategies for nerve repair and regeneration in ischemic stroke:neural stem cell therapy

Ischemic stroke is a major cause of mortality and disability worldwide,with limited treatment options available in clinical practice.The emergence of stem cell therapy has provided new hope to the field of stroke treatment via the restoration of brain neuron function.Exogenous neural stem cells are beneficial not only in cell replacement but also through the bystander effect.Neural stem cells regulate multiple physiological responses,including nerve repair,endogenous regeneration,immune function,and blood-brain barrier permeability,through the secretion of bioactive substances,including extracellular vesicles/exosomes.However,due to the complex microenvironment of ischemic cerebrovascular events and the low survival rate of neural stem cells following transplantation,limitations in the treatment effect remain unresolved.In this paper,we provide a detailed summary of the potential mechanisms of neural stem cell therapy for the treatment of ischemic stroke,review current neural stem cell therapeutic strategies and clinical trial results,and summarize the latest advancements in neural stem cell engineering to improve the survival rate of neural stem cells.We hope that this review could help provide insight into the therapeutic potential of neural stem cells and guide future scientific endeavors on neural stem cells.

Gap junctions enhance the antiproliferative effect of microrna-124-3p in glioblastoma cells

OBJECTIVE MicroR NA(miR NA)holds promise as a novel therapeutic tool for cancer treatment.However,the transfection efficiency of current delivery systems represents a bottleneck for clinical applications.Here,we demonstrate that gap junctions mediate an augmentative effect on the antiproliferation mediated by mi R-124-3p in U87 and C6 glioblastoma cells.METHODS The functional inhibition of gap junctions using either si RNA or pharmacological inhibition eliminated the mi R-124-3p-mediated antiproliferation,whereas the enhancement of gap junctions with retinoic acid treatment augmented this mi R-124-3p-mediated antiproliferation.A similar effect was observed in glioblastoma xenograft models.RESULTS More importantly,patch clamp and co-culture assays demonstrated the transmission of mi R-124-3p through gap junction channels into adjacent cells.In further exploring the impact of gap junction-mediated transport of mi R-124-3p on mi R-124-3p target pathways,we found that mi R-124-3p inhibited glioblastoma cell growth in part by decreasing the protein expression of cyclindependent kinase 6,leading to cel cycle arrest at the G0/G1phase;moreover,pharmacological regulation of gap junctions affected this cell cycle arrest.CONCLUSION Our results indicate that the″bystander″effects of functional gap junctions composed of connexin 43 enhance the antitumor effect of mi R-124-3p in glioblastoma cells by transferring mi R-124-3p to adjacent cells,thereby enhancing G0/G1cell cycle arrest.These observations provide a new guiding strategy for the clinical application of mi RNA therapy in tumor treatment.

Specific CEA-producing colorectal carcinoma cell killing with recombinant adenoviral vector containing cytosine deaminase gene

AIM: To kill CEA positive colorectal carcinoma cells specifically using the E coli cytosine deaminase (CD) suicide gene, a new replication-deficient recombinant adenoviral vector was constructed in which CD gene was controlled under CEA promoter and its in vitro cytotoxic effects were evaluated. METHODS: Shuttle plasmid containing CD gene and regulatory sequence of the CEA gene was constructed and recombined with the right arm of adenovirus genome DNA in 293 cell strain. Dot blotting and PCR were used to identify positive plaques. The purification of adenovirus was performed with ultra-concentration in CsCl step gradients and the titration was measured with plaque formation assay. Cytotoxic effects were assayed with MTT method, The fifty percent inhibition concentration (IC(50)) of 5-FC was calculated using a curve-fitting parameter. The human colorectal carcinoma cell line, which was CEA-producing, and the CEA-nonproducing Hela cell line were applied in cytological tests. An established recombinant adenovirus vector AdCMVCD, in which the CD gene was controlled under CMV promoter, was used as virus control. Quantitative results were expressed as the mean +/- SD of the mean. Statistical analysis was performed using ANOVA test. RESULTS: The desired recombinant adenovirus vector was named AdCEACD. The results of dot blotting and PCR showed that the recombinant adenovirus contained CEA promoter and CD gene. Virus titer was about 5.0 X 10(14)pfu/L(-1) after purification. The CEA-producing Lovo cells were sensitive to 5-FC and had the same cytotoxic effect after infection with AdCEACD and AdCMVCD (The IC(50) values of 5-FC in parent Lovo cells, Lovo cells infected with 100 M.O.I AdCEACD and Lovo cells infected with 10 M.O.I AdCMVCD were 】15000, 216.5+/-38.1 and 128.8+/-25.4 micromol.L(-1), P【0.001, respectively), and the cytotoxicity of 5-FC increased accordingly when the m.o.i of adenoviruses were enhanced (The value of IC(50) of 5-FC was reduced to 27.9+/-4.2 micromol.L(-1) in 1000 M.O.I AdCEACD infected Lovo cells and 24.8+/-7.1 micromol.L(-1) in 100 M.O.I AdCMVCD infected Lovo cells, P【0.05, P【0.01, respectively). The CEA-nonproducing Hela cells had no effect after infection with AdCEACD, but Hela cells had the cytotoxic sensitivity to 5-FC after infection with AdCMVCD (The IC(50) of 5-FC in parent Hele cells and Hela cells infected with AdCMVCD at 10 M.O.I was 】15000 and 214.5+/-31.3 micromol.L(-1), P【0.001). AdCEACD/5-FC system also had bystander effect, and the viability was about 30 percent when the proportion of transfected cells was only 10 percent. CONCLUSION: The recombinant adenovirus vector AdCEACD has the character of cell type-specific gene delivery. The AdCEACD/5-FC system may become a new, potent and specific approach for the gene therapy of CEA-positive neoplasms, especially colon carcinoma.

Typical Cell Signaling Response to Ionizing Radiation: DNA Damage and Extranuclear Damage

To treat many types of cancer, ionizing radiation （IR） is primarily used as external-beam radiotherapy, brachytherapy, and targeted radionuclide therapy. Exposure of tumor cells to JR can induce DNA damage as well as generation of reactive oxygen species （ROS） and reactive nitrogen species （RNS） which can cause non-DNA lesions or extracellular damage like lipid perioxidation. The initial radiation-induced cell responses to DNA damage and ROS like the proteolytic processing, as well as synthesis and releasing ligands （such as growth factors, cytokines, and hormone） can cause the delayed secondary responses in irradiated and unirradiated bystander cells through paracrine and autocrine pathways.

Altered Expression of Connexin-43 and Impaired Capacity of Gap Junctional Intercellular Communication in Prostate Cancer Cells

Connexin-43 (Cx43) expression in prostate cancer (PCa) cells and the potency of gap junctional intercellular communication (GJIC) in the cells were investigated, with an attempt to elu- cidate the reason why the so-called 'bystander effect' mediated by thymidine kinase (TK) suicide gene therapy on PCa cells is not of significance and to explore the role of GJIC in PCa carcinogenesis. mRNA and protein expression of Cx43 in a PCa cell line PC-3m was detected by re- verse-transcription polymerase chain reaction (RT-PCR) and strapt-avidin-biotin-enzyme complex (SABC) immunohistochemical staining, and inherent GJIC of PC-3m cells was assayed by scrape-loading and dye transfer (SLDT) assay. The expression of Cx43 in human normal and malig- nant prostate tissues was determined by SABC immunohistochemistry as well. It was found that Cx43 mRNA and protein expression in PC-3m cells was slightly reduced as compared with positive controls and the location of Cx43 protein was aberrant in cytoplasm rather than on membrane. As- sessment of paraffin sections demonstrated that the expression of Cx43 protein in PCa cells was ab- normally located and markedly diminished as compared with normal prostatic epithelial ones, dis- playing a negative correlation to the pathological grade (χ2=4.025, P<0.05). Additionally, capacity of inherent GJIC in PC-3m cells was disrupted, which was semi-quantified as (+) or (-). It was indi- cated that both down-regulated expression of Cx43 mRNA and aberrant location of Cx43 protein par- ticipated in the mechanisms leading to deficient GJIC in PC-3m cells. Lack of efficient GJIC is a molecular event, which may contribute not only to limited extent of 'bystander effect', but also to initiation and progression of prostatic neoplasm.

Research progress of radiation induced bystander and abscopal effects in normal tissue

Radiation-induced bystander effect(RIBE)and abscopal effect(RIAE)are non-target cellular responses outside the radiation field.It has been recognized that these effects are of great significance to both radiation protection of environmental low-dose radiation and clinical radiotherapy in which the anti-tumor abscopal effect is even beneficial to patients.However,the mechanisms of them are still obscure.This review briefly introduced the inflammatory signaling factors and immune regulation in RIBE in vitro and RIAE on normal tissues and organisms,and emphasized the genetic consequences of RIAE.Based on a large number of investigation results,we suggest that it’s time to incorporate RIBE and RIAE into the concept of“classic”radiation biology.

The role of gap junctions in cell death and neuromodulation in the retina

Vision altering diseases,such as glaucoma,diabetic retinopathy,age-related macular degeneration,myopia,retinal vascular disease,traumatic brain injuries and others cripple many lives and are projected to continue to cause anguish in the foreseeable future.Gap junctions serve as an emerging target for neuromodulation and possible regeneration as they directly connect healthy and/or diseased cells,thereby playing a crucial role in pathophysiology.Since they are permeable for macromolecules,able to cross the cellular barriers,they show duality in illness as a cause and as a therapeutic target.In this review,we take recent advancements in gap junction neuromodulation(pharmacological blockade,gene therapy,electrical and light stimulation)into account,to show the gap junction’s role in neuronal cell death and the possible routes of rescuing neuronal and glial cells in the retina succeeding illness or injury.

The use of suicide gene systems in vascular cells in vitro

To investigate the efficiency of suicide gene systems on vascular cells, HSV-tk/GCV and EC-CD/5-FC systems were established on vascular endothelial cells in vitro by retroviral transduction. Both modified cell lines were highly sensitive to prodrugs, the IC50 for GCV was less than 0.4 μM, and IC50 for 5-FC was less than 75 μM,while the parental endothelial cells were insensitive even at the highest concentrations of prodrugs in this experiment. Mixed cellular assay showed that significant bystander effect was exhibited in modified endothelial cells.When only 10% or 30% of the mixed cells were tk positive and exposed to 20 μM GCV for 6 days, more than 60% or 90% of the whole population was killed. Similar result was also found in CD positive cells. These results indicated that both HSV-tk/GCV and EC-CD/5-FC systems could efficiently suppress endothelial cell growth in vitro.

Recombinant adenoviral vector expressing the tumor necrosis factor-related apoptosis-inducing ligand gene suppresses human pancreatic cancer growth

Objective: To investigate the antitumor effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene transfection mediated by adenovirus into human pancreatic carcinoma cell line Panc-1, and the mechanisms involved in this effect. Methods: TRAIL gene was transfected into pancreatic cancer cell line Panc-1 by an adenovirus vector (Ad-TRAIL). Level of TRAIL mRNA expression was determined using RT-PCR, and TRAIL protein synthesis was evaluated with Western blot. Cell-growth activities were determined by MTT assay. The bystander effect was observed by co-culturing the Panc-1 cells with the transfected TRAIL gene at different ratios. Apoptosis in pancreatic cancer cells was detected by flow cytometry. Procaspase-8 and procaspase-3 were determined by Western blot. Results: The stable overexpression of TRAIL was de-tected in Panc-1 cells transfected by Ad-TRAIL. Ad-TRAIL significantly inhibited of cell viability of Panc-1 cells. Furthermore, co-culture of cancer cells transfected with TRAIL with that nontransfected resulted in the cell death of both cells by bystander effect. Moreover, the percentage of apoptotic cells was significantly higher in the Ad-TRAIL-treatment group compared to the control groups (P < 0.01). And there was a diminished amount of procaspase-8 and procaspase-3 after infection with Ad-TRAIL. Conclusion: The overexpression of TRAIL gene in Panc-1 cells by Ad-TRAIL exerts its antitumor effects, and the mechanisms involved in this effect may be proapoptosis and bystander effect.

The impact of radicals in cold atmospheric plasma on the structural modification of gap junction:a reactive molecular dynamics study

Cold atmospheric plasmas are currently gaining increasing attention for cancer therapy.However,very limited studies regarding the interaction mechanisms between plasma species and tissues are available.We report the interaction of plasma produced species(^(*)OH and HO_(2)^(*))with gap junction by employing reactive molecular dynamics simulations.Our results indicate ^(*)OH and HO_(2)^(*) radicals can chemically react with N-terminal of gap junction resulting in its structural damage.There are two breaking mechanisms being identified:C-N peptide bonds and C-C bonds can be damaged by ^(*)OH and HO_(2)^(*) radicals,respectively.Our findings could be particularly important for understanding the plasma-generated reactive species triggering bystander effect based on gap junction intercellular communication.

Overcoming tumor antigen heterogeneity in CAR-T cell therapy for malignant mesothelioma(MM)

Malignant mesothelioma(MM)is a rare,aggressive solid tumor with limited therapeutic options and poor therapeutic response.The role of immunotherapy in MM is now well established and therapeutic options,such as checkpoint inhibitors,are increasingly being approved.Chimeric antigen receptor(CAR)-T cell therapy is successfully implemented in several hematologic cancers,but currently has inadequate effect in solid tumors,owing to several limitations,such as trafficking and infiltration,limited T cell persistence and exhaustion,the immunosuppressive TME and tumor antigen heterogeneity.The lack of uniform and universal expression of tumor-associated antigens(TAAs)on tumor cells,as well as TAA heterogeneity following tumor editing post-therapy,are issues of significant importance to CAR-T cell and associated antigen-targeting therapies.Our review discusses the concept of tumor antigen heterogeneity in MM,the consequences for CAR-T cell therapies and the strategies to overcome it.