The coronavirus disease 2019 (COVID-19) first came to the Unites States in January 2020. Though adult and pediatric vaccines became available to the public, vaccine uptake among youth and particularly younger children has been gradual. This explanatory study aimed to better understand parents’ attitudes and beliefs of the pediatric COVID-19 vaccine and the barriers and facilitators to vaccine uptake in a rural community through a brief, online demographic survey, and in-depth qualitative interviews. Forty-one in depth interviews were conducted with parents (31-English and 10-Spanish-speaking) residing in rural and frontier counties in Colorado between September 2021 and February 2022. Six emergent themes related to COVID-19 pediatric vaccine uptake were identified among the population. These themes spanned the three levels of influence in the Social Ecological Model . The six themes were identified as such; 1) Vaccine accessibility was associated with pediatric COVID vaccine uptake in rural communities, 2) Previous pediatric vaccine behaviors were not associated with COVID-19 pediatric vaccine uptake, 3) Perceived health status of a child or family member influenced pediatric COVID-19 vaccine uptake, 4) COVID-19 health seeking behaviors, like COVID pediatric vaccine uptake, are influenced by an individual’s prosocial or individualistic perspectives, 5) Child autonomy and “age of consent” frames vaccine decision making behaviors in parents, and lastly 6) Social networks impacted COVID-19 pediatric vaccine decision making. These findings inform next steps for COVID-19 pediatric vaccine uptake including targeted and tailored messaging for communities (cues to actions), engaging youth stakeholders, and identifying trusted sources to build rapport and trust between health professionals and community members. The growing vaccine hesitancy among parents has serious implications for disease eradication and future viral outbreaks. Understanding the perceived barriers and facilitators to pediatric vaccine uptake is important to maintain the health of our youth and communities. The first case of coronavirus disease 2019 (COVID-19), the illness caused by the severe acute respiratory syndrome coronavirus 2 virus (SARS-CoV-2), was reported in the U.S. on January 20, 2020 . COVID-1nd, 32% of children aged 5–11 are fully vaccinated against COVID-19, while nearly 61% of youth aged 12–17 are fully vaccinated [On October 29, 2021, the United States Food and Drug Administration (FDA) authorized the emergency use of the Pfizer-BioNTech COVID-19 vaccine for children five years of age and older. Over seven million children have tested positive for SARS-CoV-2  with thiccinated . These dccinated . While tThis study utilized an explanatory study design, collecting both quantitative and qualitative data, with a larger emphasis on the qualitative findings. The quantitative component consisted of a brief survey with a demographic questionnaire and questions relating to adults’ attitudes and beliefs surrounding the COVID-19 vaccine and virus. Following the brief questionnaire, semi-structured qualitative interviews were conducted to further explore these phenomena.Approval for this study was obtained from the Colorado Multiple Institutional Review Board (COMIRB # 21–3731). Participants were recruited from nine rural and frontier (fewer than six people per square mile)  southernAfter completing an online consent form through Research Electronic Data Capture (REDCap) , eligiblFollowing completion of the brief survey, semi-structured qualitative interviews averaging 30-minutes in length were conducted in English or Spanish through a secure, online video platform. A semi-structured interview guide was developed using the Health Belief Model  (HBM) anEnglish interviews were conducted by members of the study team trained in qualitative interviewing  with Spanish interviews conducted by Spanish-speaking, trained interviewers from the community. All Spanish speaking interviewers attended a qualitative interviewing training conducted by members of the research team. Interviews were audio-recorded, transcribed verbatim, translated (if conducted in Spanish), and stored on a password-protected hard drive. After completion of the interview, participants were compensated with a $20 electronic gift card.The qualitative portion of this study met the four criteria for trustworthiness of qualitative data: credibility, transferability, dependability, and confirmability . Credibia priori codes related to the HBM and the SEM as well as inductive coding to explore and discover emergent themes outside of these two models’ constructs. Sampling decisions were guided by saturation that was reached at 41 interviews, after no novel themes or ideas emerged from participants in either the English or Spanish-speaking subgroups. Each transcript was coded separately by two research team members (RL and MT), and 15% of the transcripts were independently coded by a third researcher (DL) to establish consensus on coding structure and theme development. This third coder summarized themes and subthemes separate from the other two coders with the three coders then meeting to discuss themes and to reach consensus if there were differences in codes or emerged themes. To further explore these emergent themes, the interviews were grouped by vaccinated (76%) and unvaccinated (24%) and Spanish- (76%) and English-speaking (24%) parents. Sub analyses were conducted amongst these subgroups with an intention to compare and contrast the differences in these subgroups. All methodology was reviewed and approved by an investigative team trained in both quantitative and qualitative research methodologies, who were not involved in data collection or interview coding processes.Qualitative data analyses followed best practices for qualitative research and utilized data-driven and theory-informed approaches. Transcripts were reviewed multiple times to develop familiarity with the data, and then qualitative analysis of the transcripts was conducted using ATLAS.ti software. Constant comparison methodology was utilized to systematically reduce the data to codes . Themes Questionnaire data was exported from REDCap, and all quantitative analyses were conducted using Statistical Analysis Software version 9.4. Descriptive statistics were run to understand the demographics of the sample, including age, gender, number of children, race, ethnicity, household income, political affiliation, education level, and COVID-19 vaccination status. Each response for the four PACV-4 items was scored as hesitant or non-hesitant, and parents were identified as vaccine hesitant if they endorsed a hesitant response for ≥2 items. This data was used to create a dichotomous PACV-4 variable, identifying parents as vaccine hesitant or non-vaccine hesitant. Due to the relatively small sample size, a Fisher’s exact test was used to examine the association between parental vaccine hesitancy via the PACV-4 and parental COVID-19 vaccination status.A total of 41 parents were interviewed (31 English-speaking and 10 Spanish-speaking) of which 38 (92.7%) were female with an average age of 38.5 years old. Similar to the ethnic breakdown of the rural community, 21 (51.2%) of our parents were Hispanic or Latino. Additionally, 33 (82.5%) reported an income less than $75,000, and over 36 (87.6%) had obtained a high school degree or higher. A Fisher’s exact test was used to examine the association between parent attitudes about childhood vaccines (PACV-4) and parental COVID-19 vaccination status with a significant association identified (p < 0.001), with 16 (39%) parents identifying as vaccine hesitant. Additional demographics are described in Cross-tabulations and findings presented in Tables The HBM and SEM provided the guiding frameworks for our semi-structured interview guide. We were most interested in overlaying how rural parents utilized their interpersonal, intrapersonal, and community networks to make COVID-19 pediatric vaccine and virus decisions through the lens of the HBM’s constructs; perceived susceptibility, severity, benefits, barriers, cues to action, and self-efficacy.When initially rolling out FDA-approved vaccines for adults 18+, availability in rural Colorado was limited. At the time of these interviews, the availability of the vaccine was not cited as a significant barrier. However, perceived barriers related to access of the vaccine included: lack of knowledge regarding where and how to find a preferred vaccine brand, time off from work to account for common adverse side effects, time spent scheduling appointments and traveling to clinics, and distance from home/work to a vaccine location. Despite these barriers, high self-efficacy was perceived in vaccinated parents on obtaining a pediatric vaccination.Numerous parents previously vaccinated their children on a routine childhood vaccine schedule but elected to not vaccinate against COVID-19. However, these individuals displayed higher perceived severity and viewed the COVID-19 vaccines as “different” or “riskier” than other established and well-known pediatric vaccines. Parents reported heightened concerns regarding the COVID-19 vaccines including the belief that vaccines can alter DNA, the vaccine came out “too fast”, uncertainty of the vaccine ingredients, mistrust of the vaccine effectiveness due to variants, and unknown short- and long-term outcomes of the vaccine.Confidence in the health of the family unit influenced vaccine uptake. Numerous parents held the belief that those who are healthy , participate in regular physical activity, eat healthy, and have a strong immune system) are less susceptible to SARS-CoV-2. The lack of perceived susceptibility due to lifestyle choices negatively influenced pediatric vaccine uptake. Parents also related their health status to a decreased likelihood of hospitalization or severe lasting impacts, suggesting low perceived severity, if infected with the virus and therefore viewed the vaccine unnecessary due to their or their child’s good health. Conversely, parents viewed the pediatric vaccine as an additional component in increasing their health confidence and overall health status.Parents fell on a spectrum from prosocial to individualist beliefs. Parents varied among their definitions of “health responsibility”. Several felt that to best protect themselves and their family, an individualistic approach to health was necessary while others demonstrated a prosocial view towards the health of others with voluntary behaviors intended to benefit the community . Parents who held individualistic views regarding the COVID-19 pediatric vaccine displayed a heightened level of vaccine hesitancy and concerns. Conversely, parents with a prosocial attitude indicated higher readiness and likelihood to vaccinate their children once eligible.Parents allowed children to influence vaccine behavior and decision-making. Children aged 12–17 were viewed as old enough to decide to vaccinate or not and to weigh the potential consequences of these decisions. Numerous parents perceived these conversations related to a child’s body autonomy as an important learning opportunity for the child. While these discussions were not the sole deciding factor in parents’ decision to vaccinate their children, this concept of childhood body autonomy heavily influenced decision making. Children aged 11 and younger were perceived as having fewer capabilities to make an informed decision for themselves. Many parents felt that until a child could fully consent, they were uncomfortable with vaccinating their child. These beliefs were highest among parents with children aged five or younger.Parents relied heavily on their social networks to inform vaccine decision-making and sought information from a variety of trusted sources. Parents brought their own perceived knowledge, attitudes, and skills to the decision-making process and leveraged their interpersonal, organizational, and community social networks. Most parents viewed their interpersonal social networks, such as personal connections with vaccinated healthcare workers, as a valuable and trusted information source. Parents described triggers to their decision making (cues to action) such as a family member or friend having a positive vaccination experience and/or a provider’s recommendation to vaccinate themselves of their children.Vaccinated and Unvaccinated Parents. Notable differences existed between vaccinated and unvaccinated parents. Vaccinated parents spoke more frequently about their responsibility to protect their children, the value of science, and the benefits of vaccinations. Vaccinated parents often mentioned external cues to actions like provider’s recommendations or stories from personal healthcare connections of the severity of contracting the COVID-19 virus influencing their decision-making processes. Although these parents wanted more information surrounding childhood vaccines, vaccinated parents tended to trust the public health institutions such as the CDC, WHO, and were more inclined to seek trusted scientific journals. Additionally, vaccinated parents documented less perceived barriers to the COVID-19 pediatric vaccination and increase perceived benefits of the vaccine.Unvaccinated parents were more likely to trust interpersonal connections in their decision making . Unvaccinated parents often cited frustrations with inconsistent messaging surrounding the COVID-19 vaccines and virus. These parents were highly focused on perceived associated risks of the vaccine  and downplayed the protective benefits of vaccines and described less perceived benefits than vaccinated parents.English speaking and spanish speaking parents. Similarities between these groups involved perceived benefits of the vaccine, concerns regarding short and long-term effects of vaccines, and a need for more consistent and robust information regarding COVID-19 vaccine and the virus. The Spanish-speaking parents reported more trust in their local medical community and cited similar providers whom they felt trustworthy and knowledgeable than English-speaking parents. Spanish-speaking parents also requested a community forum with chosen health care professionals to discuss the associated risks and benefits of the pediatric vaccine. Spanish-speaking mothers also mentioned their trust in their husbands when making medical decisions and the importance of a community-centered approach for eliminating the virus.“Vaccination boosts the body’s natural defenses against disease to keep you free of infection” and “Vaccination helps you take personal control of your life and allows you to be free to live a healthy life” for communities rooted in liberty (27).There were multiple key themes which influenced pediatric vaccine uptake among our rural sample. Individual levels of influence were informed by parents’ perceived susceptibility based on their overall general health status. While a recent global survey, where almost half of American adults resided in a rural community, indicated that general health was not a significant predictor in COVID-19 vaccine uptake,  our respAdditional individual influences on vaccine uptake centered on beliefs each parent held on the continuum from prosocial to individualism. Parents who described more individualistic beliefs reported that the governing bodies should not mandate the vaccine in school-aged children and each community member should “take ownership of their own health”. This finding is consistent with extant literature in which 82% of rural parents opposed a vaccine mandate for eligible students, compared to 57% of urban parents . MoreoveIn addition to the individualist beliefs parents are bringing to the vaccine decision making process, parents in our study also encouraged their children to participate in the vaccine decision-making process. Particularly, our findings suggest parents with children aged 12–17 desire to engage their child in the healthcare decision-making process. Parents reported children refused the vaccine for various reasons . This shift of decision making responsibility may be in part due to omission bias, where parents consider the risks of vaccinating to outweigh the risks from vaccine refusal . Many paIn addition to the beliefs that each parent and child brings to the decision process, other influences are interacting. Our sample indicated a lack of perceived benefits of the pediatric vaccine  as well as lower perceived severity , and perceived susceptibility . Rooted in these perceptions, many parents remain hesitant to vaccinate their children against COVID-19 despite 79% of children in the U.S. vaccinated with the combined 7-vaccine series by 24 months of age . In part“very” or “somewhat concerned” regarding serious side effects of the vaccine (high perceived severity of the vaccine) [definitely not” vaccinate their children 5–17 years of age [Parents across the U.S. have expressed concerns related to vaccine safety and potential side effects of the pediatric COVID-19 vaccine, particularly for younger children . A survevaccine)  with 50%s of age . Similars of age . In a res of age  with cles of age . Our SpaOther trusted community experts were schools, particularly teachers and school nurses. In a nationwide survey of parents, schools that encouraged childhood vaccination saw increased vaccine uptake, particularly among children aged 12–17 . DespiteRural communities have historically been faced with barriers surrounding availability of healthcare services . AlthougLimitations of this study include the different stages of federal vaccine approval throughout the interview timeframe. The opinions expressed by parents could have been impacted by FDA approval of the pediatric vaccine for their children’s ages, yet this is unavoidable due to the ever-changing landscape of the pandemic. Additionally, our sample included a higher number of vaccinated than unvaccinated parents, potentially suggesting more favorable opinions towards vaccinations, in general. While we aimed to have a sample representative of the population, the polarizing nature of COVID-19 created challenges in recruiting non-vaccinated individuals and 33 of the parents had received at least one dose of the COVID-19 vaccine at the time of the interview. Additionally, an oversampling of highly educated parents occurred with 84% of our sample reporting having at least some college or trade school, while only 25% of the rural population aged 25+ have completed some college . EducatiThe landscape of COVID-19 is constantly changing, with new COVID-19 variants emerging. Information and strategies to reach vaccine hesitant populations are critical. Ensuring that access to vaccines is equitable in low-resourced communities is an important step in increasing vaccination rates. Identifying trusted community partners to advocate and spread awareness of the benefits of vaccines, while inviting youth and unvaccinated individuals to partake in the conversations, could be effective in increasing vaccine uptake and uniting communities to address public health initiatives. COVID-19 has highlighted the need for increased relationship building within communities, particularly in rural areas where multi-generational families often reside. Generating buy-in of health initiatives among younger individuals could impact the amount of knowledge in familial units.S1 FileSeme-structured interview guide used among interviewee participants.(PDF)Click here for additional data file.


Localization of small-sized pulmonary nodules is challenging during video-assisted thoracoscopic surgery. Several preoperative strategies have been developed to mark these targets. We describe our localization strategy using a preoperative computed tomography-guided near-infrared dye marking. Minimally invasive surgery, including video-assisted thoracoscopic surgery (VATS), has been ideal for the resection of small nodules because it results in minimal postoperative morbidity and mortality, less pain, and a better quality of life than with an open thoracotomy , 2.Localizing small-sized pulmonary nodules is challenging during VATS. Thus, several preoperative strategies have been developed to mark these targets , 4. InadWe present a case of a no smoker 62-year-old man diagnosed with two metachronous left lung metastases (15 and 12 mm) 20 months after surgery for colorectal adenocarcinoma . After dWe describe and discuss the technical steps of our localization strategy of nodules localization by using a preoperative CT-guided near-infrared dye marking (NIDM).Written informed consent was obtained from the patient. The first step of the surgery consists of a preoperative CT-guided injection of indocyanine green (ICG). ICG is powder at 50 mg/10 ml and diluted in 20 ml of human albumin 20% (2.5 mg/ml). A 1 ml syringe was filled with the ICG/albumin mixture and connected to a CT-guided percutaneous 21-gauge needle, and the needle lumen was filled with the marking solution prior to the marking procedure. After local anesthesia with lidocaine, a marking solution (1 ml) was injected near the nodules . CT scanDetection of small pulmonary nodules has increased with the widespread use of chest CT screening , 9. SimiVATS is the minimally invasive procedure of choice in cases involving unsuccessful diagnosis through percutaneous transthoracic needle biopsy or the need for the resection of pulmonary nodules. However, two significant limits are strictly linked to the VATS procedure: the inability to visualize or palpate the target lesions. Furthermore, ground-glass nodule lesions do not alter the surface of the visceral pleura, and the elevation of tumors cannot be perceived in the deflated lung during VATS .To overcome these problems, several localization methods have been developed to mark small-sized nodules before VATS –7. The mKleedehn et al. reported that hook–wire needle localization has major complications like pneumothorax and pulmonary hemorrhage. The consequent dislodgement of the wire may make the surgical resection even more complex . The injOur technique presents several specific advantages. First is the reproducibility and the absence of specific skills for an interventional radiologist who usually performs percutaneous TC lung biopsies. Second, the risk of radiation exposure is not present. Furthermore, target localization is usually not complicated by displacement or diffusion. ICG injection is well tolerated by the patient, which can freely move without the risk of any landmark dislocation. By the injection of other radiotracers like methylene blue or lipiodol is not easily identified the precise resection margin while has been described that dyes rapidly diffuse and do not remain in the target lesion. Finally, NIDM does not need immediate surgery because the marking persists from hours to days .On the other side, NIDM presents some disadvantages. In cases of severe pulmonary emphysema, there is concern that liquid markers do not form a distinct spot but rather diffuse into pulmonary cysts; therefore, ICG may not be ideal in such cases. Moreover, the VATS equipment required to visualize ICG-FL is expensive and is available only in a limited number of institutions. The above-described procedure is the first European experience, having been exclusively described in oriental series , 4. The In our experience, ICG diluted human albumin effectively overcame the diffusion and limitations of most common markers, facilitating accurate localization and resection of pulmonary nodules during VATS. Further investigation, including larger series, could confirm our initial report.


The spatial organization of chromosomes is involved in regulating the majority of intranuclear processes in higher eukaryotes, including gene expression. Drosophila was used as a model to discover many transcription factors whose homologs play a key role in regulation of gene expression in mammals. According to modern views, a cohesin complex mostly determines the architecture of mammalian chromosomes by forming chromatin loops on anchors created by the CTCF DNA-binding architectural protein. The role of the cohesin complex in chromosome architecture is poorly understood in Drosophila, and CTCF is merely one of many Drosophila architectural proteins with a proven potential to organize specific long-range interactions between regulatory elements in the genome. The review compares the mechanisms responsible for long-range interactions and chromosome architecture between mammals and Drosophila. Drosophila based on the available models and experimental data.The sets of transcription factors (TFs) assembled on regulatory genome elements, enhancers and promoters, determine cell specialization in higher eukaryotes. Promoters are up to 150 bp in size and contain motifs for DNA-binding TFs that open chromatin and ensure recruitment of general TFs that determine start site and direction of transcription . EnhanceWhole-genome Hi-C studies in various organisms and cell lines showed that chromosomes are divided into large domains wherein contacts between distant chromatin regions occur at similar frequencies . On averDrosophila, TAD boundaries most often coincide with housekeeping gene clusters and lack sites to preferentially bind a single architectural protein, such as CTCF in mammals  were identified as new regulatory elements in d by TEs . Only paMany modern studies have shown that at least some functionally interacting enhancers and promoters do not form stable contacts . To explThe cohesin complex is highly conserved in eukaryotes. Its main function is to hold sister chromatids together during mitosis and meiosis . There aIn mammals, the cohesin complex, together with CTCF, is involved in organizing long-range enhancer–promoter interactions and forming the TAD boundaries in interphase nuclei . A charaA common model suggests that the cohesin complex, after binding to chromatin, begins DNA extrusion with the formation of a chromatin loop. CTCF-binding sites block its progress and fix the chromatin domain boundaries . When CTDrosophila initially revealed the role of the cohesin complex in regulating long-range enhancer–promoter interactions (Drosophila remains unstudied how the inactivation of the cohesin complex affects the formation of TAD and long-range interactions between regulatory elements. Drosophila CTCF (dCTCF) lost some of its functions, and ctcf null mutants are consequently viable , Pita, and CTCF. For example, the Mcp insulator harbors the binding sites for Pita and dCTCF, and the Fub insulator has one binding site for dCTCF, one for Pita, and two for Su(Hw)  , 2020. Ise assay .Drosophila C2H2 architectural proteins also work cooperatively to form active promoters. A large group of architectural C2H2 proteins, including M1BP, CTCF, Su(Hw), and Pita, ensures the promoter localization of the TF CP190  conserved protein Ying Yang 1 YY1,  has fourDrosophila lines (Drosophila. It is very likely that there are many mammalian C2H2 architectural proteins, and most of them have not yet been studied. It can be assumed that architectural C2H2 proteins in cooperation with associated chromatin proteins can initially form chromatin loops as a result of the restriction of DNA extrusion by cohesin and stabilize the newly created chromatin loops using protein-protein interactions between homodimerizing domains (The views of chromosome architecture and long-distance enhancer–promoter interactions in higher eukaryotes substantially changed with recent development of more precise techniques . It becola lines . To func domains . New stu domains  has show


Chemotherapeutics drugs play a pivotal role in the treatment of cancer. However, many issues generate by chemotherapy drugs, including unfavorable harm to healthy cells and multidrug resistance (MDR), persist and have a negative impact on therapeutic outcomes. When compared to monotherapy, combination cancer therapy has many advantages, like improving efficacy through synergistic effects and overcoming drug resistance. Combination treatment may comprise several chemotherapeutics drugs and combinations of chemotherapeutic drugs with some other therapeutic options such as surgery or radiation. Cancer treatment that utilizes co-delivery strategies with siRNA and chemotherapeutic drugs has been shown to have highly effective antitumor effects in the treatment of many cancers. However, the highly complex mechanisms of chemotherapeutic drugs-siRNA pairs during the co-delivery process have received little attention. The ideal combination of chemotherapeutic drugs with siRNA is very crucial for producing the desirable anticancer effects that would greatly enhance therapeutic efficiency. This review puts an emphasis on the logic for choosing suitable chemotherapeutic drug-siRNA combinations, which may open the way for the co-delivery of chemotherapeutic drugs and siRNA for treating cancer in the clinic. This review summarizes recent breakthrough in the area of diverse mechanism-based chemotherapeutic drugs-siRNA combinations in cancer treatment.  •Chemotherapeutic drugs in combination with siRNAs.•Overcoming multidrug resistance using the combinatorial approach.•Use of Combinatorial therapy to promote apoptosis.•Extracellular barriers to RNAi mediated siRNA therapy.•Nanocarrier chemotherapeutics-siRNA co-delivery system. Cancer progression is characterized by dynamic genetic alterations and a complex system of interactions between cancer cells, which include a range of distinct cellular processes that form tumors. The complexity of signaling pathways, associated with various mechanisms to avoid programmed cell death, makes healing cancer a difficult task . TherefoChemotherapy is one of the most popular therapeutic approaches used in cancer treatment today. The first chemotherapeutic agent given for the treatment for cancer was a compound called nitrogen mustard during World War II, which was later categorized as DNA alkylating agent. Following the success of the former in the treatment of lymphoma, several other chemotherapeutic agents with similar mechanisms that interfere with cellular or metabolic processes have been also identified . The cheRNA interference (RNAi) technology as shown in c and regulates survival of cancer cells over apoptosis through the non-pump mechanism, could improve chemotherapeutic efficacy  can be selected . The benOncology research is currently focused on identifying and targeting specific genetic changes in cancerous cells, probably leading to cancer and understanding of genetic mutations in cancerous guides selection of chemotherapeutic drugs alongwith a development of advanced non-traditional gene-based therapeutics . The non3Combination therapies based on chemotherapeutic agents and RNAi technology drastically enhance cancer treatment efficacy by targeting distinct cellular signal pathways, which enhance the genetic hurdles for cancerous cells mutations. As a result, this strategic approach can retard the tumor adaptation system thereby improving therapeutic efficacy. In order to combine chemotherapy and RNAi technology, it is necessary to select a suitable gene target. The following genes have been targeted using this combinatorial therapy: (1) genes which play a role in cancer drug resistance  (2) genes responsible for survival of tumors  (3) genes expressed selectively in cancerous tissues  .3.1A single drug therapy is generally not effective in treating cancer because of the heterogeneity and complexity of the mechanisms involved in the regulation of the progression of the diseased state. Owing to its complex pathophysiology, cancer is prone to multidrug resistance (MDR), which is characterized by concomitant resistance to more than one drug . Decreas3.2Drug resistance in cancer patients could also be attributed to reduced apoptosis  in cancer cells which in turn reduces the drug potency and restriction in its full use. Reduction of apoptosis enables survival of cancer cells and their accentuated resistance towards cellular stress , leads to a progression of cancer. Transformation of the apoptotic signaling pathway could be counted for the resistance of cancer cells towards chemotherapy, accentuated expression of anti-apoptotic genes (B-cell CLL/lymphoma 2 (BCL2), BCL2-like 1 (BCLXL), Livin/ML-IAP/KIAP), X-linked inhibition of apoptosis, and survivin represent the members of the inhibitor of apoptosis protein family , KUNZE . BCL2 an3.3Cancer cells are supposed to vanquish the immune system of the patient, and inadequacy of chemotherapy, radiation, and surgery add to the distress of the patients. At the genetic level, many genes play a vital role in protecting cells from cancer, and any mutation in such genes acts as a setback in the management of cancer. Now it is well established that genes related to immune response could be a potential target, and reversal of immunosuppression mediated by cancer cells, could be an efficient approach in the treatment of cancer . The app3.4. Several experiments have shown that, in addition to its role in angiogenesis, VEGF can inhibit cell apoptosis by stimulating specific kinases or signaling pathways, such as phosphoinositide 3-OH kinase, p44 protein kinase, c-jun-NH2-kinase, the PKB pathway, and Bcl-2. Sometimes, the existence of VEGF in the tumor environment can interfere with the delivery of chemotherapeutic drugs due to increase in the permeability of blood vessels. Furthermore, due to interstitial tumor hypertension, drugs could be effluxed out of the tumor cells, resulting in chemotherapy resistance. Therefore, VEGF downregulation could enhance the effect of chemotherapeutics, and anti-VEGF siRNA can be used to boost the efficacy of chemotherapeutic drugs  activity resulted in the down-regulation of several cancer-causing factors such as endothelial cell proliferation, angiogenesis, and tumorigenesis . Combinaic drugs . This waic drugs . VEGF si4Nucleic acid-based drugs require sufficient cellular transfection efficiency, which can only be achieved with the use of suitable delivery platforms. Nucleic acids, including siRNAs, are water-soluble and negatively charged so it is difficult to deliver them through the cell membrane. Furthermore, they must escape in the intact form from endosomes and lysosomes before being delivered to the site of action to exert their effect. Another challenge is protecting them against degradation by the endogenous enzymes and media along with the formulation excipients . The extVirus-based delivery typically uses retroviruses, lentiviruses, adenoviruses as delivery vectors. Although virus-based delivery systems have a high transfection efficiency, they frequently have negative side effects, most notably the stimulation of immune response and toxic cellular effects. Because of the numerous drawbacks of virus-based delivery vehicles, there has been increased interest in the usefulness of non-viral-based delivery techniques that use nanoparticles as a delivery vehicle . siRNA dThe use of nanoparticles as a carrier for siRNA delivery have the several benefits: (1) To overcome barriers, particle size is favorable for siRNA delivery (2) They are not immunogenic because they are inert (3) they can augment interferon production and natural killer (NK) cell activity, resulting in antitumor activity and increase therapeutic efficacy (4) They have a longer circulation time, which allows them to much more effectively permeate and accumulate in tumor cells (5) they can be easily imaged and tracked. On the other hand, they have some drawbacks like low water solubility and limited bioaccumulation. However, these issues can be solved by choosing the right polymers. As a result, the benefits outweigh the drawbacks .4.1To overcome the issues related to delivery of siRNA, several bioengineered nanoparticles (NPs) have been investigated and optimized. Some bioengineered siRNA conjugated nanocarriers which have been studied are lipidic NPs, polymeric NPs, inorganic NPs, and RNA-based NPS. Physico-chemical properties of nanocarriers such as size, shape, surface chemistry, and ligand-based targeting of the nanocarriers can affect biodistribution, cell-specific internalization and stability of these carriers.4.1.1The cellular uptake and tumor accumulation via endocytosis are primarily determined by the shape and size of siRNA conjugated nanoparticles. A study demonstrated efficient cellular uptake and gene silencing along with prolonged circulation of siRNA loaded in micellar nanoparticles (MNPs) 90/siRNA with higher retention in tumor cells MDA-MB-231 murine xenograft model as compared to MNP-90/siRNA. In contrast, larger particles showed rapid clearance from the circulation with low gene silencing efficacy and antitumor efficacy . SimilarIn addition, silicon-based nanoparticles demonstrated shape-dependent distribution of discoidal particles which showed higher penetration in organs than quasi-hemispherical and cylindrical shapes . Likewis4.1.2Surface charge (negative or positive) on the nanoparticles influence the pharmacokinetic and biodistribution pattern of these carriers. The negatively charged nanoparticles undergo electrostatic repulsion by the cells leadings to the non-delivery of contents into the cells as compared to positively charged nanoparticles , Li and. The catAlthough positively charged and negatively charged particle has capability to reach target site by following various targeting mechanism for delivery of siRNA. Both kinds of nanoparticle have some advantages and disadvantages, so developing a suitable nanocarrier is major challenge. Some literature has shown cationic nanocarrier as good for delivering siRNA with higher cellular uptake and cytotoxicity . Some haStudies reported the stearylamine (positively charged) decorated Poly Lactic acid—Poly Ethylene Glycol (PLA-PEG) nanoparticles showed higher internalization compared to PLA-PEG nanoparticles in HeLa cells indicated that positively charged nanoparticles possess better cellular uptake capability than negatively charged nanoparticle with less cytotoxicity. The cationic nanoparticles undergoes micropinocytosis and clathrin/caveolin mediated endocytosis mechanism whereas, anionic nanoparticles undergoes clathrin-dependent/caveolin-independent endocytosis for their cellular uptake .In contrast, some authors have reported the lower cellular uptake and induced toxicity inspite of greater capacity of positively charged nanoparticles to electrostatically interact with negatively charged phospholipidic cellular membrane due to agglomeration of cationic nanoparticles. A comprehensive study on cationic carbon nanoparticles by assessing the in vitro and in vivo lung toxicity on THP-1-derived macrophages by evaluating zeta potential value and surface charge density clearly reveals that the surface charge density of a cationic carbon NP is a relevant descriptor of cytotoxicity rather than zeta potential. Higher Charge density of carbon nanoparticle has shown the high viability loss of THP-1-derived macrophages . SimilarThe immunogenicity, carcinogenicity, and inflammation restricted clinical efficacy of viral vectors. The non-viral gene carriers like Lipidic nanoparticles, polymeric nanoparticles, and inorganic nanoparticles are a good choice due to less immunogenicity with tunable size, surface and charge properties for delivering siRNA. The surface charge of non-viral vectors provide a safe and biocompatible strategy for siRNA delivery with enzymatic protection, efficient cellular uptake . Various4.1.3Polyplex is a polymeric system consists of condensed complexed gene or siRNA interacted through electrostatic interactions between cationic groups of the polymer and the negatively charged nucleic acids. Rapid degradation and poor cellular uptake and inability of transfection are the major challenges that can be overcome by synthesizing a polyplex with polyethylene glycol (PEG)-modified l-arginine oligo grafted with poly(ethyleneimine) (PEI) for siRNA delivery. The altered surface chemistry provides stability, facilitating cellular uptake and minimizing cytotoxicity .Surface chemistry has significant impact on pharmacokinetics and tumor delivery. Studies have shown that 20 kDa polyethylene glycol (PEG) and 20 kDa Poly(2-(methacryloyloxy) ethyl phophorylcholine) (PMPC) block protein absorption in vitro with higher stability in presence of salt or heparin with 5-fold higher circulation half-life as compared to 5 kDa PEG. Further, zwitterionic PMPC-based polyplexes showed in vivo silencing of luciferase with 3-fold higher cell uptake as compared to PEG (5 kDa) polyplexes. These experiments showed that zwitterionic polyplexes with high molecular weights drastically enhanced pharmacokinetics and tumor uptake . InorganSurface modification of nanocarriers using covalent and non-covalent approaches can effectively increase the biocompatibility and facilitates active and passive cellular uptake drug and siRNA. Specific protocol needed to be followed to achieve surface modification with desired characteristics: addition of larger molecule could lead to change in size and 3D conformation of nanocarriers. The conjugation of molecule on surface of nanocarriers delivering siRNA conjugation density and ligand orientation are an important aspect to influence cellular uptake. Thus, modification in surface chemistry of nanocarriers can yield smart platforms for treatment of cancer by incorporating therapeutic agents to provide controlled release therapy at specific site and decreases the toxicity .4.1.4The study suggested that siRNA loaded lipidic nanoparticles (LNP) are found stable and potent in the gastrointestinal (GI) tract at pH 1.2with “fasting” pepsin and bile salts concentration . Additio4.1.5The siRNA delivery undergoes rapid enzymatic degradation with limited translocation across cellular barriers, and endosomal release is the major challenge for siRNA therapy. Desirable gene delivery nanocarriers must have low toxicity and high transfection efficiency across the various cellular extracellular and intracellular barriers. The intracellular barriers and extracellular barriers comprise of biological , chemical , and physical barriers . The main extracellular barriers faced by siRNA to arrive at site of action are endogenous nuclease, renal elimination, biological membrane impermeability, reticuloendothelial system entrapment, opsonization, plasma protein sequestration and endothelial crossing. The intracellular barriers such as endosomal trap, cytosol and off target effects also influenced the release of siRNA from nanocarriers . These bco-glycolic acid (PLGA), Polyethylenimine (PEI) have the capacity to increase endosomal escape efficiency and cellular uptake. Stimuli-responsive systems are derived from inorganic nanoparticles, lipids, and polymers are capable to deliver siRNA in a site-selective manner accomplished by endogenous stimulus  to trigger the controlled release of API that primarily focusing on the principle of membrane destabilization  transports the siRNA conjugated nanoparticles RES rich organs (spleen and liver) eliminating them from the general circulation of body, recognizing them as unwanted elements leading to accumulation of siRNA in liver and spleen instead of the target organ. Incorporation of polymers like poly lactic-lization . The stalization .4.1.63 Da), termed as ABP polyplex, showed enhanced transfection efficiency, high cellular penetrating ability, and good nuclear localization ability  acid (PLGA) is an attractive polymer that has been regulated by the Food and Drug Administration (FDA) for the delivery of bioactive molecules because of its excellent biocompatibility, biodegradability, and excellent controlled release properties. The PLGA matrix designed to protect siRNA from nuclease degradation and allowed for a burst release of surface-localized siRNA accompanied by a triphasic controlled delivery over two months , Kaur e. PLGA coCationic nanoparticles are formed by use of several cationic components such as cationic initiators, cationic monomers, cationic polymers, and cationic surfactants . Cationi4.2Chemotherapeutic drugs are typically loaded into nanoparticles (NPs) using the “like dissolves like” phenomena while siRNA is typically encapsulated into nanoparticles using the interaction between a positively charged polymer and negatively charged siRNA . NPs havCombination treatment therapies in which a sufficient amount of siRNA and chemotherapeutic agents are delivered to the same tumor cell contribute a significant role in the treatment results. Numerous chemotherapeutic drug/siRNA co-delivery nanoparticulate systems have been developed for cancer treatment.Liposomes are amongst the most effective drug delivery carriers, as well as co-delivery carriers for chemotherapeutics and gene agents. Recently, scientists have found success with co-delivery systems utilizing modified cationic liposomes; the most popularly used cationic lipid is 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) , Kumar . In theiPolymeric NPs have been used for the specific delivery of chemotherapeutic drugs and nucleotides. They have been employed to co-deliver chemotherapeutic drugs and siRNAs together due to their biocompatibility, stability, restricted size distribution, controlled drug release profile, and highly effective cellular absorption , Wang e. Poly L. Biotin-demonstrated increased anticancer activity in resistant JC  cancer cell line, particularly in comparison to NPs and PTX when used alone. Biotin-functionalized siRNA-PTX NPs given intravenously to tumor-bearing mice models demonstrated efficient antitumor activity .Polymeric micelles, which have a nanoscale core/shell structure formed by amphiphilic block copolymers, are gaining popularity as chemotherapeutic drug/siRNA co-delivery carriers for cancer treatment. Polymeric micelles are not only effective in encapsulating water-insoluble drugs and releasing siRNA in a sustained manner, but they also have other beneficial attributes, such as a unique core/shell arrangement, passive tumor internalization via enhanced permeability and retention (EPR) effect, and additional protection of entrapped drugs from degradation and metabolism . Zhu et Mesoporous silica nanoparticles (MSNs) are also being used extensively for drug and gene-targeted delivery applications. MSNs are non-toxic, biocompatible, and chemically stable. They are ideal for co-delivery of siRNA and chemotherapeutic drugs because of their physicochemical properties. In a recent study, functionalized MSN was developed for the co-delivery of siRNA and chemotherapeutic drugs by using PEI-PEG-modified MSNs for encapsulating epirubicin and BCl2 siRNA. BCL2 siRNA discharge from MSN was pH-dependent and the combinatorial formulation was more cytotoxic in vitro and more effective in vivo than free epirubicin and MSN carrying epirubicin, or siRNA alone. The therapeutic effect was synergistic .Dendrimers are monodispersed and hyperbranched nano molecules that are composed of a single micellar molecular unit. They can be prepared in a variety of shapes and sizes, and the nature of their peripheral functional groups evaluate their physicochemical characteristics, such as drug entrapment efficiency, solubility, and toxic effects . PAMAM . Low-mol5Gene therapy is a valuable means for the treatment of severe ailments, including cancer, despite the barriers involved in the delivery of siRNA in the body. Various factors such as fast enzymatic breakdown, short biological half-life, and low permeability across the cell membranes because of negative charge, instability, and reticuloendothelial system (RES) uptake affect delivery of siRNA. However, the development of conjugated biocompatible nanocarriers via physical and chemical treatment is possible to overcome these barriers . These bMoreover, the modification of nanocarriers using targeting moieties could be a suitable option for co-delivering siRNA and chemotherapeutic drugs limiting the expression of target protein and proliferation of cancerous cells . The maiAdditionally, the long half-life of the siRNA-mediated therapeutics is also pivotal to the EPR effect. The size of formulation, electrostatic nature of surface, formulation stability and lipophilicity are essential properties that protect the siRNA nanocarriers from phagocytic cells . The neg6RNA interference (RNAi) is booming as a promising cancer treatment strategy. siRNA is a significant RNAi method that can be used to down regulate the genes responsible for drug resistance and chemotherapeutic ineffectiveness. Co-delivery increases therapeutic efficacy by providing synergistic or additive effects and by overcoming MDR. When combined with other anticancer agents, siRNA can be used to enhance therapeutic response by targeting many pathways and regulatory proteins related to cancer cell growth, metastatic spread, and drug resistance. Despite considerable progress over the past few decades, there are still a number of obstacles to be overcome before siRNA and drugs can be successfully delivered together, which significantly reduce the clinical application. Thus, combination delivery of chemotherapy drugs and siRNA as a cancer treatment therapy still requires further research. Gene-based cancer therapy has gained momentum as a result of this prospect. In the coming years, personalized therapy based on genetic makeup would be feasible, and siRNA is currently one of the most promising therapeutic approaches for the same.Krishan Kumar: Formal analysis, Conceptualization, Project administration, Writing – original draft, Writing – review & editing, Methodology, Validation. Varsha Rani: Writing – original draft, Writing – review & editing. Mohini Mishra: Writing – original draft. Ruchi Chawla: Project administration, Supervision, Visualization.The authors declare that they have no known competing financial interests or personal relationships, which have, or could be perceived to have, influenced the work reported in this article.


Immunity by Erttmann et al., an unexpected beneficial role of gut microbiota in protecting mice against invading viruses by promoting low levels of systemic innate immune activation via type I interferon (IFN) priming was revealed.1In a recent study published in 2 In turn, the immune system maintains and regulates this delicate, symbiotic balance. However, the underlying molecular mechanisms are poorly understood. Innate immune activation by microbiota is mediated by a dedicated, germ-line encoded set of innate immune sensors, so-called pattern recognition receptors. Microbials breaching the barriers of the gut epithelium are classically sensed by Toll-like receptors (TLRs). Subsequent inflammatory responses provoke local antibacterial immune responses but also cause and/or contribute to the pathogenesis of many diseases.2 However, the recent study by Erttmann et al. showed that non-invading commensal bacteria activated beneficial systemic type I IFN innate immune responses, independent of TLRs and direct bacteria-cell contacts. Removal or disturbance of the gut microbiota by antibiotic treatment reduced overall type I IFN induction in vivo. Conversely, fecal transplants after antibiotic treatment restored levels of immune activation. The authors show that membrane vesicles (MVs) derived from Gram-negative and Gram-positive gut bacteria  deficient in cGAS. To exclude that TLR-dependent detection of microbiota contributes to elevated type I IFN levels, the authors used a set of knockout mice, deficient in TLR and/or STING. Their data shows a significant reduction of basal type I IFN levels only for the STING knockout mice indicating only a minor role of TLRs in systemic priming. Notably, the authors show that MVs containing bacterial DNA can pass the gut epithelial barrier. Consequently, these MVs were also detected in feces as well as in the blood serum of mice, indicating induction of a cGAS-dependent type I IFN beyond a local response. Analyses of the spatial distribution of ifnb1 levels in mice after E. coli or E. coli-derived MV priming also support a systemic elevation of type I IFNs. Again, this systemic response is abrogated in cGAS KO mice. As a consequence of low level systemic type I IFN induction, antiviral defenses are primed, and replication of viral pathogens like the DNA virus Herpes simplex virus 1 (HSV-1) or the RNA virus Vesicular stomatitis virus (VSV) is reduced in mice with a balanced microbiota (Fig. This adds to accumulating evidence of a positive impact of balanced gut microbiota on shaping and training both the adaptive and innate immune responses.ria Fig.  were sufria Fig. . The secListeria monocytogenes infection.4 Pathology of many diseases is driven by elevated levels of inflammatory IFNs, most prominently including a set of monogenic inflammatory diseases called type I interferonopathies.5 Whether and how the low level “chronic”, but beneficial inflammation induced by bacterial MVs are tolerated in the long run remains unknown. It is tempting to speculate that these elevated levels of systemic type I IFN are typically below a pathogenic threshold, thus avoiding detrimental effects. This begs the question how the microbiota or the host regulates and limits this immune activation. Most importantly, the conclusions of this study may need to be considered for current antibacterial therapies using broad-spectrum antibiotics. Erttmann and colleagues suggest that disturbance or depletion of balanced gut microbiota by antibiotics may—besides other health issues—also convey an elevated risk of viral infections.In summary, the study by Erttmann et al. demonstrates a role of microbiota in priming systemic type I IFN responses via the cGAS-STING axis, thereby protecting local and distal host cells from external threats: viruses. This conceptual framework that microbiota promote antiviral immunity is supported by mechanistic evidence highlighting commensal-derived MVs as a systemic communication platform between the microbiota and the innate immune system. Although microbiota-derived MVs were previously suggested to mediate host–microbe interactions, their mode of action had been unclear so far. As this study highlights new mechanistic aspects of the mutually beneficial relationship between commensal bacteria and their host, interesting questions are prompted. Is the situation in humans similar to mice? How systemic is the MV-induced type I IFN response, and which tissues/cell types are affected? Would elevated type I IFN levels also affect pathogenic bacteria and to some extent a local response also target the gut microbiota themselves? Is there an evolutionary advantage for bacteria to promote the antiviral capacities of their host? Of note, the other way around—viruses being beneficial in host immune reactions—has already been shown. Herpesviruses were reported to protect mice against pathogenic


Owing to the impending global scarcity of high-quality sources of phosphate (Pi) fertilizers, lowering its use in crop production requires improved insights into factors stimulating Pi uptake from the soil as well as the efficacious use by plants. Following decades of extensive research on plants’ adaptation to Pi deficiency with mitigated success in the field, a better understanding of how plants exposed to zinc (Zn) deficiency accumulate much more Pi provides a novel strategy in comparison to when plants are grown in Zn-rich soils. In this context, we review current knowledge and molecular events involved in the Pi and Zn signaling crosstalk in plants that will bear great significance for agronomical and rudimentary research applications. For their growth and development and to face changing environments, plants rely on a constant supply of phosphorus (P). Plant root systems primarily uptake P in their inorganic forms (Pi) ,2,3,4,5,In soil, Pi interacts with cations such as zinc (Zn) and forms immobilized complexes . Therefore, facilitating a progressive liberation of these elements into the soil and to avoid chelation/precipitation of Pi by Zn together with a constant improvement in fertilizer formulas are needed ,9. The eIn general, a significant amount of Pi is found in soils, although plants are only able to gain access to bioavailable Pi . Plants When plants access high levels of Pi, for instance, after Pi replate, the cell metabolism quickly reverts to conditions that impede Pi uptake. We know that under these conditions (P-sufficiency) PHR1 interacts with proteins containing SPX domains , which iStimulating Pi uptake without completely triggering the Pi starvation-signaling pathway could be used to improve plant growth capacity under a unfavorable nutritional (P) environment.When identifying novel ways to regulate Pi uptake, it is important to avoid the classical comparison of Pi starvation versus Pi-rich conditions. Instead, future studies need to leverage intriguing observations where plants over-accumulate Pi during biotic or abiotic stresses, including in the presence of Pi concentrations that are elevated.As aforementioned, Zn deficiency causes an overaccumulation of Pi. Notably, since the expression of Pi transporters is not induced by other macro- or micronutrients tested in several plant species, this Zn/Pi relationship appears to be specific . The indPHT1 expression in low Zn conditions remain poorly understood [Investigations into the molecular basis of the Zn/Pi interaction in plants have begun recently, while the mechanisms explaining the unexpectedly enhanced derstood . Recent derstood . Perhapsderstood .PHT1 expression in the whole plant but mostly in the leaves, whereas Pi deficiency induces PHT1 expression throughout the plant but predominantly in the roots. The Pi uptake stimulation triggered by Zn and Pi deficiencies appears to rely on the induction of PHT1;1 for Zn deficiency [PHT1;1 is solely responsible for the increased P uptake in low Zn, or if it also involves post-translational regulations.Importantly, it remains unknown why Pi uptake stimulation in roots remains activated in low Zn environments, even when internal Pi levels are high. The increase in Pi uptake due to Zn deficiency stimulates ficiency ,22, and ficiency . FurtherA. thaliana accessions grown in a low Zn condition, Lyso-Phosphatidyl Choline AcylTransferase 1 (LPCAT1) proven to be a mitigating factor that curtails Pi accumulation and the induction of PHT1;1 triggered by Zn deficiency [PHT1;1 deregulation remains to be identified even though these studies highlight an important role for PHT1;1 in the Zn deficiency response.By employing a Genome-Wide Applications Studies (GWAS) analysis of the Pi content variation among ficiency  -specific Pi transporter genes in various plants species including Lotus japonicus, tomatoes, and potatoes [Our knowledge of phospholipids (PL)-derived signals in plants has been scant until the recent past; but physiological and molecular studies have demonstrated that some PL classes might act as precursors to generate diverse signaling molecules ,23. The potatoes ,25. Aparpotatoes . Anotherpotatoes ,28,29. FNutrient–nutrient interaction along with its ensuing impact on their accumulation in plants has elicited widespread recognition. The interaction between Pi and Zn homeostasis demonstrates the way in which the deficiency of one element can lead to the over-accumulation of the other element. This observation opens up new avenues to take measures in making improvements in the plants’ Pi response. However, there is ambiguity on whether these responses have an underlying genetic/signaling or nutritional basis. However, except for the example of P/Zn interaction, more studies need to be conducted to understand how plants sense levels of nutrients not only in the surrounding soil but also within their cells before adjusting various transport steps that then lead to nutrient accumulation in cells. This is critically important to augment our existing knowledge of how plants regulate/coordinate these processes to facilitate the design of strategies to improve plant nutrition, particularly Pi nutrition.


Antimicrobial resistance (AMR) is a global problem that also includes countries of the Arabian Peninsula. Of particular concern, is the continuing development of extended-spectrum β-lactamases (ESBLs) in the countries of this region. Additionally, antibiotic treatment options for ESBL-producing bacteria are becoming limited, primarily due to the continuing development of carbapenem resistance (CR), carbapenems being frequently used to treat such infections. An overview of recent publications (2018–2021) indicates the presence of ESBL and/or CR in patients and hospitals in most countries of the Arabian Peninsula, although the delay between microbial isolation and publication inevitably makes an accurate analysis of the current situation rather difficult. However, there appears to be greater emphasis on CR (including combined ESBL and CR) in recent publications. Furthermore, although publications from Saudi Arabia are the most prevalent, this may simply reflect the increased interest in ESBL and CR within the country. Enhanced ESBL/CR surveillance is recommended for all countries in the Arabian Peninsula. Enterobacteriaceae family, which are part of the normal gut microbiota. However, these bacteria may become pathogenic when associated with urinary tract and bloodstream infections  and K. pneumoniae [8.1%]) isolated in 2020 from three government hospitals showed ESBL resistance, stating that patients with UTIs in the Najran region are at a high risk of antibiotic resistance, which may be leading to significant problems in outpatient department treatment outcomes  OR "extended-spectrum beta lactamase"[Title] OR "extended spectrum beta-lactamase"[Title] OR "extended-spectrum beta-lactamase"[Title] OR "extended spectrum β lactamase"[Title] OR "extended-spectrum β lactamase"[Title] OR "extended spectrum β-lactamase"[Title] OR "extended-spectrum β-lactamase"[Title] OR "ESBL"[Title] OR "ctx-m"[Title] OR "carbapenem"[Title] OR "carbapenemase"[Title]) AND ("Arabian peninsula"[Title] OR "bahrain"[Title] OR "iraq"[Title] OR "jordan"[Title] OR "kuwait"[Title] OR "oman"[Title] OR "qatar"[Title] OR "saudi arabia"[Title] OR "united arab emirates"[Title] OR "uae"[Title] OR "yemen"[Title]) AND ("2018/01/01"[Date - Publication]: "3000"[Date - Publication]). This generated 56 results, including one corrigendum. All publications were included in the current study and some publications  described research performed prior to 2018. Only English language articles were included , and wheThere is an inevitable lag between the initial isolation of microbial strains and the eventual publication of AMR results. Therefore, most of the studies published in 2018–2021 relate to isolates obtained several years before their publication. This makes an accurate analysis of the current state of affairs regarding the epidemiology of ESBL and CR in the Arabian Peninsula difficult, although this aspect of AMR epidemiological reporting is not restricted to this region of the world. However, recent publications have shown that there is an increasing interest in the epidemiology of CR in this region, most likely due to the increasing number of CR isolations from patients. Further, it is inevitable that the increasing use of carbapenem antibiotics to treat ESBL-related infections will lead to an eventual increase in CR globally, including in the Arabian Peninsula. As such, the steps required to help prevent or slow down the global emergence of CR infections include: i) recognition of the impact of carbapenem prescribing on regional infections and ii) ensuring that appropriate measures are taken towards the responsible prescription of antibiotics. In this respect, the need for a doctor’s prescription, a consultation with infection control teams, an accurate diagnosis of bacterial infections, and antibiotic sensitivity is essential . It is eFrom this review, it is obvious that most country-specific publications related to ESBL and CR infections were from Saudi Arabia. However, it should be remembered that this does not mean that Saudi Arabia has a larger AMR problem than other countries in the Arabian Peninsula, but suggests that the problem is potentially being more thoroughly investigated in this country . Focusing on publications in PubMed may mean that a few relevant publications could have been omitted from this review. However, we described data obtained from 56 relevant publications, and it is unlikely that the impression obtained by this review would be altered by the addition of any additional publications that may have been missed.Based on the current review, continued and expanded surveillance of ESBL and CR in hospitals and the environment is recommended for all countries in the Arabian Peninsula. Increased funding and the implementation of relevant AMR policies directed at non-specific and specific attributes associated with countries of the Arabian Peninsula, such as migration, religious pilgrimages, and food imports, are particularly recommended.


The COVID-19 pandemic introduced significant disruptions in the learning environment for many post-secondary students. While emerging evidence suggest mental health has declined during the pandemic, little is known about how the pandemic has affected students academically. This study investigates how COVID-19 psychological distress impacts academic performance among a Canadian sample of post-secondary students (n = 496). Path analysis findings suggest that greater levels of COVID-19 distress was associated with lower self-reported predicted GPA. Metacognitive, motivational, and social and emotional challenges emerged as the most salient challenge areas that fully mediated the relationship between COVID-19 psychological distress and self-reported predicted GPA. Specifically, COVID-19 distress predicted greater levels of metacognitive and motivational challenges which, in turn, predicted lower self-reported GPA. Similarly, greater levels of COVID-19 distress predicted more social and emotional challenges but these challenges were associated to higher perceived GPA. Findings warrant future research to help students manage and cope with academic challenges that may be exacerbated under stressful conditions. The COVID-19 pandemic has created a significant shift in daily behaviors worldwide with many adhering to social distancing guidelines by primarily working and studying from home. In response to ongoing pandemic restrictions, most Canadian post-secondary institutions transitioned to full-time online learning in order to ensure that students remain safe while completing their studies. However, this shift in students’ learning environment may introduce challenges that impede their academic engagement. Psychological distress, feelings of isolation, technology-related issues , and limited access to research resources and faculty pose challenges to students’ motivation to learn and to their academic performance . Such disruption to the student experience can ultimately contribute to an increase in dropout rates. In addition to reporting disruptions to their learning such as not being able to secure courses or completing their degrees on time , the panIncreasing worries related to income, housing, social and health uncertainties can exacerbate academic challenges which may be compounded under pandemic restrictions as students transition to remote online learning . While eWhile distance learning is not a novel approach in the post-secondary setting, the abrupt shift from face-to-face learning to remote online learning during the COVID-19 outbreak has globally introduced unique challenges to students who are unfamiliar with self-regulated learning in an online environment. In an Algerian sample of post-secondary students, While emotional distress resulting from academic difficulties are normative for students, the abrupt shift to remote online learning can exacerbate existing worries related to uncertainties with graduating on schedule and employment prospects . For stuEmerging evidence suggests that the increased amount of stress introduced by the pandemic may be impacting students’ academic performance and engagement. Students have reported a significant reduction in work performance and time dedicated to studying during the pandemic , as wellHowever, there is substantial heterogeneity in academic challenges experienced by post-secondary students that differentially moderate academic success yet few studies have accounted for these differences. For example, for some students, cognitive challenges related to difficulties with processing course content and metacognitive challenges related to problem-solving, planning and monitoring of tasks may be more salient e.g.,  than motSelf-regulatory learning (SRL) competencies may help to mitigate some of these academic challenges , but strParticipants (N = 496) were enrolled in a Western Canadian post-secondary institution. Participants were recruited from three domains including students who were (a) registered in an academic course on learning strategies for university success (N = 95), (b) included in the psychology research participation pool (N = 369), or (c) accessing a learning strategies library resource available to all students at the university (N = 32). Participants who were registered in the learning strategies academic course were required to participate in the study in order to receive course credit while participants who were drawn from the psychology research participation pool received course credit  after completing the survey. Participants who were drawn from the online learning strategies resource were entered into a draw for a gift card as compensation for their participation.Participants completed an online survey that included measures of COVID-19 psychological distress, self-reported predicted GPA, and academic challenges. After completing the survey students were sent a personalized academic challenge report that allowed students to understand their learning habits and challenges. The study was approved by the Institution's Human Research Ethics Board.COVID-19 psychological distress was measured using the COVID Stress Scales  which coAcademic performance was measured using self-reported predicted GPA which asked students to report on what overall GPA (average grade) they expected to get this year. Academic challenges were measured using the challenges subscale from the Hadwin et al.'s   SRL-acaPath analyzes was used with Mplus v.8  to estimrs range from −.15 to −.27; ps <.05) and positively correlated with COVID-19 psychological distress . GPA was negatively correlated with COVID-19 distress .Means and standard deviations of the variables in the study are provided in p < .01). A composite of all the academic challenges was subsequently entered into the model in order to assess whether it served as a mediator in these associations. As shown in p = .327) suggesting the possibility of a full mediation. A significant indirect effect  confirmed that the negative association between COVID-19 distress and GPA can be fully explained by academic challenges. Specifically, COVID-19 distress predicted more academic challenges  which in turn predicted lower GPA .A direct path between COVID-19 distress and GPA was first examined before the addition of academic challenge mediators. COVID-19 distress negatively predicted GPA . Specifically greater COVID-19 distress predicted higher levels of metacognitive challenges effect between  which in turn predicted lower GPA . Similarly greater COVID-19 distress predicted more motivational challenges  which in turn was related to lower expected GPA . However, while greater COVID-19 distress predicted more social and emotional challenges , this increase in turn was related to higher expected GPA .Finally, specific academic challenges, cognitive, motivational, social and emotional, behavioral, and metacognitive were analyzed as mediators to determine their unique contribution to COVID-19 distress and GPA. As shown in The current study examined the impact of psychological distress related to COVID-19 on post-secondary students’ academic performance. Consistent with previous research, greater COVID-19 psychological distress was associated with poorer academic performance as indicated by students’ self-reported predicted GPA e.g., . SpecifiDaily stressors associated with the post-secondary learning environment such as problems related academics, financial concerns, interpersonal challenges, as well as risky health behaviors involving substance use, physical inactivity, poor sleep habits and nutrition are common impediments to students’ academic engagement. The multitude of stressors experienced by post-secondary students place them at greater risk for mental health problems  may provide further insights into how students adapt to their changing learning environments and challenges. Another limitation in the study surrounds the use of self-reports of academic performance  which can be subjected to respondent biases. Moreover, students’ psychological state could affect their confidence or ability to predict how they are performing academically particularly among students with poorer metacognitive awareness. Institutional GPA and self-reported GPA would offer additional insights into how pandemic-related academic challenges impact students’ overall academic functioning. Regardless of self-reported predicted GPA, self-report of different types of challenges provides a meaningful assessment of students’ overall academic functioning including their motivation processes and perceptions of how well they monitor, set goals, and adapt effectively to challenges particularly if measures are sensitive to the academic task at hand .Despite these limitations the study highlights the need to tailor academic intervention efforts to help students manage and cope with their specific academic challenges. Self-regulated learning models posit that academic challenges provide opportunities for students to exercise self-regulatory control by deploying strategies, monitoring progress, and adapting accordingly to the academic challenges they face. During the pandemic, students with fewer SRL skills may be less equipped to cope with the demands of new academic challenges and pandemic stressors as such challenges necessitate more intensive SRL capacities . Enhancing post-secondary students’ SRL skills through SRL training interventions could help ameliorate academic challenges. Further research is needed to examine the role of self-regulated practices in mitigating the impact of those challenges on academic performance and whether SRL targeted interventions can be leveraged to assist students in overcoming specific academic challenges. Academic intervention programs that help students develop strategic self-regulated learning habits have demonstrated utility in improving academic engagement and performance in both traditional face-to-face  and onli


Moyamoya disease is often diagnosed after intracranial hemorrhage in adult patients. Here, we report a case of unilateral moyamoya disease treated with indirect revascularization combined with cranioplasty after treatment for acute subdural hematoma and subcortical hemorrhage. A middle-aged woman with disturbed consciousness was transferred to our hospital. Computed tomography (CT) revealed an acute subdural hematoma with left temporoparietal subcortical hemorrhage. Three-dimensional CT angiography indicated a scarcely enhanced left middle cerebral artery (MCA) that was suspected to be delayed or nonfilling due to increased intracranial pressure. Subsequently, hematoma evacuation and external decompression were performed. Postoperative digital subtraction angiography (DSA) revealed stenosis of the left MCA and moyamoya vessels, indicating unilateral moyamoya disease. Forty-five days after the initial procedure, we performed encephalo-arterio-synangiosis (EAS) using the superficial temporal artery simultaneously with cranioplasty for the skull defect. The modified Rankin Scale score of the patient one year after discharge was 1, and the repeat DSA showed good patency of the EAS. Revascularization using EAS in the second step can be an option for revascularization for hemorrhagic moyamoya disease if the patient required cranioplasty for postoperative skull defect after decompressive craniotomy. Moyamoya disease is a known cause of intracerebral hemorrhage in adult patients, and acute subdural hematoma associated with moyamoya disease sometimes requires decompressive craniotomy, as reported in previous studies , 2. ReceA middle-aged woman with sudden consciousness disturbance was transferred to our institute. Initially, her Glasgow Coma Scale score was 4, and anisocoria and decorticate position of extremities were observed. Computed tomography (CT) demonstrated an acute subdural hematoma associated with left temporoparietal subcortical hemorrhage . Three-dAs a treatment for unilateral moyamoya disease to reduce the stroke recurrence, we planned to conduct surgical revascularization at the same step as cranioplasty for the postoperative skull defect. Forty-five days after the initial surgery, we performed a combined procedure. In the first step, we attempted direct revascularization between the parietal branch of the STA and the MCA. However, because of the adhesion of surrounding tissues and the small size of recipient arteries, it was difficult to achieve successful direct anastomosis. We then decided to perform indirect revascularization. We simply placed the end of the STA onto the surface of the M4 segment of the MCA with sutures using 10-0 nylon . After iThe postoperative course was uneventful, and her symptoms gradually improved. The modified Rankin Scale score of the patient one year after the treatment was 1, and regular magnetic resonance angiography (MRA) indicated increased blood flow in the revascularized STA ; there wIn this case report, we describe the case of a patient with unilateral moyamoya disease who presented with an acute subdural hematoma associated with subcortical hemorrhage. According to previous studies, moyamoya disease might cause severe intracranial bleeding leading to acute subdural hematoma –8. BeforRecently, several articles have stated that surgical revascularization has the potential to reduce the risk of recurrent stokes in patients with moyamoya disease , 11. SpeThis case of hemorrhagic moyamoya disease mimicked the delayed or nonfilling phenomenon and was successfully treated with the step-by-step surgical treatment including indirect revascularization. We found that EAS combined with cranioplasty may be a viable option for the second procedure.


The use of liquid biopsy for tumor genomic profiling to identify genomic biomarkers for targeted therapies has revolutionized the clinical practice in oncology management. In this review, we have summarized the recent advancements of liquid biopsy-based genomic profiling that have led to their approval for treatment selection in advanced cancer patients and highlighted the major factors that should be considered to choose the most appropriate genomic profiling assay for different patients under different clinical conditions.Genomic profiling using tumor biopsies remains the standard approach for the selection of approved molecular targeted therapies. However, this is often limited by its invasiveness, feasibility, and poor sample quality. Liquid biopsies provide a less invasive approach while capturing a contemporaneous and comprehensive tumor genomic profile. Recent advancements in the detection of circulating tumor DNA (ctDNA) from plasma samples at satisfactory sensitivity, specificity, and detection concordance to tumor tissues have facilitated the approval of ctDNA-based genomic profiling to be integrated into regular clinical practice. The recent approval of both single-gene and multigene assays to detect genetic biomarkers from plasma cell-free DNA (cfDNA) as companion diagnostic tools for molecular targeted therapies has transformed the therapeutic decision-making procedure for advanced solid tumors. Despite the increasing use of cfDNA-based molecular profiling, there is an ongoing debate about a ‘plasma first’ or ‘tissue first’ approach toward genomic testing for advanced solid malignancies. Both approaches present possible advantages and disadvantages, and these factors should be carefully considered to personalize and select the most appropriate genomic assay. This review focuses on the recent advancements of cfDNA-based genomic profiling assays in advanced solid tumors while highlighting the major challenges that should be tackled to formulate evidence-based guidelines in recommending the ‘right assay for the right patient at the right time’. Technological advancements and reduction in sequencing costs have enabled genomic profiling of solid tumors to be performed routinely, which promoted the incorporation of precision oncology into the standard of care for advanced cancer patients . MoleculLiquid biopsies, which involve genomic profiling of tumors using circulating biomarkers in the bodily fluid have emerged as a promising tool to complement and overcome the challenges of tissue-based CGP ,14,15. AThe compilation of evidence that supports the use of ctDNA to identify actionable alterations from both analytical and clinical studies in recent years have advocated the FDA approval of several single-gene and multigene assays to be used as companion diagnostics matched to specific targeted therapies . DespiteThis review summarizes the recent advancements and supporting studies of the use of plasma ctDNA for genomic profiling in patients with advanced solid malignancies. In addition, we will also highlight the major challenges that should be tackled and factors that are to be considered to formulate evidence-based guidelines for the routine use of plasma-based genomic profiling in clinical settings.Recent developments in sequencing technologies have increased the sensitivity of detecting the minute ctDNA present in plasma cfDNA with a higher level of accuracy and confidence . Each meEGFR exon 19 deletions and L858R, ALK fusions, BRAF V600E) [PIK3CA, ESR1, AKT1, and HER2 [KRAS, NRAS, and BRAF—showed near 100% ctDNA sensitivity compared to tissue-based CGP [BRCA1/2 alterations detected by tissue CGP were also detected from plasma cfDNA, highlighting the clinical utility of liquid biopsy CGP for the detection of clinically actionable alterations [Extensive work has been performed to compare the mutation detection concordance between tumor and ctDNA as an approach to evaluate the feasibility of utilizing ctDNA NGS-based assays for CGP of tumors in advanced cancer patients. The reported level of concordance by different studies has been greatly variable, ranging from 8.3% to 93% across different cancer types . The difF V600E) . A similand HER2 . A high ased CGP . In a ren = 1687) or using tumor tissues (n = 5621) [In addition to concordance analysis, several large cohort studies have been conducted in recent years to assess whether ctDNA NGS-based assays could benchmark against the current gold standard of tissue-based assays in detecting biomarkers for molecular targeted therapies. The detection of actionable alterations using ctDNA NGS-based assays has been consistent across studies, with approximately 40% of advanced cancer patients harboring at least one actionable target, comparable to tissue-based assays ,60,61,62 = 5621) . The aut = 5621) . The var = 5621) . However = 5621) ,64.ESR1 mutations from plasma had improved progression-free survival (PFS) after being treated with fulvestrant (estrogen receptor antagonist) compared with exemestane (aromatase inhibitor), while patients with wildtype ESR1 had similar PFS after receiving either treatment [PIK3CA-mutated breast cancer, detected using plasma ctDNA, was associated with a better response to alpelisib plus fulvestrant than the fulvestrant arm [Recent clinical trials have incorporated exploratory objectives to evaluate the performance of ctDNA in identifying genomic markers for the prediction of treatment response for molecular targeted therapies. Several studies have shown patients with biomarkers of interest detected from cfDNA tend to exhibit a better prognosis than patients without the biomarker of interest, indicating their predictive value for treatment response ,77,78,79reatment . This warant arm . The prerant arm . CUP reprant arm . In the rant arm . The autrant arm .The promising results from the exploratory studies suggested the potential of utilizing ctDNA CGP in clinical settings for genomic biomarker identification. The clinical benefits of ctDNA CGP were validated in recent large retrospective studies with cohort sizes of over 1000 patients. No significant differences were observed in the PFS and overall survival (OS) of patients selected based on ctDNA or tissues across several studies ,66,68. TAKT1, ESR1, HER2, and PTEN) identified from plasma. A total of 34% of the sequenced patients had targetable mutations for cohort entry, and 13% of the patients entered one of the treatment arms. The HER2 and AKT1 arms reached the primary end point and exceeded the target number of responses where the response rate achieved by ctDNA-selection was comparable to that observed when guided by tissue testing [ESR1 and PTEN arms did not reach the target number of responses, with only 8% and 11% response rates, respectively, similar to that previously reported [ALK rearrangements [ALK-positive, and 3.9% of patients were enrolled and received the mutation-matched treatment alectinib. The ALK-positive cohort met its primary end point with an overall response rate of 87.4%, comparable to previous reports using tissue-based profiling [In the past 2 years, several ongoing prospective phase II interventional clinical trials that were aimed to assess the accuracy and validity of ctDNA testing to select patients for genomic-directed therapies across different solid tumors have released their early results. All studies have shown over 99% of ctDNA sequencing success rates ,82,83. P testing . Howeverreported . A similThe overall high sequencing success rate and fast turnaround time (TAT) of ctDNA-based CGP are some of the key advantages of liquid biopsy over tissue profiling. Several studies have compared the TAT from sample collection to reporting results between the two CGP approaches. The median TAT for ctDNA-based NGS is 9 days (ranging 2–15 days) compared to 15 days for tissue CGP (ranging 12–20 days) ,82,84,85EGFR mutations to identify NSCLC patients that are eligible for erlotinib [EGFR ctDNA testing provided a rapid and noninvasive method to detect clinically relevant genomic markers for treatment selection and has been proven to be reliable in clinical settings [EGFR mutations, both approved ctDNA-CGP panels could not achieve the same level of sensitivity as the RT-PCR-based Cobas system, highlighting the difficulty to maintain the high sensitivity of mutation detection in large multiplexing systems.In 2016, the U.S. Food and Drug Administration (FDA) approved the first ctDNA plasma-based genomic testing as a companion diagnostic for the detection of rlotinib . The Cobrlotinib   and the low concentrations of ctDNA present in the blood. The detection of rare somatic mutations from such limited genomic material input is highly challenging . Target The current reported limit of detection by different ctDNA-targeted NGS platforms ranged from 0.004 to 2% . HoweverALK, RET, and ROS1 fusion from tumor tissues were concordantly detected from plasma cfDNA [ROS1 and NTRK) [ALK, ROS1, and RET fusions than a cfDNA-based NGS system  [The detection of targetable fusions or gene rearrangements from cfDNA remains technically challenging with inconsistent sensitivity across studies owing to the low prevalence of fusions in common solid tumors. To address this, Esagian et al. compiled 38 published studies that assessed the concordance of fusion detection between tumor tissues and plasma cfDNA in NSCLC patients . A totalma cfDNA . Most ofnd NTRK) ,118. Thend NTRK) ,118. Curnd NTRK) . Previound NTRK) ,121. In nd NTRK) . In a rectively) . Furtherctively) ,123. LarBesides the technical limitations, biological factors such as the location, size, and vascularity of the tumor may affect the release of ctDNA into the circulation, thereby compromising its detectability . ctDNA fKRAS, EGFR, and PIK3CA [Non-tumoral variants detected from plasma contribute to the false-positive results detected from ctDNA-based CGP. Clonal hematopoiesis (CH) is a normal process of aging with the accumulation of somatic mutations in hematopoietic cells . The detd PIK3CA . The VAFd PIK3CA . These fDue to the continuously growing evidence and recognition of the clinical use of ctDNA-based CGP for treatment selection, several leading professional organizations have included recommendations for the use of liquid biopsy in their clinical management guidelines. However, these recommendations were limited to NSCLC, breast cancer and prostate cancer patients. Furthermore, different multidisciplinary bodies also released contradicting suggestions as to whether a ‘plasma-first’ or a ‘tissue-first’ approach should be adopted in clinical settings. For the management of advanced breast cancer, both National Comprehensive Cancer Network (NCCN) and The European Society for Medical Oncology (ESMO) recommend ctDNA profiling as an alternative and complement to tissue CGP, while The American Society of Clinical Oncology (ASCO) recommends cfDNA as the specimen-of-choice for CGP ,138,139.Based on the observations from the current studies, the false-negative rate of plasma samples is higher than that of tissue-based assays . In caseKRAS, NRAS, and EGFR mutations were observed in metastatic colorectal cancer patients who were treated with anti-EGFR therapy than the treatment-naïve patients  [EGFR-T790M alterations is preferred in patients that have developed progression from EGFR tyrosine kinase inhibitors (TKIs) [At initial diagnosis, tissue-based CGP assays are likely to be more beneficial than ctDNA profiling for treatment-naïve advanced cancer patients with resectable tumors, owing to the lower sensitivity of ctDNA-based assays. However, for treatment selection at the time of recurrence or during disease progression, ctDNA-based genomic profiling should be preferred . Severalctively) . The clis (TKIs) ,141,143.s (TKIs) .The use of liquid biopsy is increasingly being incorporated into the clinical protocol for targeted treatment guidance; however, there are still several areas that require further research. Here, we provide our perspectives on the key challenges that should be attended to optimize the use of ctDNA-based CGP in oncology management.One of the main challenges for the clinical use of ctDNA-based CGP is the lack of standardized methods to determine the origin of the alterations detected from plasma and the exclusion of CH-related mutations. Most of the studies conducted so far utilize paired-sequencing of the matched white blood cells to a comparable depth as cfDNA to filter out CH mutations ,44,154. EGFR mutations below or above ctDNA VAF of 1% [EGFR clonal dominance determined by plasma cfDNA was observed to be independently associated with improved efficacy of EGFR-TKIs in patients with advanced NSCLC [EGFR mutation was the dominant clone across 300 treatment-naïve advanced NSCLC patients [EGFR as a dominant clone than those nondominant clones, according to plasma ctDNA NGS results [Current technological developments have been mainly focusing on improving the limit of detection to improve the ctDNA detection sensitivity. However, limited research has been conducted to evaluate the clinical outcomes of targeting alterations that are detected at low VAF using ctDNA profiling. There are no cutoff values or thresholds from the guidelines of the approved companion diagnostics to help guide clinicians on whether treatments should be initiated based on the reported VAF. It is unclear whether targeting alterations detected at low VAF from plasma could result in clinical benefits. Two recent exploratory studies have observed no significant differences in treatment response between NSCLC patients detected with AF of 1% ,67. The AF of 1% ,67. On ted NSCLC . In the patients . The obj results . The aut results .RAS/BRAF ctDNA before initialization of anti-EGFR re-challenge have a significantly longer OS compared to patients with mutated ctDNA  [RAS/BRAF/EGFR using ctDNA for selection of re-challenge therapy [RAS/BRAF/EGFR clones and to guide the initiation of anti-EGFR re-challenge therapy for maximal clinical benefits [A series of observational and interventional clinical trials have demonstrated that monitoring of clonal dynamics and the development of resistance mutations using serial ctDNA analysis may assist in treatment optimization. However, the frequency of ctDNA monitoring and optimal sampling timepoints to achieve maximal clinical benefit remains unclear. Previous studies have shown that ctDNA CGP before the initialization of re-challenge therapy could be effective in predicting clinical benefit ,159. In ctively) . Similarbenefits .ESR1 mutation became detectable from ctDNA [Early detection of resistance mutations through serial ctDNA analysis during treatment has been suggested as an indicator for treatment intervention to prevent or delay tumor progression ,163,164.The use of a large cfDNA panel or a targeted approach to longitudinally monitor patients with advanced-stage disease should also be further investigated. It has been suggested that large ctDNA CGP panels may be more beneficial in cases where resistance mechanisms of the drugs are not known, while in settings where resistance mechanisms are well described, longitudinal ctDNA monitoring using a targeted approach may be more appropriate and cost-effective . Large sTumor mutation burden (TMB) and microsatellite instability (MSI) have shown to be effective genomic biomarkers in identifying patients who are likely to benefit from immune checkpoint inhibitors . HoweverResearch developments and the accumulation of analytical and clinical evidence for the use of ctDNA genomic profiling from the past decade have transformed our clinical practice in oncology. The approval of ctDNA-based assays for CGP and as companion diagnostic tools have allowed more cancer patients to gain access to targeted therapies and supported the realization of precision oncology. Developing evidence-based guidelines for the use of ctDNA profiling and addressing the current limitations, such as the exclusion of CH alterations, will further optimize the clinical usage of liquid biopsy for treatment selection. Future studies should focus on expanding the current roles of plasma ctDNA to improve patient access to precision medicine and thereby improve patient outcomes.