Liquid biopsy, including both circulating tumor cells and circulating tumor DNA, is becoming more popular as a diagnostic tool in the clinical management of breast cancer. Elevated concentrations of these biomarkers during cancer treatment may be used as markers for cancer progression as well as to understand the mechanisms underlying metastasis and treatment resistance. Thus, these circulating markers serve as tools for cancer assessing and monitoring through a simple, non-invasive blood draw. However, despite several study results currently noting a potential clinical impact of ctDNA mutation tracking, the method is not used clinically in cancer diagnosis among patients and more studies are required to confirm it. This review focuses on understanding circulating tumor biomarkers, especially in breast cancer. Breast cancer (BC) is the most prevalent malignancy affecting women worldwide. The 5-year survival rate in individuals with a localized breast cancer diagnosis and no nodal involvement is 99% compared to 27% for patients with distant metastatic disease –3. BreasThus, a breast cancer prognosis worsens with the absence of young women’s early screening programs and efficient diagnostic tools in general . These dCirculating tumor cells (CTCs) and circulating tumor DNA (ctDNA) detection as well as their clinical applications as complementary tools to improve the outcome of patients with breast cancer.The purpose of this review is to summarize technologies for CTCs and ctDNA are new, noninvasive, and multifunctional biomarkers used in the liquid biopsy which enable early diagnosis, precise prognosis, therapeutic target selection, spatiotemporal monitoring of metastasis, and monitoring of therapeutic response as well as resistance. Different tumor types at different stages release CTCs and ctDNA, which could provide complementary information for clinical decisions ..CTCs, inCTC isolation at single-cell resolution. In order to assess the genetic heterogeneity of ctDNA and CTCs in patients with lung and breast cancer, they actually constructed a standardized sample processing methodology which allowed for the concurrent separation of CTC and ctDNA followed by targeted amplicon sequencing. To isolate CTCs at single-cell resolution, they used CD45-antibody negative selection employing a single-cell capture. The results of this study indicated that CTCs and ctDNA had a higher degree of concordance with the metastatic tumor than with the primary tumor. As a conclusion, a standardized sample processing and data analysis workflow for the concurrent analysis of CTCs and ctDNA was successful in separating the heterogeneity of metastatic tumors that were circulating as well as the progressive genomic changes that may guide the selection of an appropriate therapy against evolving tumor clonality [Kong et al.  used thelonality .RUBYchipTM (size-based microfluidic device) system’s performance vs. that of the Cell Search® system for CTC capture. In isolated CTCs, the expression of HER2 was evaluated and compared to tissue biopsy. The study found that, on average, the RUBYchipTM was up to ten times more effective at isolating CTCs compared to the Cell Search® system. Additionally, a precise assessment of various CTC subpopulations, including HER2+ CTCs, was given. The study’s results indicated that liquid biopsy, when used in the clinic with the RUBYchipTM, can overcome the limitations of histological testing and determine a patient’s HER2 status in real-time, allowing for more precise treatment planning as the disease progresses [Based on cell size and deformability, Lopes et al. evaluated and contrasted the ogresses .CTC detection has been proven in patients with both early-stage and metastatic breast cancer [CTC are ease of collection, non-invasiveness, the possibility of continuous evaluation, as well as analysis of the total tumor burden rather than a limited part of a tumor. Currently, CTC is used in breast cancer to gain prognostic information, monitor the effectiveness of treatment and therapeutic interventions, detect the disease stages early, investigate drug sensitivity, and discover new drugs for personalized treatment [CTCs is limited due to their rarity and heterogeneity, as well as problems with initial culture, necessitating the development of easier detection methods compared to the existing ones [CTC count is a criterion for evaluating treatment efficacy, and patients with metastatic breast cancer who had a higher CTC count experienced more metastases over time [CTC cells appear to be a better model for studying the malignant behavior of breast cancer than existing cell lines. Zhao et al. revealed that the CTC-3 cell line grows more aggressively in vitro and in vivo than the commonly used MCF-7 breast cancer cell line [t cancer ,46. Amonreatment . Howevering ones . It appever time . Also, Cell line .CTCs in the diagnosis and early treatment of breast tumors, the presence of CTC clusters in primary breast cancer may be considered an important risk factor for disease progression [CTC in the early stages of breast cancer [Given the numerous benefits of gression . Furthert cancer .Kroll et al. discovered the peripheral blood circulation of CTCs in patients with primary non-metastatic breast cancer, shortly after diagnosis and prior to surgical intervention for therapeutic purposes .CTCs in the early stages of breast cancer. However, the Reduzzi et al.’s study showed that marker-independent approaches for CTC evaluation would improve diagnosis and that CTC is more common in patients with early-stage breast cancer than in patients with metastatic breast cancer [Using conventional and epithelial-based methods, it is difficult to analyze and identify t cancer .CTCs, collected in vivo by the Cell Collector method in Chinese patients with metastatic breast cancer, showed prognostic significance. It seems that the increase of CTCs in the blood is a sign of the progression of cancer and the worsening of the disease. This is because, as the number of CTCs in the blood increases, so do the genetic heterogeneity of these cells and their interaction with the internal environment of the body [HER2 status matching between CTCs and primary tumors or metastatic sites can reach 77% or 67%, implying that studying CTC HER2 expression can guide clinical HER2-targeted therapy [CTC status of 264 patients with metastatic breast cancer before and after 4 weeks of a new line of palliative systemic therapy. According to the findings, HER2-targeted therapy appears to reduce the overall CTC count in patients with metastatic breast cancer [Elevated levels of CTCs have been confirmed as an independent prognostic factor in metastatic breast cancer. Xie et al. discovered that CTC had a prognostic value in patients with metastatic breast cancer. In this study, 38 patients with metastatic breast cancer were enrolled. the body . Stefano therapy . Deutscht cancer .ctDNA.ctDNA are: droplet digital polymerase chain reaction (ddPCR), beads, emulsion, amplification, and magnetics (BEAMing), tagged-amplicon deep sequencing (TAm-Seq), cancer personalized profiling by deep sequencing (CAPP-Seq), whole genome bisulfite sequencing (WGBS-Seq), whole exome sequencing (WES), and whole genome sequencing (WGS) [The main techniques employed to evaluate ng (WGS) ,64–66.DNA detection or RNA expression [HER2 amplification levels [HER2 status with a sensitivity of 90.9% and a specificity of 100% [An important method in translational cancer research is droplet digital polymerase chain reaction (ddPCR), because of its superior sensitivity and precision for genomic pression ,68, and n levels . Howevern levels . In brea of 100% .BEAMing combines PCR with flow cytometry and is an alternative sensitive approach which provides molecular information about mutations with a frequency of 1 over 10000 ,70.Tagged-amplicon deep sequencing (TAm-Seq) enhanced the identification of low frequency mutations in ctDNA . The maictDNA analysis method that achieves both an ultralow detection limit and broad patient coverage at a reasonable cost and low ctDNA input level, thus allowing the quantitation of ctDNA from early-stage tumors [ctDNA/cfDNA using large genomic libraries and individual patient sample sequence signatures. It statistically assesses well-characterized tumor alterations with DNA oligonucleotides to find patient-specific alterations. It can identify multiple mutations in patients with the same type of cancer and assess tumor heterogeneity. It was previously shown to be capable of identifying tumor burdens prior to medical imaging. It can identify many major mutation types including insertions, deletions, single nucleotide variants, copy variants, and rearrangements, but cannot identify fusions [CAPP-Seq is a recent NGS-based e tumors –74. CAPP fusions ,66.DNA methylation profiling [Whole genome bisulfite sequencing (WGBS-Seq) is the gold-standard approach to acquiring comprehensive base-pair resolution and quantitative information at most genomic methylated cytokines, allowing for unbiased genome-wide rofiling . WGBS isrofiling . Its senrofiling .ctDNA by detecting genome-wide rearrangements, somatic chromosomal aberrations and Copy Number Variations [ctDNA in clinical settings [Whole Genome Sequencing (WGS) and Whole Exome Sequencing (WES) technologies provide novel opportunities for comprehensive profiling of riations . These tsettings ,78.DNA in the blood is a result of various mechanisms including apoptosis, necrosis, and circulating tumor cell lysis causing a DNA leak into the bloodstream [Tumor odstream . Variousodstream ,32,80–85odstream .ctDNA to be detected in early-stage disease, including early-stage breast cancer [ctDNA clearance has been associated with higher rates of complete pathological response after neoadjuvant treatment and with fewer recurrences after radical treatments [ctDNA for breast cancer involves real-time monitoring of tumor response [ctDNA for breast cancer diagnosis, variations of ctDNA over time do seem to reflect the burden of the disease, determine the prognosis of cancer patients, and help predict therapeutic response [Recent advancements in ctDNA technologies have improved sensitivity and selectivity, allowing t cancer . In earleatments . Generalresponse , detectiresponse ,91, asseresponse , identifresponse , detectiresponse  and screresponse ,95. Sincresponse .ctDNA can aid in selecting the optimal sequencing of treatments [ctDNA is a reliable tool for monitoring tumor burden dynamics in patients with metastatic breast cancer undergoing systemic therapy [CA 15-3 or circulating tumor cells, ctDNA levels have a wider dynamic range and exhibit a stronger correlation with changes in tumor burden. Additionally, ctDNA analysis provides an early measure of treatment response, with up to 53% of patients showing a response within a few weeks of treatment initiation [ctDNA can also help track clone evolution and predict the development of resistance to therapy, enabling clinical decisions to be made in a timely manner and sparing patients from ineffective treatments. Since ctDNA is believed to be shed by all tumor sites, it has the potential to be a useful tool for addressing tumor heterogeneity and therapeutic resistance in both the adjuvant and metastatic settings, guiding therapy decisions more effectively [In metastatic disease, eatments . Studies therapy . Compareitiation . Serial ectively .ctDNA alterations (levels 3–4) were associated with an increased likelihood of liver metastasis. In addition, a novel ctDNA-level RECIST (ctle-RECIST) was developed to evaluate treatment response based on ctDNA alteration levels and variant allele frequency.Liu et al.  demonstrCTCs and ctDNA, has increased dramatically to enhance early diagnosis, detection of recurrence, and personalized treatment. CTCs and ctDNA have great potential for determining prognosis, monitoring therapy, and integrating data processing methodologies. Combining ctDNA and CTC analysis may further improve their prognostic value and clinical utility. As precision and personalized medicine become increasingly important, CTC and ctDNA monitoring can help detect and subtype the disease and identify patients at high risk of relapse. However, the clinical utility of ctDNA mutation tracking needs further investigation before it can become a standard of care for patients with early-stage breast cancer. Standardization of the blood collection process to enhance sample stability, defining ctDNA quantification methods, standardizing ctDNA isolation, and enhancing the sensitivity of ctDNA detection for rare molecular alterations are among the challenges that need to be addressed.One of the most pressing challenges in breast cancer treatment is how to better manage patients with early disease. In recent years, research on the applications of liquid biopsies, including In conclusion, the next generation of liquid biopsy studies will be crucial in establishing the clinical applicability of blood-based genomic profiling. Liquid biopsy techniques offer physicians a relatively inexpensive and non-invasive method for detecting and monitoring early-stage cancers, but it remains to be seen whether treatments based on liquid biopsies will lead to better outcomes.


Diabetic kidney disease (DKD) is a significant complication of diabetes that requires innovative interventions to address its increasing impact. Precision medicine is a rapidly emerging paradigm that shows excellent promise in tailoring therapeutic strategies to the unique profiles of individual patients. This abstract examines the potential of precision medicine in managing DKD. It explores the genetic and molecular foundations, identifies biomarkers for risk assessment, provides insights into pharmacogenomics, and discusses targeted therapies. Integrating omics data and data analytics provides a comprehensive landscape for making informed decisions. The abstract highlights the difficulties encountered during the clinical implementation process, the ethical factors to be considered, and the importance of involving patients. In addition, it showcases case studies that demonstrate the effectiveness of precision-based interventions. As the field progresses, the abstract anticipates a future characterized by the integration of artificial intelligence in diagnostics and treatment. It highlights the significant impact that precision medicine can have in revolutionizing the provision of care for DKD. Diabetic kidney disease (DKD) is a prominent microvascular complication of diabetes mellitus, playing a significant role in the worldwide prevalence of chronic kidney disease (CKD). With the global surge in diabetes cases, there is a growing prevalence of DKD, which calls for a thorough reassessment of its management strategies . This seDKD: prevalence and challengesDiabetes mellitus is a medical condition characterized by persistent high blood sugar levels, known as chronic hyperglycemia. It encompasses a range of metabolic disorders that can lead to damage in various organs of the body. Among these, DKD manifests as a standard and incapacitating complication, ultimately progressing to end-stage renal disease (ESRD) if not properly managed . The epiPrecision medicine: an exemplary approach for customized interventionsAlternatively referred to as personalized medicine, precision medicine represents a significant paradigm shift in healthcare, moving away from a universal approach and embracing a customized, patient-focused strategy. This approach is based on recognizing that individuals demonstrate diverse susceptibility to diseases, and genetic, molecular, and environmental factors influence their responses to treatments . PrecisiThe relevance of precision medicine extends beyond genetic factors. Technological advancements, such as proteomics, metabolomics, and transcriptomics, have enabled the thorough profiling of patients. This allows for identifying subtle molecular signatures that indicate the onset or progression of diseases. Similar to diagnostic fingerprints, these signatures provide unparalleled insights into disease mechanisms and facilitate timely therapeutic interventions . The funGenetic and molecular basis of DKD: an investigation into susceptibility, pathways, and therapeutic implicationsDKD is a commonly observed complication of diabetes mellitus that arises from a multifaceted interaction between genetic susceptibility and intricate molecular mechanisms. Gaining a comprehensive understanding of the genetic and molecular underpinnings of DKD is paramount in discovering new therapeutic approaches . This knGenetic Factors Influencing DKD SusceptibilityThe heritability of DKD is apparent based on familial aggregation studies, indicating a significant genetic factor contributing to the susceptibility of this disease. Genome-wide association studies (GWAS) have played a crucial role in identifying genetic loci associated with the risk of DKD. These genetic loci frequently intersect with genes that have been implicated in the pathogenesis of diabetes, renal function, inflammation, and vascular homeostasis. An example of such a gene is the solute carrier family 12 member 3 (SLC12A3), which encodes the thiazide-sensitive sodium-chloride cotransporter. The presence of variants in the SLC12A3 gene has been associated with the development of diabetes and DKD . These fInvestigating Molecular Pathways in the Development and Progression of DKDThe pathogenesis of DKD involves a complex interplay of molecular pathways, including inflammation, oxidative stress, fibrosis, and endothelial dysfunction. Elevated glucose levels initiate intracellular signaling pathways, such as protein kinase C (PKC), nuclear factor-kappa B (NF-κB), and advanced glycation end products (AGEs), resulting in the development of inflammation and oxidative stress. Inflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), are crucial in coordinating immune cell infiltration and intensifying renal injury . This paExploring the Prospects of Targeted Therapies Utilizing Genetic and Molecular FindingsExploring genetic and molecular factors underlying DKD deepens our comprehension of the mechanisms behind the disease. It presents opportunities for targeted interventions in precision medicine. Targeted therapies leverage these insights to improve specific disease components, ensuring optimal effectiveness and minimal adverse effects. Therapies targeting the RAAS demonstrate the promising capabilities of precision medicine . The preThe emergence of precision medicine aligns with the notion of personalized therapeutic regimens. Genetic profiling can identify patients who stand to benefit most from specific treatments, mitigating the trial-and-error approach that characterizes conventional therapies. In addition, identifying molecular signatures that indicate the onset and progression of diseases could inform early interventions, thereby helping prevent irreversible kidney damage . The genExploring insights into omics technologies: biomarkers for early detection and risk stratification in DKDBiomarkers are essential in understanding the complex nature of DKD, as they facilitate the early identification, risk assessment, and tailored patient interventions. This section provides an in-depth analysis of the importance of biomarkers in predicting the onset and progression of DKD, including both well-established and emerging candidates . In addiOnset and Progression of DKD: Established and Emerging FactorsBiomarkers are essential tools used to monitor the progression of diseases, predict outcomes, and inform therapeutic choices. Within the field of DKD, several well-established biomarkers have attracted significant attention due to their consistent correlation with the development and progression of the disease. Urinary albumin excretion, commonly referred to as albuminuria or proteinuria, is a fundamental biomarker used to assess the onset and progression of DKD . IncreasOmics Technologies: Deciphering the Complexity of BiomarkersThe advent of omics technologies, including genomics, proteomics, and metabolomics, has significantly transformed the field of biomarker discovery. These advanced technologies have provided researchers with the means to thoroughly investigate the molecular foundations of DKD. The field of genomics explores the genetic variations that may make individuals more susceptible to DKD -16. On tAdvanced mass spectrometry techniques in proteomics allow for identifying differentially expressed proteins in urine and serum. Studies in urinary proteomics have successfully identified potential biomarkers that indicate glomerular injury, including podocyte-derived proteins, such as nephrin and podocin. Furthermore, proteomics provides insights into the importance of inflammation, oxidative stress, and endothelial dysfunction in the pathogenesis of DKD, thereby identifying potential targets for therapeutic interventions. The metabolomics process, which involves analyzing metabolite concentrations, reveals disturbances in metabolic pathways crucial to the progression of DKD . AlteredThe Importance of Early Detection and Tailored InterventionsThe incorporation of biomarkers into clinical practice facilitates the prompt identification of early DKD, enabling timely interventions to impede or alleviate the advancement of the disease. Early detection is pivotal, as interventions during the early stages of DKD hold the potential to preserve renal function and avert irreversible damage. Utilizing biomarker-driven risk stratification is instrumental in identifying high-risk individuals who stand to gain the most from intensified glucose and blood pressure control . In addiBiomarkers play a crucial role in the fight against DKD by facilitating early detection, risk assessment, and tailored interventions. Integrating well-established and emerging biomarkers with advanced omics technologies, such as genomics, proteomics, and metabolomics, enables a comprehensive comprehension of disease mechanisms . This coRole of pharmacogenomics in understanding drug responses in DKD: customizing treatments based on genetic insightsPharmacogenomics, a field of study investigating the impact of genetic variations on an individual's response to medications, represents a groundbreaking paradigm in treating DKD. This section explains the concept of pharmacogenomics and its implications for drug responses in patients with DKD. By examining drugs frequently utilized in treating DKD, we can ascertain the role of genetic variations in influencing diverse therapeutic outcomes . In concPharmacogenomics: The Influence of Genetic Variations on Drug ResponsesPharmacogenomics is based on the premise that genetic variations contribute to a wide range of drug responses, including differences in effectiveness and safety. These genetic variations can potentially impact drug metabolism, transport, receptor interactions, and subsequent cellular pathways . PharmacGenetic Influences on Drug Responses: Illustrative Cases and ImplicationsNumerous pharmaceutical agents have proven effective in the management of different aspects of DKD, such as the regulation of blood pressure, glucose levels, and reduction of proteinuria. However, the diversity in treatment responses can be ascribed to genetic variations that regulate drug metabolism and targets. One noteworthy illustration involves the utilization of RAAS inhibitors. ACE inhibitors and ARBs are widely recognized as essential treatments for managing DKD . These mIn addition, it is worth noting that sodium-glucose cotransporter 2 (SGLT2) inhibitors, which are innovative antidiabetic medications known for their renoprotective properties, have been found to elicit diverse responses that genetic factors can influence. The SLC5A2 gene is responsible for encoding SGLT2, and genetic variations in this gene have been found to affect the response to certain drugs . VariantCustomizing Drug Regimens Based on Individual Genetic ProfilesThe potential of pharmacogenomics resides in its ability to provide valuable insights for treatment decisions, enhancing therapeutic outcomes while mitigating adverse effects. Within the domain of DKD, this concept involves customizing drug treatment plans according to the specific genetic profiles of individuals. Genetic testing to evaluate pertinent polymorphisms enables healthcare professionals to predict how patients will respond to specific medications . PatientIn addition, pharmacogenomic insights can be applied to effectively manage the comorbidities frequently observed in patients with DKD. Given the interconnection between cardiovascular complications and DKD, it is essential to consider the impact of genetic factors on the effectiveness and safety of medications used to manage cardiovascular risk factors. This includes lipid-lowering agents and antihypertensives . PharmacAdvancements in therapeutic approaches and precision medicine for DKD: harnessing innovation for enhanced patient careThe management of DKD is undergoing significant changes as it embraces innovative strategies to address its complex challenges . This sePresent Standard Treatments for DKDThe treatment of DKD typically focuses on achieving glycemic control and managing blood pressure to minimize the advancement of renal damage. ACE inhibitors and ARBs are widely recognized as fundamental components of therapy. These medications exert renoprotective effects by modulating the RAAS . The priInvestigating Innovative Therapeutic StrategiesThe development of new therapeutic paradigms presents innovative approaches for managing DKD to implement precise and targeted interventions to modify the progression of the disease.Targeted mAbs: mAbs have been developed to selectively target specific molecules that play a role in the pathogenesis of DKD. Agents, such as anti-vascular endothelial growth factor (VEGF) mAbs, have been shown to mitigate the vascular abnormalities associated with DKD, thereby preserving renal function -28. In aClinical trials and precision medicine-based interventions are subjects of interest and importance in healthcare. These trials and interventions involve rigorous scientific research and analysis to evaluate the effectiveness and safety of medical treatments and interventions. Precision medicine, in particular, focuses on tailoring medical interventions to individual patients based on clinical trials, which are essential platforms for evaluating the safety and effectiveness of emerging therapeutic strategies, serving as catalysts for innovations in the field . The triBardoxolone methyl: Bardoxolone methyl, a recently developed antioxidant inflammation modulator (AIM), has attracted significant interest due to its potential to reduce inflammation and oxidative stress in DKD. Various clinical trials, such as the BEACON study, have provided evidence of positive outcomes in terms of eGFR enhancement and reduction of albuminuria . The sigSGLT2 inhibitors: SGLT2 inhibitors, initially developed to manage diabetes, have demonstrated renoprotective properties. The medications empagliflozin and canagliflozin have been shown to reduce cardiovascular events and improve renal outcomes in patients with DKD based on the EMPA-REG OUTCOME and CREDENCE trials, respectively. Genetic markers, such as the polymorphism SLC5A2 rs311057, have been found to impact the responses to SGLT2 inhibitors. This discovery helps to uncover the genetic basis underlying the effectiveness of these treatments .Anti-fibrotic therapies: Emerging anti-fibrotic agents, such as pirfenidone and nintedanib, are revolutionizing the treatment field. Pirfenidone, a pharmacologically approved anti-fibrotic agent used to treat idiopathic pulmonary fibrosis, has demonstrated potential in mitigating renal fibrosis by inhibiting the TGF-β pathway. Nintedanib, known for its anti-fibrotic properties in multiple organs, shows potential to slow down the progression of DKD . ClinicaThe convergence of emerging therapies and precision medicine-driven approaches is shaping the future of treatments for DKD. The shift from traditional standards to targeted mAbs, gene therapies, and RNA-based interventions signifies a significant change toward personalized and mechanistically nuanced treatments. The effectiveness of these new interventions is supported by clinical trials, highlighting the importance of incorporating genetic knowledge to enhance therapeutic results -16. The Challenges and ethical considerations associated with the implementation of precision medicine for DKD: addressing the intricate landscapePrecision medicine represents a transformative phase in healthcare, offering customized interventions to achieve the most favorable results for patients. However, the implementation of this approach within the context of DKD poses a variety of complex challenges and ethical considerations. This section explores the complex challenges that hinder the implementation of precision medicine for DKD. These challenges encompass issues, such as data privacy, cost, and accessibility, as well as the ethical considerations surrounding genetic testing, data sharing, and informed consent. By acknowledging and addressing these challenges and ethical dilemmas, we are laying the foundation for a more thoughtful and fair incorporation of precision medicine into managing DKD .Challenges in the Implementation of Precision Medicine for DKDData privacy and security are crucial considerations in precision medicine, as it heavily relies on the meticulous gathering, safekeeping, and examination of patients' genetic and medical information. Preserving privacy and security for this susceptible information presents a considerable challenge. It is imperative to protect patient confidentiality by implementing solid data encryption, implementing controlled access measures, and adhering to privacy regulations, such as the Health Insurance Portability and Accountability Act (HIPAA) in the United States . AchieviEthical Considerations in Precision Medicine for DKDThe utilization of genetic testing plays a crucial role in precision medicine, as it allows for the identification of markers related to disease predisposition and therapeutic response. Genetic testing raises ethical concerns, specifically about the issue of informed consent. Patients must have a comprehensive understanding of the implications associated with genetic testing. This includes being aware of the potential insights it can provide into other health conditions and the possibility of uncovering non-paternity or non-maternity situations. Ensuring the provision of comprehensive, lucid, and culturally sensitive information during the consent process is of utmost importance . Data sharing and privacy: The progress of precision medicine relies on exchanging genetic and medical data among various institutions, researchers, and consortia. The acceleration of discoveries through data sharing has brought to light ethical concerns regarding patient consent for using data beyond the original study's intended scope. Finding a harmonious equilibrium between facilitating advancements in research and upholding patients' autonomy regarding their data presents a multifaceted ethical dilemma . The disEquity and inclusivity: Equity and inclusivity are crucial considerations in advancing precision medicine, as it is imperative to ensure equal access to its benefits ethically. It is imperative to ensure proper representation of diverse populations in research studies in order to prevent the perpetuation of health disparities. Insufficient representation can result in biased treatments and algorithms that disproportionately benefit specific demographics, amplifying pre-existing inequalities. Incorporating precision medicine into managing DKD involves various challenges and ethical considerations . ResolviIn summary, precision medicine can potentially transform the field of DKD management significantly . HoweverClinical implementation and patient engagement in precision medicine for DKD: facilitating the journey toward individualized careImplementing precision medicine in clinical practice significantly advances DKD management. This section explores the strategies utilized to incorporate precision medicine approaches into clinical care, focusing on patient education and engagement in the decision-making process . MoreoveStrategies for the Implementation of Precision Medicine in Clinical PracticeThe successful implementation of precision medicine, moving from theoretical concepts to practical clinical advantages, requires establishing a comprehensive framework. This framework facilitates the comprehensive integration of genomics, omics data, and clinical parameters to customize treatment regimens for individual patients. Health informatics systems, such as electronic health records (EHRs), play a crucial role in facilitating the smooth data exchange . This caPatient Education and Engagement: Facilitating Empowered Decision-Making Through InformationIt is crucial to provide patients with comprehensive information regarding precision medicine's principles, benefits, and limitations to empower them and facilitate informed decision-making. Patient education facilitates the development of comprehension regarding genetic testing, personalized treatment alternatives, and potential results. Patients who possess this knowledge are more prepared to actively participate in shared decision-making actively, thereby enhancing the optimization of treatment plans that align with their values, preferences, and goals . The praUtilizing Case Studies and Real-World Examples to Harness the Potential of Precision MedicinePrecision medicine has emerged as a promising field with the potential to revolutionize healthcare . To fullPrecision medicine's utilization in managing DKD as demonstrated through compelling case studies and real-world illustrationsCase Study 1: Selection of SGLT2 InhibitorsIn this case study, we will discuss selecting SGLT2 inhibitors. A patient diagnosed with DKD arrives at the clinic. Genetic testing results indicate the presence of the SLC5A2 rs311057 polymorphism, which has been linked to an increased response to SGLT2 inhibitors. Based on this information, clinicians decide to prescribe an SGLT2 inhibitor, which significantly decreased blood glucose levels, proteinuria, and blood pressure [Case Study 2: Personalization of Anti-Fibrotic TherapyA patient diagnosed with DKD, who has not shown a positive response to traditional treatment methods, undergoes a thorough analysis of omics data. The results of the metabolomic analysis indicate an elevation in the concentrations of specific biomarkers associated with fibrosis. With the knowledge gained from these insights, clinicians commence anti-fibrotic therapy customized to match the patient's molecular signature . Over tiFuture directions and outlookCutting-edge advancements and continuous research endeavors are currently illuminating the field of precision medicine in DKD.AI-Driven Diagnostics and TherapiesAI enhances the process of diagnostics by efficiently analyzing intricate omics data, providing insights into disease mechanisms, and identifying potential targets for therapeutic interventions . AI-drivCurrent Research and Unexplored AreasContinual research efforts consistently reveal novel genetic markers, molecular pathways, and therapeutic targets, thereby enhancing the profound influence of precision medicine. Emerging fields, such as epigenomics, single-cell sequencing, and systems biology, present promising opportunities for managing DKD. Continued research into the genetic and molecular foundations of different subtypes of DKD, along with their corresponding responses to treatment, can potentially transform the field significantly .In summary, the transition of precision medicine from a conceptual idea to a practical approach represents a significant milestone in the care for DKD. Implementing strategies focused on integration, patient education, and engagement contributes to a comprehensive approach to personalized care, ultimately enhancing the effectiveness of treatments and improving patient experiences. Case studies highlight the concrete advantages of precision medicine, influencing treatment modifications and resulting in favorable results . The conThe integration of precision medicine into clinical practice has the potential to significantly transform the management of DKD, effectively bridging the divide between conventional methods and personalized care. Patient education and engagement are recognized as crucial elements, promoting a collaborative partnership that empowers patients and enhances treatment outcomes. The case studies presented provide vivid depictions that effectively illustrate the tangible benefits of precision medicine. These examples substantiate precision medicine's efficacy in tailoring interventions and ultimately improving patient well-being. With the horizon in sight, the incorporation of AI-driven diagnostics and the advancement of research avenues enhances the potential of precision medicine to revolutionize care for DKD further. Within the expansive realm of healthcare, precision medicine represents a significant shift in approach. It embodies a patient-centric and scientifically sophisticated form of care that promises to transform the treatment of DKD and improve patient outcomes. This journey exemplifies the coming together of scientific progress, empathetic healthcare, and patient empowerment, with the potential to improve the quality of life for numerous individuals grappling with DKD.


As a porphyrin precursor, ALA is metabolized in the heme biosynthetic pathway to produce protoporphyrin IX (PpIX), a fluorophore and photosensitizing agent. ALA administered exogenously bypasses the rate-limit step in the pathway, resulting in PpIX accumulation in tumor tissues. Such tumor-selective PpIX disposition following ALA administration has been exploited for tumor fluorescence diagnosis and photodynamic therapy (PDT) with much success. Five ALA-based drugs have now received worldwide approval and are being used for managing very common human (pre)cancerous diseases such as actinic keratosis and basal cell carcinoma or guiding the surgery of bladder cancer and high-grade gliomas, making it the most successful drug discovery and development endeavor in PDT and photodiagnosis. The potential of ALA-induced PpIX as a fluorescent theranostic agent is, however, yet to be fully fulfilled. In this review, we would like to describe the heme biosynthesis pathway in which PpIX is produced from ALA and its derivatives, summarize current clinical applications of ALA-based drugs, and discuss strategies for enhancing ALA-induced PpIX fluorescence and PDT response. Our goal is two-fold: to highlight the successes of ALA-based drugs in clinical practice, and to stimulate the multidisciplinary collaboration that has brought the current success and will continue to usher in more landmark advances. Light therapy has been performed for a few thousand years, dating back to the ancient civilizations of India, Greece, Egypt, and China . SunlighAround the turn of the 20th century, Oscar Raab, a student working in the lab of Herman von Tappeiner in Munich, Germany discovered that paramecia would die after receiving acridine red dye and exposure to light. Later, Raab performed experiments that led to the discovery of the fluorescent property of molecules such as acridine. The term photodynamic action was coined from the discovery of Raab and Tappeiner. Another hallmark experiment of photodynamic action was demonstrated in 1913 by Friedrich Meyer-Betz, a German doctor who studied the phototoxic effects of hematoporphyrin, a mixture of porphyrins crudely extracted from the blood. He self-administered 200 mg of hematoporphyrin and exposed himself to the sunlight, resulting in swelling, edema, and hyperpigmentation for several months [Porphyrins such as heme in animals and chlorophyll in plants are heterocyclic macrocycle chemicals with strong absorption within the visible light spectrum . They arThe approval of ALA and its derivatives for tumor diagnosis and treatment represents a landmark in the field of photodiagnosis and PDT. It has brought tremendous success to the management of some very common pre-cancerous and cancerous conditions. However, there is still much room for further improvement to make it more effective, more precise, and more widely used. An optimized ALA protocol may deliver better outcomes by inducing higher and more selective PpIX accumulation in the target tissue. A personalized ALA protocol based on a patient’s tumor genotype and phenotype will increase the precision of this procedure. Further expanding its application to other diseases will bring benefits to more patients. In this review, we would like to summarize all U.S. FDA-approved applications of ALA and its derivatives and discuss strategies for therapeutic enhancement. Since the use of ALA-based drugs for tumor diagnosis or therapy depends on preferential PpIX tumor accumulation, we begin this review with an overview of PpIX biosynthesis from ALA and end with the prospect of combining the diagnostic and therapeutic capabilities of ALA-induced PpIX to become a fluorescent theranostic agent.ALAS1 and ALAS2 encoded by two different genes ALAS1 and ALAS2, respectively. ALAS2 located on the X-chromosome is expressed only in the erythroid precursor cells, whereas ALAS1 on chromosome 3 is expressed in all other types of cells. As the first intermediate in the heme biosynthesis pathway, ALA is a simple amino acid with no fluorescence and photosensitizing activity. Since the production of ALA is the rate-limiting step of heme synthesis, exogenous ALA given orally or topically bypasses this step, resulting in increased porphyrin synthesis.Heme biosynthesis in mammalian cells is a finely tuned process that involves eight enzymes with four in the cytoplasm and four in the mitochondrion , as showThe second step of heme biosynthesis is the formation of porphobilinogen (PBG) from two molecules of ALA catalyzed by ALA dehydratase (ALAD), also called porphobilinogen synthase (PBGS). This step as well as the following three enzymatic reactions all happen in the cytosol. ALAD is a tetramer of homodimers that utilizes zinc to both stabilize the protein structure and catalyze the reaction. Once four molecules of PBG are synthesized, they are linked together via deamination to form a linear tetrapyrrole named hydroxymethylbilane (HMB) by the enzyme porphobilinogen deaminase (PBGD), also called hydroxymethylbilane synthase (HMBS). HMB will be cyclized to produce the cyclic tetrapyrrole molecule uroporphyrinogen III (URO III) by uroporphyrinogen III synthase (UROS). There is a possibility that spontaneous cyclization of the HMB molecule may occur, forming uroporphyrinogen I, which will not proceed to form heme. Decarboxylation of URO III by uroporphyrinogen decarboxylase (UROD) leads to the formation of coproporphyrinogen III (CPO III), which completes the cytosolic process of heme biosynthesis. 2+) is inserted into the tetrapyrrole core of PpIX by ferrochelatase (FECH) to form heme [CPO III is then imported into the mitochondrial intermembrane space where it is converted into protoporphyrinogen IX, a reaction catalyzed by coproporphyrinogen III oxidase (CPOX). Protoporphyrinogen IX crosses the mitochondrial inner membrane and is aromatized by protoporphyrinogen oxidase (PPOX) to produce PpIX, a porphyrin metabolite with fluorescent and photosensitizing properties. Heme biosynthesis ends in the mitochondrial matrix where ferrous iron . With high reactivity, singlet oxygen can oxidize various biological substances including proteins, lipids, and nucleic acids, causing tissue oxidative damage. Due to the short lifetime and diffusion distance of singlet oxygen, the site of oxidative damage is in close proximity to the localization of the photosensitizer [PDT is a light-based therapeutic modality that involves a photosensitizer, a source of light with a specific wavelength, and molecular oxygen. These components individually are not harmful but become cytotoxic when combined due to the generation of reactive oxygen species (ROS) via type I and II photochemical reactions. Upon the absorption of light energy, photosensitizer molecules at the ground singlet state are excited to the excited singlet state. Molecules at the excited state are very short-lived and can decay back to the ground state by energy dissipation through fluorescence emission and internal conversion to heat, or undergo intersystem crossing to the excited triplet state with longer lifetime. Photosensitizer molecules at the triplet state may directly transfer electrons to substrate molecules to produce free radicals (Type I reaction) or, more commonly, transfer energy to molecular oxygen (Type II reaction), which is often abundantly available in the tissue of oxygen-consuming species, to generate single oxygen (nsitizer . As a nonsitizer . The mosFECH gene) than in normal human lymphocytes and could be further enhanced by an iron chelator, suggesting the importance of FECH and ferrous iron in regulating PpIX level. Photoactivation of endogenous PpIX induced by ALA results in the elimination of erythroleukemia cells as Malik et al. showed in 1987, which is the first report of using ALA-PDT for cell inactivation [ALA was known to cause endogenous PpIX accumulation in human lymphocytes in the 1970s . In thistivation . The fintivation . In 1990Multiple phase II and III clinical trials followed the initial promising study by Kennedy et al. In phase III trials of 241 patients with head AK, topical application of ALA solution on the lesion area followed by blue light treatment induced a 72% complete response rate at 12 weeks after PDT, whereas only a 20% response rate was seen in patients treated with vehicle and light only . PatientTo increase transdermal tissue penetration for thick skin lesions, the methyl ester of ALA methyl aminolevulinate (MAL) with higher lipophilicity than ALA was evaluated for PpIX production . AlthougAnother successful approach to enhancing ALA tissue penetration is to formulate it with BF-200 nano-lipid vesicles, which increases the fusion between ALA nano-vesicles and the phospholipid bilayers of skin cells . The res2+ release [2+ release, triggering apoptosis through ER-stress signaling [The high efficacy of ALA-PDT in clearing AK and BCC lesions, both characterized by abnormally activated cell proliferation, highlights its effectiveness in inactivating unwanted cells. ALA-PDT is very effective in inducing cell death largely due to the preferential PpIX accumulation in mitochondria, the powerhouse and stress sensor of the cell. Light activation of PpIX in mitochondria induces the formation of ROS species such as hydroxyl radicals, hydrogen peroxide, and superoxide radicals, which causes direct mitochondrial structural damage and Ca release . Consequ release ,26,27,28ignaling . Furtherignaling .ALA-PDT has been shown to promote necrotic cell death as well, which is highly dependent on the cell line, PpIX localization, and PDT dose. While some cell lines undergo rapid apoptotic cell death within hours of PDT, others are more prone to die by necrosis at later times . PpIX loAlthough necrosis in general is often considered to be a type of unprogrammed passive cell death due to extreme cell injuries, certain forms of necrosis including necroptosis, pyroptosis, and ferroptosis are highly regulated by specific pathways or are associated with damage to specific targets . In LN18Lastly, ALA-PDT is also known to induce autophagy, the degradation of cellular components by lysosomes. However, instead of being a cell death mechanism, autophagy is often exploited by tumor cells to survive cancer chemotherapy and radiation therapy . This isAlthough ALA itself is non-fluorescent and possesses no photosensitizing capacity, PpIX produced after ALA administration exhibits red fluorescence and photosensitizing activity upon light activation. With the maximum absorption at around 405 nm in the soret band, PpIX also has multiple absorption peaks in the visible Q-bands, enabling its excitation with longer wavelengths of light to achieve deeper tissue penetration . FollowiGlioma is the most prevalent central nervous system tumor and is classified into four grades, grades I, II, III, and IV, by the World Health Organization . Most glThe first clinical trial of using ALA for PpIX fluorescence-guided glioma resection was reported by Stummer et al. in 1998 . Out of 18F-fluoroethyl-l-tyrosine (18F-FET) PET uptake, suggesting a distinction in tumor cell biology and tumor microenvironment [Although most high-grade gliomas show strong PpIX fluorescence, low-grade gliomas typically do not exhibit PpIX fluorescence ,51. Neveironment ,53. Partironment . Due to ironment . There aironment  or delayBladder cancer is among the top 10 most common cancers in the U.S. and the 4th most common cancer in men . TreatmeThe first report showing that ALA is valuable for detecting bladder cancer came out in 1994 when Kriegmair et al. observed bright red fluorescence in all tumor lesions after intravesical instillation of ALA in 68 patients . ALA-indSince topically applied ALA has slow and limited tissue penetration due to its hydrophilicity, hexaminolevulinate (HAL) with increased lipophilicity was developed to overcome these limitations . A compaVarious approaches have been explored to enhance the use of ALA and its derivatives for PpIX-mediated tumor diagnosis and treatment. Since PpIX derived from exogeneous ALA can be converted to heme (with no fluorescent and photosensitizing property) in the heme biosynthesis pathway and effluxed via active transporters, suppressing these two predominant biological processes that reduce intracellular PpIX has been extensively evaluated for increasing PpIX tumor disposition . The recThe FDA approval of ALA and its derivatives includes accompanying devices for PpIX activation and imaging. Blue light illuminator (centered at ~417 nm) is accompanied by 20% ALA solution (Levulan) for PDT of skin lesions in the U.S., whereas red light (centered at ~635 nm) is predominately used in Europe. PpIX has stronger absorption of blue light than of red light, while red light penetrates deeper into the tissue. ALA-PDT with blue light shows equal efficacy to PDT with red light in clearing BCC lesions at 6 months after treatment . Althoug2 at 4 and 6 h after topical ALA application yields a much better clinical response than conventional single 75 J/cm2 illumination at 4 h after ALA. Compared with single-illumination PDT, double-illumination PDT resulted in a significantly higher complete response rate at one year after treatment in patients with AK [PDT protocols with fractionated light illumination have been shown to enhance PDT outcomes . Particu with AK  or super with AK  lesions. with AK . Althoug with AK ,69. The standard protocol of ALA for fluorescence-guided resection of high-grade glioma is oral administration at a dose of 20 mg/kg body weight about 4 h before surgery, which is based on the assumption that PpIX fluorescence will be at a maximum at this time . HoweverAlthough blue and red light-emitting diode (LED) illuminators are commonly used as the light source to activate ALA and MAL for PDT of AK lesions, natural daylight is emerging as an attractive and convenient alternative. Multiple clinical trials have shown no significant difference in complete response rate between patients treated with daylight-PDT or conventional PDT with LED light . DaylighImprovements in current PpIX imaging devices have been made to better visualize PpIX fluorescence in gliomas. PpIX fluorescence is usually visualized with the BLUE 400 observation filter in a surgical microscope . The sysConventional surgical microscopes also struggle to show PpIX fluorescence in gliomas deep inside surgical cavities due to limited tissue penetration with blue light and hindered light illumination. Endoscopes with angled lenses are able to increase PpIX detection by providing focused light illumination within the cavity, resulting in the diagnosis and resection of glioblastomas that were not visible by the microscope in 10 out of 12 (83.3%) patients . IncorpoNew imaging devices are being developed and tested in clinical settings for easier and more sensitive visualization of PpIX fluorescence. As current glioma surgery is often performed with the aid of surgical loupes, a loupe device with three LED light sources has been developed for PpIX fluorescence-guided glioma resection . This trAdditionally, current microscope-based PpIX visualization technology aims to reveal perceived differences in fluorescence intensity between tumor and normal tissues, which is sensitive to background signals and variations in tissue optical properties. To overcome the limitation of this intensity-based detection, new imaging technologies based on fluorescence lifetime  and tissPpIX generated after ALA administration will continue to be metabolized in the heme biosynthetic pathway to produce heme with no fluorescent and photosensitizing properties. Thus, inhibition of PpIX to heme bioconversion may cause more PpIX accumulation, resulting in enhanced PpIX fluorescence and PDT response. Since this bioconversion step is catalyzed by the enzyme FECH, which uses ferrous iron as another substrate to form heme, inhibiting PpIX to heme bioconversion can be achieved by two pharmacological approaches, FECH inhibition and iron chelation . Iron chelators are commonly used to inhibit PpIX to heme bioconversion by chelating ferrous iron. Non-specific and membrane-impermeable metal ion chelator ethylenediaminetetraacetic acid (EDTA) is the first chelator shown to increase PpIX levels in cells  and sensAlthough not yet FDA-approved, 1, 2-diethyl-3-hydroxypyridin-4-one CP94) is another iron chelator that has been extensively explored for enhancing PpIX fluorescence and PDT response. It is more effective than DFO in increasing ALA- or MAL-induced PpIX accumulation in skin cancer cells likely due to its smaller size and higher lipophilicity, resulting in greater cellular uptake . Its eff is anothFECH expression [Despite these promising results from many in vitro studies, EDTA does not increase PpIX production in a mouse tumor model  and BCC pression ,107. ParFECH significantly increases ALA-PpIX fluorescence in multiple cell lines [FECH [In addition to iron chelators, inhibition of FECH activity is another approach to inhibiting PpIX to heme bioconversion. Although genetic silencing of ll lines ,110,111 ll lines , pharmacll lines ,114,115,ll lines . Vitamines [FECH . HoweverAbcg2 knockout study [Abcg2-knockout mice developed protoporphyria with increased PpIX in erythrocytes and plasma, suggesting the involvement of Abcg2 in PpIX transport. A subsequent study determined the effects of ABCG2 overexpression and transporter inhibition on intracellular and extracellular PpIX, providing compelling evidence that PpIX is an ABCG2 substrate [PpIX produced after ALA administration can be effluxed out of cells, resulting in reduced intracellular PpIX levels. Therefore, inhibiting PpIX efflux may increase intracellular PpIX accumulation and sensitize cells to ALA-PDT . Being lut study . Abcg2-kubstrate .Identification of PpIX as a substrate of ABCG2 opens the door to using ABCG2 inhibitors to enhance ALA-PpIX fluorescence and PDT response. Fumitremorgin C (FTC) is the first selective ABCG2 inhibitor shown to significantly increase ALA-PpIX fluorescence and PDT-induced cytotoxicity in ABCG2-transfected cells . It signKo143, unfortunately, is also not suitable for in vivo application due to its instability in the plasma and rapid inactivation in the liver ,130. In While ALA-PDT targets mitochondria and other cellular targets to induce cell death, it also activates pro-survival cell signaling pathways including nuclear factor (NF)-κB, phosphoinositide 3-kinase (PI3K), and mitogen-activated protein kinase (MAPK) signaling pathways . TargetiNitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) is another pro-survival signaling molecule generated after ALA-PDT . PDT wasALA-PDT not only directly kills tumor cells but also elicits potent immune responses with important implications in the long-term therapeutic outcome. It induces the release of inflammatory cytokines such as TNFα, which promotes the infiltration of polymorphonuclear cells particularly neutrophils that can kill tumor cells directly by ROS release and indirectly by the subsequent antitumor immunity ,150,151.In addition to stimulating inflammation and adaptive antitumor immunity, ALA-PDT also causes immunosuppression ,155. VarIt is worth noting that ALA-PDT is able to selectively kill activated T cells including CD4 and CD8 cells. This is because PpIX is preferentially accumulated only in activated T cells but not resting T cells without activation after ALA incubation, resulting in the selective killing of activated T cells by PDT ,160. HowTheranostics is a general term used to denote any therapeutic agents or modalities that combine disease diagnosis with therapy . NuclearSince the first approval in 1999, five ALA-based drugs have been approved by the U.S. FDA. These drugs are approved not only in the US but also in many other countries . They arEven though ALA-induced PpIX has the potential for being a fluorescent theranostic agent, it is currently utilized as either a PDT therapeutic agent or a fluorescent diagnostic probe. Combining its tumor diagnostic and therapeutic capabilities is appealing for the management of high-grade gliomas such as glioblastoma. As mentioned above, current PpIX visualization with wide-field surgical microscopy is highly sensitive for revealing the tumor core for bulky tumor resection, but not good for tumor visualization at the tumor margin due to low tumor cell density and surgical cavities because of the tissue hindrance to light penetration. To visualize tumor tissues in these regions, more sensitive imaging technologies such as probe-based sectioning microscopy and spectroscopy need to be deployed to aid precision surgical resection. After bulky tumor resection followed by precision tumor surgery at the tumor periphery, PDT is added to target residual infiltrating tumor cells or tumors in eloquent regions that are not safe to resect. Clinical trials are needed to determine whether such a combined procedure results in any additional benefits in patient survival compared with current standard practice.All successful uses of ALA for tumor therapy and diagnosis depend on selective PpIX tumor disposition and there are currently substantial efforts going on to further increase tumor PpIX. However, despite two predominant biological processes that reduce intracellular PpIX having been identified, i.e., PpIX bioconversion and efflux, it remains to be seen whether targeting these two PpIX-reducing processes will result in any clinical benefits. Furthermore, the question regarding the biological basis of selective ALA-induced PpIX tumor disposition is yet to be addressed. Reduced tumor FECH activity is so far the most common answer to this question, although recent studies have failed to establish a correlation between tumor PpIX levels and FECH or any particular heme biosynthesis enzyme and porphyrin transporter ,127,166.Finally, the success of ALA and its derivatives as tumor diagnostic and therapeutic agents comes from decades of research by biochemists, bioengineers, clinicians, photobiologists, photo-physicists, and pharmaceutical scientists. It is this multidisciplinary collaboration that culminates in the development of ALA-based drugs that are formulated in different preparations and activated by light from stable light sources to elicit biological and physical responses for therapeutic purposes. It is expected that continued collaborative research on ALA among professionals from different disciplines will bring more successes in the years to come.


The response of aquatic and terrestrial organisms to climate change can depend on biological sex. A key challenge is to unravel the interactive effects of sex and climate change at the individual and population levels and the cascading effects on communities. This new understanding is essential to improve climate adaptation and mitigation strategies. Challenges posed by climate change threaten ecosystems, the crucial benefits they provide to people, and, ultimately, human livelihoods and lives. Global change impacts ecological systems and processes across continents and oceans, resulting in changes to the phenology, distribution and behavior of organisms, persistence and dynamics of populations, and structure and function of species assemblages and ecosystems. In the Anthropocene, a deeper understanding of the biological impacts of climate change, as well as innovative solutions, are needed to reverse climate-induced biodiversity loss.2. Sex is frequently not reported or tested, likely because researchers are not aware of sex-based differences, assume that including sex-specific information would not influence experimental results or are unable to obtain sex-specific data3. Here, we discuss examples of sex-based differences across aquatic and terrestrial domains. We argue that studying sex-based differences from organisms to populations and communities may generate new insights into resilience and vulnerability to climate stressors that may otherwise not be recognized.One largely ignored dimension in research and conservation is how organisms respond differently to climate change depending on biological sex4. The broad range of sex-specific responses to different climate stressors is illustrated through selected examples in Fig. Sex-specific responses to climate change have been documented across a broad range of ecosystems and taxa, from plants to invertebrates, fish, birds and mammalsOncorhynchus keta) populations in the North Pacific5  were found to increase in adult body size due to earlier snowmelt more than males6 7 and more males than females in tuatara (Sphenodon guntheri)8 8 Fig. .Acartia tonsa), males showed significantly lower survival than females9  segregate spatially in the Arctic, with females depending on sea-ice cover for breeding and nursery more than males. A climate-induced reduction in sea ice in the Chukchi Sea forced females to use terrestrial haul-outs instead of sea-ice sites to rest and nurse while foraging in offshore grounds15  segregate spatially following different strategies to maximize reproductive success: males to maximize pre-rut energy reserves and promote successful competition with other males for mates, and females to promote offspring growth and survival18. A reduction in high-quality forage owing to climate change means female caribou are likely to move northward to support breeding and lactation, which exposes them to greater predation during migration19  in the South Atlantic , population viability was significantly overestimated when sex ratio and mating system were ignored21, and in a theoretical model of a two-sex spreading population, population invasion was overestimated when sex-specific differences in demographic and dispersal parameters, and in mate-locating mechanisms were overlooked22.Ignoring sex-based differences leads to underestimates of the relative influence of a changing environment on aquatic and terrestrial populations. More critically, pooling sex-specific data may result in not detecting any effect of environmental drivers or in overestimating effects at a population level. In the case of Northern giant petrels, survival effects were not apparent when females and males were considered together but evident when considered separatelyPandalus borealis) are protandric hermaphrodites functioning first as males, then transitioning to become females. Ocean acidification and warmer temperatures slow Northern shrimp development such that their transition from male to female no longer coincides with the spring phytoplankton bloom, the major food source for larvae23. This situation prolongs larval vulnerability to predation, with negative impacts on recruitment rates. The Greenland shrimp fishery relies on Arctic Northern shrimp stock availability24. These climate-induced changes might underlie the decreased shrimp catch rates in this region and the subsequent effects on local employment and economies where fishery processing facilities have already been closing due to a decrease in catch rates since 201224. To fully understand biological responses to climate change, community-level consequences of sex-specific differences at individual and population levels need to be investigated for the higher-order patterns they reveal25 28 that segregate by sex in warmer years . For example, MPAs may not successfully support population viability if they unintentionally protect only one sex of a species, such as Black-vented Shearwaters  considering species distribution not including (map a) and including knowledge about climate-induced sexual segregation (map b).Decision-makersRevising spatial prioritization criteria for new protected areas in relation to sex-specific knowledge on the response of species and ecosystems to climate change;Coordinating management and monitoring in relation to sex-specific knowledge for climate adaptation and mitigation.ResearchersDisentangling the interaction between sex and climate drivers to understand their relative contribution and effects.Understanding how sex-specific responses to climate change at an individual level will propagate across systems affecting populations and community interactions.Considering sex and sex-based differences in laboratory and in field experiments across species and systems.Funding agenciesElaborating funding schemes to support basic research on sex-based differences in species in response to climate change.Example of the identification of a new MPA considering sex-specific responses to a changing climate. Some species, such as Black-vented Shearwaters (Puffinus opisthomelas) in coastal waters off Baja California, Mexico28, segregate by sex for foraging in warmer years. Sexual segregation only in warmer years might be a foraging strategy during years characterized by lower productivity, leading to sex-specific interannual variation in foraging areas. If a new MPA (in red) is selected without this information (map a), in a warming scenario, the new MPA would unintentionally protect foraging grounds for one sex but not for the other , and consequently impact population dynamics and viability.Supplementary Information


Cells are constantly assaulted by endogenous and exogenous sources of DNA damage that threaten genome stability. To maintain genome integrity, organisms have evolved a myriad of responses, including transcriptional programs, cell cycle regulation, lesion bypass, and direct repair—collectively called the DNA Damage Response (DDR). While much of the research describing these responses has utilized mammalian cell culture systems, studies performed in model organisms have also provided critical insights into the organismal regulation of damage responses ,4,5,6,7.Many of the articles in this Special Issue focus on the repair of a particularly toxic type of DNA lesion, the DNA double-strand break (DSB). DSBs are primarily repaired through non-homologous end joining (NHEJ) and homologous recombination (HR). While HR is typically considered error-free due to the use of a homologous sequence to repair the event, NHEJ can be error prone due to the insertions and deletions of nucleotides at the break site. The choice between these two pathways is dictated by several factors, including phase of the cell cycle and genomic location of the break. The eukaryotic genome is packaged into different domains that impact nuclear structure, gene expression, and DNA DSB pathway choice. Of note is silenced chromatin, which includes heterochromatin. Heterochromatin domains are typically transcriptionally inert and often contain repetitive DNA. Two review articles in this issue synthesize current understanding of the response to DSBs within silenced chromatin. These genomic domains require a unique response to DNA damage, which is described by Kendek et al. in a review describing the preference for DSB repair pathway choice and post-translational modifications related to the DDR in heterochromatin . The recIn addition to genome structure, the cell cycle also contributes to regulating the DNA damage response. In their review, Clay and colleagues describe how model organisms have shaped our understanding of the DDR, in particular within the context of the cell cycle . They diAgrobacterium-mediated transformation (AMT) of T-DNA and gene targeting (GT), both of which require DNA DSB formation. Practically, these lesions can lead to cell death if unrepaired or genome rearrangements if repaired incorrectly. Thus, understanding the mechanisms that regulate the repair of these DSBs in plants is critical.Lastly, Shen et al. provide a review focusing on the use of plants as a model system . GeneticDrosophila melanogaster. Several unique features in Drosophila allow experimental manipulations in this model organism to address specific questions regarding DSB repair pathway choice. For example, Bergerson et al. aimed to address whether the increased incidence of cancer in long-shift workers was associated with a shift from repair of DNA DSBs from a more error-free HR to error-prone NHEJ. They utilized the ability to disrupt circadian rhythms in Drosophila by experimentally shifting the light/dark cycle and measuring the repair outcome of a DNA DSB reporter assay [The original research articles featured in this Special Issue focus on new findings related to DNA damage repair in er assay . This ster assay ) may be Drosophila with CtIP deletions are viable [Drosophila CtIP is involved in homology-directed repair using DSB reporter assays that measure both HR and single-strand annealing (SSA), a third DSB repair mechanism that is homology-directed and can require extensive end resection. This work demonstrated a decrease in HR as well as “short” (500 bp) and “long” (3 kb) SSA, confirming a role for CtIP in end resection [In addition to applications of experimental manipulations, non-murine model organisms also play an integral role in determining the function of proteins that may be essential in mammals. For example, null mutations in mammalian C-terminal Interaction Protein (CtIP) are lethal, thus preventing classical reverse genetics experiments; however, e viable . Yannuzzesection .Drosophila, which affords the tracking of several mutant alleles in relatively short generation times. Thomas et al. investigated the contributions of a variety of helicases to the repair of DNA double-strand gaps following transposon excision [helq, fancm, and blm single and combination mutants suggested that the FANCM and HELQ helicases function upstream of BLM to create recombination intermediates that are then unwound by BLM. Without BLM, these structures are processed in a way that creates large genomic deletions.Using classical genetic techniques in model organisms allows for the ability to determine whether proteins exhibit epistasis or function in redundant pathways. This is a common approach using excision . Analysimutagen sensitive 109 (mus109) as the DNA2 gene, which encodes a helicase/nuclease protein [mus109 mutants as sensitive to agents that cause DNA adducts as well as DNA DSBs, thus establishing the first transgenic allele set of Drosophila DNA2 and, importantly, contributing to our understanding of the conservation of DNA2 across species.Lastly, model organisms can be utilized in forward genetic screens to identify proteins involved in the DDR. Mitchell and colleagues used classical genetic mapping techniques to identify  protein . They chThus, this Special Issue has demonstrated that model organisms continue to provide essential information about how the eukaryotic DDR maintains the integrity of our genome and reveal insights that would not be possible in cell-based systems.


The COVID-19 pandemic pushed governments worldwide to implement unprecedented mitigation measures, including safe-distancing, lockdowns, disruption of non-essential services, border closures and travel restrictions, with both potential to affect rural and urban service-users differently and unintended consequences including reductions in sexual and reproductive health (SRH) services. We aimed to explore rural–urban differences in progress and challenges in SRH services provision in Cambodia, particularly during initial months of the COVID-19 pandemic.We used a mixed-methods study design, including a household survey of 423 adolescents and women aged 18–49 and semi-structured interviews with 21 healthcare providers. We analysed survey data using multivariable logistic regression to identify associations between rural–urban setting and contraceptive perceptions or access. We analysed interview data thematically.Rural–urban residence was significantly associated with reported perceptions about and access to contraceptives. Rural participants had higher odds of stating it was possible to change contraceptive methods early in the COVID-19 pandemic, compared with urban participants. Qualitative data showed that although SRH services continued, health-workers faced differential challenges in rural and urban areas, e.g. service-users not attending due to job losses in urban areas and not complying with safe-distancing and mask-wearing requests in rural areas.COVID-19 and inadequate mitigation responses differentially affected rural and urban SRH service providers and service-users, exacerbating existing socioeconomic stressors while adding new fears of infection, transport constraints, and reduced livelihoods. Added financial support could help mitigate challenges in both rural and urban areas. Since the COVID-19 pandemic began, countries have adopted mitigation measures, including safe distancing, reducing non-essential services, lockdowns, border closures, and travel restrictions to curb disease spread. Unintended consequences of these measures have included economic disruption, job and education losses, and overwhelmed health systems due to people seeking pandemic-related services , 14, 32.Cambodia’s historically poor SRH indicators, including a 2000 maternal mortality ratio of 488 per 100,000 live births that was among the highest in the Southeast Asia region, improved significantly with government-led interventions . HoweverDespite a growing body of research focusing on SRH, few studies target young people aged 18–24, resulting in significant knowledge gaps. Adolescent SRH is of particular concern in Cambodia due to rapid increases in the adolescent population ,CambodiaWe aimed to explore progress and challenges in SRH services provision in Cambodia, especially during the first six months of the COVID-19 pandemic and identify any rural–urban differences. Objectives were to: (i) assess reported perceptions and experiences of SRH needs and service use among young people of both genders (aged 18–24) and women (aged 25–49) in urban and rural areas of Cambodia; (ii) explore SRH provider perspectives of SRH service effectiveness and use; and (iii) inform government and partner agency support for SRH services during the pandemic.We used a concurrent mixed-method design, including a cross-sectional household survey and semi-structured key informant interviews (KIIs) with SRH service providers in one urban (i.e. Phnom Penh city) and two rural provinces (i.e. Kampong Cham and Mondulkiri).Survey. We used 2020 population projection data from the National Institute of Statistics  to calcuInterviews. We conducted 21 KIIs with providers at the same time, three at provincial health department level, two at operational district level, three at referral hospital level, seven at health centre level, and six at village level . The questionnaire included three sections: (i) socioeconomic and demographic characteristics, (ii) general health  and (iii) SRH and maternal health . Participants answered questions on the pre-pandemic (December 2019) and early-pandemic periods (July–August 2020). The questionnaire was developed in English, translated into Khmer, and back-translated into English by another translator to ensure original 'content and spirit' were maintained. Once finalised, the questionnaire was transferred to tablets running KoBoToolbox software. Each questionnaire took approximately 45 min to complete. Experienced enumerators collected data July–August 2020 after piloting the questionnaire face-to-face with seven women aged 18–49 years recruited from Phnom Penh to ensure wording and contents were socio-culturally suitable and comprehensible .Interviews. We developed the interview guide in English, and translated it into Khmer, in consultation with national and regional experts to capture key challenges to SRH services provision during the pandemic. Experienced interviewers, trained in qualitative methods, conducted face-to-face interviews with health providers at their health facilities. Interviews were conducted in July–August 2020, concurrently with survey data collection. Each interview took approximately 45–60 min, was audio-recorded, transcribed in Khmer, and translated into English by the research team. Personal information and identifiers were removed from transcripts and field notes to ensure anonymity and confidentiality.2 test or Fisher's exact test  for categorical variables and Student's t-test for continuous variables. Two-sided p-values < 0.05 were deemed statistically significant. Significant χ2 test results with a degree of freedom ≥ 2, or category with adjusted residual ≥ 1.96, were treated as contributing to statistical significance. We used multivariable logistic regression to analyse associations between age, setting, household income, contraceptive perceptions, and changes in access to health services and contraceptives. Predictor variables were age group (18–24 or 25–49), setting , and household income . Logistic regression analyses of binary outcome variables included: (i) perceived safety of contraception use during the pandemic; (ii) possibility of changing contraceptive method during the pandemic; (iii) necessity of contraceptives in preventing unwanted pregnancy during the pandemic; and (iv) access to contraceptives.Survey. We analysed survey data using Stata 15 . We used descriptive statistical tests to compute means and standard deviations for numerical variables and frequencies for nominal and ordinal variables. We used χInterviews. As survey and interview data were collected and analysed concurrently, qualitative interview findings primarily served to add nuance to quantitative survey findings. We analysed qualitative data thematically using deductive and inductive coding as described by Braun and Clarke . This coWe obtained ethics approval from the National Ethics Committee for Health Research (NECHR-162) in Cambodia and the National University of Singapore Saw Swee Hock School of Public Health departmental ethics committee (SSHSPH 093-1).Table Table Table Rural participants had no significant differences in adjusted odds of reporting increased physical aggression toward household members , family tension , depressive symptoms , or tension towards children  as compared to urban participants.Table We included one deductive theme (i.e. SRH services access and use) and two inductive themes  that best described our data. While qualitative interviews were primarily intended to clarify and triangulate survey results, we noted that most interviewees focused primarily on their own experiences as SRH service providers during the initial months of COVID-19 and have reported these findings accordingly."We have all services, prescription services, health check-ups, and maternal and child-related vaccination available. We offer vaccination in the villages and every day at the centre. We offer other services such as delivery, postpartum check-up, birth control methods such as condoms, injections, pills, intrauterine devices, natural contraception like calendar contraception, and condoms." Views on the impact of the pandemic on access and use of SRH services varied. Participants indicated that rural areas were more affected by mitigation measures than urban areas, as existing transport shortages to connect households to nearby health centres were exacerbated by the public transport ban. However, some interviewees indicated that services for women and adolescents remained relatively unaffected. A rural interviewee from Kampong Cham province indicated that health providers continued to offer maternal and child-related vaccination services and antenatal and postnatal services in village health centres."It's really a challenge! Firstly, we are so scared of that public health emergency and afraid we would get that disease from clients because most of them who come here, we don't know if they have that disease or not. Some of them cooperated with us by wearing masks and keep social distancing. Some of them don't wear mask and they are in groups, big groups … when there is someone sick, there are at least three to four persons to take care of a patient. It's hard because of different traditions." "[We are] worried about COVID, afraid it would transmit. Afraid it would transmit in the community. It must be difficult, and we also do not have enough materials. We are the healthcare providers, so we protect ourselves, but the community people come without wearing masks. We tell them to wear masks, but some still do not wear masks. They act normal and are not afraid of the virus" Particularly in rural areas, fear of interacting with patients was exacerbated by poor adherence to safe-distancing recommendations among service-users and the concomitant fear of infection. Interviewees described how large numbers of patients and caretakers continued to gather in groups within health facilities."During this COVID, the numbers of clients have been decreasing, they are afraid to come. The rates of those who are the existing service-users also decrease." Urban interviewees additionally highlighted how fear of infection hindered service-users from accessing available healthcare services."Most of the women who come here complained about their financial crisis. High unemployment. Many women who work at Karaoke used to come here before, but now they seem not to have any income… we are worried that it would be less and less if the public health emergency keeps going." Some urban providers reported fewer service-users seeking SRH services from March to May 2020, mainly due to fear of infection and lack of sufficient money. As many service-users lost their employment during the lockdowns, especially those working for the private sector at karaoke clubs and garment factories, they were unable to afford out-of-pocket payments for healthcare services.“We cannot do awareness-raising because we are seeking for where the children are to offer vaccination and women who need to get pregnancy check for prenatal and postnatal. Doing that we cannot offer all services because it's a long way, we cannot carry all materials to all communities as they need, the road is an unpaved road, it's difficult to travel." Transportation and physical access to services remained a long-term challenge for SRH in rural areas, even prior to the COVID-19 pandemic. Participants reported that awareness and training sessions were temporarily halted in some villages where unpaved roads prevented transportation of essential supplies and equipment."Lack of staff…because of the epidemic, we sent two staff to the border. So when there are many patients, we face difficulties, we are so busy… We provide service to everyone, but it is a bit slow." Health-worker experiences during the initial phase of the pandemic varied. Some rural providers reported being overwhelmed by the number of service-users and insufficient medical staff, while others reported a decrease in their workload. Reduced staffing and services made it difficult for service-users to access required SRH services, and if services were available, service-users could expect delays or interruptions due to staffing shortages.'We are always affected by COVID. It's also been decreasing related training because we are not allowed to gather as big group, and the supervision activities also reduced, and the patients have been decreasing too." All providers described feeling overwhelmed by the lack of social and professional support. They reported being quarantined away from their families, which negatively affected their psychological wellbeing, consequently predisposing some to depression and anxiety. On a professional level, the COVID-19 pandemic decreased opportunities for training and supervision, leading to a decrease in professional support and training that was particularly noted by rural participants."[I have] no [supplementary] income. It's because the evaluation was stopped, so no income. There is no evaluation program, so the income has been decreased… During the public health emergency, I couldn't attend any workshops, meetings, so no income… [I have lost] about 20% of salary" Some rural health-workers also mentioned loss of income due to the cessation of secondary activities such as evaluations and training workshops. Supplementary income from activities organised by development partners provided a significant portion of health-workers’ monthly wages, so this was a significant loss at a time when salary disbursements were also affected by COVID-19 related staffing shortages."I got paid less during the March to April (2020) period." Most providers reported that working hours had doubled between December 2019 and July 2020, with many indicating their compensation had also increased in accordance with the hours worked. Some attributed the longer working hours to the increased number of service-users. However, others reported seeing fewer patients and therefore being paid less.While this pattern was not consistent, it appeared that the pandemic contributed to increased service use in some rural facilities and reductions in other, primarily urban, facilities."We are more careful. When clients come, all staff wear masks and wash hands with alcohol, keep hygiene all the time. We wear gloves. Everything we need to be more careful than before…." .During the first year of the pandemic, health-workers in rural areas reported the quality of their communication with service-users worsened due to the precautionary measures they took when interacting with service-users, such as wearing facemasks and personal protective equipment. All providers described being more careful around service-users."When we need to communicate with village or commune chiefs, we invite them. Sometimes, the village [chief] doesn't update the commune [sub-district] or police, so they contact us. It's difficult because there is no budget to travel to village or commune." Some rural interviewees reported a lack of collaboration between frontline health-workers and governmental actors, due to preferences for in-person communication and budget constraints."We have always prepared alcohol for hand sanitizer. We tell them to wear masks, and we keep them far from each other one meter, sitting far from each other, and educate them to wear a mask" .Besides their usual clinical duties, providers described the added burden of training and enforcing service-users to wear facemasks and maintain safe-distancing when they attended health facilities."A lot of Khmer Muslims […] so they said they are not afraid of COVID, they are afraid of not worshipping..." Both urban and rural interviewees highlighted that public risk communications about COVID-19 did not effectively engage with minoritized communities. While the reasons why were not clarified, several interviewees described perceived discrimination from the government and the public toward minority communities, which risked isolating and depriving these communities of healthcare services during the pandemic. However, some also demonstrated their own lack of awareness in describing these marginalised communities.This unique mixed-method examination of sexual and reproductive health services access and use in urban and rural Cambodia in the early months of the COVID-19 pandemic showed urban–rural differences in reported perceptions about and access to contraceptives during the initial phase of the COVID-19 pandemic in Cambodia.Survey findings showed that, compared to urban counterparts, rural participants had higher odds of suggesting that it was possible to change contraceptive methods and higher odds of reporting that accessing contraceptives was both more difficult and easier during the pandemic. This may be because in July 2020 the COVID-19 situation in Cambodia was well controlled, with relatively few cases  compared with neighbouring countries. Most cases were reported in the capital city, while in rural study sites there were no reported COVID-19 cases or movement restrictions. Thus, rural populations still had relatively easy access to contraception, especially condoms and pills that are generally available at local pharmacies .Qualitative findings indicated that SRH services remained roughly at pre-pandemic levels, with rural–urban differences mentioned in access, workload, and remuneration. User-related factors, such as fears of COVID-19 infection in crowded places and reduced overall income, contributed to reduced SRH service accessibility—a finding common in many countries . HoweverThe pandemic put health-workers globally under unprecedented strain, demanding they balance personal and family physical and mental healthcare needs with those of patients, while working under extreme pressure with constrained resources . Many quThe Cambodian health system generally recognised that pandemic-related constraints, such as quarantines and travel restrictions, could challenge community structures and access to quality SRH services. An important aspect in any governmental response to crises is communication . PandemiSeveral limitations should be noted. First, we focused on women and young people, so older men's views are not directly represented. Second, we treated women as homogenous in the survey due to resource and access constraints, despite studies suggesting that women and adolescents with additional potential vulnerabilities, such as those belonging to indigenous populations, living with HIV, or with pre-existing physical or mental disabilities, would be disproportionately disadvantaged by pandemic-related disruptions to SRH services. Third, cross-sectional surveys present prevalence and cannot identify causality. Fourth, this survey included approximately 400 households, so findings should not be generalised. However, inclusion of qualitative interviews allowed additional exploration of issues that help inform our findings. Fifth, we were restricted to recruiting interview participants who could communicate in Khmer or English, due to the language capacities of interviewers, so minoritized people who did not speak Khmer could not be included. Finally, the situation is dynamic and has continued to change since data were collected.COVID-19 and initial mitigation responses negatively affected SRH service access and provision in Cambodia, with rural–urban differences predominantly exacerbating existing socioeconomic stressors while adding new fears of infection risk, such as transport constraints between rural villages, lack of health-worker funding and training, and insufficient safe-distancing and mask-wearing among service-users—particularly in rural areas.