Kidney cancer accounts for 2% of new cancers diagnosed in Australia annually. Partial and radical nephrectomy are the treatment of choice for kidney cancer. Nephrectomy is also performed for living donor kidney transplantation. Nephrectomy is a risk factor for new-onset chronic kidney disease (CKD) or deterioration of pre-existing CKD. Understanding the risk factors for new-onset or deterioration of existing CKD after nephrectomy is important in developing preventive measures to provide better care for these patients. There is also a need to understand the incidence, natural history, management trends, and sequelae of radiofrequency ablation as well as surveillance of small renal cancers or small renal masses (SRMs). Clinical registries are critical in providing excellent patient-centre care and clinical research as well as basic science research. Registries evaluate current practice and guide future practice. The Flinders Kidney Health Registry will provide the key information needed to assess various treatment outcomes of patients with kidney cancer and patients who underwent nephrectomy for other reasons. The registry aims to provide clinical decision makers with longitudinal data on patient outcomes, health systems performance, and the effect of evolving clinical practice. The registry will also provide a platform for large-scale prospective clinical studies and research.Patients above the age of 18 undergoing nephrectomy or radiofrequency ablation for any indication and patients with SRMs will be included in the registry. Demographic, clinical and quality of life data will be collected from hospital information systems and directly from the patient and/or caregiver.The Registry will report a summary of patient characteristics including indication for treatment, clinical risk profiles, surgical and oncological outcomes, the proportion of patients who progress to CKD and end stage kidney disease, quality of life post treatment as well as other relevant outcomes for all patients who have undergone nephrectomy for any indication, ablation or surveillance for SRMs. The registry will record the follow-up practice after nephrectomy and patient on active surveillance, which will help to develop and enhance a best practice protocol. The collected prospective data will provide a platform for ongoing patient-orientated research and improve patient-centred healthcare delivery. Kidney cancer accounts for about 2% of malignant diseases in adults with an increasing worldwide incidence of over 430,000 new cases and 170,000 deaths per year [Kidney cancer is not a single disease but consists of a number of different cancers, each with a different histology and different clinical course. Approximately 90% of the kidney cancers are renal cell carcinomas (RCC) and the remaining 10% of kidney cancers are transitional cell carcinomas (TCC) and others. RCC has multiple subtypes including clear cell RCCs (ccRCC), papillary RCCs (pRCC), and chromophobe RCCs (crRCC).Radical nephrectomy or partial nephrectomy remains a cornerstone of the treatment of localised RCCs. In the past, survival times for patient with advanced or metastatic RCC rarely exceeded one year. However, there is trend towards earlier stage diagnosis with incidental imaging findings. Furthermore, there has been an explosion of therapies, including targeted therapies against angiogenesis and immunotherapy that target immune checkpoints such as programmed cell death–1 (PD-1) in the past ten years  which haOpen or laparoscopic radical nephrectomy is the recommended treatment for stage T1b-T4 RCC. Nephron-sparing surgery or partial nephrectomy is the preferred treatment for smaller renal cancers (< 5 cm), which has equivalent oncologic outcomes and better renal function outcomes compared with radical nephrectomy. This is important in patients with a solitary kidney, bilateral renal cancers or pre-existing significant renal impairment [Radical or partial nephrectomy for RCC is a risk factor for both new-onset and deterioration of existing chronic kidney disease (CKD) –9. In coProspective data is required to examine the long-term impact of nephrectomy on the development of CKD and ESKD and the potential bidirectional and causal relationship between RCC and CKD. More studies are needed to explore the pathophysiology of developing CKD after nephrectomy, especially the role of compensatory renal hypertrophy, and to quantify the perioperative risks, and the role of other factors at a disease, patient, health care provider, institute and socio-demographic level in determining patient outcomes.Small renal masses (SRMs) are increasingly diagnosed incidentally during the investigation of other medical conditions with abdominal ultrasound or CT scan. This has caused management challenge because many SRMs are either benign tumours such as oncocytoma or are renal cancers with relatively indolent behaviour . There hClinical quality registries play an increasingly significant role in modern evident based clinical practice and research. Currently, there is no nephrectomy registry in Australia. The Flinders Kidney Health Registry within the Southern Adelaide Local Health Network (SALHN) will provide the key information needed to assess various treatment outcomes of patients with kidney cancer or patients who underwent for nephrectomy for any indication. The Registry aims to provide clinical decision makers with longitudinal data in the real-world setting on patient outcomes, health systems performance, health-care usage, the effect of evolving clinical practice, risk stratification based on patient risk characteristics and practice guideline development and improvement. Data are needed for development of protocols and guidelines. This registry will also provide a platform for large-scale prospective patient-orientated clinical studies and research.The Registry represents both a clinical quality initiative within Departments of Urology and Renal Medicine in Southern Adelaide Local Health Network (SALHN) (Primary Aims), and a clinical research activity (Secondary Aims).Long-term oncological and non-oncological outcomes after radical and partial nephrectomy for kidney cancersLong-term outcomes after radical and partial nephrectomy for non-neoplastic kidney disease indicationsLong-term kidney and non-kidney outcomes after nephrectomy for living kidney transplant donationLong-term outcomes of patients with kidney cancer treated with ablative therapiesLong-term clinical outcomes of patients with SRMs who receive active surveillanceQuality of life post nephrectomyThe primary aim of the Registry is to monitor patient outcomes, health system performance, health-care usage and the effect of evolving clinical practice within the Department of Urology and Department of Renal Medicine in SALHN. Specifically, the Registry aims to evaluate:Risk factors for new-onset CKD or deterioration of pre-existing CKD after nephrectomy for kidney cancers or non-neoplastic kidney indicationsThe clinical significance of post-nephrectomy acute kidney injury (AKI) on long-term outcomesThe incidence and effects of coexistent non-neoplastic histopathology changes and score in the nephrectomised kidney on long-term kidney function and other outcomesCompensatory renal hypertrophy in the contralateral kidney after radical and partial nephrectomyComparison of oncological and non-oncological outcomes of different treatment modalities  for small localized renal malignanciesEstablishment of a clinical risk score to predict the risk and clinical outcomes post nephrectomy which can be used in clinical consultation with patients before nephrectomy and guide the post nephrectomy follow up and managementThe secondary aims of the Registry are to examine the prevalence, extent and pathophysiology of compensatory renal hypertrophy of the remaining kidney, new-onset or deterioration of existing CKD and the efficacy of different treatment modalities in post nephrectomy care. Specifically, the secondary aims are to evaluate:Flinders Medical Centre (FMC) is a 750-bed tertiary referral teaching hospital accepting about 55,000 admissions per year. It provides medical services for nearly 350,000 people living in the southern metropolitan area of Adelaide, South Australia. We perform about 80 nephrectomy procedures per year. We also have retrospective data on 680 nephrectomy cases over the past 10 years.Patients undergoing radical nephrectomy or partial nephrectomy at SALHN for any indicationAged > 18 years at the time of nephrectomyInclusion criteria include:Any T1 tumours (size up to 10 cm)Tumours can be solid or cystic Referred to Department of Urology in SALHNBiopsy is not necessary for inclusion in the active surveillanceMust have a least two cross sectional imaging encountersPatients with SMRs managed with radiofrequency ablation and/or active surveillance are also eligible providing that they meet the following criteria:An opt-out consent model will be employed to ensure this registry can function effectively and maintain quality. The data on outcomes generated by this registry is likely to be compromised if the participation rate is not near complete, and the requirement for explicit consent would compromise the necessary level of participation. Involvement in this registry carries minimal risk.Provide prospective participants with written plain language information explaining the nature of the information to be collected, the purpose of collecting it, the confidentiality and the simple procedure to decline participationAllow a reasonable time period between the participant receiving information and the collection of their data so that they have opportunity to withdrawAllow participants to opt-out of the registry at any time without any effect on their clinical care and without costDevelop a website which will provide participants further information and register their intension for non-participationIn order to satisfy the requirement for opt-out consent, this registry will.While participants can opt-out at any time, after a period of two weeks has elapsed patient data will be collected into the registry. If participants opt-out after this two-week period they can nominate to have all clinical details removed or simply not receive any follow up calls.The Registry Officer will manually collect data on Registry case report forms (CRFs) from hospital medical records, electronic health information systems and directly from the patient or patients’ family/caregiver, as appropriate.Patient identifiers: Unique Unit Record Number (URN), name, gender, date of birth, ethnicityContact details: Address, emailPhysical characteristics: Height, weight and body mass index (BMI) Funding: Medicare number, category of funding Comorbidities: Charlson comorbidities, Charlson score, American Society of Anaesthesiologists (ASA) grade, other comorbidities, smoking statusMedications: Including all medications especially antihypertensive medications, angiotensin-converting enzyme (ACE) inhibitor, angiotensin receptor blocker (ARB), sodium‑glucose co‑transporter 2 (SGLT2) inhibitors antiplatelet agents, lipid lowering medications, perioperative antibiotics usagePreoperative presentation and diagnosisBiopsy confirmation and details if availableStaging investigationLaboratory investigations: full blood count (FBC), biochemistry results including serum creatinine, estimated glomerular filtration rate (eGFR), liver function test results, urinary albumin-to-creatinine ratio (ACR) or urinary protein-to-creatinine ratio (PCR)Procedure details: type of surgery, technique used, lymph node dissection, explorations of vena cava, blood loss, ischemic time, operative time, surgeon involvedPost-operative course: post-operative complications, need for Intensive Care Unit (ICU) admission, length of hospital stays, readmissionPathology report including type of cancer, tumour diameter, number of tumours, margin, stage of cancer, grade, presence of necrosis, sarcomatoid transformation, presence of invasion, growth pattern, Fuhrman score, International Society of Urologic Pathologists (ISUP) score, non-neoplastic pathology includes: diabetic nephropathy, hypertensive changes, glomerulonephritis, global and segmental glomerulosclerosis (GS), tubular atrophy (TA), interstitial fibrosis (IF) and arteriosclerosisFollow-up assessments such as date of follow-up, patient status, laboratory & radiology reports, presence of recurrence, need for dialysisSerial measurements of serum creatinine, eGFR, proteinuria and haemoglobinSerial images in detection of recurrence or metastases, remaining kidney size and volume and evidence of compensatory renal hypertrophyThe time for follow up visits, blood tests, medical images and data collection points are summarised in Table Data on surveillance such as date commenced, follow up information and need for treatment conversionAblation treatment data such as date of ablation, subsequent ablation, follow up dataRadiotherapy detailsSystemic therapy details such as type and duration of therapy, complications, response to systemic therapyValidated instruments (AQoL-6D and NCCN-FACT FKSI) will be used to assess quality of life. Patient Reported Outcome Measures will be requested at baseline (before treatment) and then at 12 and 24 months after diagnosis.Death Registry. This will provide the date and cause of death for patients in the registry. Deaths captured by the South Australian Births Deaths and Marriages Registry will be linkedMedicare Benefits Schedule (MBS) and Pharmaceutical Benefits Scheme (PBS). These two data sources will provide greater definition around medications and procedures relating to each participant.The patient information sheet makes provision for the following data linkage activities.The Registry will be subject to ongoing quality assurance audits. Annually, 10% of eligible patient records will be reviewed to ensure clinical and outcomes data abstracted by the Registry Officer remain consistent with the clinical record. The Quality Assurance (QA) Plan, acceptable discrepancy rates and remedial actions will be detailed in an appropriate standard operating procedure.Tumour stage will be determined according to the tumour, nodes and metastases system (TNM) and the cellular grade as grades 1–4 using the Fuhrman system.Detailed examination of glomerular, tubular, interstitial and vascular pathology will be performed on all nephrectomy specimens by SA pathology service in accordance with the Royal Australasian College of Pathologist guidelines . The co-2.The estimated glomerular filtration rate (eGFR) is calculated according to the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) study equation . The uni2 which developed after nephrectomy and persists for > 3 months, regardless of aetiology. The time to development of CKD after nephrectomy is defined as the date of nephrectomy to the date of the first eGFR < 60 mL/min/1.73m2.The development of new onset CKD is determined by an eGFR < 60 mL/min/1.73mIncrease in serum creatinine > 30 µmol/L within 48 hIncrease in serum creatinine > 1.5 times baseline in 7 daysUrine volume < 0.5 ml/kg/hour for 6 hAcute kidney injury (AKI) is defined based on Kidney Disease: Improving Global Outcomes (KDIGO) guideline:AKI is assessed by comparing the highest serum creatinine level within 7 days post-nephrectomy with the pre-operative serum creatinine level.the number and proportions of patients undergoing partial and radical nephrectomysummary of patient characteristics including indication for nephrectomy and clinical risk profilesthe oncological outcomesthe proportion of patients who progress to CKD and ESKDcomplication ratein hospital, 30 day and long-term mortalitylength of hospital stays, 7 day and 30 day readmission rateQuality of life at 12 months post treatmentthe outcomes of patients underwent active surveillanceThe Registry will report annually and provide data on the following metrics:Clinical outcomes monitoring is critical in ensuring that the patients are receiving the highest quality of care and is the key component of evidence-based practice. This requires high quality, prospective data. There is little literature in terms of perioperative and longer-term outcomes on kidney cancer treated with partial or radical nephrectomy or other modalities such as active surveillance or radiofrequency ablation within Australia. Furthermore, there is no registry data to monitor the clinical outcomes of nephrectomy for renal cancer or other indications to evaluate the current practice and improve the best practice guideline.Best practice guidelines are essential feature of good clinical care. Guideline development requires access to patterns of care, quality of care, and a platform for research , 22. AssThere is no evidence regarding the best management strategies of SRMs  in patient with pre-existing CKD or other comorbidities. Active surveillance is increasingly accepted as a treatment modality. This is particularly true of the elderly who may not be fit for surgery or dialysis. It is important to collect data prospectively to monitor the progression of a mass, oncological outcomes and trends in renal function. Immunotherapy has significantly changed the treatment landscape for patients with advanced or metastatic RCC. A clinical quality registry, with its data collection, feedback and practice improvement loop is the ideal tool for monitoring and improving guidelines relating to nephrectomy in Australia; as well as providing a basis for research studies .Health related quality of life (HRQoL) measures are increasingly being recognised as important determinants of treatment outcomes. HRQoL measures include patient reported symptoms, assessment of general function including satisfaction or dissatisfaction with areas that are deemed important to the patient. HRQoL measures provide information about the impact of disease processes and treatments to individual patients, the health care systems and to society.Patients with localised kidney cancer usually enjoy an excellent prognosis. Recently there has been increased interest in this cohort of patients to measure HRQoL outcomes as treatments for kidney cancer like nephrectomy can reduce quality of life for many years subsequent to the treatment exposure especially if the patient develops advanced CKD or ESKD requiring renal replacement therapy such as haemodialysis.There have been a number of studies examining patient reported outcome measures (PROMs) and quality of life in kidney cancer . The EurThere are several limitations for this registry: (a) These routinely collected data points are not for a specific research project so their timing, quality and completeness may not meet all needs; (b) The catchment area for the registry is local rather than national at this stage and (c) This registry does not enrol and collect data from patients who are managed in the private hospital and private practice. Despite these limitations, this protocol describes a unique registry providing a valuable quality improvement and research resource.


Cicer arietinum L. is the third greatest widely planted imperative pulse crop worldwide, and it belongs to the Leguminosae family. Drought is the utmost common abiotic factor on plants, distressing their water status and limiting their growth and development. Chickpea genotypes have the natural ability to fight drought stress using certain strategies viz., escape, avoidance and tolerance. Assorted breeding methods, including hybridization, mutation, and marker-aided breeding, genome sequencing along with omics approaches, could be used to improve the chickpea germplasm lines(s) against drought stress. Root features, for instance depth and root biomass, have been recognized as the greatest beneficial morphological factors for managing terminal drought tolerance in the chickpea. Marker-aided selection, for example, is a genomics-assisted breeding (GAB) strategy that can considerably increase crop breeding accuracy and competence. These breeding technologies, notably marker-assisted breeding, omics, and plant physiology knowledge, underlined the importance of chickpea breeding and can be used in future crop improvement programmes to generate drought-tolerant cultivars(s). After the world . Chickpehe world . Mexico,he world ,7. In sehe world ; howeverhe world ,10. In ahe world ,12,13. Whe world  at vegethe world . Droughthe world ,17,18.The chickpea is also termed as the “poor man’s meat” , since iChickpea is mostly cultivated in the post-rainy season , using s2 concentrations and temperatures, more erratic precipitation, and changes in humidity. Due to the increased variability in precipitation during the growing season and more so in soils with low water holding capacity, these climate changes may result in an increase in the atmospheric water demand by crops and an increase in the potential for limitations in the availability of water in the soil. In the long run, breeding cultivars with high water use efficiency (WUE) is a more realistic and cost-effective strategy for raising yields in drought-prone locations. WUE promotes modest water absorption while maintaining elevated WUE, which is a crucial component of breeding programmes because of its yields in drought-prone areas. Any WUE is impacted by changes above the soil surface because they have an impact on the soil water balance by the evaporation and penetration of soil water. The majority of the chickpea crop is grown on residual moisture; however, additional irrigation can increase yields. At some sites in India, irrigation during the pre-flowering stage and at the beginning of the pod fill led to an increase in yield. Chickpeas’ reproductive cycle was prolonged by irrigation, which also increased plant biomass and increased the number of pods per plant.The four climatic elements that are changing will have an impact on how much water plants consume . These eThe greatest sustained surface winds of tropical storms range from 39 to 73 mph, and they are fast rotating storm systems with an organized centre over warm tropical oceans. These storms have a wide range in size and can cause a variety of dangers for the impacted areas, including tornadoes, catastrophic winds, coastal floods, and inland flooding. The effects of tropical cyclones on drought have been extensively studied, but less research has been conducted on how smaller tropical storms affect the severity of drought. According to research, rainfall is not necessarily inversely correlated with the strength of a tropical cyclone; therefore, tropical storms can sometimes provide more rain than expected. The question of whether tropical storms can help to lessen and mitigate drought conditions is now being researched. Water deficit and surplus are related to drought and tropical storms (TS), respectively. When it comes to monitoring dryness, soil moisture is a crucial element of the hydrological cycle, since it reflects the water that TS rainfall has penetrated or stored. Soil moisture data can be used to determine whether TS can alleviate extremely severe drought situations . The autDrought has a substantial influence on crop growth and photosynthesis, both of which are directly related to production ,35. DrouAdvances genomics has made it possible to tag genes  associatNext-generation sequencing (NGS) is a segment of revolutionary biology being a frontier area in crop science and produces correct data, with the results of significant throughput  and reduTranscriptomics is the technology used to study the transcriptome of an organism . TranscrProteomics is the study of whole protein complement in a cell, tissue or organism in detail . Mass spRole of various genes of plants under drought stress conditions have been recognized clearly . Drought−1 on a global scale, which is quite low [Ascochyta blight, Fusarium wilt, and Helicoverpa are biotic factors that have been recognised as key drivers of yield reduction in chickpea [Chickpea productivity has been found to be around 995 kg hauite low . Droughtuite low , excessiuite low , whereaschickpea . DroughtSeveral factors are responsible for complexity of drought stress , includiDue to the overabundance of wheat in irrigated areas in India, chickpea growth is primarily limited to rainfed areas. Crops in rainfed areas are experiencing water shortages, particularly during the sowing and terminal growth periods.Soil and plant management are important for minimizing water stress. For this purpose, various experiments have been conducted with the applications of different agents. Gypsum can enhance overall plant growth, since it is a moderately soluble source of the crucial plant nutrients, calcium and sulphur. Gypsum supplements can also enhance the physical and chemical characteristics of soils, hence lowering nutrient concentrations in surface water runoff and reducing soil erosion losses. The most often used addition for reclaiming sodic soil is gypsum, which can also be found in synthetic soils used in nursery, greenhouse, and landscaping applications. Gypsum can be used for a variety of purposes in agriculture and horticulture, which could be advantageous to users. There are currently no recognized standards that outline the broad best management practices for using gypsum in agricultural applications.Drought tolerance is a complex phenomenon that involves defence mechanisms as well as stress-induced signal responses ,115. DroDrought escape is the capacity of the plant to complete its life cycle before experiencing a major water deficit. Drought escape causes early flowering and maturity, as well as better yield potential, allowing plants to finish reproduction before drought strikes . Crop loDrought escape is a critical strategy for preventing chickpea crop from drought . Water sDrought avoidance is explained as a plant’s aptitude to retain a high tissue water potential contempt in a lack of soil moisture . ProcessRoot biomass plays major role in absorbing water , as it i2 fixation and photosynthesis due to the reduction in the internal CO2 concentration of the leaf (Ci). All of these factors have their role on the reduction in yield due to the reduced rate of photosynthesis [One of the important impacts of drought stress is stomatal closure. Drought stress reduces the stomatal conductance and transpiration rate. This declines the COynthesis .The reduced rate of photosynthesis is directly related to extreme drought stress, and it is a result of the decreased chlorophyll content. Because of the lowered chlorophyll content, continuous poor moisture availability reduces light collecting capacity, triggering the generation of reactive oxygen species due to excessive energy absorption . This isAscochyta blight, increased seed size, and suitability for mechanised harvesting [The leaf surface is also an imperative characteristic of plants in relation to drought stress. As tiny leaf surfaces lose less water , waxy lervesting .The drought tolerance refers to a plant’s ability to maintain its metabolism in a water shortage  conditioIn recent years, the role of reactive oxygen species (ROS) in stress signalling has been widely researched and evaluated ,154. ThePushpavalli et al.  emphasisPlants have multi-level systems of antioxidant defence  with maiThe antioxidant defence system has both enzymatic and non-enzymatic components. The enzymatic component involves superoxide dismutase, catalase, peroxidase, ascorbate peroxidase, and glutathione reductase. However, the non-enzymatic component involves cysteine, reduced glutathione, and ascorbic acid ,165.2 and H2O2 [In plants, ascorbate peroxidase is an important antioxidant enzyme, and glutathione reductase is important for sustaining the reduced glutathione pool during stress . In variand H2O2 . The droand H2O2 .Proline appears to play a variety of activities under stress situations as a multifunctional amino acid, including stabilizing proteins, membranes, and subcellular structures as well as defending cellular functioning by scavenging reactive oxygen species (ROS). The functional diversification of proline metabolism is more complicated as a result of the compartmentalization of proline production and degradation in the cytosol, chloroplast, and mitochondria. When the electron transport chain is saturated under stressful circumstances, the increased rate of proline production in the chloroplast can help to stabilize the redox balance and maintain cellular homeostasis by dissipating the excess reducing potential. Proline is one of the most widely dispersed suitable solutes and a key component of plant stress resistance that increases in plants under adverse environmental conditions. Proline serves as a superb osmolyte and also has important functions as a metal chelator, antioxidant defence molecule, and stress signalling molecule. By regulating mitochondrial activity, affecting cell growth, inducing certain gene expression, and stabilising membranes, it promotes stress tolerance by reducing electrolyte leakage, bringing ROS concentrations back into normal levels, and promoting stress recovery.One of the elements driving drought resistance in the chickpea is proline build up in different plant sections due to the increased activity of proline synthesising enzymes . DroughtPlant growth regulators, basically known as phytohormones, can be administered externally or synthesised inside the plant . Auxins,Abscisic acid and cytokinin are thought to play opposing functions in drought stress. Under water stress, an upsurge in abscisic acid and a diminution in cytokinin levels favour stomatal closure and minimise water loss by transpiration . AbscisiEthylene is a growth inhibitory hormone that acts as a part in both inhibiting and stimulating growth in response to environmental factors. The plants can maximise growth and resist abiotic challenges such as drought to avoid this adversity, and this response also requires ethylene synthesis .Plant growth and development are known to be affected by polyamines. There has been an increasing interest in the role of polyamines in the plant defence against environmental stressors, and substantial research energies have been conducted in the last twenty years. The overexpression of the apple spermidine synthase gene, for example, results in high amounts of spermidine synthase, which enhances abiotic stress resistance, for instance, drought tolerance .Breeders commonly employ traditional breeding techniques such as introduction, selection, hybridization, and mutation . HybridiArabidopsis ecotypes [ERECTA gene [ESKIMO1 gene [Arabidopsis, which have been examined well. Similarly, some biosynthesis genes in the chickpea [The evaluation of different genotypes of a plant species in response to the drought controlled condition is needed . In diffecotypes . In thisCTA gene  responsiMO1 gene , governschickpea  have beeNew cultivars, landraces, wild relatives, or a new crop species for the region could all be introduced. By using this technique, it is feasible to find a desirable genotype with a higher yield and better environmental tolerance, while also increasing the genetic variety. Through the international exchange of the germplasm and the inclusion of the crop of wild relatives and landraces, significant progress has been made in recent decades in enhancing the genetic diversity of the cultivated chickpea. Landraces are an important resource of novel genes in crop breeding. Landraces may possess genes for resistance against various biotic as well as abiotic stresses. For the identification of drought tolerant chickpea landraces, a field study was conducted by Kumar et al. . The expComplex abiotic stress such as drought requires a large group of genetic resources  to studyIn comparison to molecular markers, i.e., the hybridisation-based RFLP, the PCR-based RAPD and ISSR, SSR, and biochemical markers, i.e., isozyme, have a low polymorphic ability, which could be related to the decreased polymorphism in structural genes in the chickpea genome .Cicer species [Cicer arietinum [In some of the previous studies, the RAPD markers were utilised to detect genetic relationships amongst  species . The non species . However species . Amplifirietinum . Howeverrietinum  and the rietinum  proved tMicrosatellite and STMS (sequence-tagged microsatellite site) markers are numerous, scattered throughout the genome, and highly polymorphic . STMS raMore than 3000 microsatellites ,203, 15,desi genotypes  performed fine.A crucial requirement for identifying and integrating genes in linkage maps for marker–aided selection (MAS) is the knowledge of the agronomic trait inheritance . The marFusarium wilt resistance [The advanced genomics involves a genome-wide association study (GWAS) that helps researchers in the screening of a wide range of genotypes ,227 withsistance ,231.In some of the earlier studies, a combined analysis of the GWAS and sequencing of candidate gene  has beenScientific efforts made on the improvement of the chickpea crop made it possible to generate not only a bi-parental plant population but also multi-parent populations . The neeOne of the drought tolerant high yielding Ethiopian chickpea varieties,‘Geletu’, was developed and released in the year 2019 through the marker-assisted back-crossing after multi-location trials. During the development of this variety, the‘QTL-hotspot’ linked to drought tolerance was introgressed into an Indian chickpea cultivar JG11 from ICC4958 (gldc.cgiar.org). Recurrent selection is a crucial breeding technique used to increase crop plant populations. It is a productive method used in plant breeding to enhance the quantitative traits through repeated crossing and selection. Among the genomics-assisted selection methods, the marker-assisted back crossing has been found better for the introgression of the targeted region of the genome into a desired genotype. Consequently, the introgression of the ‘QTL hotspot’ region for the development of the drought-tolerant chickpea through molecular breeding has been found effective. Similar to this, numerous drought tolerance characters were introgressed into three elite Indian chickpea varieties: Pusa 372, Pusa 362, and DCP 92-3from ICC 4958. Recently, drought tolerant root traits have been introgressed into Kenyan chickpea varieties using the marker-assisted backcrossing approach .https://www.icrisat.org/new-climate-resilient-disease-resistant-chickpea-varieties-coming-farmers-way/, accessed on 20 October 2022).Initially, the Pusa 372, chickpea variety was released as a drought tolerant variety for cultivation in the central, north-east, and north-west plains zones. However, under drought conditions, this variety’s output has decreased in recent years. To enhance drought tolerance in this variety, the MABC approach was adopted to introgress the ‘QTL-hotspot’ region from ICC 4958 into ‘Pusa 372′. Recently, the Pusa 372 was released with improved drought tolerance under the name ‘Pusa 10216′. This improved chickpea variety is the example of the first enhanced drought tolerant chickpea variety developed through the MABC approach. ICRISAT in collaboration with other research institutes in India is in the process of the development and release of drought tolerant chickpea varieties, i.e., IPC L4-14 and BGM 4005.Both of these varieties were developed by transferring a ‘QTL-hotspot’ from ICC4958 into DCP92-3 and Pusa362, respectively  based on a drought tolerant ICC 4958 cultivar [The RNA-Seq technique has also been used to analyse differential regulation of genes under drought stress in the chickpea . Kumar ecultivar . The finArabidopsis thaliana, various genes were identified with their similar contributions under both salinity and drought stresses. The production of similar metabolites under both abiotic stresses indicates a common tolerance mechanism for drought as well as in the salinity in plants.The regulation of metabolic activities plays a major role in maintaining the osmotic potential of the cell under drought stress . With thThe proteomics’ analysis has also been performed in the chickpea for the identification of changes at a protein level under abiotic stresses. Earlier, a comparative proteomics analysis was conducted on one chickpea cultivar (JG-62), and novel dehydration-responsive proteins were detected . In thisPhenomics is an emerging tool in plant research, and is used to describe the use of genomics in phenotyping . PhenomiAKIN), DREB2A, dehydrin (DHN), CAP2, and Myb transcription factor (MYB) [A contender or candidate gene is thought to be linked to a specific disease or phenotypic character , and theor (MYB) , Table 5Arabidopsis thaliana genome, which have their part in stress-responsive gene expression.Transcription factors induce the cis-elements in the promoter provinces of different stress-responsive genes to accelerate the countenance of various downstream genes, which have their part in the stress tolerance  of plantDREBs (dehydration responsive element binding proteins) are key plant transcription factors [Arabidopsis thaliana [AtDREB1a transgenic chickpea lines under water stress conditions. factors . They arthaliana , with itthaliana  reportedDHN) are stress-responsive proteins that are found when the temperature is low or when the body is dehydrated [DHN than wild-type plants [DHN gene in the Pusa1103 and Pusa362 genotypes of the chickpea has been found to be linked with the drought tolerance. Furthermore, in comparison to other genotypes, these genotypes were recognised as drought tolerant due to their better response [Dehydrin (hydrated . Proteinhydrated . A bettee plants  have beeresponse .STPK), tyrosine protein kinase (TPK), and histidine protein kinase (HPK) are the three types of eukaryotic protein kinases [STPK family gene AtSnRK2.8 is reported with an enhanced degree of drought tolerance [Serine/threonine protein kinase  are a new type of transcription factor that are only found in plants. The ERF domain, a highly conserved DNA binding domain, is the protein family’s distinguishing trait. Primers for the chickpea were constructed using a contig sequence that was found to be identical to the esponses .AMADH gene has been reported with its association to osmotic stress tolerance [AMADH gene in Arabidopsis [In some crops, an olerance  by detoxolerance . On the bidopsis , the rolERECTA gene has a part in leaf organogenesis, lowering the density of the stomata on the leaf underside and thereby lowering evapotranspiration. It can also control the transpiration by the alteration of the leaf epidermal cell expansion, proliferation of mesophyll cells, and cell–cell interactions. The ERECTA gene has been demonstrated to regulate the growth and development of the organ and flower in Arabidopsis via encouraging cell proliferation [Arabidopsis plants confirmed the involvement of the ERECTA gene to the water usage competence [ZmERECTA genes from maize, which were implicated in the drought tolerance in crop plants.The feration . Complemmpetence . Pioneer3, and MeJA all significantly increased the CarLEA4 expression. CarLEA4 is a LEA assembly 4 protein that may participate in a variety of plant developmental processes as well as abiotic stress responses [The attainment of dehydration tolerance and the behaviour of plants to drought have been linked to late-embryogenesis abundant (LEA) proteins. Increased LEA and Dehydrin expression in genotypes during the vegetative, flowering, and podding stages could represent an adaptation to assist the plant survival by supplying the energy for growth and survival . Leaf agesponses .SFL) encodes for MYB, which works as a central factor responsible for the regulation of the numbers of flowers in chickpea inflorescence.Plants have a big transcription factor (TF) family, called the myeloblastosis (MYB) gene . It playSAM) is a precursor in the production of polyamines and ethylene [SAM decarboxylase is responsible for spermidine and spermine production. The exogenous polyamine administration or overexpression of polyamine production genes has been demonstrated to improve abiotic stress tolerance. Primers were developed using a contig sequence that was comparable to the S-adenosylmethionine synthetase 1 (SAM1) gene of Arabidopsis thaliana for the isolation of the S-adenosylmethionine synthetase 1 gene homologue in the chickpea. The PCR amplification revealed amplicons of roughly 300 bp in eight chickpea genotypes [S-adenosylmethionine (ethylene . In planenotypes .SAM gene in pigeon pea (Cajanus cajan L.) was evaluated under drought, heavy metal (CdCl2), and cold stresses. The enhanced up-regulation of SAM gene in the leaves were recorded after three days [Expression of ree days .ASR) gene plays a critical role in controlling various plant stresses. The ASR gene has been reported in plants, and is induced by abscisic acid and different abiotic stresses during the process of fruit ripening [ASR genes with their responses in different plant species under drought, salt, and cold stresses [Arabidopsis demonstrated the over-expression of the ASR gene in response to drought and salt stresses [ASR gene expression [ASR1 gene in the common bean. In a recent study conducted on the chickpea under drought stress, increased ASR gene expression was observed. The increased expression may have helped the drought-tolerant chickpea genotypes function better under stress. This hypothetical ASR protein could have boosted the activity of the ASR gene as a transcription factor mediating drought responses in chickpeas.Among many other genes, the abscisic acid stress and ripening (ripening . Reportsstresses  confirm stresses . Genotyppression . Similarpression  reportedABREs), which use bZIP-type AREB/ABF transcription factors to regulate the gene expression. ABA and water stress upregulate the expression of the AREB/ABF gene. Expression of AREB gene under drought stress has been reported by Yoshida et al. [Arabidopsis thanliana.Various genes that are activated by abscisic acid (ABA) have been discovered to be drought stress-inducible. Such ABA-regulated genes have conserved cis-elements in their promoter regions known as ABA responsive elements  is a crucial enzyme that hydrolyzes sucrose directly to provide substrates for plant metabolism. It is also used as a bio-marker for plant sink strength [Sucrose synthase (strength . Plant sstrength ). In an Sucrose synthase activity has a major role in chickpea seed growth. The supremacy of the sink, as measured by the sucrose synthase movement most of the time, hinges upon genetic features of a genotype along with the accessibility of water obtainable at seed filling . In both2 subgroup of proteins has two copies of the DNA-binding domain (BD), detached by an insertion province [2CAP is a C-Repeat binding factor (CBF) that muddles to the DRE/CRT (dehydration responsive element/C-repeat element)(CCGAC) found in the promoters of abiotic-stress responsive genes. Dehydration, excessive salinity, and exogenous ABA treatment all enhanced 2CAP gene expression. The incidence of roughly 60-amino-acid long AP2/ERF DNA-binding realms in these transcription regulators allows them to connect directly with GC-rich cis-acting elements (GCC box/C-repeat) in the promoter of their target genes. Ectopic expression of CAP2 in tobacco resulted in increased drought, salinity, and heat tolerance, as well as improved transgenic plant growth [2CAP in chickpea leaves. 2CAP activates the CaZF promoter through interacting with C-repeat elements (CRTs) in CaZF promoter, according to the gel mobility shift and transient promoter-reporter tests. The 2CAP protein interacts with the CaZF promoter in vivo, according to a chromatin immunoprecipitation (ChIP) test [The APprovince . CAP2 ist growth . The enhIP) test .SPS) gene has an important function in the sucrose production in different plant species. It regulates sucrose metabolism in drought sensitive and tolerant genotypes [SPS activity in maize, potato, soybean, and some other crops, however, some reports advocate the increment in the SPS activity in rice, wheat crops, and Arabidopsis [SPS gene has been studied in chickpea under a low temperature by Sharma et al. [The sucrose phosphate synthase (enotypes . Sucroseenotypes . Some rebidopsis . The siga et al. .namely RVE7 and 4CL, have been identified in the chickpea and their association with drought tolerance.The CRISPR/Cas9-mediated editing of the chickpea protoplast was reported for the first time by Badhan et al. [The genome editing approach, specially CRISPR-Cas9, has proved their efficiency in the development of climate resilient cultivars of different crops . Two genn et al. , where tn et al. . Genome As previously stated, the changes in the plant shape and internal biochemical characteristics during drought stress have been extensively characterised in previous studies. Plant drought stress techniques can help us better use scientific means to improve plant tolerance to water shortage environments and increase crop yields, allowing us to play a larger role. As a result, by thoroughly examining and summarising the mechanisms of the chickpea plant response to drought, this study provides essential background knowledge and theoretical framework for selective breeding, cross breeding, and molecular breeding of the chickpea in the future. Drought tolerant land races/germplasm lines may be employed in classical as well as molecular breeding programmes to breed drought tolerant cultivars in future by using the available scientific data.


Rare diseases affect more than 300 million people worldwide. Diagnosing rare diseases is a major challenge as they have different causes and etiologies. Careful assessment of clinical symptoms often leads to the testing of the most common genetic alterations that could explain the disease. Patients with negative results for these tests frequently undergo whole exome or genome sequencing, leading to the identification of the molecular cause of the disease in 50% of patients at best. Therefore, a significant proportion of patients remain undiagnosed after sequencing their genome. Recently, approaches based on functional aspects of the genome, including transcriptomics and epigenomics, are beginning to emerge. Here, we will review these approaches, including studies that have successfully provided diagnoses for complex undiagnosed cases. Epigenetics plays an important role in pathogenicity since it regulates basic cellular functions, such as gene expression, DNA damage, chromatin topology, and chromosomal organization. DNA in the eukaryotic cell nucleus is wrapped around two copies of each of the core histones  to form chromatin. Among other epigenetic mechanisms, modifications of DNA and histones play critical roles in gene expression regulation. The level of chromatin compaction has important consequences for gene transcription as it influences the accessibility of DNA sequences to transcription factors and other regulatory proteins. Modifications of DNA and histones regulate the level of chromatin compaction, either directly or by facilitating the binding of remodeling proteins that recognize modified sites.Genetic alterations can have an important impact on epigenetic regulation. Mutations might affect the function of genes involved in histone or DNA modifications or even affect histone genes. These alterations typically have a broad impact on gene expression. Alternatively, mutations can be located in regulatory elements or alter the conformation of chromatin affecting the expression of particular genes.A disease is considered rare if it affects fewer than 1 in 2000 people . The larAlterations in chromatin properties and structure are common in rare diseases and can be used as diagnostic tools. These alterations can be caused directly by mutations in genes that encode proteins involved in the regulation of chromatin. In addition, other alterations not involving epigenetic factors directly can affect the epigenome. For example, chromatin-related factors are very often recruited to chromatin through transcription factors and, therefore, mutations in transcription factors, their binding sites, or components of signal transduction pathways that control their activity can also lead to alterations in the cellular epigenetic landscape .Haploinsufficiency in chromatin-related factors frequently causes neurodevelopmental syndromes. Although most of these proteins are ubiquitously expressed, the nervous system appears to be particularly vulnerable to the alteration of their activities. Next, we review critical aspects of epigenetic regulation and its alterations .DNA methylation is catalyzed by DNA methyltransferases (DNMTs), typically at cytosines (5mC) . DespiteDNMT1 are associated with neuropathies, mutations in DNMT3A cause overgrowth syndromes with intellectual disability, and DNMT3B mutations are involved in immunodeficiency and intellectual disability . Si. Si16]. In addition to histone modifications and its effector readers, gene expression and repression entail the remodeling of chromatin, making it more or less accessible to transcription factors and the transcriptional machinery. Chromatin remodelers utilize energy from ATP hydrolysis to alter nucleosome spacing/density or to facilitate histone variant exchange. Several activities with ATP-remodeling activity or that are components of ATP remodeling complexes have been identified in patients with rare diseases, the most well-known being the Coffin–Siris syndrome caused by loss-of-function mutations of different subunits of the SWI/SNF chromatin remodeling complex involved in transcriptional activation .Recently, it has been described that mutations in histone H3 tails can also contribute to rare neurologic dysfunctions and congenital anomalies. These mutations likely cause disruptions of H3 interactions with DNA, other histones, and histone chaperone proteins, and result in altered histone modification patterns .Patients affected by rare diseases can spend an average of 5 years looking for a diagnosis . InitialWES can capture protein-coding regions of the genome, and in some cases, untranslated regions (UTRs) and intron-exon boundaries. It has lower costs than WGS and its analysis is more straightforward. However, WES covers only about 1–2% of the entire genome and has difficulties in detecting structural variants (variants that are greater than 50 base pairs and up to 3Mb), tandem repeats, and pathogenic variants in deep intronic regions and regulatory non-coding regions. Some of these challenges can be addressed by WGS; however, this technique also has its limitations, such as higher costs, similar limitations in structural variant detections, and more complex analyses.Despite the great advances in diagnostic achieved by WES and WGS, more than 50% of patients might not receive definitive diagnoses after applying these technologies . ComplexGenetic studies often reveal a large number of VUS. More recently, functional approaches to identify or confirm variants involved in diseases have been developed. Some of them are focused on correlating variants with alterations in gene expression or epigenomic marks.Gene expression and its regulatory mechanisms, different from genetics, vary from tissue to tissue; therefore, the choice of tissue or cell type to carry out functional approaches is critical. However, certain tissues might be difficult to access or might be unrealistic for undiagnosed disease programs covering hundreds of patients with different phenotypes. The most common tissues collected from patients for diagnostic purposes are blood followed by skin. Although most studies analyze whole blood, purifying mononucleated cells or other subpopulations of cells might bring some advantages. When analyzing whole blood, it is important to take into account that expression and epigenetic patterns are cell-specific; therefore, the differences found in patients might reflect changes in cellular composition. Blood offers the possibility to generate patient-derived B-lymphoblastoid cell lines (LCLs), which consist of transforming B lymphocytes with the Epstein–Barr virus (EBV). LCLs are immortalized cell lines and can be used for follow-up studies. Skin offers the opportunity to stablish primary cultures of fibroblast or keratinocytes. In any case, long passages of patient-derived cell lines should be avoided to minimize the chance of introducing genetic aberrations.Other tissues that are relatively easy to access are skeletal muscle or fat, but others are more difficult to obtain and might be only available if a therapeutic surgery is performed. One way to preserve access to such precious material is to establish organoid cultures, self-organized three-dimensional tissue cultures that replicate much of the complexity of an organ and that can be indefinitely expanded. Importantly, protocols for the establishment of organoids from a large variety of human tissues have been reported . ChanginTranscriptomics profiles have been successfully used in the diagnosis of rare diseases. The sequencing of transcripts (RNA-seq) allows the identification of aberrantly expressed genes, aberrant splicing, monoallelic expression, and variant identification, including structural variants. Therefore, RNA-seq can improve the interpretation of VUS identified by genotyping.Regarding aberrantly expressed genes, RNA-seq has been useful for the identification of underexpressed genes most commonly affected by frameshifts, truncations, and splicing mutations that induce mRNA nonsense-mediated decay. Missense mutations appear less likely to be correlated with altered mRNA levels. Additionally, unexpected increases in mRNA levels have been reported for some mutant genes not producing proteins and might reflect a compensatory mechanism in response to absent protein . RNA-seqTherefore, compared to WES or WGS, RNA-seq can provide a functional assessment of genetic variation but it also implies additional challenges that will be discussed next.An essential limitation of RNA-seq approaches is the fact that each tissue expresses only a subset of genes. Analysis of the expression of disease-associated genes in different human tissues has shown that mitochondrial disease genes are the most ubiquitously expressed, but other disease-associated genes have more pronounced tissue-specific expression profiles, such as neurological genes in the brain . Analysihttps://www.gtexportal.org/ accessed on 25 June 2022) [An additional challenge of transcriptomic approaches for diagnosis is the need to compare patients’ samples with healthy controls. In some scenarios, the inclusion of a reasonable number of healthy samples is possible. For example, Hong et al. compared patients’ muscle biopsies with muscle control samples obtained from healthy individuals undergoing plastic surgery . Howeverne 2022) . Interesne 2022) ,34,35,36ne 2022) . A similne 2022) . An altene 2022) ,38.LRPPRC, a gene that regulates the stability of mature mitochondrial transcripts, in the patient phenotype [LIG3, a gene causing mtDNA depletion when mutated. Therefore, the analysis of pathways and function enrichment in DEGs can be helpful to support diagnostics. This might be particularly relevant when mutations affect transcription factors, chromatin-related factors, or activities involved in signal transduction that impinge on transcriptional pathways  can allow the detection of transcripts expressed in rare populations of cells, evaluate the abundance of different cell types, or tackle mosaicism in a biopsy. However, its implementation for diagnostic purposes is not feasible at present regarding cost and analysis efforts. Related approaches have aimed to extrapolate cellular components from bulk RNA-seq using deconvolution methods. Hong et al., applying these methods, deconvoluted cell type abundances in muscle biopsies from patients with neuromuscular diseases and captured clinical and pathological aspects of the diseases. For example, the abundance of fibro–adipogenic progenitor cells estimated from the deconvolution of the bulk RNA-seq data correlated with muscle fibrosis in patients .Overall, RNA-seq used to identify expression and/or splicing outliers has been reported to provide diagnoses for about 10–20% of undiagnosed cases with negative WES or WGS and confirmed diagnosis in around 50% of cases with a candidate variant identified by genome sequencing ,37,38. TA number of publications have described changes in DNA methylation profiles associated with genetic syndromes. This has allowed the development of strategies that allow the classification of undiagnosed patients in one particular disease according to their methylation profile in blood. Simple and cost-effective genome-wide DNA methylation arrays, such as the Illumina Infinium HumanMethylation450 or HumanMethylationEPIC BeadChip array, assess the methylation status of approximately 450,000 and 850,000 CpGs.Pipelines that allow the classification of patients into a number of syndromes according to their DNA methylation profiles have been developed. The EpiSign classifier is based on 100–500 differentially methylated probes that best separate the case samples from controls and allow the diagnosis of undiagnosed cases based on those episignatures . The pathttps://epigen.ccm.sickkids.ca/ accessed on 25 June 2022), have been recently developed [In addition, public resources for the analysis of DNA methylation data, such as the EpigenCentral portal . Compared to DNA methylation arrays, ChIP-seq is a far more tedious and variable technique, which so far has not been implemented for rare disease diagnosis routines.Studies investigating the genetic basis of rare diseases (focused on coding variants) have failed to provide clear answers for more than 40% of the studied cases , suggestEnhancers are distal regulatory units that participate in the regulation of gene expression by establishing contacts with promoters favoring the recruitment of RNA polymerase II. Enhancers contain docking sites for transcription factors (TFs) that in many cases are tissue-specific. These transcription factors participate in the recruitment of HATs and KMTs that maintain high levels of histone acetylation and monomethylation of lysine 4 of histone H3 (H3K4me1) . The abuPAX6 that disrupts an autoregulatory PAX6 binding site [SOX9 transcriptional enhancer caused the aberrant gonadal expression of SOX9, causing a disorder of sex development [PTF1A were found in patients with isolated pancreatic agenesis [TBX5 has been reported in patients with congenital heart disease [SHH causing preaxial polydactyly [Enhancer dysfunction might be caused by alterations in chromatin-related factors causing global deregulation of chromatin and gene expression. Alternatively, patients might carry mutations that genetically disrupt the enhancer region, including SNVs that alter TFBSs, and that affect, in this way, histone modifications and/or DNA methylation . Geneticing site . Structuelopment . Recessiagenesis . A homoz disease . Moreoveydactyly .Most of the reported regulatory alterations mentioned above were identified by focusing on the regulatory regions of well-known disease-causing genes in patients with very specific pathologies. Unfortunately, identifying disease-causing non-coding variants at enhancer regions in large cohorts of undiagnosed patients with variable symptoms might be challenging. Eventually, larger variants may be more disruptive to regulatory elements than SNVs and, therefore, easier to predict their pathogeny. Turró et al. focused on identifying large deletions likely to disrupt enhancer functions in a cohort of patients with hematopoiesis-related disorders . First, It has been estimated that 1–3% of neurodevelopmental patients without a diagnostic coding variant carry pathogenic de novo mutations in fetal brain-active enhancers . HoweverGenetic variation can also influence the 3D organization of the genome in the nucleus, resulting in the dysregulation of gene expression and, consequently, might cause disease. Groups of adjacent coregulated genes, often targeted by common enhancers, have been described to cluster within megabase-scale topological associating domains (TADs) . TADs arHere, we discussed epigenetic and functional strategies for the diagnosis of complex cases of rare diseases. It is becoming clear that the implementation of multiple strategies is typically needed to reach a diagnosis in complex cases. In this sense, epigenetic strategies are intended to complement genomic techniques, such as WES and/or WGS. These techniques can be used to confirm the pathogenicity of a VUS already identified after genome sequencing or identify variants that are not prioritized after the genomic analysis. However, several aspects need to be improved before they can reach their full diagnosis potential. A better description of altered patterns of DNA methylation, histone modifications, and gene expression changes for each disease is needed to improve the classification of patients into one particular syndrome. Still, the functions of many human genes are unknown and the transcriptional or epigenetic phenotypes resulting from their perturbation remain undescribed. The interpretation of VUS in non-coding regions is more challenging. In this regard, a better description of the regulatory regions that control the expressions of disease-relevant genes in each tissue or cell type will be fundamental to anticipate the consequences of their malfunctions. In addition, there is a need for improved sequencing methods with better coverage and accuracy, but also with lower costs and more accessibility. Making the best of sequencing patients’ data requires improved machine learning techniques for more successful classifications of patients. However, the success of these approaches might be limited by the small number of patients affected by each pathology. Data sharing and collaborative efforts should be oriented to overcome this limitation. Novel methods, such as scRNA-seq, can improve our understanding of rare diseases but are far from being used as routine diagnosis methods due to cost and analysis challenges. Overall, there is a need for realistic approaches that can be implemented by diagnostic programs around the world that deal with multiple complex cases with variable pathologies. For that, affordable, sustainable, and accessible standardized methods need to be developed to ensure equal access to diagnosis for all patients.


Skeletal muscle is a dynamic organ that responds to various stimuli by altering its size, strength, and composition. Maintenance of skeletal muscle mass and strength is critical for well-being and overall health. Conversely, ageing, and age-related diseases are associated with the loss of muscle mass and strength, termed sarcopenia. The exact molecular mechanisms of sarcopenia are poorly understood, which hamper the development of effective interventions to boost muscle mass and strength in ageing.Sarcopenia is accompanied by functional disability and decreased quality of life. Skeletal muscle exhibits several characteristic alterations in sarcopenia, including diminished mitochondrial respiratory activity  contractSarcopenia may be physiological and accompanied by healthy ageing without comorbidities. Nevertheless, sarcopenia is associated with alterations in skeletal muscle gene expression involving damage accumulation and compensatory mechanisms. An ambitious study by Tumasian III et al. investigated this question by extracting RNAs from muscle biopsies of healthy individuals over a wide age range. Altogether, 1,134 RNAs changed significantly with advancing age out of the 57,205 protein-coding and non-coding RNAs .Skeletal Muscle in Age-Related Diseases: From Molecular Pathogenesis to Potential Interventions” several studies address important questions in the field as well as examine potential molecular targets to treat ageing skeletal muscle.After its onset, sarcopenia is difficult to treat because it involves complex molecular interactions and is a risk factor for developing other diseases. Consequently, developing pharmaceutical strategies for sarcopenia remains a critical challenge due to multifactorial etiology and interface of skeletal muscle with multiple organs in the context of ageing. In our Research Topic “During skeletal muscle ageing, mitochondrial dysfunction contributes to reactive oxygen species (ROS)-induced oxidative damage , while eRuan et al. examined the role of the long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), which was found to have decreased expression in aged skeletal muscle. Previous studies have suggested that changes in lncRNA expression can influence muscle growth and differentiation by incompletely understood mechanisms  versus degradation (catabolic). Interestingly, in aged mice, the tibialis anterior (TA) muscle displayed resistance to denervation-induced atrophy. It was found that aging promotes protein synthesis rates and ribosomal RNA (rRNA) biogenesis in denervated TA, which is independent of mTORC1 regulation but via enhanced signaling through the Akt-GSK-3β pathway.The study by Lo et al. cut to the chase and first examined whether fetal skeletal muscle extract could benefit muscle health of aged mice. After 8 weeks of treatment, the aged mice showed enhanced muscle performance with increased lean mass, heightened grip strength, and isometric force generation. While the authors have not yet identified the factors in the fetal serum providing beneficial effects on aged muscle, the study is intriguing. Further analysis may yield one or multiple curative factors that can be replaced by direct injection or other administration methods to treat sarcopenia.Another line of thought in treating sarcopenia in aged individuals is replacing molecular factors lost during aging. While iTRAQ analysis yielded a lengthy list of peptide changes, the study by Falqueto et al. highlight the pros and cons of using anabolic-androgenic steroids to increase muscle function and mass. Sarcopenia is driven by diverse pathological processes in ageing patients, including elevated muscle catabolism than anabolism. The combination of anabolic hormones and exercise training can reverse muscle wasting in aged individuals. However, in some studies, the type of exercise mattered—with resistance training yielding favorable results by minimizing fatigue and exercise intolerance while increasing muscle strength, hypertrophy, and power. Although these above outcomes appear favorable, the discernable observed may depend on differences in participant exclusion criteria. Overall, it is promising that administration of anabolic hormones to some patients bolstered the exercise effect on sarcopenia, at least in some patients.Exercise training accompanied by nutritional approaches remains the best non-pharmaceutical treatment strategy to counter sarcopenia. However, in the elderly or immobilized, these may not be plausible treatment strategies. Experimental studies highlighted in this edition provide new and provocative findings that represent novel targets and potential as alternative therapies for sarcopenia. These studies further our understanding of the role of lncRNAs in protecting against muscle senescence, the effect of innervation and ageing, and beneficial properties of fetal muscle extract in sarcopenic muscle.


Surgery is currently a mainstream treatment modality for various solid tumor indications. However, aggressive resection of tumor tissues frequently causes postoperative complications, which severely undermine the well-being of patients. Moreover, the residue tumor cells may substantially increase the risk of local and distant tumor relapse. The recent development in black phosphorus (BP)-based nanomaterials offers a promising opportunity to address these clinical challenges. BP is an emerging nanomaterial with excellent biocompatibility and versatile functionality, which has already demonstrated great potential for a variety of biomedical applications including tumor therapy and tissue engineering. In this review, the recent advances in BP-based nanobiomaterials for the post-surgery treatment of solid tumor have been summarized, while specific emphasis was placed on their capability to continuously inhibit residue tumor growth at the surgery site as well as stimulating various healing mechanisms, aiming to preventing tumor relapse while promoting the healing of surgery-induced traumatic soft/hard tissue injuries. It is anticipated that the nanoengineered BP-based materials may open new avenues to tackle those clinical challenges in surgical treatment of solid tumors. Cancer is one of the leading causes of death around the globe, which accounts for around 10 million deaths in year 2020 alone and the incidence and mortality rates are still on a rapid rise, imposing great burden on the patients, families and society . Solid t2 . T. T2+, Znethasone –122. NevHydroxyapatite (HA), the major mineral in mature bones, is a highly crystalline form of calcium phosphate , 124. CoBP nanosheet is an emerging 2D inorganic nanomaterial with many unique and exciting capabilities, which shows promise to revolutionize various fields in biomedical technology. Specifically, BP nanosheets possess a direct and thickness-dependent band-gap rarely found in other typical 2D materials, which is correlated to a wide light absorption range from visible light to infrared range. The unique optoelectrical properties of BP nanosheets make them an exceptional candidate for many biomedical applications such as biosensing, in vivo imaging and light-dependent therapies. Meanwhile, BP nanosheets also presents large surface-to-volume ratio, high anisotropic structural and functional properties and inherent bioactivity, which prompts their application not only as an efficient drug carrier, but also a versatile platform for the realization of novel therapeutic concepts including chemotherapy, photothermal/photodynamic therapy, immune therapy, radiotherapy, bioactive therapy and combinational therapies. Furthermore, BP nanosheets are completely degradable in biological milieu by reacting with oxygen and water molecules, of which the degradation products are non-toxic and can readily join the endogenous phosphorus metabolism to meet the increasing demand for nutrients during the regeneration of both solid and hard tissues. These inherent and functional merits have tremendously spurred research and application of BP-based biomaterials in almost all fields of biomedical technology and substantiated their extraordinary potential for postoperative tumor treatment.Nevertheless, it is worth noting that the research and development of BP-based biomaterials are still in an early stage compared to other well-studied 2D inorganic nanomaterials such as graphene and MXenes, as there are many issues and challenges regarding their synthesis, modification and implementation that remain to be solved. Currently, few-layer BP nanosheets are predominantly synthesized by time-consuming exfoliation method with limited yield, and the product quality is largely determined by the bulk BP crystal seeds . As the


Mycobacterium tuberculosis. Drug resistance in this species is usually due to point mutations in the drug target or in proteins required to activate prodrugs. However, another interesting and underexplored aspect of bacterial physiology with important impact on drug susceptibility is represented by the changes in transcriptional regulation following drug exposure. The main regulators involved in this phenomenon in M. tuberculosis are the sigma factors, and regulators belonging to the WhiB, GntR, XRE, Mar and TetR families. Better understanding the impact of these regulators in survival to drug treatment might contribute to identify new drug targets and/or to design new strategies of intervention.Bacterial drug resistance is one of the major challenges to present and future human health, as the continuous selection of multidrug resistant bacteria poses at serious risk the possibility to treat infectious diseases in the near future. One of the infection at higher risk to become incurable is tuberculosis, due to the few drugs available in the market against  One of the most fascinating aspects of bacteriology is the extremely fast and efficient responsivity of bacteria to external stimuli, which is translated in a fast and precise variation of their transcriptional profile. This capacity is founded on complex regulatory networks based on sigma factors, transcriptional repressors/activators, two component systems, small RNAs or riboswitches able to reshape bacterial physiology allowing the cells to adapt in real time to any external challenge.Antibacterial drugs can be considered atypical stressors, both causing a direct interference with cellular physiology and inducing secondary stress due to this interference, as in the case of the oxidative stress induced by bactericidal drugs . ConsequTo be noted that whereas DR is usually associated with genetic mutations , in some cases DR can be developed without chromosomal abnormalities (defined as phenotypic resistance) . TranscrM. tuberculosis.In this paper, we review the impact of transcriptional regulation on drug susceptibility in M. tuberculosis genome encodes 13 sigma factors and it is the obligate pathogen with the higher amount of sigma factor genes per megabase  is induced upon treatment with ethambutol (EMB), rifampin (RIF), streptomycin (STR), and cycloserine (CS)  , while at to RIF . However strains .I (ii) directly regulates the expression of the structural gene of KatG, an enzyme required for the activation of isoniazide (INH)  sigma factor σ of KatG .H (iii) is involved in oxidative stress response. One of the genes most induced by σH in response to oxidative stress is rv2466c , a member of the primary-like sigma factors, and the ECF sigma factor σE (v) have been shown to be induced upon exposure to vancomycin (VAN) , STR and INH (both mutants). These data support the hypothesis that σE represents a bistable switch involved in persistence development (Both σin (VAN) , and invin (VAN)  showed thoxazole . Intereselopment .E-mediated activation of surface stress response is essential for pyrazinamide (PZA) susceptibility. PZA is only active when bacteria are exposed to low pH, while its derivative pyrazinoic acid is active also at circumneutral pH. However, a mutant overexpressing sigE due to the deletion of the gene encoding its anti-sigma factor RseA  motif, while their putative role is that to sense O2 and nitric oxide  clinical isolates. Many ycosides .whiB7 as the unique gene responsible for increased susceptibility to ribosome-targeting drugs STR and linezolid (LZD) (rv2416c (eis) encodes an aminoglycoside acetyltransferase which inactivate the drugs, whereas 23S rRNA methylation by Rv1988 (Erm37) confers resistance to macrolides. Another gene found to be associated with DR to different drugs including STR is rv1258c (tap), which encodes for an efflux pump. All these DR genes are part of the WhiB7 regulon (whiB7 observed by Reeves and colleagues leading to enhanced expression of rv2416c (eis) (low-level KAN and amikacin resistance) and of rv1258c (tap) (low-level STR resistance)  and rv1655 (argD) genes were associated with increased survival during RIF exposure, as well as with minimum inhibitory concentration (MIC) increase to clarithromycin, again related to the up-regulation of whiB7. Modern Beijing isolates and one phylogenetically intermediate Beijing isolate harbored a loss-of-function mutation in tap; thus whiB7 mutations would not translate into low-level STR resistance, but would lead to low-level KAN and AMK resistance only, despite this epistatic interaction has not been proven in clinical isolates yet  . Among tistance) . Interese to KAN . These mates yet . Interesates yet .A, and mutations in either whiB7 or sigA preventing their interaction, have been found to cause multidrug susceptibility  ultimately fine-tuning mycobacterial cell division and adaptation to stress response, including increased mutation rates during antibiotic challenge  encoding for ergothioneine, which together with mycothiol, plays an important role in maintaining the oxidoreduction balance within the bacterial cell . Ms0535 acts as a transcriptional activator for the expression of its own structural gene and a major facilitator superfamily permease gene ms0534 in the same operon, thus triggering INH resistance. Interestingly, the two genes are not responsive to INH, although their over-expression increases INH resistance. Both ms0535 and ms0534 are absent in M. tuberculosis, thus they can contribute to explain the differences in INH resistance between the two species.Among the techniques used to identify transcription factors potentially relevant for DR, over-expressing libraries for transcriptional regulators under selection on high drug concentrations have been shown to be successful tools. For example, Hu and colleagues  identifiM. tuberculosis there are at least eight putative GntR-like proteins: Rv0043c, Rv0165c (Mce1R), Rv0494, Rv0586 (Mce2R), Rv0792c (MoyR), Rv1152, Rv3060c, and Rv3575c (M. smegmatis (Ms2173). M. smegmatis mutants for Ms2173 showed altered INH and RIF susceptibility (its over-expression led to increased INH and RIF susceptibility), likely due to the regulatory activity of this transcription factor on membrane-associated transporter genes  established increased sensitivity that could be restored by complementation with rv1152 , Rv0474, Rv1129 (PrpR), Rv2017, Rv2021, and Rv3849 (EspR).XRE response element are among the most widespread regulatory elements in bacteria. They are characterized by a conserved HTH DNA binding domain at their N-terminus and a highly variable C-terminal region. The + regulation, Gupta and coll. explored the role of Rv0023 in INH and ETO tolerance. The overexpression of Rv0023 conferred increased INH and ETO tolerance in M. smegmatis by downregulating the expression of the ndh gene, which encodes for a NADH dehydrogenase. This leads to increased NADH cellular concentration and subsequent inhibition of drug-NAD+ adducts formation, which are essential for INH activity , and Rv1129 (PrpR) are involved in the regulation of propionate and acetate metabolisms, respectively. A link between the two metabolic pathways and conditional drug tolerance has been established; however, its biological meaning remains unclear and needs further elucidation .rv2017 was found to be deleted or disrupted by IS6110 in several drug resistant isolates; however, the link between this gene and specific drug resistant phenotype has yet to be defined , which are involved in cell wall biosynthesis and over-expressed in response to INH treatment (M. tuberculosis so far.Rv2327 has been hypothesized to participate in INH antibiotic response given its role in the regulation of reatment . Howeverrv3065 (mmr), a small multidrug resistance (SMR) efflux pump  Rv0275 (InbR) is able to directly interact with INH, and its overexpression is associated with increased resistance to this drug, whereas knockout mutants showed increased susceptibility to several anti-TB drugs  Rv3855 (EthR) is well-known for its regulatory role on ethA (rv3854c), which encodes a Baeyer-Villager monooxygenase involved in the activation of ETO (ethA in ETO resistance is evident (iii) Rv0273c (EtbR) is a transcriptional repressor of the inhA gene, which encodes the target of INH. EMB can bind EtbR, increasing its repressing activity on inhA transcription, thus increasing susceptibility to INH  Ry to INH .M. tuberculosis have to be discovered yet. Of note, Rv0302, Rv1816, and Rv3249c are predicted to regulate mmpL3 and mmpL11, two relevant drug target candidates , Rv0474, Rv1129 (PrpR), Rv2017, Rv2021, Rv3082c (VirS), and Rv3849 (EspR). Mutations in rv3082c (virS) were found to mediate resistance to a new putative antimicrobial compound (defined as thiooxadiazole 3) and ETO by affecting the expression of the mymA operon, which is responsible for the activation of these molecules , Rv1994c (CmtR), Rv2358 (SmtB), and Rv3744 (NmtR), have been described in olerance , no furtexplored .rv3597c gene) is involved in several regulatory functions involving cell wall biosynthesis, transport, and responses to antibiotic treatment. Lsr2 represses INH-mediated induction of iniBAC and efpA   . Similarrculosis . Anothersistance . An addisistance .egt operon), is under the strict regulation of the serine/threonine-protein kinase PknD, and its phosphorylated form fails to catalyzes ergothioneine biosynthesis  under grant agreement no 853989. RS was supported by the Italian Ministry of Health “Ricerca Finalizzata 2016” under grant agreement GR-2016-02364014 to PM.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Respiratory tract exacerbations play a crucial role in progressive lung damage of people with cystic fibrosis, representing a major determinant in the loss of functional lung tissue, quality of life and patient survival. Detection and monitoring of respiratory tract exacerbations are challenging for clinicians, since under- and over-treatment convey several risks for the patient. Although various diagnostic and monitoring tools are available, their implementation is hampered by the current definition of respiratory tract exacerbation, which lacks objective “cut-offs” for clinical and lung function parameters. In particular, the latter shows a large variability, making the current 10% change in spirometry outcomes an unreliable threshold to detect exacerbation. Moreover, spirometry cannot be reliably performed in preschool children and new emerging tools, such as the forced oscillation technique, are still complementary and need more validation. Therefore, lung imaging is a key in providing respiratory tract exacerbation-related structural and functional information. However, imaging encompasses several diagnostic options, each with different advantages and limitations; for instance, conventional chest radiography, the most used radiological technique, may lack sensitivity and specificity in respiratory tract exacerbations diagnosis. Other methods, including computed tomography, positron emission tomography and magnetic resonance imaging, are limited by either radiation safety issues or the need for anesthesia in uncooperative patients. Finally, lung ultrasound has been proposed as a safe bedside option but it is highly operator-dependent and there is no strong evidence of its possible use during respiratory tract exacerbation. This review summarizes the clinical challenges of respiratory tract exacerbations in patients with cystic fibrosis with a special focus on imaging. Firstly, the definition of respiratory tract exacerbation is examined, while diagnostic and monitoring tools are briefly described to set the scene. This is followed by advantages and disadvantages of each imaging technique, concluding with a diagnostic imaging algorithm for disease monitoring during respiratory tract exacerbation in the cystic fibrosis patient. Althougphy (CT) . The frephy (CT) . Moreovephy (CT) .via sputum sample, treatment may be complex. While invasive approaches, such as bronchoalveolar lavage (BAL), provide higher sensitivity and specificity  and overgrowth of preexisting airway flora (mostly bacteria) can determine the nature of acute infection and, since pathogen determination is not always possible cificity , they ar1), and relief of symptoms  (The treatment of RTE is not standardized either  and therduction) .For all these reasons, early diagnosis of RTE is crucial for timely initiation of therapy, as well as optimizing treatment efficacy. As recently highlighted in national guidelines , imagingAlthough evidence for its use remains controversial, chest radiography (CR) is the most frequently used imaging technique for RTE detection . The golConversely, magnetic resonance imaging (MRI) offers a useful and safe alternative, especially for both inflammation and functional assessment , while lThis state-of-the-art review summarizes the clinical challenges of RTE in PCF with special focus on imaging. A short description of the definition of RTE, available diagnostic and monitoring tools, and current treatment options are firstly given to illustrate the clinical context. The review then discusses the advantages and disadvantages of respective imaging techniques, which incorporates a diagnostic imaging algorithm for monitoring PCF during RTE.The Cystic Fibrosis Foundation defines RTE as “acute worsening of respiratory symptoms” requiring medical treatment, mainly represented by antimicrobial therapy and airway clearance through physiotherapy, often under hospitalization . The occNonetheless, a shared and objective definition of RTE, exploitable as an outcome in clinical trials, is still lacking.The easiest way to define RTE is to consider the beginning and end dates of antimicrobial treatment on the patient medical record, assuming that the treatment correlated with the onset and resolution of RTE. The choice of drug administration route  and intervention type  depend on the RTE severity , 17.The need for more objective criteria for identifying RTE has been driven by the need to avoid variability in clinician assessment.The dornase alfa Phase 3 study  defined A challenging aspect of using the Fuchs criteria in clinical practice is that prescribed antibiotic therapy forms part of the diagnostic criteria for RTE as well as its treatment.. . However, evaluating FEV1 decline and subsequent RTE management in non-cooperative children can be challenging  symptoms. However, these findings are frequently subject to low interobserver repeatability, e.g., changes on auscultation . The oddllenging .Physical examination of the chest is a Fuchs criterion for RTE diagnosis  and partMost scoring tools are barely used in a clinical setting, while lung examination findings remain central to the clinical practice of any clinician. However, the last two years of the COVID-19 pandemic have reduced reliance on traditional diagnostic criteria, especially the physical examination. The impossibility of visiting patients made telehealth more prevalent, and RTE had to be defined without examination or spirometry .In the absence of any physical examination, symptoms recalled by PCF or caregivers are a direct and fundamental source of information. Patient-reported outcomes represent the first attempt at standardizing subjective symptoms and their incorporation as additional endpoints in scoring tools and clinical practice is becoming a developing area in RTE definition and monitoring .1) is the most used parameter to assess the severity of lung disease, measure outcomes in clinical trials, evaluate therapeutic response and refer patients for lung transplantation  in PCF. In particular, the forced expiratory volume in the first second . The LCI is a parameter of ventilatory inhomogeneity which is currently recognized as the most sensitive index for identifying small airway obstruction –45. Despring RTE –49. HowePseudomonas aeruginosa, accelerate lung function decline in PCF, eradication therapy must be quickly initiated.Respiratory tract exacerbations in CF are treated with antibiotics directed against microorganisms found in the airways. Because infections by microorganisms, such as Microbiological cultures from airways are therefore taken regularly and represent an important biomarker for the management of CF lung disease; samples can be collected from BAL, sputum or the upper airways, with decreasing reliability of accuracy for finding microorganisms in the lower airways , 51. BroPseudomonas cultures in PCF  and increased neutrophils in BAL infants are risk factors for developing bronchiectasis at a later age , 54. ResNumerous studies have investigated sputum as a source of biomarkers in CF. Existing sputum biomarkers with higher scientific reliability include interleukin (IL)-8 and NE, since their levels increase during RTE and decrease after treatment –58. A reP. Aeruginosa infection, FEV1 levels, or hospital admission in the last year  that could predict RTE when measured in EBC and four other metabolites  with a high sensitivity for RTE diagnosis  64)..64).Regarding blood biomarkers, a recent meta-analysis concluded that there is reasonable evidence to support their application in RTE diagnosis but further studies are needed to demonstrate the usefulness of this approach . AlthougIn conclusion, the most promising predictive inflammatory biomarkers for RTE in PCF appear to be NE, IL-8, tumor necrosis factor alpha, and IL-1 β in sputum and calprotectin in serum. The metabolomic analysis of EBC, sputum, and serum has shown interesting results but needs further investigation.Imaging plays a key role in RTE management by its ability to provide structural and functional information. The advantages and disadvantages of the imaging techniques currently available are discussed in the following paragraphs and summarized, along with the other diagnostic methods, in CR is usually the first imaging tool utilized for detecting RTE due to its ready availability, low cost, and lowest radiation exposure compared with CT . A recenHowever, CR has low sensitivity and specificity in both detecting and following up on new lung alterations. Since lung abnormalities may not differ between patients with and without RTE, nor before and after therapy , becauseMore recently, dynamic CR has been proposed for RTE monitoring. FitzMaurice et al. demonstrated improving speed and range of the diaphragmatic movement in PCF during RTE following treatment . DynamicGiven its ability to provide a detailed view of regional airway and parenchymal changes, CT remains the tool of choice for assessing structural changes in CF lung disease alongside CR. The technique currently offers the best spatial resolution of all imaging modalities. Common CT scanners have a resolution in the order of one millimeter (mm), which allows detection of airways as small as 2 mm in diameter. For an adult without lung disease, this degree of resolution means that during an inspiratory CT scan airways up to the 13th generation can be detected. This is less for preschool children when only more central airways up to the 9th generation can be seen , 74. HowDavis et al., using the modified Brody score, demonstrated that CT can reveal areas of active inflammation during RTE (which is sometimes used to guide sampling) which improves after treatment . MoreoveCurrent low and ultra-low CT protocols have further reduced the risk of radiation exposure , especiaAnother major advantage of CT is its scan speed: high image-quality – thanks to fast acquisition strategies and iterative reconstructions – can also be achieved in non-collaborative patients, such as infants and young children , i.e., aFinally, CT imaging is far beyond the capability of MRI in terms of automatic quantification of lung abnormality. Automatic scoring systems for CT in PCF have been proposed and tested in large cohorts , 83. TheMagnetic Resonance Imaging might become the “one-stop-shop” for diagnosing and monitoring RTE in PCF. In facts, thanks to several MRI techniques, information on structure, inflammation and function may be collected during a single examination and without radiation exposure.Morphological changes during RTE can be evaluated using conventional sequences such as T1-, T2- and proton density-weighted images , 85. Acc3 against the current 0.2 mm3 capability of new Photon Counting CTs  sequences, which are currently the MRI sequences with the best resolution and signal-to-noise ratio (SNR) , were teting CTs , 88. Furting CTs , 90. How., by using a semi-quantitative DWI score on high b-value images, analyzed PCF with and without RTE and those with RTE before and after treatment, interestingly showing significant differences between the groups  and T2-weighted scans –93. Ciete groups  ..., by usAlthough data on inflammatory activity can be also obtained by positron emission tomography (PET)–CT, the radiation exposure is even higher than with standard CT. As an alternative, PET–MRI may offer the same information at a much lower radiation dose, but there is a lack of RTE data using this technique .Functional information on ventilation are obtained using 3D spoiled-gradient echo, which may show areas of air trapping at end-expiration. In common with CT, this technique can be only used in cooperative patients as it necessitates the ability to breath hold for 5–10 s at both end-inspiration and, especially, end-expiration .Moreover, Fourier decomposition (FD) MRI and derivations such as Phase-Resolved FUnctional Lung (PREFUL) MRI sequences, both ventilation and perfusion can be assessed without intravenous or gaseous inhaled contrast administration . PREFUL .  MRI protocol proposed by Tiddens et al.  might beIn fact, the main factor limiting the use of chest MRI in clinical practice and for RTE monitoring is the need for sedation or general anesthesia in uncooperative patients, both of which require specialized personnel, while the general anesthesia conveys some risks for brain development , 100. HiLung ultrasound is a low-cost, bedside and safe imaging technique used both in pediatric and adult patients with pulmonary diseases. In recent years, interest in this technique has increased, making LUS a valuable point-of-care option.. demonstrated that LUS is comparable with CT in detecting some pulmonary findings during RTE, especially consolidations and air bronchogram , if the CR outcome is negative or showing no improvement under therapy, further examination is warranted, especially if a recent (<2 years) CT or MRI scan is not available. The choice of the imaging technique depends upon the patient age and compliance. In uncooperative patients or children under 5 years old, a free-breathing CT may be suitable since it is usually conducted without sedation. Fast-acquisition strategies, such as high-pitch with dual source scanning, or restraining tools like vacuum mattresses, often provide sharp image quality without needing sedation or anesthesia . Since fIn cooperative PCF, generally more than 5 years old, MRI can offer a valid alternative to assess both structural and functional information related to RTE. Chest MRI provides relevant information about ventilation and perfusion, both of which are important in the assessment of RTE. Moreover, MRI can be used repeatedly to monitor the effects of treatment without any limitation in terms of radiation exposure. In centers with no experience of chest MRI, a paired non-contrast-enhanced end-expiratory and contrast-enhanced end-inspiratory CT can be considered.Physical examinations and PFTs have several limitations in respect to RTE diagnosis and monitoring. Current sputum and blood biomarkers of infection and inflammation during RTE are not sensitive nor robust enough for the reliable diagnosis of RTE. Therefore, lung imaging is still crucial for diagnosing RTE and monitoring treatment response. While chest radiography can exclude gross abnormalities or complications, it cannot sensitively diagnose new structural changes induced by RTE, especially in PCF with severe lung disease. Computed tomography allows a detailed assessment of RTE-related structural changes and represent the current standard for CF lung disease evaluation. However, short-term CT serial imaging during RTE is limited by the risk of radiation exposure, and it might be only advised with the introduction of ultralow-dose CT protocols. On the other hand, magnetic resonance imaging (MRI) remains behind CT in terms of morphologic assessment capability but, thanks also to recently developed morphological sequences , could become a one-stop-shop for obtaining both structural and functional information in a single examination. Current limitations of MRI include the need for sedation or anesthesia in uncooperative PCF, the lack of image-quality standardization and the necessity of developing dedicated post-processing tools.


ICVTS, Prinzig et al. [In this issue of the g et al.  report aEarly functional and haemodynamic results are certainly important for an initial evaluation;the absence of regurgitation coupled with excellent gradient and larger effective orifice areamight indicate that satisfactory mid- and long-term results could later be expected. However,comparing valve repair versus valve replacement can be tricky because many confounding factorsmight play a role. Normally functioning BAV is intrinsically mildly stenotic by definition andit is certainly not surprising that the repair procedure would exacerbate this aspect.Furthermore, the authors have almost exclusively used the sub-commissural annuloplasty toreduce the dimension of the annulus. This technique, nowadays generally abandoned for itsunsatisfactory mid-term results , might aet al.are of little help in this direction. While properly selected bicuspid valve, when treatedfollowing an anatomical repair [Whether the AVNeo procedure can be considered a good option in the treatment of un-reparablebicuspid valve is difficult to say and the data reported by Prinzig l repair  and/or bl repair  are knowl repair , still mInnovation often comes at the price of patience, perseverance and continuous analysis andevaluation of the results. As always, time will tell.