Several epidemiological studies have consistently reported inverse associations between cardiorespiratory fitness and the risks of cardiovascular disease and mortality ,2, leadiConsequently, cardiac rehabilitation (CR), with exercise training as a core component, is widely proposed for primary and secondary cardiovascular prevention ,7. CR reCardiopulmonary exercise testing permits the simultaneous evaluation of the ability of the cardiovascular and respiratory systems to perform gas exchange in order to support the increase in muscle respiration required to perform exercise. Knowing the physiology or even the pathophysiology of exercise performance and/or limitations is crucial for safe cardiac rehabilitation, to establish exercise training protocols and to assess the final response ,11.Based on the above, the aim of this Special Issue is to explore new insights into cardiopulmonary exercise testing and cardiac rehabilitation, provided below in three brief topic chapters summarising the main ”take home messages”.transcatheter aortic valve replacement (TAVR) who were transferred to an inpatient cardiac rehabilitation (CR group) or discharged home (DH group) to evaluate the impact of CR in TAVR patients during a 1-year follow-up period. Data are scarce in this field. The CR consisted of exercise prescriptions, psychosocial interventions and lifestyle modifications, overseen by a multidisciplinary team . Data show that CR led to significant improvements in clinical performance and Quality of life in the TAVR patients, offering the opportunity to provide specific recommendations. Moreover, the idea to periodically offer CR to TAVR patients is suggested by evidence that the outcomes were attenuated after 1 year of follow-up.Kleczynski and colleagues  measuredCOVID-19 patients, mainly due to the positive effects of exercise training on not only the immune system and inflammation but also on the multifactorial features characterizing post-COVID-19 patients’ clinical conditions, including pulmonary, cardiac, vascular and musculoskeletal dysfunction.It is now widely demonstrated that SARS-CoV2 infection does not end with control over pulmonary inflammation, as indicated by a negative virology test. Rehabilitation centres could be now useful for -COVID-19 patients (very little data);-Severe acute respiratory syndrome (SARS) patients (who likely have similar damage to lungs and other organs as COVID-19 patients);-Frail patients;-Patients with interstitial lung disease and, in particular, idiopathic pulmonary fibrosis;-Patients with chronic obstructive pulmonary disease ;-Patients with pulmonary hypertension ;-Patients with heart failure .We therefore reviewed  previousTherefore, the “new combined COVID-19 exercise protocol” was tailored to post-COVID-19 patients who are imagined as frail subjects with interstitial lung disease complicated by a mixture of pulmonary, cardiac and vascular diseases .Resistance exercise (RE) remains underused, with poor specific guidelines and persistent safety doubts about the enhancement of cardiovascular risk. In this Special Issue, Kambic and colleagues [lleagues  publisheaerobic threshold, usually measured via cardiopulmonary exercise testing, is crucial [Training zone classification is a cornerstone for the assessment of functional capacity for CR exercise protocols, and the identification of the  crucial . In pati crucial  demonstrexertional pathophysiology conditions, specifically evaluating oxygen extraction measurements following rehabilitation in chronic obstructive pulmonary patients. The same intriguing comparison between pulmonary and cardiac patients’ pathophysiologies was also performed by evaluating different parameters, such as minute ventilation for a given carbon dioxide production, which is known to also have a relevant prognostic power in heart failure patients [Physical exercise involves the interaction of the pulmonary, cardiovascular and muscle systems. Peak oxygen consumption, the gold standard for performance evaluation, is measured via cardiopulmonary exercise testing as ventilation multiplied by the inspired oxygen concentration minus the expired oxygen concentration, reflecting the total oxygen extraction relating to cardiopulmonary–muscle crosstalk in the body. Moreover, peak oxygen consumption, following the Fick principle, corresponds to the product of cardiac output and the arteriovenous oxygen difference . Previoupatients .Telerehabiliation is a term used for a set of rehabilitation services provided remotely via telecommunications systems and the Internet which were widely used in the recent era of pandemic restrictions. In a prospective, single-centre, two-arm randomized controlled trial, Batalik and colleagues [The home-based model of CR is a feasible proposal for an alternative rehabilitation model, and lleagues  supporteMoreover, and in more detail, a systemic review and meta-analysis  publishescore based on cardiopulmonary capacity and left ventricular systolic function with the main goal, of identifying low-risk patients who can safely complete CR in a setting other than the hospital, making CR more accessible to suitable patients.Finally, knowing the importance of risk stratification scores in cardiac patients ,23, CabrIn conclusion, no matter the reason, whether for measurement via a cardiopulmonary exercise test or for a training session as a part of a cardiac rehabilitation program, it’s time to run!


A rhythmic expression of clock genes occurs within the cells of multiple organs and tissues throughout the body, termed “peripheral clocks.” Peripheral clocks are subject to entrainment by a multitude of factors, many of which are directly or indirectly controlled by the light-entrainable clock located in the suprachiasmatic nucleus of the hypothalamus. Peripheral clocks occur in the gastrointestinal tract, notably the epithelia whose functions include regulation of absorption, permeability, and secretion of hormones; and in the myenteric plexus, which is the intrinsic neural network principally responsible for the coordination of muscular activity in the gut. This review focuses on the physiological circadian variation of major colonic functions and their entraining mechanisms, including colonic motility, absorption, hormone secretion, permeability, and pain signalling. Pathophysiological states such as irritable bowel syndrome and ulcerative colitis and their interactions with circadian rhythmicity are also described. Finally, the classic circadian hormone melatonin is discussed, which is expressed in the gut in greater quantities than the pineal gland, and whose exogenous use has been of therapeutic interest in treating colonic pathophysiological states, including those exacerbated by chronic circadian disruption. Clock” for gene or messenger RNA, and “CLOCK” for the protein.Biological rhythms that persist on a roughly 24-h cycle under stable environmental conditions, can be synchronized by external cues (zeitgebers), and retain constancy across varying physiological temperatures are classified as circadian . Howeverlocal circadian clocks of the brain centres that control cognition, mood, behavioural rhythms such as sleep-wakefulness and feeding-fasting, and autonomic and neuroendocrine circadian rhythms . See omplexes . Succeedociation , possiblociation . A decreomplexes  lead to omplexes . Genomicbundance , whilst bundance . The cormplitude , and thrmplitude . Togethemplitude .peripheral clocks”) such as in the gut, liver, bladder, adipose tissue and skeletal muscle  and a decreased occurrence during the “inactive period,” or “rest period” . In humaThe SCN drives the activation of sympathetic nerves in the superior cervical ganglia that project to the pineal gland, evoking nocturnal melatonin synthesis and release into the circulation . MelatonThe large intestine receives from the ileum undigested content as well as endogenous secretions, metabolites and dead epithelial cells. Undigested material may be fermented by microbiota in the caecum and proximal colon. In the more proximal regions, intraluminal content is an amorphous semi-liquid. Water, electrolytes, and microbial products are absorbed along the colon as the content forms a stool that is released on defecation . These pDefection is an overt indication of colonic motility that shows daily rhythmicity, peaking in the active period. This has been reported in numerous species, including diurnal humans  and non-ad-libitum food access. However, the persistence of defecation patterns during the active period under constant lighting conditions has also been identified in mice  study of human colonic smooth muscle electrical behaviour distinguished long and short burst of spiking activity . HoweverTaken together, the available data suggest the human colon and rectum show complementary daily rhythmicity favouring increased diurnal motility in colon and nocturnal motility in the recto-sigmoid region. Food intake promptly enhances colonic motility but does not appear to fully account for daily rhythmicity, nor does ambulation. We speculate the daily rhythms in human colonic and rectal motor activity represent true circadian rhythms but this remains to be shown in temporally-isolated subjects.in vivo, were significantly more prominent in the early day period compared to other periods  period  period , reminis) period . Importa) period . Sympath) period  are pred) period . Interes) period , suggest) period . This ef) period , similar) period .ex vivo  in mouse distal colon continued to show daily rhythms following 24 h of constant darkness and fasting  abolished their daily rhythm of fecal pellet output, total colonic pressure and cholinergic agonist sensitivity in continuous dark conditions (120 h), leading to the conclusion that daily colonic motility rhythms are regulated by Period genes  knockout mice  suggestireported . This gereported  and selereported . This clreported  and genereported . Thus, v2-receptors , have received attention as potential circadian entraining factors. Microbes and their metabolites are themselves subject to daily rhythms, highlighting a major potential source of variability in studies of the microbiome . Partly in vitro  is a functional gastrointestinal disorder characterised by recurrent abdominal pain and altered bowel habits: -induced colitis mice delayed colon epithelium regeneration via disruptions to rhythms of cell proliferation  in rat colonic epithelium showed circadian rhythmicity under constant lighting conditions, peaking in the night (active) period  period , paralleNhe3 in intestinal epithelia  in response to luminal nutrients such as glucose, potentiating pancreatic glucose-evoked insulin secretion while inhibiting glucagon secretion  and contA daily rhythmicity of GLP-1 secretion was suggested by the observation that identical meals consumed at different times evoked significantly different plasma GLP-1 responses in humans, favouring higher GLP-1 secretion in the morning, compared to evening . A circaDaily rhythmicity in pain perception in humans is commonly reported, with peak and nadir timing varying across sensory modalities and pathophysiological conditions . The firColonic spinal afferents and their function have been reviewed extensively elsewhere . In brieIn the distal colon, potential circadian regulation of colonic afferents could be inferred through measurements of visceromotor responses (VMRs), that can be assessed by recording abdominal EMG activity, evoked by colonic distension. Distension of hollow visceral organs evoked VMRs that may serve as an indirect indication of visceral afferent activity, and, at noxious distensions (>40 mmHg) VMRs are used as surrogate measure of pain . An earlMelatonin arises from multiple sources, of which the best known is nocturnally generated pineal melatonin. However, extra-pineal melatonin is a far greater source of melatonin in the body, much of which may be generated in mitochondria where it controls oxidative processes and which may represent its original site of synthesis in evolution   . MelatonI system .q to stimulate phosphoinositide hydrolysis and increased cytosolic Ca2+  improves abdominal pain associated with both IBS-C and IBS-D . Howevertagonist  which suAs previously mentioned, disruptions to circadian rhythms can exacerbate UC signs and pathology. In UC-circadian disrupted mice, treatment with melatonin reduced the signs and severity of inflammation in the colon  which waA wide array of colonic functions shows circadian rhythmicity optimized to the period of food intake. Disruptions of these rhythms can cause organ disorders or exacerbate pre-existing ones. Multiple neural, hormonal and intraluminal mechanisms may contribute to the entrainment of circadian variation in colonic functions, but their full details remain to be elucidated. Gut melatonin, in contrast with pineal melatonin, may be principally arrhythmic in function but nevertheless may have therapeutic potential in its exogenous application for treatment of gut disorders that are exacerbated by circadian disruption.


Editorial on the Research TopicAdvances in the care of the pediatric pulmonary hypertension patient: from the neonate to the adolescent-young adult patientIn pediatric pulmonary hypertension (PH), occlusion of small pulmonary arteries leads to increased right ventricular afterload and risk of right heart failure. In recent years, therapeutic options have increased for young patients, however, in the United States most treatments are given “off-label” without formal approval from the Food & Drug Administration. Despite treatment advances, PH remains a life-altering and life-limiting diagnosis for many infants and children. This Research Topic presents novel approaches to the diagnosis, treatment, and risk assessment of PH across the pediatric spectrum. Contributions address novel medical and transcatheter interventions, multidisciplinary care, and risk scoring in pediatric PH. The collection of manuscripts increases awareness of the complexity of this disease and advances the goal of improving functional status, quality of life, and survival in children with this serious condition.de novo and transition cases) at two North American centers. These encouraging data are especially relevant in young children with PH who cannot tolerate enteral tablets due to age, developmental status, or feeding tube requirements. Domingo and colleagues describe novel use of riociguat, an oral soluble guanylate cyclase stimulator, in 2 infants with genetic mutations and severe, nitric oxide-dependent pulmonary arterial hypertension who did not respond to sildenafil. This experience should prompt further study of riociguat, especially in neonatal populations with respiratory failure and high utilization of nitric oxide, in order to expand therapeutic options in the sickest children. Three manuscripts add to knowledge about selexipag, an oral selective prostacyclin receptor agonist, with clear benefits in adult PH but limited published experience in pediatric PH. In a case report, Hasan and colleagues describe a neonate with developmental lung disease related-PH refractory to phosphodiesterase type 5 inhibitor and endothelin receptor antagonist treatment who demonstrated clinical and echocardiographic improvement with selexipag. In a second manuscript, Youssef's group describes a heterogeneous group of 24 PH patients treated with selexipag at 3 Canadian centers. In short term follow-up (12 months), most patients maintained clinical stability with expected gastrointestinal side effects. Faircloth and colleagues describe their experience initiating, transitioning, and titrating selexipag in 7 children  younger than 10 years of age with weights 10–30 kg. Their institution-specific algorithm provides practical insight for other centers and supports continued successful use in the pediatric population. These studies also highlight the reliance of pediatric PH treatment on case reports and observational studies given the challenges of performing randomized clinical trials in children. However, continued observational work and novel trial designs are critical to test pulmonary vasodilator therapies in the pediatric population and increase access to medications traditionally limited to adult use.Several manuscripts present critically important pediatric experience with off-label pulmonary vasodilator therapies. Tadalafil is a once-daily, long-acting phosphodiesterase type 5 inhibitor with a favorable side effect profile but limited data in children. Youssef and colleagues describe improvement in right ventricular function and minimal side effects in 154 children treated with tadalafil suspension  with frequent transcatheter re-intervention on the graft or stent. These data further inform our understanding of the longer-term outcomes of this palliative alternative for patients with severe PH and exhaustion of treatment options other than lung transplantation.The breadth and complexity of pediatric PH is highlighted in several manuscripts detailing a need for comprehensive, multidisciplinary care in this population. Yung and colleagues outline their center-specific, systematic approach to care of infants with bronchopulmonary dysplasia related-PH (BPD-PH). With optimization of respiratory support, low threshold for closure of intracardiac and interarterial shunts, and initiation of pulmonary vasodilators only after diagnostic confirmation by cardiac catheterization, they report universal resolution of PH. In particular, these findings emphasize the need to consider the impact of shunts in patients with developmental lung diseases and limited pulmonary vascular beds. Sullivan and colleagues also augment the understanding of BPD-PH through their description of cardiac catheterization data from 34 patients with BPD, 32% of whom had left atrial hypertension defined as a pulmonary capillary wedge pressure >10 mm Hg. Left atrial hypertension was associated with increased risk of tracheostomy and/or death, suggesting that left atrial hypertension (from left ventricular diastolic dysfunction or intracardiac shunts) may play a role in pathogenesis and patient outcomes.Comprehensive pediatric PH care requires many subspecialists including geneticists, nutritionists, laboratory medicine specialists, rehabilitation teams, and other experts. Ishizuka and colleagues report the burden of heritable pulmonary arterial hypertension at a large PH referral center. Among 66 patients who underwent genetic testing, 14% were found to have a pathogenic mutation, most commonly BMPR2 mutations, with severe hemodynamic findings. These “real-world” single-center clinical data support larger scale population studies to characterize the burden of heritable pulmonary arterial hypertension in the pediatric population in order to develop targeted treatment approaches. Ruland and colleagues demonstrate that malnutrition is underappreciated and underdiagnosed in pediatric PH but that treatment by a registered dietitian improves malnutrition status in many patients at their center. The need for close monitoring and individualized nutrition recommendations can be delivered through a multidisciplinary team at a comprehensive pediatric PH center. Wieland and colleagues report acquired von Willebrand syndrome with bleeding complications in a series of patients with severe PH requiring bilateral lung transplantation, highlighting the importance of considering this diagnosis in patients undergoing surgical interventions or experiencing trauma. Avitabile and colleagues reported associations between skeletal muscle deficits and worse exercise performance on cardiopulmonary exercise testing and exercise cardiac magnetic resonance imaging in adolescents with PH. These data may support interventions to improve muscle mass and strength as a potential means to improve functional status and quality of life in this population.Finally, Lokhorst and colleagues present a systematic review of risk stratification in adult and pediatric PH, which is critical to determining treatment strategy. They identified the most common variables in risk stratification models as: World Health Organization functional class, 6-minute walk test distance, N-terminal pro brain natriuretic peptide level, and mean right atrial pressure, cardiac index, and mixed venous oxygen saturation on cardiac catheterization; however, they identified very limited data on risk stratification in pediatric PH. Future studies must test these adult-focused scoring systems in large pediatric PH registry populations and identify new pediatric-specific imaging parameters, serum biomarkers, and physical activity metrics that predict mortality in children.In conclusion, this Research Topic presents important advances in pediatric PH, but we are reminded that much work remains to be done. This collection provides a framework for identifying future areas of research for pediatricians, advanced practice providers, and PH providers who share the common goal of advancing the care of this vulnerable population from the neonatal period to adolescent-young adulthood.We are grateful to the many pediatric PH experts who contributed their science to this Research Topic collection and to the peer reviewers and editors who provided critical feedback which further improved the quality of the publications.


Lung cancer as the leading cause of cancer related mortality is always one of the main global health challenges. Despite the recent progresses in therapeutic methods, the mortality rate is still significantly high among lung cancer patients. A wide range of therapeutic methods including chemotherapy, radiotherapy, and surgery are used to treat lung cancer. Doxorubicin (DOX) and Paclitaxel (TXL) are widely used as the first-line chemotherapeutic drugs in lung cancer. However, there is a significant high percentage of DOX/TXL resistance in lung cancer patients, which leads to tumor recurrence and metastasis. Considering, the side effects of these drugs in normal tissues, it is required to clarify the molecular mechanisms of DOX/TXL resistance to introduce the efficient prognostic and therapeutic markers in lung cancer. MicroRNAs (miRNAs) have key roles in regulation of different pathophysiological processes including cell division, apoptosis, migration, and drug resistance. MiRNA deregulations are widely associated with chemo resistance in various cancers. Therefore, considering the importance of miRNAs in chemotherapy response, in the present review, we discussed the role of miRNAs in regulation of DOX/TXL response in lung cancer patients. It has been reported that miRNAs mainly induced DOX/TXL sensitivity in lung tumor cells by the regulation of signaling pathways, autophagy, transcription factors, and apoptosis. This review can be an effective step in introducing miRNAs as the non-invasive prognostic markers to predict DOX/TXL response in lung cancer patients. Lung cancer is the most frequent cancer and is responsible for the highest number of cancer-related deaths in the world . DespiteMiRNAs are involved in DOX/TXL response of lung tumor cells via the regulation of signaling pathways Fig. . PI3K/AKJAK/STAT pathway has a critical role in cell proliferation, inflammation, and apoptosis. IL-6 activates the JAK2 that promotes the STAT3 dimerization and nuclear transportation to regulate the JAK/STAT target genes . STAT3 iNOTCH is a developmental signaling pathway that has critical roles in embryogenesis and tumor progression. It can be triggered by activation of NOTCH receptors that releases the NICD into the cytoplasm. Subsequently, NICD enters into the nucleus and regulate the NOTCH target genes by MAML/CSL transcriptional machinery . There wMiRNAs are involved in DOX/TXL response of lung tumor cells via the regulation of transcription factors and DNA binding proteins Fig. . EMT hasCathepsin L (CTSL) belongs to the papain-like cysteine protease family that is associated with the tumor progression –107. It MiRNAs are involved in DOX/TXL response of lung tumor cells via the regulation of protein ubiquitination Fig. . ROS is Arachidonic Acid (AA) pathway regulates the cell proliferation, immunity, and homeostasis . COX-1 oG protein-coupled receptor kinase 5 (GRK5) belongs to the serine/threonine kinase protein family that is involved in sensing various internal stimuli and regulation of the subsequent signaling pathways . There wTumor cells develop paclitaxel resistance through the various processes such as increased DNA repair, cell cycle regulation, and anti-apoptotic pathways –152. MiRMiRNAs are involved in DOX/TXL response of lung tumor cells via the regulation of DNA repair factors Fig. . PARP1 hMultidrug resistance (MDR) is the ability of tumor cell to resist against the chemotherapy drugs . MDR is Doxorubicin and Paclitaxel are widely used as the first line chemotherapeutic drugs in lung cancer patients. However, a significant percentage of patients show resistance to these drugs. Therefore, considering the DOX/TXL side effects in normal body tissues, it is required to introduce the novel prognostic markers to predict the Doxorubicin and Paclitaxel responses in lung cancer. The present review is an effective step towards introducing miRNAs as the non-invasive markers to predict DOX/TXL response in lung cancer which improves the therapeutic strategies to prolong the survival rates in these patients. However, the introduction of miRNAs as the non-invasive prognostic markers in lung cancer patients requires more clinical studies. In this context, it is required to assess the circulating levels of miRNAs in body fluids to clinically use them as the non-invasive markers in screening programs among lung cancer patients and healthy people with a positive familial history. Considering that the miRNAs mainly promote the sensitivity of lung tumor cells to Paclitaxel and Doxorubicin, microRNA mimics strategies can have the promising therapeutic effects in these patients. However, more animal studies and clinical trials are needed to be able to clinically use the microRNA mimics to treat the DOX/TXL-resistant lung cancer patients.


Despite decades of research in NS, the underlying causes are still not fully understood, and effective therapeutic agents that promote complete remission are still limited.et al. performed several computational analyses of a NEPTUNE Study database (GSE200828) to identify shared molecular pathways across patients with FSGS, membranous nephropathy (MN), and minimal change disease (MCD).5 FSGS, MN, and MCD are distinct forms of glomerular diseases that all manifest NS. The identification of common molecular pathways in NS will potentially establish known and novel mechanisms of podocyte injury that may translate into the development of novel therapeutic targets to arrest proteinuria and preserve kidney function.In this issue, Li To attain the goal, the authors first identified glomerular differentially expressed genes (DEGs) in FSGS, MN, and MCD and compared with the healthy kidney by using GEO2R, an interactive web tool that allows users to compare and identify DEGs among two or more groups of samples. Next, R packages and machine learning were used to identify enriched pathways in the Kyoto Encyclopedia of Genes and Genomes database. At last, the authors implemented the Cytoscape, Database for Annotation, Visualization and Integrated Discovery (DAVID) Bioinformatic, and STRING tools to reveal the most significant shared pathways contributing to FSGS, MN, and MCD.ZYX) as the most important contributor to these glomerulopathies, followed by actinin 4 (ACTN4), CRK, and integrins (ITGA). Finally, to determine which type of glomerular cells express these genes, the authors used a single-cell RNA-seq dataset, obtained from glomerular-enriched cells in normal kidney tissue from a patient who underwent nephrectomy. Single-cell RNA-seq was processed with the Leiden algorithm from the Phyton Scanpy package. The findings revealed that ZYX, ACTN4, CRK, and ITGA3 are expressed in most known kidney cell types, especially podocytes.Their results suggest that these three types of NS share more than a thousand DEGs. The enrichment analysis identified focal adhesion as the most significative shared dysregulated pathway and established zyxin  to confirm whether zyxin expression in glomerular podocytes in patients with NS is increased, (2) to correlate zyxin expression with disease status and progression, and (3) to create strategies that modulate zyxin function on kidney injury. Studies investigating zyxin as a potential therapeutic target will be highly valuable.A limitation of the study presented by Li et al. applied a machine learning framework to determine the molecular characterization of primary MN that distinguished it from FSGS and MCD. The authors found that MN-specific genes were enriched in the nuclear factor kappa B signaling pathway and were predominantly expressed in podocytes.11 In a similar study, Zambrano et al. performed single-cell sequencing and observed that endothelial cells played an important role in the recruitment of immune cells in the pathogenesis of IgA nephropathy.12 Furthermore, urinary single-cell transcriptomics have revealed that urine samples from patients with FSGS contains increased numbers of immune cells and a higher expression of epithelial–mesenchymal transition-associated genes in numerous kidney epithelial cells when compared with the MCD counterparts.13 The presence of all kidney cell types and immune cells in urine has also been demonstrated by single-cell transcriptomic analysis in patients with diabetic kidney disease.14Although it is very useful to identify shared pathways for proteinuric kidney diseases, several investigations have also attempted to determine, at the cellular level, the distinctive dysregulated pathway in each glomerular ailment. Sealfon 15 where they provide workflow recommendations throughout the entire gene sequencing and raw data processing . Making use of these guidelines will have a substantial positive effect on the outcome.As illustrated in the previous paragraphs, the immense potential of kidney transcriptomics and bioinformatics has attracted researchers and nephrologists seeking to identify shared and distinct dysregulated molecular pathways in proteinuric kidney diseases. During the last decade, kidney transcriptomics using human and mouse models has become progressively more feasible and accessible to the scientific community. To take full advantage of these rich datasets, efficient gene sequencing and computational methods are required. Hence, we recommend following guidelines developed by experts, such as the one published by Luecken and Theis,et al. performed in this issue, is a promising and more approachable strategy for understanding cell type–specific and cell heterogeneity changes in NS. The knowledge gained will greatly facilitate future research and clinical work.In sum, kidney transcriptomics has been increasingly used to understand genome-wide transcriptomic variations in different conditions such as disease states. Tissue (bulk) RNA-seq, widely used in human patients and animal models, generates an output for the average expression of genes, reflected by cell type–specific gene expression differences and changes in cell composition. The emerging technology, single-cell RNA-seq, has enabled cell type–specific transcriptome profiling and identified the specific cell types involved in disease development and progression. However, its application in clinical studies still has some limitations to overcome, such as high cost and issues with cell dissociation in solid tissues. Combining tissue RNA-seq with single-cell profiling methods, as Li


Diseases are evolving as living standards continue to improve. Cancer is the main cause of death and a major public health problem that seriously threatens human life. Colorectal cancer is one of the top ten most common malignant tumors in China, ranking second after gastric cancer among gastrointestinal malignant tumors, and its incidence rate is increasing dramatically each year due to changes in the dietary habits and lifestyle of the world’s population. Although conventional therapies, such as surgery, chemotherapy, and radiotherapy, have profoundly impacted the treatment of colorectal cancer (CRC), drug resistance and toxicity remain substantial challenges. Natural products, such as dietary therapeutic agents, are considered the safest alternative for treating CRC. In addition, there is substantial evidence that natural products can induce apoptosis, inhibit cell cycle arrest, and reduce the invasion and migration of colon cancer cells by targeting and regulating the expression and function of miRNAs. Here, we summarize the recent research findings on the miRNA-regulation-based antitumor mechanisms of various active ingredients in natural products, highlighting how natural products target miRNA regulation in colon cancer prevention and treatment. The application of natural drug delivery systems and predictive disease biomarkers in cancer prevention and treatment is also discussed. Such approaches will contribute to the discovery of new regulatory mechanisms associated with disease pathways and provide a new theoretical basis for developing novel colon cancer drugs and compounds and identifying new therapeutic targets. Cancer remains one of the most alarming health issues humanity faces, and according to information from the National Cancer Database, in 2023, approximately 153,020 people in the United States will be diagnosed with colorectal cancer (CRC), and 52,550 will die from the disease, including 19,550 cases and 3750 deaths in individuals under the age of 50 [CRC begins in the inner layers of the colon, rectum, and appendix and arises due to microsatellite and chromosomal instability , the accNatural products are compounds or small molecules produced from natural and biological resources , isolateThe discovery of ribonucleic acid (RNA) brought about major findings in the field of molecular biology in the second half of the twentieth century, and one of the most important discoveries was that of many types of small RNA molecules (sRNAs), later categorized as non-coding RNAs (ncRNAs), such as tRNAs, rRNAs, uRNAs, snoRNAs, snRNAs, and siRNAs. This group also includes many of the microRNAs (miRNAs) commonly found in living organisms . Ambros Nuclear miRNA biogenesis begins with the production of primary miRNA transcripts ~300–1000 nucleotides in length by RNA polymerase II or III . After iDespite significant progress in understanding the basic mechanisms of miRNA biosynthesis, little is known about the mechanisms controlling miRNA biogenesis and how this process is dysregulated in carcinogenesis. According to recent findings, the mechanism of miRNA dysregulation involves the following processes: amplification or deletion of miRNA genes, abnormalities or epigenetic dysregulation of transcription factors, and defects in genes in the miRNA biogenesis pathway . InitialIn the following sections, we will highlight the role of miRNAs and natural products in human cancer, analyze their regulatory elements, and discuss the emerging mechanisms of action underlying the natural product-targeted regulation of miRNA and their contribution to cancer pathogenesis.Natural drugs, such as curcumin and bitter ginseng bases, have long been used to prevent and treat cancer . NaturalTumor-associated macrophages are traditionally divided into two types, the immunosuppressive/anabolic M2 phenotype and the classical inflammatory M1 phenotype; M1 macrophages produce pro-inflammatory cytokines and reactive oxygen/nitrogen species essential for host defense and tumor cell killing , while MAll studies on natural products have indicated that they can affect apoptotic cell death, cell proliferation, migration/invasion, and angiogenesis by modulating the activity or expression of their molecular targets or targeting multiple oncogenic signaling pathways simultaneously . NaturalIn recent years, many miRNAs have been identified as potential therapeutic regulators of CRC. Apoptosis is an active programmed cellular process that occurs in response to some stimuli and allows cells with damaged DNA to “commit suicide” before they become cancerous; thus, apoptosis is an important basis for tumorigenesis and an effective therapeutic target in cancer . DysreguRosmarinus officinalis L.) extract and its active ingredients have been shown to be natural and potent antitumor suppressors in colon cancer cells.Bitter ginseng bases were found to target the mRNA 3′-untranslated region of ERBB3 and MECOM in colon cancer cells, upregulating miR-22. Bittersweet has been suggested to induce apoptosis and G0/G1 cell cycle blockade in colon cancer cells and downregulate the Wnt/β-catenin and MEK/ERK pathways, thereby directly or indirectly exerting therapeutic effects . RosemarRosemary extract downregulated miR-15b in SW480 cells, and an electronic analysis predicted that miR-15b targets glucosaminyltransferase 3 (GCNT3), a tumor suppressor . These rQuercetin is found in many plants and foods and is thought to be capable of preventing and treating CRC . ImportaMany other natural products derived from plant seeds, flowers, leaves, and stems have been assessed in different studies and found to help prevent tumorigenesis and development. Curcumin is an orange−yellow crystalline still powder, a natural product isolated from the ginger plant of the family Tennantaceae . Its actSimilarly, baicalin is commonly used to treat intestinal diseases, such as diarrhea and colon cancer, and a previous study on the antitumor mechanism of baicalin in colon cancer revealed that it induces apoptosis through the miR-217/DDK1-mediated Wnt signaling pathway . In anotC. alba and C. purpurea, and several fruits; UA is an antitumor compound that targets oncogenic proteins and their associated miRNAs [Ursolic acid (UA), a pentacyclic terpenoid, is derived from medicinal plants, such as d miRNAs . There id miRNAs . UA has d miRNAs .Lignocaine has been found to inhibit the epithelial-to-mesenchymal transition of CRC cells by inhibiting CREB1 expression . LignansIt is widely believed that CRC development requires a sequence of genetic changes to drive the transformation of normal colonic epithelial tissue to malignant CRC. If patients with colon cancer could be detected and treated before they develop advanced-stage disease, patient mortality rates would subsequently be reduced . Thus, nThe mechanism of CRC metastasis is shown in Another group exposed colon cancer cells to soy extract for 72 h and observed inhibited protein kinase C and cyclooxygenase-2 (COX-2) activity and expression . The denGiven that regulatory miRNAs targeted by natural products are being investigated as potential therapeutic targets, understanding the basis of miRNAs in CRC development and promotion may help to further understand the pathways that can be targeted for cancer prevention. Predicting cancer development and investigating more accurate biomarkers could advance our understanding of the disease and greatly improve patient prognosis and overall survival.Although the prognosis of advanced CRC remains discouraging, the disease is curable in its early stages, highlighting the importance of prevention and early detection. Many drugs and dietary components have demonstrated efficacy in preventing CRC in animal and cellular models, with natural products demonstrating the fewest side effects. Numerous recent studies have revealed that natural products can target various miRNAs, and these miRNAs can also regulate molecular targets that alter disease onset and progression processes. For example, certain natural products can act as chemopreventive agents by upregulating miRNAs that silence proto-oncogenes . PreventNotably, grape seed extract (GSE), a by-product of commercial grape juice and winemaking processes, contains proanthocyanidin, a flavonoid comprising dimers, trimers, other catechins, and epicatechin oligomers . ProanthThere is growing evidence for aberrant miRNA expression in numerous malignancies, which can also affect chemoresistance. In recent years, natural product active ingredients have been found to play a role in reducing the toxic effects of chemotherapy, improving chemoresistance, and enhancing tumor resistance . ScholarEmerging studies have shown that almost all colon cancer patients develop drug resistance, which limits the therapeutic effect of anti-cancer drugs, ultimately leading to chemotherapy failure . HoweverCinnamomycetes spp. (AC), a Taiwanese endemic species that grows as a parasitic fungus in Cinnamomycetes hardwoods of the camphor family [Among multiple ncRNAs, miR-24 was the first chemoresistance-associated miRNA to be identified and was found to promote methotrexate resistance through binding site polymorphisms in the dihydrofolate reductase gene . Traditir family , induce r family .Ant cinnamic acid has been reported to maximize the therapeutic effect in colon cancer patients by upregulating the tumor suppressor inflammatory factor miR-142-3p, thereby downregulating many cancer stem cell-related genes, enhancing 5-FU chemosensitivity, and inhibiting colon carcinogenesis and progression . PreviouDespite decades of efforts toward preventing and fighting cancer, it remains one of the three deadliest diseases worldwide. Natural products have been described as an irreplaceable source of medical therapies for human oncology because of their multiple pharmacological activities, multi-targeting capabilities, and diverse chemical structures. Over the past three decades, a series of studies have shown that natural products can play an important role in CRC cell proliferation, metastasis, and chemoresistance by indirectly modifying CRC-related signaling pathways, EMT, and angiogenesis by targeting miRNAs. Abnormal miRNA-targeted regulation of proto-oncogene and oncogene expression serves as a biomarker for CRC diagnosis, treatment response assessment, and prognostic evaluation, which provides new clinical ideas for the early diagnosis of CRC patients and could prolong their survival and reduce suffering. In this review, we summarized the anti-CRC effects of some natural products from different sources and their role in TME antagonization and immune regulation; for example, curcumin, lignan, and rosemary extracts can alter the miRNA expression profile and target multiple genes simultaneously. We also discussed some natural products that target and regulate miRNAs that act as potential markers of CRC and drug resistance; these miRNAs inhibit cell migration and metastasis.Despite the exciting progress that has been made in investigating natural products as modulators of colon cancer therapy in various studies and the broad benefits in miRNA regulation, such as the classical downregulation of cancer cell-associated miRs  enabling tumor suppressor genes , the potential to modulate treatment-resistant cells, and the use of natural products to treat colon cancer through epigenetics, numerous issues remain to be addressed. First and foremost, natural products will demonstrate individual patient and TME differences and could be affected by cancer heterogeneity. Second, a deeper and more comprehensive exploration of the immune system signaling pathways associated with CRC is needed to aid in the selection of more effective natural products. Third, most natural products have a wide range of pharmacological effects; however, their targets and molecular mechanisms associated with tumor immunity have not been fully elucidated. Many studies have found that bioactive compounds modify epigenetics in a dose-dependent manner. Therefore, it is necessary to determine the effective doses and concentrations of these compounds for preventing or treating cancer in the future. Overall, natural products play a major role in the regulation of miRNAs in various cancers, and targeting miRNAs has shown promising therapeutic effects. However, further efforts are required to unravel ncRNA functions and understand their precise mechanisms.


Cervical cancer is the fourth most diagnosed cancer among women globally, with much of the burden being carried by women in limited-resource settings often worsened by the high prevalence of HIV. Furthermore, the COVID-19 pandemic disrupted organized screening efforts and HIV management regimens worldwide, and the impact of these disruptions have not been examined in these settings. The purpose of this paper is to describe whether uptake of cervical cancer screening and HIV management changed before, during, and since the COVID-19 pandemic in North-Central Nigeria.Longitudinal healthcare administration data for women who obtained care between January 2018 and December 2021 were abstracted from the AIDS Prevention Initiative Nigeria (APIN) clinic at Jos University Teaching Hospital. Patient demographics, pap smear outcomes, and HIV management indicators such as viral load and treatment regimen were abstracted and assessed using descriptive and regression analyses. All analyses were conducted comparing two years prior to the COVID-19 pandemic, the four quarters in 2020, and the year following COVID-19 restrictions.p = 0.015) compared to the other periods examined in this study. Conversely, the average viral load for women who sought care during that period was significantly higher in adjusted models (p < 0.0001). Finally, we determined that the average viral load at each clinic visit was significantly associated with the period in which women sought care.We included 2304 women in the study, most of whom were between 44 and 47 years of age, were married, and had completed secondary education. About 85% of women were treated with first line highly active retroviral therapy (HAART). Additionally, 84% of women screened using pap smear had normal results. The average age of women who sought care at APIN was significantly lower in Quarter 3, 2020 (Overall, we found that COVID-19 pandemic mitigation efforts significantly influenced women’s ability to obtain cervical cancer screening and routine HIV management at APIN clinic. This study buttresses the challenges in accessing routine and preventive care during the COVID-19 pandemic, especially in low-resource settings. Further research is needed to determine how these disruptions to care may influence long-term health in this and similar at-risk populations. Cervical cancer is one of the most commonly- diagnosed cancers among women worldwide . Recent HPV DNA testing has become the global standard of care for cervical cancer screening in high-income countries (HIC) for its superiority to cytology-based approaches to screening . HoweverFurthermore, many LMICs have a high prevalence of HIV, an important risk factor for cervical cancer . In 2022The COVID-19 pandemic brought notable disruptions to the management of chronic diseases globally. The cancer care continuum, and prevention and control programs in the United States and globally experienced disruptions due to nation-wide lockdowns imposed to reduce the spread of the virus and were among the most effective public health measures instituted by governments worldwide , 6, 19. In low-resource settings, studies evaluating the impact of COVID-19 pandemic and the ensuing lockdowns on the cancer care continuum are scarce, with one study reporting changes in cancer treatment but not screening . This gaIn Nigeria, the lockdowns were instituted nation-wide, limiting access to food, transportation, houses of faith, and work, and the lack of adequate telecommunication infrastructure limited access to tele-work opportunities. For healthcare professionals who needed to physically be present at work, limited availability of personal protective equipment (PPE) reduced the services healthcare providers could offer to patients, created additional barriers in access to healthcare facilities including routine HIV care, access to highly active antiretroviral therapies (HAART), and cervical cancer screening. The goal of the present analysis, therefore, is to assess the use of cervical cancer screening and routine HIV management before, during, and after the lockdowns in response to the COVID-19 pandemic at a tertiary healthcare facility in North-Central Nigeria.We conducted a secondary analysis using data from the AIDS Prevention Initiative Nigeria (APIN) clinic at Jos University Teaching Hospital between January 2018 and December 2021. Various services that cater specifically to people living with HIV are available at no direct cost to the patients receiving care within APIN including HIV counseling, laboratory services including viral load and CD4 count monitoring, tuberculosis (TB) screening and treatment, pharmacy drug pickups, sexually transmitted infection (STI) screening, and cervical cancer screening offered through Operation Stop Cervical Cancer (OSCC). OSCC began opportunistic screening and treatment for cervical precancer in 2006 through support provided by Exxon Mobil, Texas, USA, through the African Organization for Research and Training in Cancer (AORTIC) . OSCC alAll the departments within APIN maintain detailed physical and electronic medical records for all patients who are enrolled in routine care at APIN including demographic characteristics , clinical factors pertaining to adherence to HIV treatment , risk factors , and clinical outcomes (outcomes of pap smear) of women who were screened at the clinic, and other relevant outcomes. Women received all necessary HIV care services at APIN during a routine clinic day and were referred for cervical cancer screening by a healthcare provider or self-referral. We included data for all women living with HIV who were screened for cervical cancer between January 2018 and December 2021. As our primary outcomes of interest were adherence to HIV care and cervical cancer screening access, men and people who were not infected with HIV were excluded from these analyses. Additionally, women with HIV viral load above 100,000 copies per mL of blood were excluded from the analyses as this viral load was greater than two standard deviations above the mean.p ≤ 0.05 for descriptive analyses. Data for these analyses were aggregated and analyzed in 2020 using STATA v.16 .A de-identified dataset was created of records from women living with HIV who were screened for cervical cancer between January 2018 and December 2021. The database included demographics, clinical, HIV management, history of STI, and pap smear outcomes. The primary analyses were conducted on all women meeting the inclusion criteria of these analyses. Data were categorized as follows: women screened in 2018 and 2019 (classified as the pre-COVID pandemic period), women screened between January and March 2020 (Quarter 1 2020), women screened between April and June 2020 (Quarter 2 2020), women screened between July and September 2020 (Quarter 3 2020), women screened between October and December 2020 (Quarter 4 2020), and finally women screened during 2021 (post-COVID pandemic period). Descriptive analyses were conducted using the six groups described above; means and standard deviations were used for continuous variables, and analyses of variance for categorical variables. Statistical significance was set at APIN maintains longitudinal records of all patients using a unique patient identifier, that we were able to assess the impact of COVID-19 pandemic mitigation efforts on patient access to routine HIV management and cervical cancer screening cross-sectionally and longitudinally. We used the cross-sectional approach to identify the number of women who obtained care (pap smear and HIV management) at APIN before, during, and after COVID-19 pandemic mitigation efforts. All eligible women were included in these analyses. However, to obtain a cross-sectional sample of these data, we identified the “first” visit for each woman who obtained care between 2018 and 2021 as indicated by the earliest date at which each woman was seen at APIN. A dataset was created which included demographic and clinical data for each woman on the earliest date she obtained care between 2018 and 2021. The primary analysis assessed two questions; first, how many women obtained routine care for HIV and cervical cancer screening before, during, and after COVID-19 mitigation efforts were instituted, and second, to describe how demographic factors such as age, level of education, marital status of women, and pap smear use during these periods changed. The analysis results displayed in Tables Previous literature suggests that people living with HIV in this region may have increased use of healthcare services. To account for this, we also examined these data longitudinally to assess whether the relevant patient outcomes changed significantly among women who were able to obtain care multiple times between 2018 and 2021. To achieve this, we compiled a second dataset consisting of data from every visit for all women who obtained care between 2018 and 2021. Using linear regression models, we sought to describe how, if at all, COVID-19 pandemic mitigation efforts impacted patient adherence to HIV management regimens as measured by changes in HIV viral load across the periods previously described. To account for potential confounding between more severe illness and greater number of clinic visits, we stratified these analyses by number of clinic visits, enabling us to identify changes in HIV viral load over the periods in question, stratified by frequency of clinic visits which serves as a proxy for disease severity. The analysis results displayed in Table Table We found that the number of women who were able to obtain routine care for HIV decreased in 2020, with the fewest number of women obtaining care between April and September 2020 Table . A similp = 0.01, see Table Level of education and marital status were not related to obtaining care across the six periods assessed in this study (data not shown), but age was associated with obtaining care. Specifically, the average age for women who were seen for routine care for HIV between January and September 2020 decreased significantly compared to the average age of women who obtained care before the COVID-19 pandemic, as is shown in Table p < 0.0001) and 107.3 vs. 488.3 copies/mL of blood (p = 0.04), respectively, as displayed in Table Concerning changes in patient HIV viral load, we identified that compared to the average viral load of women who obtained care at APIN before the COVID-19 pandemic, women who obtained care between July and September 2020 and in 2021 had significantly higher average viral loads, with 107.3 vs. 2207.4 copies/mL of blood (p < 0.0001). We observed a second peak in the average viral load among women who obtained care after COVID-19 pandemic restrictions, but that increase was not statistically significant (p = 0.11).As shown in Table p = 0.72) followed by increased average viral load for the women who obtained care twice in the following quarter (p = 0.89) compared to women who obtained care before the COVID-19 pandemic. This increase was followed by two quarters in which the average viral load decreased, though both changes were not significant compared to women who obtained care before the COVID-19 pandemic. However, among women who obtained care twice in 2021, we observed a significant increase in average viral load compared to women who obtained care before the COVID-19 pandemic, with the average viral load of 5088 copies/mL of blood (< 0.0001). The average viral load among women who obtained care three times within the study period did not vary significantly in 2020 or in 2021 compared to the pre-COVID pandemic period. However, we observed increased average viral load for women who obtained care three times between July and September (p = 0.42). We observed a similar trend among women who obtained care four times during the study period, where trends suggest a non-significant increase in average viral load compared to the period before the COVID-19 pandemic, but these women remained virally suppressed regardless of the period in which they sought care (data not shown).Finally, we observed decreased average viral load among the women who obtained care twice between January and March 2020 (Overall, we found that COVID-19 pandemic lockdowns had a significant impact on women’s ability to obtain routine cervical cancer screening and HIV management at APIN in Jos University Teaching Hospital. On average, younger women received care between April and September 2020, the periods immediately following COVID-19 pandemic lockdowns, as compared to the period before lockdowns began. We have also shown that cervical cancer screening uptake decreased significantly during these periods. Finally, we have shown that on average, patient viral load at clinic visits increased between April and September 2020 and then again in 2021, with the greatest increases in average viral load being observed among women who obtained care once between July and September, and women who obtained care twice in 2021.As mentioned above, women who sought care in the periods immediately following the COVID-19 pandemic restrictions were on average younger than women who sought care before the COVID-19 pandemic restrictions. One possible explanation for this finding is that much of the early public health messaging emphasized that older individuals were at increased risk for COVID-19 infection. As such it is possible that younger women perceived that their risk for infection was lower and were more likely to seek routine care. One study conducted to assess risk perception in ten countries found that perceived lower risk of COVID-19 infection influenced their active engagement in local COVID-19 pandemic mitigation efforts . To our Next, we learned that changes in patient viral load in our study sample depended on the period at which viral load was measured. More specifically, patients who had the first viral load assessed in the pre-COVID period had on average, lower viral load for the remainder of the study period while patients who had the first viral load assessed between July and September 2020 on average had higher viral loads for the remainder of the study period. While the literature around this finding in this setting is sparse, this finding seems to point to the impacts of delays in accessing HIV care because of COVID-19 pandemic lockdowns. In Nigeria, as in many parts of the world in March 2020, nationwide lockdowns were instituted and enforced to minimize the spread of COVID-19. However, in Nigeria, unlike most places in the world, this brief lockdown period was followed by a period in which the initial restrictions were scaled back to allow people to purchase food and attend to their businesses. This period also allowed patients to go to healthcare centers to obtain necessary care. To be in alignment with WHO requirements of social distancing, the APIN clinic significantly limited the number of patients who could be seen on the days when it was open, per local government regulations.These institutional, national, and global COVID-19 pandemic mitigation policies may be a significant contributing factor to delays in patient care that resulted in the increased average viral load observed in this study. Studies examining the influence of the COVID-19 pandemic on HIV care in northeastern Nigeria, Ethiopia, and South Africa observed similar trends, providing supporting evidence to suggest that lockdown efforts negatively impacted initiation of and adherence to HIV care , 25, 28.Furthermore, we found that the number of clinic visits, and the period in which those visits fell also significantly influenced patient viral load. It is expected that increased access to care, as measured by number of clinic visits within the study period, would lead to better HIV management outcomes using viral load as a surrogate marker. However, our study findings highlight that the benefit of increased access to care was attenuated by the COVID-19 pandemic mitigation efforts, with various COVID-19 periods showcasing statistically and clinically significant higher viral loads among patients who were able to obtain care more than once within that period. This finding is difficult to explain given how quickly policies and guidelines regarding COVID-19 pandemic mitigation changed in the months following global lockdowns in March 2020. However, one possible explanation is that delays in accessing ARTs early in the pandemic period led to drug resistance in some HIV treatment regimens, which is a consequence observed in other contexts, leading to an increased need for follow-up care that we observed , 30. RegThis study has many notable strengths. First, the data collected during healthcare administration at APIN had very limited missing data, enabling us to conduct analyses cross-sectionally and longitudinally to compare patient outcomes before and after the COVID-19 pandemic. Additionally, this study adds to existing literature that illustrates specific interruptions to HIV care and cervical cancer screening due to the COVID-19 pandemic. However, the limitation on external validity to populations outside the study setting is duly acknowledged. All patients represented in this database are women living with HIV, thus the interpretations of these findings are limited to this population. However, we believe that these findings maybe applicable to similar low-resource settings that serve people living with HIV.The novelty of the COVID-19 pandemic and the variations in the policy measures and recommendations that were taken to mitigate the spread of SARS-CoV-2 potentially posed additional barriers to HIV care and cervical cancer screening uptake in North-Central Nigeria. This added difficulty in obtaining care may have had dire consequences in Nigeria because of the high prevalence of HIV infection, and on UNAIDS 95-95-95 targets for minimizing the burden of HIV globally . Additio


Dairy cattle systems have targeted improvements in feed efficiency by selecting animals that can convert less feed into more products. Residual feed intake (RFI) has been the index of choice when selecting dairy cattle for feed efficiency. Nonetheless, RFI studies have focused on lactating cows, and the crucial importance of pre-weaning efficiency on farm profitability and cow productivity has been mostly neglected. This review discusses the current knowledge of how RFI divergence relates to nutrient metabolism in pre-weaning dairy calves, including the advantages and limitations of evaluating RFI in this phase. Existing literature indicates that nutrient utilization, energy metabolism, protein metabolism, vitamin metabolism, intestinal development, and hindgut bacterial populations may be implicated in RFI divergence between pre-weaning calves. Techniques developed to date to evaluate RFI in this phase are still evolving to better adapt to the unique characteristics of this phase, and more research is needed to fill in the gaps in our current understanding of early-life feed efficiency divergence in cattle. However, current results suggest great potential for selecting high-efficiency calves while in pre-weaning to accelerate the progress of genetic selection in dairy cattle. Feed expenses have accounted for 30 to 70% of the total costs of milk production over the past two decades . Hence, 0.75 [Ruminants have been intensively selected for a combination of feed efficiency and milk production traits. Residual feed intake (RFI) has been included in genetic selection programs in some countries ,4 as a m0.75 . Negativ0.75, such as body maintenance, possible pregnancy, milk production, parity, and stage of lactation [Most RFI studies in dairy cattle have focused on lactating cows. Because in lactation, orchestrated adaptations prioritize a certain physiological state, estimates of RFI in lactating cows need to account for additional sources of variation aside from ADG and BWSome studies observed a repeatability of RFI from growing to lactating phase ,9, indic0.75) per day for pre-weaning Holstein and crossbred Holstein × Gyr calves, with an efficiency of utilization of metabolizable energy for maintenance at 66% [The energy expenditure for body maintenance is a key component in feed efficiency variations among different animals . Maintene at 66% . It has e at 66% . HoweverRFI divergence in pre-weaning dairy calves was first investigated in 2018  using HoAlbeit tissue catabolism and anabolism, activity, and thermoregulation play a significant role in feed efficiency divergence in cattle , feed efFeed intake is mainly driven by maintenance requirements; hence, the amount of energy spent on feed digestion, or simply heat increment, is proportional to feed intake. Thus, heat increment increases as feed intake increases . It has 2) uptake and carbon dioxide (CO2) and methane (CH4) production by animals using open-circuit respiration chambers [2 uptake is directly related to feed intake [2 uptake is lowest in efficient animals. Indeed, RFI was positively correlated with heat production (r = 0.48), O2 uptake (r = 0.48), CO2 production (r = 0.48), and heart rate (r = 0.40) in pre-weaning dairy calves [2, and produced less CO2 compared to high-RFI animals.Traditionally, heat increment has been estimated through indirect calorimetry, which measures oxygen , especially during the afternoon (1200 to 1800 h), but eating for a longer time during the night (0000–0600 h) than the least-efficient animals . NonetheIn pre-weaning dairy calves, it has been suggested that differences in feed utilization between RFI-divergent groups may be related to greater intestinal activity due to the large contribution of intestinal digestion . In broa2 uptake by the portal-drained viscera can reach 25% [Small intestine mass (g) and relative small intestinal mass (g/kg BW) were positively correlated with dry matter intake and RFI in young cattle , and higeach 25% . These oeach 25% . In facteach 25% .The efficiency of N utilization in ruminants is typically low due to the several steps involved in peptide degradation in these animals . The intRFI was moderately and positively correlated with nitrogen intake (r = 0.50) and fecal losses (r = 0.60) in pre-weaning calves , supportRecent results obtained with lactating cows show an association between RFI and milk protein efficiency, expressed as dietary protein captured in milk . Cows clChanges in rumen fermentation products have been reported in pre-weaning calves classified as low or high efficiency  from 28 The lack of correlation between rumen fermentation parameters and RFI in the pre-weaning phase is probably related to insufficient rumen function in milk-fed animals, as neonatal calves have an underdeveloped rumen until close to weaning , and difIn lactating cows, low and high efficiency of milk production has been correlated with variations in the abundances of certain rumen bacterial communities . BecauseOdoribacter, Cyanobacteria, Ruminiclostridium 9, Prevotellaceae UCG-001, and Eubacterium nodatum, indicating superior hindgut functionally in these animals. In addition, high-efficient calves had a greater number of functional genes involved in VFA biosynthesis. The analysis of the hindgut metabolome also showed significant differences in the metabolic profile between low- and high-efficiency calves at birth. High-efficient calves had greater enrichment of key metabolites involved in energy-generating pathways, including the citric acid cycle, gluconeogenesis, and pyruvate metabolism, potentially enhancing the supply of energy to the calf and the ability to activate metabolic pathways for amino acid, vitamin, and fatty acid metabolism, which could benefit hindgut development and function [High-efficient calves had a lower abundance of pathogenic bacteria at birth, such as function . On the Interestingly, microbiome communities shifted in response to RFI divergence during the pre-weaning period . The resThe microbiome of low- and high-efficiency calves also exhibited distinct vitamin and amino acid metabolism . SynthesPrevotella, Bacteroides, Lactobacillus, and Faecalibacterium, which were among selected abundant bacteria, was significantly different among the GIT regions and between mucosa- and digesta-associated communities. The rumen contained the most diverse bacterial population, comprising 47 genera in total and 16 rumen-specific genera. Additionally, it has been acknowledged that bovine ruminal bacterial populations change from birth to adulthood [A study investigating mucosa and digesta bacterial communities throughout the gastrointestinal tract (GIT) of pre-weaned calves  has idenThe growth requirements of dairy calves change daily during the evaluation period. However, in commercial settings, calves receive fixed amounts of milk or replacer throughout the pre-weaning phase, regardless of their body weight at birth. This practice gives lighter calves an advantage, as they receive more nutrients from the liquid diet. Ideally, milk offer should be adjusted according to the calves’ metabolic weight and increased as they grow. However, this is not feasible in classic commercial settings. The use of automated milking feeders during RFI evaluations in pre-weaning calves can help overcome these issues, as different liquid diet allowances can be provided over time.Pre-weaning calves receive nutrients in both liquid and solid form, and digestion occurs in different parts of their digestive system, mainly the abomasum/intestine and developing rumen. Liquid and solid diets have different digestibilities, with liquid diets usually being more digestible. Therefore, the individual proportions of liquid and solid intake can introduce bias in RFI evaluations. Developing equations to account for this can potentially improve the accuracy of the evaluations during this phase.Since overall intake during the pre-weaning phase is low and variable, it can be challenging to detect differences in dry matter intake and other variables between RFI groups. Increasing the number of animals enrolled in efficiency trials can improve the accuracy of the evaluations.Assessing nutrient digestion before 30 days of age can be challenging due to the low intake of a solid diet and the higher incidence of digestive disorders and diseases in the first two weeks of life. In addition, digestive disorders in the first two weeks of life usually depress feed intake and add additional bias to feed intake evaluations and digestibility values. Digestibility assays have a higher chance of success when performed in animals that are 35 days of age or older.Variations in rumen fermentation are also difficult to observe in the first 30 days of life due to limited ruminal activity.Using a homogenous concentrate for solid diets is preferred over using sortable concentrates. Sorting can affect the nutrient composition of the diet and introduce bias. If forage sources are offered, providing them separately from the concentrate may be ideal, as calves offered forage and concentrate together tend to sort, leading to differences in intake composition between RFI groups.The recommended 63-day observation period for RFI evaluations is lengthy for pre-weaning calves, considering that calves are usually on a milk diet for 60 days on commercial farms. The use of technology that allows for automatic daily measurements of body weights, body composition, and accurate values for liquid and solid intakes has the potential to provide consistent measurements and reduce the time needed to observe divergence in RFI for pre-weaning calves.As mentioned in previous studies ,48, therAs previously mentioned, the advantages of assessing RFI in the pre-weaning phase lie in that pre-weaning calves are raised individually, facilitating individual measurements of feed intake. Moreover, pre-weaning RFI models are not subjected to the effects of negative energy balance, pregnancy, or tissue mobilization, evaluation costs may be lower compared to post-weaning or lactating phases, and the repeatability of RFI may accelerate improvements of the herd feed efficiency through genetic selection of young calves. On the other hand, the particularities of feeding management and nutrient digestion in the pre-weaning phase impose limitations on methods developed to date for assessing RFI and its relationship with nutrient metabolism in pre-weaning calves . These iTo our knowledge, no specific protocols for RFI evaluations in dairy cattle have been established. Research investigating RFI in dairy cattle has adjusted to general recommendations for RFI assessment in beef cattle, which are based on studies published by Archer et al. and Wang et al. in 1997 and 2006, respectively, with growing beef cattle ,50.The Australian Standard Protocol for RFI testing in beef cattle  advises The lack of specific protocols for evaluating RFI during pre-weaning is probably one of the main reasons why there is substantial literature on post-weaning and lactating phases but not on milk-fed calves. More importantly, the unique features of this phase require several adaptations to general methodologies described in studies carried out with growing and adult cattle. Based on the practical application of RFI methodologies to pre-weaning studies, we suggest the following recommendations for future evaluations of RFI in pre-weaning calves: enrolled calves should be of similar sex and breed/genetic composition; calves should be evaluated for at least 56 days; calves should be enrolled in the test after 14 days of life when intake of the solid feed has become more significant, and scours interference on feed intake and BW gain has been reduced; the solid feed should be highly homogeneous to avoid diet selection; calving dates should be as concentrated as possible to minimize age variations among contemporary calves and keep it under 60 days; due to the age differences, pre-established testing periods should be applied to calves individually. In that case, BW measurements should be taken based on the age of individual calves until the total testing period is complete; BW should be measured at least once a week to allow for proper adaptation to the test and reduction of the total test period; BW should be always be recorded around the same time, prior to the morning milk feeding; available facilities should allow individual assessment of dry matter intake and the distinction between intake of milk and the solid feed; milk offer should be adjusted for metabolic BW throughout the evaluation period.Available techniques are still being adjusted to be more suitable for evaluating RFI in the pre-weaning phase. Further studies could benefit from refined technologies that will increase the accuracy of RFI evaluations. Future research is warranted to explore the roles of different nutritional strategies, nitrogen metabolism, rumen epithelium and microbiota, and digestion sites in RFI-divergent pre-weaning calves and clarify the mechanisms distinguishing efficient animals in this phase. Physiological differences between suckling calves, bucket-fed calves, and calves fed through automatic milk feeders should also be explored. Due to inconsistent results regarding the repeatability of RFI classification over phases, additional work should also focus on investigating the genetic repeatability of RFI from pre-weaning to productive life, shedding light on whether calves selected for high efficiency in the pre-weaning phase will remain most efficient in the growing and lactating phases and, therefore, be suitable for genetic selection of this trait.


However, acquiring the ideal bioink to manufacture biomimetic constructs is challenging. A natural extracellular matrix (ECM) is an organ-specific material that provides physical, chemical, biological, and mechanical cues that are hard to mimic using a small number of components. Organ-derived decellularized ECM (dECM) bioink is revolutionary and has optimal biomimetic properties. However, dECM is always "non-printable" owing to its poor mechanical properties. Recent studies have focused on strategies to improve the 3D printability of dECM bioink. In this review, we highlight the decellularization methods and procedures used to produce these bioinks, effective methods to improve their printability, and recent advances in tissue regeneration using dECM-based bioinks. Finally, we discuss the challenges associated with manufacturing dECM bioinks and their potential large-scale applications. The growing demand for organ and tissue regeneration in patients is complicated by the dearth of suitable donors and concerns about immune rejection or biocompatibility after transplantation. Tissue engineering (TE) provides excellent prospects for overcoming the limitations of current therapeutic and organ transplantation methods 3D bioprinting (3DBP) is an additive manufacturing process for generating hierarchical scaffolds with customized geometries and structures via the placement of bioink containing living cells and biomaterials, allowing the production of patient-specific implants and structures The ECM mediates extracellular signaling to resident cells via its unique tissue-specific structure and protein composition However, it is difficult to employ dECM alone as a bioink for 3D printing due to its low viscosity and mechanical instability Figure 1). During decellularization, it is essential to preserve the native ECM structure and components, such as GAGs, collagen, and growth factors After removing any undesired tissue components, an appropriate decellularization process is required to remove cellular and immunogenic molecules from the tissues  After removing residual reagents, decellularized tissues can be lyophilized and milled to form a powder that can be dissolved in acidic environments via pepsin digestion. With physical stirring, it is possible to generate gel-like substances at a desired concentration Residual cellular debris can be harmful and may generate inflammatory or immunological responses in hosts. The primary objective of decellularization is to remove as much cellular and genetic material from tissues as possible to eliminate the risk of immunogenicity A variety of 3DBP techniques have been devised and applied to produce functional 3D structures, including extrusion-based printing techniques, digital light processing (DLP) techniques, inkjet-based printing techniques, fused deposition modeling technology, etc Table 1). Typically, these strategies can be categorized as physical or chemical. Physical strategies include support baths, sacrificial polymers, and leveraging an external supporting structure, such as polycaprolactone (PCL) Hydrogels can be extruded through nozzles to generate shape-stable gels, which are ideal for fostering cell proliferation in 3D environments. For extrusion-based 3DBP technology, bioinks are often required to have adequate rheological properties to keep the shape of extruded filaments and provide the desired printability. Printability is a nebulous topic that has been defined and quantified in recent years. The "excellent printability of a hydrogel" refers to a bioink's capacity to sustain pre-designed structures Table 1.The polymers adopted to improve dECM printability are both natural and synthetic; these are the most frequently used biomaterials for 3DBP The effects of gravity and time-dependent flow prior to cross-linking on printed filaments can be mitigated by providing support to the bioink or printing in a buoyant environment. As with conventional 3D printing, supportive polymeric materials such as PCL Figure ). With the bioprinted designs, they achieved good cell survival rates, cell line-specific gene expression, and ECM production. Similarly, dECM bioink produced from swine tracheal mucosa was successfully printed with a PCL frame support to build a functioning in vitro airway-on-a-chip connected to a vascular network Figure ). Yu et al. Figure ). In vitro and in vivo observations indicated that parathyroid-printed patches could reverse surgery-induced lifelong hypoparathyroidism. For breast reconstruction, a recent study designed a dome-shaped 3D cell-printed construct made of decellularized adipose tissue matrix bioink and a PCL framework that prevented structural collapse . The results demonstrated that the constructs promoted host cell infiltration and adipose tissue formation PCL is biocompatible, biodegradable, and possesses physicochemical and mechanical qualities such as viscoelasticity and formability Figure ). In this model, needle-like PEVA posts helped generate mechanical strain in encapsulated cardiomyocytes, which may then influence cardiomyocyte alignment. The dECM-based structures enhanced cardiomyocyte development and differentiation.PEVA, a non-biodegradable thermoplastic derived from ethylene and vinyl acetate, has an elastic modulus between 10.22 and 13.86 MPa, making it suitable for providing mechanical support to bioinks Figure ). In conclusion, the use of sacrificial methods to construct vascular networks has the potential to create complicated vascular systems in 3D TE constructs. However, the total removal of the sacrificial material is not always convenient, and any residual materials may be cytotoxic to the host Sacrificial materials have been extensively studied to improve geometrical characteristics via the molding of non-sacrificial materials around sacrificial components, which are subsequently removed from the sacrificial components. Sacrificial materials can provide the mechanical conditions for dECM bioprinting without using permanent polymer composites Figure ) Figure ) Figure ) Figure ). The FRESH gelatin microparticle support bath introduced microporosity to the scaffolds and aligned the dECM hydrogel to the wound geometry.Using reversible support baths to achieve a 3DBP construct is effective for most low-viscosity materials; moreover, it enables the printing of complex structures without the need for additional support structures To eliminate the necessity of supporting materials when generating mechanically stable structures using dECM-based bioinks, the printability of dECM bioinks can be enhanced by combining cross-linkable hydrogels Figure ) Figure ). Importantly, this kidney-specific dECM-based bioink formulation supported the maturation and tissue development of human kidney cells in a kidney-specific microenvironment. Visscher et al. Figure ). The cartilage-derived ECM was methacrylated into a photo-cross-linkable hydrogel and combined with chondrocytes to create a printable bioink. After methacrylation, the bioinks exhibited adequate mechanical properties, with a stiffness of 25050 ± 2573 Pa, and were printed into an anatomical ear shape. Lee et al. Figure ). Compared to pure alginate bioinks, composite architectures can significantly increase cellular activity. Kim et al. Depending on their origin, native tissues exhibit a wide range of mechanical properties in nature Figure ). When exposed to UV light at different times, the compression modulus of the hydrogel ranges from approximately 1.5 to 6.5 kPa. This dECM-based biomaterial for in vitro disease modeling featured biomimetic architecture and modifiable mechanical properties. For example, different bioink stiffness values were used for printing to mimic a cirrhotic liver environment and evaluate cellular behavior. When exposed to UV light at times of 10 s, 20 s, and 40 s, the compression modulus values of the hydrogel are roughly 0.5 kPa, 5 kPa, and 15 kPa, which each correspond to the softer than healthy range (soft), healthy liver range (medium), and cirrhotic range (stiff), respectively. HepG2 cells encapsulated in a stiff cirrhotic-like substance exhibited decreased proliferation and elevated invasion markers  Figure ). In vitro experiments showed that dECM significantly enhanced HepG2 cell activity and proliferation, liver function, and metabolism. In addition, the print resolution of DLP 3DBP technology was greater than that of extrusion printing. Xie et al. Figure ). The blended bioink possessed the requisite mechanical properties, swelling ratio, and printability and could produce auricular structures with high elasticity and high printing precision. Based on a multilayer biomimetic strategy, Jian et al. GelMA, a gelatin with a photopolymerizable methacrylamide group, is widely used Hyaluronic acid methacrylate (HAMA), derived from the methacryloylation of hyaluronic acid, can rapidly undergo gelation with lithium phenyl  phosphinate under UV irradiation Figure ). The printed heart construct had up to 10 layers, with mechanical stiffness comparable to that of native cardiac tissue. The dERS were cross-linked via an oxidation mechanism driven by visible light. This pathway is triggered by visible light (400-450 nm), inducing the formation of tyrosyl free radicals that form covalent dityrosine cross-links with adjacent tyrosine molecules. Tyrosine is a common amino acid that controls structural conformation changes in proteins Figure ). Ru2+ undergoes photolysis in the presence of visible light and an electron acceptor to produce Ru3+, which oxidizes aromatic residues like tyrosine. The oxidized tyrosine groups are converted into tyrosyl radicals, which are then neutralized by the formation of covalent dityrosine bonds Figure ). Validation analyses of bioprinted tubular structures as prospective intestinal models demonstrated the successful creation of intestinal tissues with high levels of enteroendocrine marker expression Vitamin B2 is a biocompatible photo-crosslinking agent Polyethylene glycol diacrylate (PEGDA) is a common synthetic polymer used in the production of hydrogel biomaterials with flexible elasticity, solute permeability, and biocompatibility. PEGDA enabled rapid photopolymerization after printing Figure ) Figure ). Gao et al. Figure ). The CPF127 solution permitted the ionic gelation of alginate by releasing Ca2+ ions Figure ). CPF-127 components were extruded into the inner layer during the printing process, whereas VdECM/alginate comprised the outer layer. Ionic cross-linking of alginate was initiated to ensure initial form fidelity. This cell/drug co-loaded vessel design resulted in exceptional neo-vessel development and limb salvage at 28 days after implantation in the ischemic limbs of mice, demonstrating VdECM's potential to induce neovascularization. Moreover, the presence of alginate could inhibit the rapid breakdown of dECM by native matrix metalloproteinases by crosslinking Alginate, a bioinert and biocompatible material, undergoes rapid cross-linking and gelation when exposed to calcium ions. Thus, this method is commonly employed to produce double-network hydrogels Figure ).In the past decade, the field of dECM biomaterials has significantly expanded. Applications have branched out from wound healing patches to scaffolds that match physicochemical properties to recreate entire organs. This extends to the recent inclusion of dECM biomaterial applications to the tumor, skin, reproductive tissues, etc. In the realm of TE, 3D cell-printing technologies have been used to generate artificial tissues or organs Figure ). In addition, the CNs-enhanced gastric dECM bioink was utilized to print a variety of 3D forms, including stomach rugae. Using dECM produced from tongue tissue, Kort-Mascort et al. Figure ). These models permit in vitro monitoring of the long-term interactions between drug-loaded nanoparticles and tumor tissue. Future research must combine 3D tumor models and vascular arteries to investigate the enhanced permeability and retention effect during tumor-selective medication delivery and metastasis.Matrix stiffness in the tumor microenvironment is associated with tumor cell behavior regulation. Most tumors are characterized by abnormal ECM deposition and increased stiffness Figure ). This skin model was examined using functional markers for each region to confirm tissue maturity . Full-thickness skin models more closely resemble natural human skin than do dermal and epidermal skin models. The manufactured skin model has a microenvironment that is more similar to that of native skin than standard skin equivalents, resulting in a more reliable and predictive platform for aesthetic testing, drug screening, and fundamental research. Bin et al. Figure ). Firstly, the bioink was created using scar ECM and alginate-gelatin hydrogels with the appropriate physical qualities to imitate the microenvironmental variables; secondly, patient-derived fibroblasts were precultured in the bioink to generate topographic cellular aggregates for future printing. Notably, these scar models represent the earliest stage of scar formation based on gene and protein expression, with the activation of inflammation- and cell-proliferation-related signaling pathways, thus mimicking the in vivo tissue dynamics of scar formation. These models can be employed for precise drug screening as well as for engraftment. In vitro and in vivo models were used to investigate the clinically observed effects of concurrent anti-scarring drug treatment. In addition, the use of materials with differing stiffnesses facilitated the spatial tissue organization of scar collagen bundles in the model. In conclusion, the scar model replicated both biochemical and biophysical characteristics, and its gene expression profile closely resembled that of scar tissue.The skin is a sophisticated organ that serves as a barrier, regulator, and messenger Figure ). The collagen/SIS villi contained epithelial cells and demonstrated various cellular activities, including considerable cell proliferation. Collagen/SIS bioink and cell-printing technology may soon be used to develop intestinal models that resemble the human intestine. Compared to the pure cell-laden collagen villus structure with comparable villus geometry, the cell-laden collagen/dECM villus structure offers a more functional intestine-like epithelium. Based on these findings, dECM-based 3D villus models will improve the accuracy of small intestine physiological models.Furthermore, Kim et al. Patients with vaginal loss suffer psychological and physical pain; thus, vaginal reconstruction techniques are urgently needed. Traditional TE technology, which uses cells and biomaterials to construct a tissue-engineered vagina, has some efficacy in vaginal reconstruction Frequently, VML is far too substantial for normal healing, resulting in functional deficits and scar formation ECM derived from tissues or whole organs possesses regenerative benefits. dECM products are gaining clinical significance and market share as a result of their consistent availability for grafting and superiority over competing options, producing greater clinical outcomes than autografts in some applications. The current shortcomings of conventional treatments can be addressed by using dECM. dECM materials have been approved for the treatment of tissue and organ abnormalities and disorders, including orthopedic and dental, cardiovascular, plastic, and reconstructive surgery ® regenerative tissue matrix, have been converted for clinical application ® is another decellularized product for skin repair, derived from decellularized porcine SIS, and primarily used for chronic wound treatment While the skin has a robust wound healing system, extreme injuries such as full-thickness third- or fourth-degree burns or diabetic wounds exceed the skin's regenerative capacity ® and Allopatch HD™. In particular, GraftJacket®, created using human dermal dECM, has demonstrated efficacy in the treatment of rotator cuff lesions and rotator cuff rupture and was approved for clinical use in 2014 by the FDA Tendons and ligaments transmit tension forces between SMs and bones or between bones. Their fundamental purpose is to improve mobility and joint stability TM is a commercially available dECM bioink made from a mouse sarcoma cell line and sold as a cell culture supplement. The complex composition of MatrigelTM, which mimics the properties of natural ECM, is one of its chief advantages TM, is that they are neither quantitatively nor qualitatively defined, and their variability across batches is substantial. MatrigelTM remains a support for bioassays despite its effectiveness for cell culture, but it is improbable that such a substance will ever be put into humans because of safety concerns  Although the decellularization and in vitro recellularization of numerous human tissues and organs have produced good outcomes in studies, there is still a great deal of work to be done. Efforts to construct human grafts of complicated organs  are exciting, as their complexity originates from tissue architectures that are difficult to achieve in vitro. The literature reports few attempts to produce bioinks originating from humans In summary, grafting is a viable alternative in situations where other therapeutic options may fail to address tissue or organ damage. Autografts are the first choice, but size constraints and the complications associated with grafts encourage alternate options. Decellularized materials have the capacity to meet the standards afforded by autografts, explaining their wide range of applications. After decellularization, allogeneic or xenogeneic tissues retain their tissue-specific features and bioactive chemicals, depending on tissue type. Hence, dECM materials have the potential to assist tissue regeneration and remodeling. Xenogeneic tissue is a practically limitless source, but it is controversial due to the risk of disease transmission resulting from a failure to optimize decellularization or from low-quality donor animals. While the number of decellularized tissue types accessible for clinical application is limited, the increasing number of pre-clinical and clinical investigations is an encouraging sign. Tissue-specific decellularization is the research ground for the future development of whole-organ decellularization and the manufacture of functional organs for regenerative medicine.In the past decade, 3DBP has become increasingly complex and has been investigated for TE and regeneration First, the biocompatibility and mechanical strength of bioinks are compromised by printability concerns. Optimal bioinks should be printable, bioactive, biodegradable, stable, reasonably priced, commercially available, and regulated for clinical usage The incorporation of living cells in the manufacturing process makes bioprinting unique. The significance of safeguarding cells has been recognized. In order to maintain structural fidelity and support the subsequent stable culture, the printed structure needs to have a certain mechanical strength . However, for many types of soft tissue (the Young's modulus of brain tissue is 1 kPa)-sourced cells, the excellent rigid hydrogels will limit their growth and functional expression The reason for using dECM is that it has various advantages, but only considering printability is not enough. For a specific cell, increasing the strength of the bioink rather causes a problem with viability, and problems such as hypoxia occur when manufacturing in large volumes. To precisely imitate biological tissues, bioink should be deposited at a cell-size resolution (510 μm). For clinical applications, thick, multi-layered tissue is necessary. 100-200 μm is the maximal nutrient/oxygen diffusion distance for cells to survive in the absence of vascularization Another trend in TE research is the integration of 3DBP with other biofabrication methods, seeking to capitalize on their respective strengths. Other new technologies, such as four-dimensional (4D) bioprinting Conventional decellularization techniques usually produce dECM with batch-to-batch variance, unstable gelation, and changed composition, including the loss of GAGs and growth factors Quantification of endotoxin contamination is also essential for assessing the appropriateness of dECM for cellular encapsulation, as endotoxin contamination can result in increased islet inflammatory cytokine production dECM is a promising material for TE. Toxicity, mechanical properties, and immune-related issues are the greatest challenges for dECM-based approaches. Particularly, additional research is needed on achieving the balance between its biological and mechanical properties. Furthermore, studies have explored various physical and chemical approaches to enhance the printability of dECM, each with its boundedness. For the industrialization of dECM bioink bioprinting, factors beyond tissue or organ selection must be considered, such as the decellularization process, sterilization, cost, yield, quality, and batch variance of dECM bioinks. Without a multidisciplinary design incorporating cell biology, material science, physics, and mechanical engineering, the clinical utility of dECM cannot be achieved. In the future, we hope to optimize the decellularization process and develop more standardized decellularization evaluation criteria to improve the quality, quantity, and reproducibility of dECM; develop dECM-based bioinks with controlled printability, degradation, and biological properties; construct tissues and organs using 3DBP technology; and create molecularly engineered dECM.