MicroRNAs (miRNAs) are a class of small non-coding RNAs (ncRNAs) that typically consist of 19–25 nucleotides in length. These molecules function as essential regulators of gene expression by selectively binding to complementary target sequences within messenger RNA (mRNA) molecules, consequently exerting a negative impact on gene expression at the post-transcriptional level. By modulating the stability and translation efficiency of target mRNAs, miRNAs play pivotal roles in diverse biological processes, including the intricate orchestration of organ development. Among these processes, the development of the kidney has emerged as a key area of interest regarding miRNA function. Intriguingly, recent investigations have uncovered a subset of miRNAs that exhibit remarkably high expression levels in the kidney, signifying their close association with kidney development and diseases affecting this vital organ. This growing body of evidence strongly suggests that miRNAs serve as crucial regulators, actively shaping both the physiological processes governing kidney function and the pathological events leading to renal disorders. This comprehensive review aims to provide an up-to-date overview of the latest research progress regarding miRNAs and their involvement in kidney development. By examining the intricate interplay between miRNAs and the molecular pathways driving kidney development, this review seeks to elucidate the underlying mechanisms through which miRNAs exert their regulatory functions. Furthermore, an in-depth exploration of the role played by miRNAs in the occurrence and progression of renal dysplasia will be presented. Renal dysplasia represents a significant developmental anomaly characterized by abnormal kidney tissue formation, and miRNAs have emerged as key players in this pathological process. By shedding light on the intricate network of miRNA-mediated regulatory mechanisms involved in kidney dysplasia, this review aims to provide valuable insights for the diagnosis and research of diseases associated with aberrant kidney development. Among them, microRNAs (miRNAs) have been the most extensively researched type of ncRNA. To date, nearly 28,000 miRNAs have been reported in almost 200 species .F.F4].Fig.Most miRNAs act as negative regulators of gene expression. miRNAs typically bind to the 3′ untranslated region (3′-UTR) of messenger RNA (mRNA), leading to gene degradation or translation repression. The inhibitory activity of endogenous miRNAs depends on their loading into the RNA-induced silencing complex (RISC). Single-stranded miRNAs are loaded onto the Argonaute (AGO) protein, forming the RISC complex. The complex targets and binds to the complementary 3′-UTR of the mRNA, thereby regulating the expression of the target mRNA . When th(1)Transcriptional regulation: miRNAs located between genes are transcribed from their independent promoters, while miRNAs located within introns can be co-transcribed with their host genes or transcribed independently. The transcription of miRNA is also regulated by transcription factors, enhancers, silencing elements, and chromatin modifications . Approxi(2)Post-transcriptional regulation: After miRNA genes are transcribed, the entire process from pri-miRNA to mature processing and assembly into the RISC complex is finely regulated. Mechanisms involved in this regulation include RNA editing, regulation of the miRNA microprocessing complex, and RNA-binding proteins specific to certain miRNAs . Key mol(3)Degradation regulation: Current studies suggest that miRNA degradation is regulated through modifications such as adenylation or uridylation of miRNA residues, the formation of RNA-protein complexes, and degradation by nucleases . Additio(4)Epigenetic regulation: It is estimated that approximately 50% of miRNA genes are associated with CpG islands, and the expression of many miRNAs is influenced by DNA methylation . ResearcThe generation and degradation of miRNAs are tightly regulated to ensure specific miRNAs are expressed at appropriate levels and times in cells. Dysregulation of miRNA expression can lead to uncontrolled downstream target gene expression and contribute to disease development . Current3Mammalian kidneys originate from the intermediate mesoderm as the nephric duct, also known as the pronephros. Kidney development begins in humans at embryonic day 18 (E18) and in mice at embryonic day 8.5 (E8.5). The process of kidney development can be divided into three stages: the pronephros, mesonephros, and metanephros. The pronephros and mesonephros are transient structures that regress during embryonic development, while the metanephros develops into the permanent kidney.At human E22/mouse E9.5, the anterior part of the nephric duct differentiates into the pronephric tubules. Subsequently, the posterior part of the nephric duct gradually forms the mesonephric tubules. At human E35/mouse E10.5, the caudal end of the mesonephric duct elongates dorsally to form the ureteric bud (UB). As the UB invades the mesenchyme, the nephric duct differentiates into metanephric mesenchyme (MM). The UB and MM interact with each other, promoting the development of the metanephros. The UB undergoes successive branching to form a complete urinary collecting system, while the MM undergoes mesenchymal-epithelial transition. Some MM cells differentiate into non-epithelialized stromal cells, forming smooth muscles, stroma, and the renal microvascular system. Another portion of the MM differentiates to form renal units, including renal corpuscles, proximal and distal convoluted tubules, loops of Henle, and distal tubules.Overall, this process describes the sequential development of the kidney from the pronephros and mesonephros to the metanephros, involving the differentiation of various cell types and the establishment of the urinary collecting system and renal units .Although there is a growing body of research on miRNAs in the kidney, the available data on the role of miRNAs in kidney development is limited, and their specific functions remain unclear. In recent years, sequencing studies have provided insights into the expression profiles of miRNAs in the embryonic kidneys of mice, which have facilitated research on miRNAs in kidney development ,12. AguiA substantial body of research suggests that miRNAs play a regulatory role in coordinating the timing of embryonic development and differentiation ,14. A reFurthermore, specific studies have investigated the role of miRNAs in kidney development by selectively knocking out miRNAs or key components of miRNA biogenesis, such as Drosha and Dicer, in kidney tissues/cells ,17. ThesStudies have shown that miRNAs may participate in early kidney development by influencing key transcription factors. Several transcription factors expressed in renal progenitor cells, including SIX Homeobox 2 (Six2), spalt like transcription factor 1 , paired Box gene 2 (Pax2), and Wilms' tumor 1 (WT1), are essential for their proliferation, survival, and subsequent differentiation . A studyThe LIM-class homeobox factor Xlim1/Lhx1 is an important transcription factor required for early renal tubule formation and nephron differentiation. It exhibits a tightly regulated dynamic expression pattern during kidney development . A studyDuring the process of posterior kidney development, the budding and branching of the ureteric bud are critical steps. The glial-cell-line-derived neurotrophic factor (GDNF)/tyrosine kinase receptor (c-Ret) signaling pathway plays a major role in inducing ureteric bud branching . StudiesBone morphogenetic proteins (BMPs) are members of the transforming growth factor-β (TGF-β) superfamily of growth factors. They play a crucial role in the normal development of the ureteric bud and nephron formation during kidney development. Mutations in the BMP4 gene can lead to kidney developmental defects. Several recent studies have provided evidence of the interplay between miRNAs and key genes in the TGF-β/BMP signaling pathway.One mechanism by which the TGF-β/BMP signaling pathway regulates miRNA levels is through the interaction of downstream effector proteins, such as Smad, with Drosha . This inNumerous studies have demonstrated that miRNAs participate in the regulation of epithelial-mesenchymal transition (EMT) through the involvement of the TGF-β receptor 2 (TGFβR2). EMT is a critical process in various physiological and pathological events, including kidney fibrosis and embryonic development. It has been confirmed that TGFβR2 is a target of miR-302. Increased expression of miR-302d in mesangial cells leads to reduced expression of TGFβR2 . MiR-590Furthermore, in studies examining the regulation of key molecules involved in kidney fibrosis by miRNAs, it has been found that miR-22 and BMP-7/6 are part of a regulatory feedback loop. MiR-22 not only inhibits the expression of BMP-7/6 but is also induced by BMP-7/6, thereby highlighting the critical role of miR-22 in BMP signaling cascades . AlthougThe renin-angiotensin system (RAS) is a major regulator of blood pressure and fluid/electrolyte homeostasis, and it plays a central role in controlling normal kidney development . The maiChromatin modification is an epigenetic mechanism that can influence gene transcription activity. Histone deacetylases (HDACs) play important roles in many cellular processes, including cell cycle, proliferation, differentiation, and cell death . Studies4As mentioned earlier, several studies have investigated the role of miRNA-mediated gene regulation in kidney development by targeting key enzymes involved in miRNA biogenesis within specific cell lineages of the kidney. The results have revealed that the kidneys of miRNA-deficient animals exhibit various congenital anomalies of the kidney and urinary tract (CAKUT) . TherefoIn recent decades, scientific research has provided a deeper understanding of developmental abnormalities in the kidneys. Studies have indicated that genetic variations and changes in the fetal environment are major factors contributing to fetal renal developmental abnormalities . ChromosAnother important factor contributing to CAKUT, and delayed kidney development is changes in the fetal environment . NumerouStudies have shown that maternal mice subjected to a low protein diet (LP) during pregnancy can result in intrauterine growth retardation (IUGR) in offspring and exhibit impaired kidney development, possibly associated with RAS inhibition and increased Na+-ATPase activity . A serieIn recent years, numerous studies have shown that miRNA dysregulation is associated with developmental defects in various organisms and organ systems, including the kidney. Some studies have provided evidence of the involvement of miRNAs in the pathogenesis of renal developmental abnormalities.Genomic sequencing techniques of miRNA genes, facilitated by genetic variations, have contributed to the research on miRNAs in disease. Currently, only a few studies have established a clear link between miRNAs and specific genetic variations in renal diseases. Jovanovic et al.  analyzedRattus norvegicus), including miR-141 (71%), miR-200a (50%), miR-200b (60%), and miR-429 (59%) [In the context of environmentally induced renal developmental abnormalities, miRNA regulation may also play a crucial role. A recent study found that administration of miRNA inhibitors to pregnant mice resulted in sustained significant reduction in miRNA levels in offspring's kidneys and other organs. This suggests that certain drugs taken during pregnancy that induce miRNA expression, such as tetracycline-based tetracycline-controlled transactivators and tamoxifen-based estrogen receptor systems, can affect miRNA expression in offspring's kidneys through maternal-placental-fetal transmission . Further29 (59%) . Althoug5Long non-coding RNAs (lncRNAs) are more than 200 bases long, transcribed by RNA Pol II, capped and polyadenylated at the 5′ and 3′ ends, respectively . Sequenc6In recent years, there has been a growing interest in exploring the role of miRNAs as essential regulatory molecules in kidney development and diseases. This field of study has garnered significant attention, and researchers are making notable progress in unraveling the complex mechanisms involving miRNAs in the kidney. Numerous studies have demonstrated that miRNAs exhibit distinct expression patterns during kidney development, indicating their active participation in this intricate process. By influencing key growth factors and signaling pathways, these miRNAs play a vital role in orchestrating the precise development and maturation of the kidney. Notably, experiments involving the knockout of critical miRNA processing enzymes, such as Drosha or Dicer, have shed light on the indispensable nature of miRNAs in kidney development. These knockout studies have further emphasized the crucial role played by miRNAs in ensuring the normal growth and functionality of the kidney. However, despite the advancements made, there are still many unanswered questions in this field. One significant challenge arises from the fact that knocking out Drosha or Dicer leads to global changes in miRNA expression, making it challenging to pinpoint the specific functions of individual miRNAs or miRNA clusters in kidney development. Moreover, while the involvement of miRNAs in kidney developmental disorders is increasingly recognized, the precise mechanisms underlying their contribution to these disorders remain elusive. Further research is needed to unravel the intricate interplay between miRNAs and the molecular pathways implicated in kidney developmental disorders. To advance our understanding, future research should focus on deciphering the precise roles of individual miRNAs or groups of miRNAs in kidney development. It is essential to uncover the intricate mechanisms through which these miRNAs regulate physiological processes during kidney development and how dysregulation can lead to pathological conditions. To facilitate such investigations, it is crucial to harness the power of advanced sequencing technologies. These technologies can provide comprehensive profiles of miRNA expression and facilitate the identification of key miRNAs that are critically involved in kidney developmental disorders. Constructing miRNA-related gene networks specific to kidney development will be instrumental in unraveling the complex interactions and regulatory networks underlying normal kidney development and related diseases. This comprehensive understanding holds the potential to not only identify early biomarkers for kidney diseases but also provide valuable insights into therapeutic targets that could revolutionize the treatment and management of kidney disorders. In conclusion, miRNAs have emerged as crucial regulators in kidney development and diseases. Despite the existing challenges and unanswered questions, ongoing research efforts are paving the way for a deeper understanding of the roles and mechanisms of miRNAs in kidney development. By leveraging advanced technologies and interdisciplinary approaches, researchers aim to unlock the full potential of miRNAs as diagnostic tools and therapeutic targets in the field of kidney diseases.Pengfei Sun and Jiaqi Wang wrote the draft and revised it. Pengfei Sun and Jiaqi Wang designed and supervised the study. Tatiana Ilyasova, Alina Shumadalova, Murad Agaverdiev, Chunlei Wang collected the data and designed the figures and tables. All the authors read the submitted version and approved it.This work was supported by the Bashkir State Medical University Strategic Academic Leadership Program (PRIORITY-2030).The authors declare that they have no conflict of interests.


BackgroundMedications for the treatment of opioid use disorder (MOUD) are effective evidence-based strategies to reduce opioid overdose deaths. Strategies to optimize MOUD availability and uptake are needed.ObjectiveWe aim to describe the spatial relationship between the estimated prevalence of opioid misuse and office-based buprenorphine access in the state of Ohio prior to the removal of the Drug Addiction Treatment Act of 2000 (DATA 2000) waiver requirement.MethodsWe conducted a descriptive ecological study of county-level (N=88) opioid misuse prevalence and office-based buprenorphine prescribing access in Ohio in 2018. Counties were categorized into urban (with and without a major metropolitan area) and rural. The county-level prevalence estimates of opioid misuse per 100,000 were derived from integrated abundance modeling. Utilizing data from the Ohio Department of Mental Health and Addiction Services, as well as the state’s Physician Drug Monitoring Program (PDMP), buprenorphine access per 100,000 was estimated by the number of patients in each county that could be served by office-based buprenorphine (prescribing capacity) and the number of patients served by office-based buprenorphine (prescribing frequency) for opioid use disorder. The ratios of opioid misuse prevalence to both prescribing capacity and frequency were calculated by county and mapped.ResultsLess than half of the 1,828 waivered providers in the state of Ohio in 2018 were prescribing buprenorphine, and 25% of counties had no buprenorphine access. The median estimated opioid misuse prevalence and buprenorphine prescribing capacity per 100,000 were highest in urban counties, particularly those with a major metropolitan area. Although the median estimated opioid misuse prevalence was lower in rural counties, all counties in the highest quartile of estimated misuse prevalence were rural. In addition, the median buprenorphine prescribing frequency was highest in rural counties.While the ratio of opioid misuse prevalence to buprenorphine prescribing capacity was lowest in urban counties, the ratio of opioid misuse prevalence to buprenorphine prescribing frequency was lowest in rural counties. Opioid misuse prevalence and buprenorphine prescribing frequency demonstrated similar spatial patterns, with highest levels in the southern and eastern portions of the state, while office-based buprenorphine prescribing capacity did not.ConclusionUrban counties had higher buprenorphine capacity relative to their burden of opioid misuse; however, access was limited by buprenorphine prescribing frequency. In contrast, in rural counties, a minimal gap was evident between prescribing capacity and frequency, suggesting that buprenorphine prescribing capacity was the major factor limiting access. While the recent deregulation of buprenorphine prescribing should help improve buprenorphine access, future research should investigate whether deregulation similarly impacts buprenorphine prescribing capacity and buprenorphine prescribing frequency. Since the early 2000s, the United States (US) has seen an alarming increase in opioid-related mortality nationwide. While there were some decreases in overdose death rates in 2018 and 2019, the onset of the COVID-19 pandemic led to a worsening in the opioid overdose crisis. More than 100,000 overdose deaths were recorded in 2021, representing the highest annual overdose death count in US history, and about two-thirds of these deaths involved fentanyl or another synthetic opioid [The overdose crisis is particularly severe in Ohio. Ohio’s age-adjusted annual death rate per 100,000, due to unintentional drug poisonings, increased from 8.9 in 2005 to 44.1 in 2017. Like national patterns, this increase was largely driven by opioid-involved overdoses . While OMedications for the treatment of opioid use disorder (MOUD) are effective evidence-based strategies to reduce opioid overdose deaths and have been endorsed as a primary strategy for addressing the opioid crisis by the US Department of Health and Human Services’ Opioid Initiative ,7. MOUD National data finds that access to MOUD is dependent on geography and treatment referral streams . StrategWe conducted a descriptive ecological study of the estimated prevalence of opioid misuse and office-based buprenorphine access for all 88 counties in Ohio in 2018.Opioid misuse prevalenceSurveillance of behaviorally linked conditions, such as substance use, is challenging. These conditions tend to vary at the local level, and formal diagnosis of a Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) substance use disorder requires accessing specific healthcare. Given multiple barriers to accessing care, which can vary by location and across time, many individuals with problematic opioid use never receive a formal opioid use disorder diagnosis. To more accurately represent the burden of problematic opioid use, county-level opioid misuse was estimated using an integrated abundance model described by Hepler et al. . UtiliziOffice-based buprenorphine accessOffice-based buprenorphine access included both buprenorphine prescribing capacity and frequency.Buprenorphine Prescribing CapacityThe Ohio Department of Mental Health and Addiction Services (OMHAS) provided information on the number of physicians (MD or DO) with an active DATA 2000 waiver certification in quarter 1 (March 31) of 2018. We calculated the prevalence of patients per 100,000 population in each county who could have potentially been treated by a prescriber with a waiver (office-based buprenorphine prescribing capacity) from January to March 2018. This outcome was generated by aggregating the legal limit of all waivered providers in a county. This measure reflects a “best case scenario” by county of available office-based buprenorphine.Buprenorphine Prescribing FrequencyThe state’s Physician Drug Monitoring Program (PDMP) was accessed to determine the actual number of patients who received a prescription for buprenorphine for opioid use disorder in 2018. We calculated the average number of patients per 100,000 population in each county receiving an office-based buprenorphine prescription (office-based buprenorphine prescribing frequency) in each quarter of 2018. This measure reflects the “real-life availability” by county of office-based buprenorphine.Opioid treatment programshttps://dpt2.samhsa.gov/treatment/). This directory lists the first full certification date for each program that was utilized to identify which of Ohio’s OTPs were certified in 2018.OTP locations were obtained from the SAMHSA OTP directory (County designationstate.edu/positioning/ohio-urban-county-directory). Urban counties were then further delineated into those with and without major metropolitan areas .Rural and urban county categorizations were gathered from the Ohio Urban County Directory  actively prescribing buprenorphine. Fifteen (17%) counties had no physicians certified to prescribe office-based buprenorphine, and an additional seven (8%) counties had physicians who had a certification to prescribe office-based buprenorphine but did not actually prescribe office-based buprenorphine in 2018.Among the 88 Ohio counties, 15 were categorized as urban and 73 as rural. Six of the urban counties contained a major metropolitan area. The median estimated opioid misuse prevalence and buprenorphine prescribing capacity per 100,000 were highest in the six urban counties that contained a major metropolitan area (Table The median ratio of opioid misuse prevalence to buprenorphine prescribing capacity Table  was loweThe county-level opioid misuse prevalence per 100,000 in 2018, as estimated by integrated abundance modeling, is mapped in Figure County-level office-based buprenorphine prescribing capacity and frequency per 100,000 are mapped in Figure The relationship between estimated opioid misuse prevalence and office-based buprenorphine prescribing capacity is shown in Figure The relationship between estimated opioid misuse prevalence and office-based buprenorphine prescribing frequency is shown in Figure For both buprenorphine prescribing capacity and frequency, counties in the highest categories demonstrate a higher burden of opioid misuse per office-based buprenorphine access. For example, counties in the highest categories have at least 25 people who are estimated to misuse opioids per each individual able to or receiving treatment, signaling a lack of prescribing access in those counties relative to the estimated potential demand for treatment. Unlike opioid misuse prevalence and buprenorphine prescribing frequency, which were geographically highest in the southern and eastern portions of the state, OTPs mapped predominantly to urban counties with major metropolitan areas.In this descriptive ecological study, we found that less than half of waivered providers in the state of Ohio in 2018 were prescribing buprenorphine, and 25% of counties had no buprenorphine access. In addition, while opioid misuse prevalence and buprenorphine prescribing frequency demonstrated similar spatial patterns, with highest levels in the southern and eastern portions of the state, office-based buprenorphine prescribing capacity did not. It is important to note that our findings represent a cross-sectional investigation of spatial patterns; changing substance misuse burden and prescribing patterns require real-time analyses of these trends .Buprenorphine access was different in urban versus rural counties. In urban counties, particularly those with major metropolitan areas, buprenorphine prescribing capacity was high, while buprenorphine prescribing frequency was relatively low, suggesting that many waivered providers were not prescribing buprenorphine or were so to a limited number of patients. In contrast, in rural counties, buprenorphine prescribing capacity and frequency were very similar, highlighting the need for additional buprenorphine-prescribing providers in these areas. While additional providers might be helpful in urban areas, unless the gap between capacity and frequency is understood, buprenorphine access may still be limited.It is worth considering how the recent removal of the DATA 2000 waiver requirement will impact buprenorphine access moving forward. It is postulated that deregulation would improve access to buprenorphine . WhetherDeregulation may also impact buprenorphine prescribing frequency as regulations reinforce stigma, constraining access and discouraging patient engagement and retention in treatment . MarginaOur mapping of OTP locations demonstrated that they were concentrated in counties with major metropolitan areas, and they did not show the spatial pattern seen for opioid misuse prevalence and buprenorphine prescribing frequency. Along with mainstreaming of buprenorphine, there have been calls for mainstreaming of methadone . The NatThe limitations of the current study should be considered. As noted above, our findings represent a one-time cross-sectional view of spatial patterns and may not represent current treatment access and opioid misuse burden in the state of Ohio. In addition, for buprenorphine capacity, we assumed that each physician only prescribed office-based buprenorphine in the county in which they registered. This assumption may be incorrect as physicians may list a county other than the one that they work in on their DATA 2000 waiver paperwork or may work in multiple counties. Additionally, these office-based buprenorphine prescribing capacity and frequency data were limited to physician prescribers, which may not reflect more recent access as more advanced practice providers became certified and began prescribing office-based buprenorphine in 2017, especially in rural areas .We also assumed that all treatment admissions or opioid overdose deaths were representative of individuals requiring treatment. While this may be a reasonable assumption, it is unlikely to be universally true, particularly as fentanyl is unknowingly added to other substances ,24. As sIn summary, in the state of Ohio, buprenorphine prescribing frequency shared a similar geographic distribution to opioid misuse prevalence, whereas buprenorphine prescribing capacity did not. Urban counties had higher buprenorphine capacity relative to their burden of opioid misuse; access was limited more by the number of providers who were actually prescribing. In contrast, rural counties had lower buprenorphine capacity relative to their burden of opioid misuse, but a minimal gap was evident between prescribing capacity and frequency, suggesting that access was limited by the number of available providers. The location of OTPs, which were predominantly in counties with major metropolitan areas, added to the complexity of MOUD access. While the recent deregulation of buprenorphine prescribing should help improve buprenorphine access, future research should investigate whether deregulation impacts buprenorphine prescribing capacity and buprenorphine prescribing frequency similarly.


Positron emission tomography (PET) with F-18 fluorodeoxyglucose (FDG) has been commonly used in many oncological areas. High-resolution PET permits a three-dimensional analysis of FDG distributions on various lesions in vivo, which can be applied for tissue characterization, risk analysis, and treatment monitoring after chemoradiotherapy and immunotherapy. Metabolic changes can be assessed using the tumor absolute FDG uptake as standardized uptake value (SUV) and metabolic tumor volume (MTV). In addition, tumor heterogeneity assessment can potentially estimate tumor aggressiveness and resistance to chemoradiotherapy. Attempts have been made to quantify intratumoral heterogeneity using radiomics. Recent reports have indicated the clinical feasibility of a dynamic FDG PET-computed tomography (CT) in pilot cohort studies of oncological cases. Dynamic imaging permits the assessment of temporal changes in FDG uptake after administration, which is particularly useful for differentiating pathological from physiological uptakes with high diagnostic accuracy. In addition, several new parameters have been introduced for the in vivo quantitative analysis of FDG metabolic processes. Thus, a four-dimensional FDG PET-CT is available for precise tissue characterization of various lesions. This review introduces various new techniques for the quantitative analysis of FDG distribution and glucose metabolism using a four-dimensional FDG analysis with PET-CT. This elegant study reveals the important role of tissue characterization and treatment strategies in oncology. Various imaging modalities have played important roles in the diagnosis, staging, and therapeutic monitoring of cancer. Positron emission tomography (PET) has recently been applied in several oncological areas. Compared to several other noninvasive imaging modalities, PET is characteristically unique for the in vivo quantitative assessment of tumor characteristics , 2. The Quantitative assessment of FDG uptake can often be used for treatment monitoring after chemotherapy or chemoradiotherapy –8. NumerWhole-body PET imaging allows the accurate staging and restaging of various cancers before and after treatment, in combination with CT or MRI. Such three-dimensional (3-D) analysis of FDG distribution using a high-resolution PET system and appropriate software enables the provision of important quantitative parameters for tissue characterization and treatment strategy. On the other hand, FDG uptake is present in many physiological conditions. Therefore, it is often difficult to differentiate pathological from physiological FDG uptake in routine static PET studies –28. is one of the most commonly used PET parameters for estimating FDG uptake. The SUV represents the radioactivity concentration in the lesion at a single time point. The SUV is simply the ratio of the activity concentration in the target tissue or lesion to the activity concentration in the whole body. The SUV can be calculated using the following formula: SUV = [tissue tracer activity concentration [Bq/mL]]/ ([injected dose [Bq]]/[body weight [g]]) –8. When Metabolic changes are often assessed using the SUV value of the tumor. The SUV accounts for the differential partitioning of injected activity within the body. When the same PET camera and scan acquisition parameters are used, reasonable reproducibility of the SUV values can be achieved –36. ThisThe maximal SUV (SUVmax) of a lesion is independent of the size of the region of interest (ROI). SUVmax is most commonly used in clinical practice because it is simple, reproducible, and readily available using widely used software , 34. SUV3 ROI surrounding the voxel with the highest activity). The SUVpeak is less susceptible to noise and scanner differences in spatial resolution. In addition, an index called SUL has been proposed [The peak SUV (SUVpeak) was introduced to overcome the shortcomings of SUVmax as a hybrid of the mean value of radiotracer uptake within the ROI surrounding the highest-intensity voxel  and total lesion glycolysis (TLG) are commonly used . MTV repMany malignant tumors tend to be naturally heterogeneous. Several mechanisms have been used to explain the heterogeneous nature of malignant tumors. First, tumors tend to develop from genetic mutations more frequently than normal cells, leading to heterogeneous biological behavior. Second, tumors grow rapidly, and develop a hypoxic region, resulting in elevated glucose consumption (Warburg effects) or severely decreased metabolism (necrosis). Third, tumor cells coexist with microenvironment cells, such as tumor-associated macrophages (TAM), cancer-associated fibroblasts (CAF), and myeloid-derived suppressor cells (MDSC). The variability of tumor proportions and activities may cause metabolic heterogeneity in cancer tissues as a whole. Generally, tumor heterogeneity may be associated with aggressiveness, growth speed, and metastatic potential, all of which are important for clinical management.Tumor heterogeneity can be described qualitatively as ‘homogeneous’ or ‘heterogeneous’ . In radiology reports of FDG PET-CT, radiologists and nuclear medicine physicians often evaluate tumors using qualitative categories; however, such expressions are subjective and poorly reproducible. Especially, the criteria to distinguish ‘homogeneous’ from ‘heterogeneous’ are unclear. In addition, it is difficult to describe the degree of tumor heterogeneity.In this context, radiomics has recently been introduced as a technique that uses a mathematical model to quantify heterogeneity by extracting numerical features from radiological images . IndividThe simplest radiometric method encloses the tumor and a region of interest (ROI) or volume of interest (VOI) if it is 3-D, creates a histogram of voxel values  within the ROI/VOI, and calculates the mean, standard deviation, energy, entropy, kurtosis, and skewness. This is typically called a first-order statistic. This may represent tumor heterogeneity to some extent; however, this method does not consider spatial relationships. In other words, this method does not distinguish adjacent from distant voxels. Therefore, when researchers apply radiomics or texture analysis to their data, they usually employ higher-order statistics that can incorporate the spatial distribution of the voxels. To calculate higher-order features, it is necessary to calculate a matrix  that represents heterogeneity, such as the gray-level co-occurrence matrix (GLCM), gray-level size-zone matrix (GLSZM), gray-level run-length matrix (GLRLM), or neighborhood gray-tone difference matrix (NGTDM). Each matrix generates several features. The imaging biomarker standardization initiative (IBSI) described 174 radiomic features with clear definitions . It may x, y, z) coordinates with the SUVs to preserve the locations of the voxels. In this example, 7206 voxels were extracted from the VOI.Figure The SUV is a continuous value and is often discretized for texture analysis. A common method is to discretize the SUV with a fixed number of bins between the SUVmin and SUVmax . An alternative method is to fix the SUVmin and SUVmax at 0 and 20, respectively.y = x).The GLCM was calculated from the discretized voxel list, which can be regarded as a correlation plot between adjacent voxels. Note that in the 3D space, there are 13 definitions of connected voxel directions whose distance is less than 2 voxels . PyRadiomics is a python library package. If a GUI is needed for PyRadiomics, 3D Slicer can work together.There are two popular free software tools for radiomics or texture feature calculation: LIFEx  and PyRaadiomics . LIFEx iAmong several problems in radiomics, robustness, and replicability seem to be the largest . RecentlWhile most of the FDG parameters are derived through static PET imaging, there are a few trials that assessed temporal changes in FDG distribution using dynamic PET studies. For instance, dual-time-point PET imaging, which obtains both early standard and delayed PET images, has been used to differentiate benign from malignant lesions –33. In aThere are new strategies for the generation of parametric images  based on graphical analysis, such as the Patlak method , 50. TheThe Patlak model can be applied when the tracer reaches a steady state between blood and tissue. This model estimates the Patlak slope (Ki), which is the rate of irreversible uptake, and the Patlak intercept, which is the apparent distribution volume (DV), of the nonmetabolized tracer Fig. . AccordiA preliminary study indicated that multi-pass whole-body PET Ki parametric imaging that utilizes robust Patlak graphical analysis may achieve equivalent or potentially, superior lesion detectability than standard-of-care SUV imaging with reduced false-positive rates in routine oncology applications . Ki paraA key issue in the kinetic analysis is obtaining a suitable input function for graphical analysis. Arterial or arterialized venous blood sampling is commonly performed, particularly for brain studies; however, this method is apparently invasive –58. An iTable These quantitative parameters have several clinical applications in clinical oncology. The 3-D quantitative analysis of FDG distribution provides important information for treatment strategies in oncology.Quantitative analyses of FDG uptake have been performed for prognostic studies in patients with lung cancer. The SUV has been used for assessing either the likelihood of malignancy or aggressiveness in lung masses and as a result, the prognosis of known lung cancer has been assessed in many studies –17. For Similar to SUV evaluation, volumetric analysis has been used as a prognostic biomarker. Despite the lack of a standardized method for its determination and its significant variability with different thresholds, MTV has a great potential for risk analysis in lung cancer , 65. MorRadiomics, such as texture analysis, has the potential for semiquantitative analysis of spatial FDG distribution. The heterogeneity of FDG distribution has been used for risk analysis in various cancers , 75–77. As FDG uptake is more sensitive to changes induced by treatments rather than morphological analysis, changes in SUV are considered fundamental components of molecular imaging response criteria. Accordingly, SUV measurements are commonly applied in multicenter trials in oncological treatment studies , 37.The assessment of treatment response using FDG PET-CT plays an important role in optimizing subsequent treatment strategies and predicting patient outcomes. Qualitative evaluation remains the most commonly used approach in clinical practice and quantitative assessment of FDG uptake has been applied for treatment monitoring in most malignancies. Recent reports and review articles evaluated the prognostic values of changes in FDG uptake (SUV change) in various cancers , 83–89. Diffuse large B-cell lymphoma (DLBCL) and Hodgkin lymphoma have been the most extensively studied cancers for risk stratification and outcomes using FDG PET/CT. Interim PET performed after two or four cycles of chemotherapy has been proposed as a tool for adapting therapy in patients with good responses , 93. In The assessment of temporal changes in FDG uptake using dynamic imaging has several clinical advantages over static imaging. Serial assessment of changes in FDG uptake, even within a short period, is useful for distinguishing pathological from physiological uptake, especially in the abdominal regions , 33, 99.In addition, most malignant lesions may show a gradual increase in FDG uptake, indicating high glucose (or FDG) metabolism, as compared to minimal change or decrease in benign lesions and physiological uptake areas. Recent reports indicated that the target-to-background ratio of Ki in the Patlak analysis was higher than the commonly used SUV values, indicating a higher contrast on the parametric PET images than on the static images , 99–101 PET has significant advantages in the quantitative analysis of tracer activity distributions. In particular, 4-D PET analysis provides new metabolic parameters. A more precise assessment of the clinical values of such parametric imaging is expected in oncology with more patient-based studies in the future.In addition to the introduction of elegant data analyses, instrumental developments have been conducted.PET-CT systems equipped with silicon photomultiplier–based detectors  have been introduced. This new system has improved detection capabilities that might contribute to enhanced diagnostic performance and reduce the activity administration or scan duration. In addition, quantitative capability has been improved significantly owing to a higher spatial resolution and lesser scatter noise –108. SucSuch digital PET with their magnetic-susceptibility tolerance makes them ideal devices for use in PET-MR systems , 110. ThFDG PET-CT can provide valuable clinical information regarding tumor metabolism and aggressiveness in various types of cancers. Various quantitative approaches have been introduced for image-based precision medicine, including tracer concentration, volume, and heterogeneity analysis using 3-D FDG distribution. Recent progress in PET imaging permits the temporal assessment of FDG uptake. Thus, a 4-D analysis of FDG PET-CT is available for the precise tissue characterization of various lesions. Such elegant studies will provide new and valuable information for tissue characterization and treatment strategies in oncology.


Transient global amnesia (TGA) is the prototypical neurologic disease for acute-onset reversible amnesia. It is currently defined by resolution of symptoms within 24-hours. In this case report we describe an atypical case of prolonged TGA, emphasizing our current lack of knowledge surrounding this disease entity and its pathophysiology.A 66-year old female presented acutely with profound anterograde amnesia and variable retrograde amnesia with no inciting event. A thorough workup to exclude alternative causes of amnesia  was normal. Her magnetic resonance imaging was consistent with TGA, with punctate diffusion restriction changes bilaterally in the hippocampi. She was also mildly hypoxemic with no discernible cause. She was ultimately diagnosed with TGA although her diagnosis remains controversial as her symptoms persisted for 72-hours.Our patients clinical and imaging features (apart from her protracted time-course and hypoxemia) were in keeping with a diagnosis of TGA. The association of hypoxemia, COVID-19, obstructive sleep apnea, and the development of TGA remains to be elucidated. Although the underlying pathophysiology for TGA is unknown several mechanisms have been postulated including cortical spreading depression and reversible hypoxic-ischemic injury. The time course for symptom resolution, could be an important clue in discerning the pathophysiology of TGA on an individual basis. Importantly, a clinician should not be deterred by amnestic symptoms lasting >24-hours, if the patients clinical/radiologic presentation is consistent with TGA. The prototypical disease with this distressing (yet short-lived) presentation is transient global amnesia (TGA). TGA is defined by profound anterograde amnesia with variable retrograde amnesia lasting up to 24-hours , mild retrograde amnesia, and perseveration with repetitive questioning. The patient had no broader cognitive deficits in the domains of language , apraxia, and executive functioning (tested with Luria hand sequencing). There were no signs of delirium or difficulties with complex attention when doing serial 7’s and WORLD bedside testing.She had no history of psychiatric disease and there was no evidence of acute drug intoxication . Her biochemical profile was normal . Brain and neck computed tomography (CT)-angiogram did not reveal cerebrovascular insult or a structural lesion. Her electroencephalogram was normal with no focal slowing, epileptiform discharges, or seizures.4 were felt to be consistent with a diagnosis of TGA.Brain magnetic resonance imaging (MRI) 2-days from symptom onset revealed punctiform diffusion weighted imaging (DWI) restriction changes bilaterally in the medial temporal lobes  with corIn addition to her acute amnesia she was found to be mildly hypoxemic for an unknown duration. She tested negative for COVID-19 and respiratory syncytial viruses on serial testing, with a normal white blood cell count, D-dimer, troponin, and arterial blood gas. Serial chest X-Rays revealed slightly prominent lung markings with no evidence of collapse or consolidation. A CT-chest did not reveal an underlying pathology for her hypoxemia/evidence of thrombus, and pulmonary function tests were within normal limits. She was weaned off oxygen prior to discharge.2 At follow-up she had returned to her cognitive baseline, with complete resolution of her clinical symptoms and MRI findings , bilateral punctate hippocampal DWI lesions, and lack of alternative causes of acute amnesia. Brain MRI in TGA can often be normal, however bilateral/unilateral punctate DWI lesions  are noted in up to 84% of patients.4 Typically these changes appear after the amnestic period has resolved, with the highest detection rate 48-hours after symptom onset.3 This differs from our patient, whose lesions were noted in the acute symptomatic phase. DWI findings typically resolve within 6-12 months, similarly to our patient.Our patients clinical and imaging features were consistent with current criterion for TGA 2 with 612 In one case-report profound amnesia lasted up to 8-days, and in a retrospective observational study of 185 TGA patients subtle reductions in cognition lasting longer than 24-hours were noted .Controversially, the protracted timeframe of her symptom resolution was not in keeping with a TGA diagnosis  could favor cortical spreading depression, while a longer TGA duration could be more in keeping with transient hypoxic-ischemic injury or even cerebral ischemia. Regardless of the underlying mechanism it is important to not strictly rely on the 24-hour criterion when the clinical/radiologic presentation is consistent with TGA.Although the pathophysiology for TGA is unknown, the symptomatic timeframe may implicate multiple underlying processes. Several pathophysiological mechanisms have been postulated in TGA including cerebrovascular insult with small vessel abnormalities, hypoxic-ischemic changes with venous congestion in memory relevant structures, or cortical spreading depression in association with migraines/epilepsy/enhanced physiological or emotional stress. Further, a study by Buratti et al (2017) highlighted a higher prevalence of obstructive sleep apnea (OSA) in TGA patients. In this study the prevalence of OSA in TGA patients was 44.8% compared to 13.8% in the general population (P = .02) for an odds ratio of 5.078 . Our patient did not have known OSA, however it is tempting to postulate an association between OSA-induced hypoxic-ischemic injury and TGA. Certainly sleep and memory are closely related, and subtle cortical damage in areas of memory processing have been noted in OSA patients.1416 Further studies on the association between systemic hypoxia/COVID-19/OSA/TGA are worth considering.Further adding to the complexity of our patients’ case was her hypoxemia. This is not a known complication of TGA although a recent case report highlighted TGA as a possible first manifestation of COVID-19 and hypoxemic respiratory failure.In summary, this case highlights a patient with typical clinical/imaging findings of TGA although her protracted symptomatology and hypoxemia made the diagnosis controversial. It is difficult to discern whether a protracted course of amnesia points towards a different pathophysiological mechanism for the development of TGA. Regardless, it is important for the clinician to not strictly rely on the 24-hour criterion if a patients presentation is in keeping with TGA as a timely diagnosis can help avoid extensive investigations, misdiagnosis of ischemia or irreversible pathology, and reassure patients and their relatives of the benign character of this dramatic disorder.


Dear Editor,We are writing to discuss the recent article titled “Efficacy of OLIF combined with pedicle screw internal fixation for lumbar spinal stenosis on spinal canal changes before and after surgery” by Xu et al.  publisheThe authors conducted a retrospective study involving sixteen patients who underwent a combination of single-segment OLIF and pedicle screw internal fixation for the treatment of lumbar spinal stenosis. They compared the patients’ pre- and post-operative data and observed various parameters such as intraoperative bleeding, duration of surgery, visual analogue score (VAS), Oswestry Disability Index (ODI), disc height (DH), cross-sectional area of the vertebral canal (CSAVC), cross-sectional area of the dural sac (CSADS), cross-sectional area of the intervertebral foramen (CSAIF), spinal canal volume (SCV), spinal canal volume expansion rate, lumbar lordosis, and sagittal vertical axis.The results of the study demonstrated that OLIF combined with pedicle screw internal fixation effectively restored disc height and increased the cross-sectional area of the spinal canal. This technique also had an indirect decompression effect. The intraoperative bleeding and duration of surgery were within acceptable ranges. Furthermore, the VAS and ODI scores significantly improved after surgery, indicating a reduction in pain and improvement in functional disability. The CSAVC, CSADS, CSAIF, SCV, and spinal canal volume expansion rate were all increased postoperatively. Additionally, there was improvement in lumbar lordosis and sagittal vertical axis. The follow-up conducted at 1 year after surgery showed that the parameter values remained statistically significant compared to the preoperative values.Heterogeneous patient population The study population in their research was heterogeneous, with different lumbar levels and pathologies. This can make it challenging to draw definitive conclusions about the effectiveness of the procedure for specific subgroups of patients.Small sample sizes Small sample sizes can limit the generalizability of the findings. Larger studies with more participants are needed to provide more robust evidence.Short follow-up duration Longer-term follow-up is necessary to assess the durability and long-term outcomes of the procedure.Lack of comparison groups There was no direct comparison between OLIF combined with pedicle screw internal fixation and other treatment approaches or control groups. Comparative studies are essential to determine the relative efficacy and safety of this procedure compared to alternative treatments.Selection bias There may have been selection bias in their patient cohorts, which can affect the generalizability of the results.While the study provides valuable insights, it is important to acknowledge certain limitations.Clinical outcomes The study found that the standalone OLIF group achieved better clinical outcomes in the early postoperative term (1 week and 3 months), but the differences disappeared by 2 years. This could be because standalone OLIF does not invade the muscle groups on both sides of the spine and does not cause intra-muscular hematoma, leading to better early recovery.Cage subsidence The rate of cage subsidence was 15.6% in the standalone OLIF group and 7.3% in the combined OLIF group. The study suggests that cage subsidence may occur after OLIF combined with pedicle screw fixation as well, but theoretically, pedicle screw fixation could add more protection. Patients who are osteoporotic or obese might be better undergoing combined OLIF with pedicle screw fixation.Fusion rate The study found no difference in fusion rate between standalone OLIF and OLIF combined with pedicle screw fixation within 2 years of follow-up. Despite being more unstable compared to OLIF combined with screw implantation, standalone OLIF with intact bilateral muscles could help stabilize the spine, resulting in a similar fusion rate.Cage subsidence and adjacent vertebral fractures The study suggests that cage subsidence and adjacent vertebral fractures could be mutually causal. Endplate damage during surgery can cause trabecular bone fracture in the vertebral body, leading to cage subsidence. The study emphasizes the importance of careful surgical technique to minimize endplate damage.In a retrospective study, He et al.  comparedIn another study, Liu et al.  providedThe authors conducted a retrospective cohort study comparing OLIF and MISTLIF in patients with L4/5 DLSS. They evaluated various perioperative factors such as operative time, blood loss, hospital stay, cost, and complications. Additionally, they assessed radiographic changes and clinical outcomes, including visual analog scale (VAS) scores, Oswestry Disability Index (ODI), and Hospital Anxiety and Depression Scale (HADS).The findings of this study are intriguing. The OLIF group demonstrated advantages in terms of shorter bedtime, reduced postoperative hospital stays, and lower intraoperative and postoperative blood loss compared to the MISTLIF group. However, the OLIF group required more intraoperative fluoroscopy, had a longer operative time, and incurred higher costs. Importantly, the complication rates were similar between the two groups.Radiographic measurements revealed that OLIF resulted in significant increases in posterior intervertebral space height (PISH), intervertebral space foramen height (IFH), and intervertebral foramen area (IFA) compared to MISTLIF. Both surgical approaches led to a significant increase in the area of the spinal canal (ASC), but the ASC increase was more pronounced in the MISTLIF group.Regarding clinical outcomes, the OLIF group exhibited significantly lower lumbar VAS scores at 1 month and 6 months post-operation. There were no significant differences in lower extremity VAS and ODI scores between the two groups. Notably, the OLIF group showed a significant decrease in HADS scores on postoperative day 3 and prior to hospital discharge, indicating a reduction in anxiety levels compared to the MISTLIF group.These findings suggest that OLIF may offer several advantages over MISTLIF in the treatment of mild-to-moderate symptomatic DLSS. OLIF appears to result in less surgical invasion, lower incidence of postoperative low back pain, faster postoperative recovery, and reduced anxiety levels. However, it is important to consider the higher cost and longer operative time associated with OLIF.In conclusion, the study suggests that OLIF combined with pedicle screw internal fixation is an effective treatment option for lumbar spinal stenosis. It not only restores disc height and increases the cross-sectional area of the vertebral canal but also provides valuable information for evaluating the efficacy and functional recovery of the lumbar spine in patients with lumbar spinal stenosis treated with OLIF surgery. These findings contribute to the growing body of evidence supporting the use of this technique in clinical practice.


Symbiotic Nitrogen Fixation (SNF) is a vital process in agriculture since it allows for atmospheric nitrogen assimilation through legume-rhizobia symbiosis. This process sustains plant growth on nitrogen-poor soils and legumes are utilized in crop rotation cultivations for limiting N-fertilizers use, resulting in enhanced plant growth. SNF is also based on a complex relationship between the host-bacteria symbiosis and environment. The symbiosis can be affected by a number of abiotic constrains as soil composition (including nutrient availability), salinity and especially drought. Moreover, in the recent decades, considerable progresses have been made to explore novel genes controlling the symbiotic signaling pathways and nodule functioning in legume plants . The aimGlycine max L.), faba bean (Vicia faba L), white clover (Trifolium repens) and Medicago truncatula. In all of them different aspects of the process of SNF were discussed and different biotic and abiotic factors influencing this process were investigated. In addition, all articles pointed on the practical application of the obtained knowledge for sustainable agriculture, environmental safety and economic competitiveness.The five research articles included in the volume presents new original data for four agronomically important legume plants: soybean  are the primary transport forms for fixed-N in soybean, and the concentration in plant tissue correlates with the fraction of N derived from Nfixation . Therefofixation . In ordede Borja Reis et al. the N-fixation process is highly susceptible to drought conditions. The regulation of SNF under drought involves diverse factors as internal oxygen availability, carbon limitation, and N-feedback regulation. Proteomic analysis suggests that plant carbon metabolism, protein synthesis, and cell growth are among the processes most altered in soybean nodules under drought stress  GmHSP17.1 was carried out. GmHSP17.1 overexpression promoted an increase of nodules number and size, and nitrogenase activity, whereas RNAi lines showed significantly impaired nodule development and N2-fixation. Furthermore, a direct interaction of GmHSP17.1 with the peroxidase GmRIP1 was demonstrated through liquid chromatography-tandem mass spectrometry, yeast-two hybrid, and bimolecular fluorescence complementation. GmRIP1 is preferentially expressed in soybean nodules where appears to be a direct target of GmHSP17.1. In fact, the ROS content greatly decreased in GmHSP17.1 overexpression lines and increased in suppression lines. These results could be very useful for genetic improvement of legume plants in the future.Additionally, small heat shock proteins (sHSPs) are largely involved in the response to different environmental stresses including drought. sHSPs are present in all organisms and involved in multiple plant developmental processes, but their role in nodule development in soybean is largely unknown . In thisWeith et al. In order to improve the quality of New Zealand pastures by limiting the application of N fertilizers, they have investigated quantitative parameters associated to genetic variations of white clover (Trifolium repens L.) families under symbiotic and non symbiotic conditions. The establishment of a vigorous growth of white clover in the first year is a critical quantitative parameter for high quality mixed pastures. A genetically structured breeding population comprising 120 half-sibling families was assessed for symbiotic traits with a commercial Rhizobium strain (TA1). Quantitative genetic analysis identified significant family additive genetic variance for dry shoot and roots, symbiotic potential, and root to shoot ratio. Although moderate correlations have been found between the phenotypic parameters analyzed and families genetic variations, the obtained results provide a promising platform to breed white clover with improved symbiotic traits and are informative for other legume crops.Another study presented in this Research Topic with the prospect of direct applications in sustainable agriculture is the one reported by Abdelkhalek et al. focused their research on nitrogen-fixing plant growth-promoting rhizobacteria (PGPR), Rhizobium leguminosarum bv. viciae strain 33504-Alex1, isolated from the root nodules of faba bean. The applications of 33504-Alex1 in faba bean plants through either soil or foliar treatments, promoted a considerable reduction in disease incidence and severity of Bean Yellow Mosai Virus (BYMV) infection, resulting in an improved growth and increased total chlorophyll content. The treatment with 33504-Alex1 determined a reduction of non-enzymatic oxidative stress markers associated to the increase of free radicals and reactive oxygen species scavenging activities as well transcriptional levels of pathogenesis-related genes. Furthermore, high-performance liquid chromatography and gas chromatography-mass spectrometry analyses indicated an accumulation of polyphenolic compounds as likely elicitor of an induced systemic acquired resistance in the treated faba bean plants. As a result, PGPR-Alex1 can be used as a simple, environmentally safe, bio-fertilize/bio-controller to protect faba bean plants from BYMV infection.In the recent years application of beneficial rhizosphere microbiota as an alternative strategy to chemical treatments to enhance plant performances and to increase resistance to biotic and abiotic stresses has become a promising option for agriculture . In thisIn summary the five articles included in this Research Topic provide new inside knowledge in specific aspects of the SNF. The important role of GmHSP17.9 in this process and the missing effect of AsA on the symbiotic performance are intriguing results. The positive effect of PGPR 33504-Alex1 for an efficient biocontrol and the models developed after analyses of the effect of environmental factors or quantitative genetic on SNF will be of future interest for sustainable agriculture.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


Droplet microfluidicsis a valuable method to “beat theodds” in high throughput screening campaigns such as directedevolution, where valuable hits are infrequent and large library sizesare required. Absorbance-based sorting expands the range of enzymefamilies that can be subjected to droplet screening by expanding possibleassays beyond fluorescence detection. However, absorbance-activateddroplet sorting (AADS) is currently ∼10-fold slower than typicalfluorescence-activated droplet sorting (FADS), meaning that, in comparison,a larger portion of sequence space is inaccessible due to throughputconstraints. Here we improve AADS to reach kHz sorting speeds in anorder of magnitude increase over previous designs, with close-to-idealsorting accuracy. This is achieved by a combination of (i) the useof refractive index matching oil that improves signal quality by removalof side scattering (increasing the sensitivity of absorbance measurements);(ii) a sorting algorithm capable of sorting at this increased frequencywith an Arduino Due; and (iii) a chip design that transmits productdetection better into sorting decisions without false positives, namelya single-layered inlet to space droplets further apart and injectionsof “bias oil” providing a fluidic barrier preventingdroplets from entering the incorrect sorting channel. The updatedultra-high-throughput absorbance-activated droplet sorter increasesthe effective sensitivity of absorbance measurements through bettersignal quality at a speed that matches the more established fluorescence-activatedsorting devices. Fluorescence-basedassays are not available for many enzymatic assays, and they requiresynthetic substrates that are typically large and hydrophobic to accommodatethe fluorophore. In a directed evolution campaign, these featurescan favor binding to the artificial fluorophore rather than the coresubstrate. Absorbance-activated cell sorting (AADS) provides practicalmeans to cover chromogenic assays,18 offering the opportunity to test a more comprehensivearray of substrates.Functional screening of millions of differentproteins in one experimentis possible at ultrahigh throughput in droplet microfluidics, wherewater-in-oil emulsions cocompartmentalize genotype and phenotype.A selection is achieved by interrogating these pico- to nanoliter-sized“test tubes” for an assay reaction that brings aboutan optically active primary or downstream product. Several successfullydirected evolution campaigns have been performed using droplet microfluidicsto improve enzyme variants and develop non-natural catalytic properties.12 The AADS dropletsorter developed by Gielen et al. has been applied to the directedevolution of phenylalanine dehydrogenase19 and glucose dehydrogenase.20 Additionalimprovements to this initial design have been made: Zurek et al. overcamesensitivity limitations in droplet absorbance measurements by thegrowth of clonal variants in droplets.17 Duncombe et al. introduced UVADS (UV–vis spectra-activateddroplet sorter), which allows the collection of the whole spectrafrom 200 to 1050 nm, including a right-angled turn at the detectioninterface to increase the droplet path length for higher sensitivity.15 However, the main limitation in all AADS designsremains the throughput at which droplets can be reliably sorted. Thehighest claimed sorting rate for absorbance detection is 300 Hz14 . This trade-off poses a limitation on the sizeof a library that can be screened in a practical time frame and constrainsthe scope of the absorbance detection technology when trying to obtainrare variants in a screening campaign.Practically AADS is attractive: the setupis more straightforwardand less expensive than FADS, as no lasers and photomultiplier tubesare needed. These factors also reduce safety requirements and meanthat the microfluidic rig can be run on a lab bench instead of ina dedicated laser room. On the other hand, detection for AADS is notas sensitive as fluorescence detection .21 surrounding the channels upstream and downstreamof the sorting junction; however, fragmentation remains an issue.A third problem is of a practical nature, namely that the scatteringcaused by droplet edges as they pass the optic fibers places limitson the minimum droplet size and minimum substrate concentration thatcan be quantitatively detected. This effect becomes more pronouncedfor lower concentrations of the absorbing medium, as scattering obscuresthe true value of absorbance for lower molarities . A key challengein directed evolution is the notion of “beating-the-odds”,such that the more variants screened, the higher are the chances ofimproving functional performance. Currently, FADS is at least ∼20-foldfaster than AADS (1–27 kHz vs 100 Hz14), implying a substantially reduced number of potentialvariants screened and leaving much sequence space unexplored.Compared to FADS, thesecond disadvantage of current AADS setupsis that absorbance is directly proportional to path length, meaningthat droplets with larger diameters and, therefore, larger volumesare needed. This, in turn, increases the amount of reagent requiredfor each droplet and limits the throughput of sorting since a higherelectric field is needed to sort larger droplets. If the electricfield is too large, droplets tend to merge and/or become fragmented.A remedy to prevent merging is to provide a “Faraday moat”l-phenylalanine (l-Phe) tophenylpyruvate.In the present work, we systematically explore various microfluidic,chemical and computational features of AADS and introduce improvementsthat collectively level its throughout with FADS, while still retaininghigh sensitivity, albeit at a higher volume than with a typical FADSsetup. Specifically, we address (i) improved microfluidic design,(ii) the use of added compounds for refractive index matching thatare mixed with the spacing oil, and (iii) development of new softwarefor signal detection and automation of droplet sorting. Finally, theutility of our improved UHT-AADS (ultra-high-throughput AADS) wasvalidated in an enrichment of phenylalanine dehydrogenase (PheDH)for the conversion of 22 The detailedfabrication protocol is provided in the SI.Microfluidic chips were fabricatedusing soft lithography in two layers. Briefly, microchannels wereobtained from polydimethysiloxane (PDMS) and were bonded to glassslides after surface plasma treatment. Device designs are depositedon our repository DropBase.nD = 1.29)23 fluorinated oil and 2% 008-FluoroSurfactant(RAN Biotechnologies). Gas-tight syringes (Hamilton Company) wereoperated with syringe pumps . Droplets were sortedinto the positive outlet channel by dielectrophoresis. Electric pulseswere applied using on-chip NaCl electrodes. Optical fibers were usedto pass light from a LED  via the detectionpoint to a photodetector . Data visualization andtriggering of sorting events were performed as previously describedby Gielen et al.14 A detailed protocolis provided in the SI. 1,3-Bis(trifluoromethyl)-5-bromobenzene wasmixed with HFE-7500 at different volume percentages, as describedin the SI.Droplets were made witha flow-focusing microfluidic junction using HFE-7500 . Aftersorting, the emulsion was broken and plasmid DNA was extracted andtransformed into E. coli. Single colonies were pickedand grown to saturation in 96-well plates. After protein expression,cells were lysed. Enzyme assays with 10 mM l-phenylalanineand 10 mM NAD (Sigma) were conducted in 96-well microplates. Absorptionat 340 nm was measured after incubation for 20 h. For calculatingenrichment factors, equations previously used by Baret et al.25 and Zinchenko et al.26 were employed.The enrichment experiment was carried out as previously describedby Gielen et al.21). The partial barrier (gapped divider)between the two outlets is designed so that the droplets do not breakapart on impact and instead “hug” the sides of the barrier.This is important for sorting of larger-sized droplets at higher speedssince a gentler impact ensures droplet stability. Large volumes inherentlylimit the sorting speed since as the volume increases, the electrophoreticforces required to move the droplet increase,14 so smaller droplet volumes were employed in our study. Since absorbanceis directly proportional to path length, in line with the Beer–Lambertlaw, decreasing droplet volumes reduces the diameter of the dropletsand therefore leads to a lower absorbance that is harder to detect.There is, therefore, a trade-off between the droplet size and themaximum sorting speed. Also, as the droplet size increases, due tothe shear forces acting on them, droplets are more likely to fragmentdue to the higher flow rate and the higher electric field needed todirect droplets to the positive channel.To achievehigher sorting frequencies, we first optimized the microfluidic sortingdevice through several iterations of design, fabrication, and testingof its performance. The design of a final device suitable for higherthroughput is shown in SI).The depth of the AADS device is determined by the width oftheoptical fibers. By using a shallower 50 μm deep droplet injectionchamber, smaller droplets can be evenly spaced, preventing two dropletsfrom being injected at the same time and sorted incorrectly. Thesesmaller droplets in a device with a depth of 100 μm are notsqueezed, and therefore, “derailing” them in the sortingjunction becomes easier so that higher sorting frequencies are achieved,which is also demonstrated in this study. The effect of a decreaseddroplet size and increased sorting speed is evaluated in the followingexperiments. Higher throughput of optimized microfluidic devices requiredimplementation of improvements in the software that was used for recordingthe signal and triggering sorting events (detailed in the 14). At the saturation limit of the photodetector,droplets with low molarity of absorbing dye can have an absorbancevalue greater than that of the saturation limit, lifting the truevalue above the saturation limit (rendering it nonquantitative). Tominimize this effect, an offset of tartrazine can be added to thedroplet contents, resulting in a deliberate absorbance increase toa level below the saturation limit. However, doing so reduced thedynamic range, as the upper limit of the detection range was reachedearlier in the presence of added tartrazine.We additionally focused on implementinga new strategy for the removal of unwanted scattering signals comingfrom light deflected by the droplet interfaces. The determinationof the true absorbance value becomes much more challenging due tothe presence of the spikes caused by the droplet edges. This makesit algorithmically harder to assign a true absorbance value correctlysee 2B,C. Thi27) as an example of a refractive index (RI) matching compound.This RI-matching oil has high miscibility with oil and does not interferewith droplet reinjection and spacing in the sorting junction. Severaladditional practical considerations determined how such a mixturebest removed the droplet edges in the signal, provided a wide dynamicrange, and allowed empty droplets to be confidently identified. TheRI-matching oil had an additional effect on the measured absorbancevalues. The percentage is the percentage volume of 1,3-bis(trifluoromethyl)-5-bromobenzeneadded to HFE-7500 oil. The choice of a suitable percentage of RI-matchingagent is determined first by trying to minimize the scattering effect,and second to have a suitable dynamic range between high and low concentrationsof absorbing compound . In an alternative approach tomitigating the issue of scatteringat droplet edges, 1,3-bis(trifluoromethyl)-5-bromobenzene was addedto the fluorocarbon HFE-7500 oil -5-bromobenzenesince this represents the best compromise between removing dropletedges, providing a high dynamic range, and allowing empty dropletsto be identified. Removing edges significantly minimizes the sortingalgorithm’s complexity  and therefore increases computationspeed.The addition of a RI-matching compound to the spacingoil alsorequired a change in the design of the microfluidic sorting junction.Since the RI-matching oil reduces emulsion stability and might causethe wetting of droplets to walls of channels or tubing, we decidedto apply a strategy of transferring positively sorted droplets backto the oil without RI. Therefore, a bias oil channel  was added at the sorting junction and duringsorting, both oils are in a laminar flow regime. This standard oil,therefore, poses a barrier to droplets from entering the positiveoutlet. Only when the electric pulse applied to the droplets fromthe sorting electrode exceeds the inertial force driving the dropletinto the negative outlet does the droplet enter the positive outlet(see 14)As previously stated, droplets with a smaller volumeare easierto sort at high frequencies due to less force needed to move themeffectively. Our results proved that modified microfluidic designand adding RI-matching oil allow for detection and sorting of dropletswith a volume of 75 pL. This is a significant improvement comparedto larger volumes of droplets in previous studies  at different frequencies. Flow rates were adjusted to allow different dropletfrequencies, and the ratio of the flow rates was kept the same betweenfrequencies .Droplets were counted as being correctly sorted only if the droplettriggering the sorting event was sorted alone, without any fragmentsfrom other droplets before or following behind. Droplets are in theRI-matching oil only for a very brief period between respacing inthe channel just upstream of the sorting junction and exiting to thepositive sorting channel (where they mix with pure oil), and so thecontact time of droplets with the RI-matching oil is miniscule. Furthermore,droplets are demulsified post-sorting.28Fragmentation of dropletsoccurs as the frequency increases due to the insufficient force requiredto move the droplet into the positive outlet, and the droplet collideswith and breaks at the barrier. Additionally, as the electric fieldincreases, this also leads to droplet fragmentation. This is alsomore pronounced because of the different oil composition at the sortingjunction. Finally, since the bias oil (coming in from theside channel) andflow rate of the droplets from the main channel are entering the junctionat different rates, this poses an additional barrier to pulling thedroplet into the positive channel. As the droplet is pulled over tothe positive elution channel, part of it is still being pulled intothe negative outlet at a higher flow rate. This stress on the dropletcauses the droplet to break apart at the barrier.When probinghigher droplet frequencies, the forces needed to pullthe droplet into the positive outlet chamber must become larger. Accordingly,sorting efficiency decreases as the droplet frequency increases 4A, and t14 is seen. A video showing sorting at 1 kHz is provided in the Supporting Information.In the light of these issues, the success of sorting was evaluatedby analyzing video traces (with ∼100 droplets after sorting)with an algorithm detecting the absorbance in droplets passing thedetector. Sorting was carried out at 1 kHz , and a 10-fold improvement of the apparatus usedby Gielen et al.26 To evaluate if UHT-AADS can beused to screen for phenylalanine dehydrogenase (PheDH) activity, weused a previously established droplet-based colorimetric cell lysateassay workflow14 and combined it with ournovel device. We generated a mock library consisting of PheDH anda glycosidase as negative control mixed in a 1:100 ratio (29). DNAwas recovered by transformation into E. coli anda small sample was analyzed in a secondary photometric assay to evaluatethe efficiency of sorting. A total of 34 of 38 clones tested showedPheDH activity  is equivalent to FADS campaigns by employingthe improvements discussed in this paper (see S14 for a table comparing the improvements to the previousdesigns). Based on this 10-fold improvement in throughput  larger libraries can be screened in the sameamount of time, increasing the chances of success by making it morelikely to identify rare functional variants as larger fractions ofsequence space are interrogated.Absorbance-activateddroplet sorting is still less frequently usedfor protein engineering9 molecules per droplet).13 In fact,thesensitivity of the setup described in this work is slightly lowerthan that introduced by Gielen et al.14 due to the path length reduction . However, the local enzyme and productconcentrations are larger in smaller droplets (2.4-fold in 75 vs 180pL droplets), partially compensating for the reduced sensitivity.In-droplet growth of enzyme-expressing E. coli cells(after single cell encapsulation) has been shown to increase the amountof enzyme in an assay, which provides means to boost the product signal.17 Increasing the sensitivity of the AADS device may be possible bychoosing a readout molecule with a high extinction coefficient  For example, Probstet al.16 used nanoparticles, a combinationof confocal optical systems and a postprocessing algorithm to achievea sensitivity of 800 nM. As a suggestion for future improvements,broadband-enhanced cavity absorption with mirrors could be added,32 although manufacturing is complex due to needing to precisely placemicromirrors on either side of the channel between the optical fibers.Other drawbacks of AADS remain:for FADS, the minimum product concentrationis >2 nM (corresponding to >2500 product molecules per droplet),whereasfor AADS the sensitivity is lower at >7.5 μM  is added tothe aqueous phase. In this case, the signal from all the dropletsis artificially increased above the baseline and therefore scatteringeffects are also minimized. The robustness of absorbance sorting isenhanced by these practical measures so that its use will make iteasier to obtain quantitative data or good quality, and an increasedsorting frequency ensures accurate sorting decisions based on highquality peak detection at lower volumes and high frequency. Postprocessinganalysis of droplet data can additionally be easily reviewed throughthe Python scripts provided, and has been written with packages thatare consistently improved by the community for easy updating of thecode. Also, since the sorting uses Arduino-like microcontrollers,improvement in hardware will result in increased computation speedwhile retaining the accessibility of being inexpensive and open-source.Complementary work by Richter et al.33 hasshown an increased sorting throughput and removal of droplet traceartifacts by using a combination of surface acoustic waves and microlensesin the form of an optical air cavity. We envision that a further upgradeto AADS designs could incorporate design improvements from both studies.On the other hand, AADS is cheaper to build (requiring no lensesand expensive laser equipment) than FADS, can be used on a benchtop(without laser protection) and provides a very accessible setup forultrahigh throughput screening.22 and in the SI) and new software described in the SI and deposited on GitHub,24 whichis immediately available as open-source material, we hope to facilitateuptake of ultra-high-throughput screening in droplets across the communityand make it the method of choice for protein engineering by directedevolution.By making device designs (deposited on our repository DropBaseas CAD files


Antibiotic resistance is a global threat caused by factors such as overuse of antibiotics, lack of awareness, development of biofilms etc. World Health Organization released a list of global priority pathogens which consisted of 12 species of bacteria categorized as expressing critical, high and medium resistance. Several Gram-negative and Gram-positive species are known to cause wide varieties of infections and have become multidrug or extremely drug resistant. Pathogens causing infections associated with invasive medical devices are biofilm producers and hence their treatment becomes difficult due to a structurally stable matrix which prevents antibiotics from penetrating the biofilm and thereby showing its effects. Factors contributing to tolerance are inhibition of penetration, restricted growth and activation of biofilm genes. Combination drug therapies has also shown potential to eradicate biofilm infections. A combination of inhaled Fosfomycin/tobramycin antibiotic strategy has been effective against Gram-negative as well as Gram positive organisms. Along with antibiotics, use of natural or synthetic adjuvants shows promising effects to treat biofilm infections. Fluroquinolone activity on biofilms is disrupted by low oxygen tension in the matrix, a strategy known as hyperbaric oxygen treatment that can enhance efficacy of antibiotics if well optimized. Adjuvants such as Ethylenediaminetetraacetic acid (EDTA), Sodium Dodecyl Sulphate (SDS) and chlorhexidine act by killing non-growing microbial cells aggregated on the inner layer of the biofilm. This review aims to list down current combination therapies used against Gram-negative and Gram-positive biofilm forming pathogens and brief about comparison of combination drugs and their efficacies. Currently, resistance has developed towards Beta-Lactams and other antimicrobials that are commonly used to cure bacterial diseases. According to a paper published in the Lancet, antimicrobial resistance poses a serious threat to global public health, killing at least 1.27 million people worldwide and being linked to over 5 million deaths in 2019 . The inatreC and sugE are the genes responsible for capsular polysaccharide production in K.pneumonieae , Type 1, curli formation (csg) and LPS formation  along with other genes (Factors that govern biofilm formation are i) systems of communication between cells, such as quorum sensing (QS), in which the production of signaling molecules by the cells controls the formation of the biofilm, ii) bacterial secondary messengers that regulate flagellar attachment that leads to attachment on the surfaces and iii) EPS Matrix components. Both manmade and natural environments are capable of supporting biofilms. Expression of certain genes triggers formation of EPS matrix; umonieae . It is wsistance . Similarruginosa . In E. cer genes . Treatme22.1Biofilms are composed bacterial cell aggregates and extra polymeric substances. These structures when formed on medical devices used for treatment purposes are hard to remove with physical and chemical shear forces. In general, there are 4 major stages of biofilm formation shown in 2.2Acinetobacter species, specifically Carbapenem Resistant Acinetobacter baumannii (CRAB). The most common types of biofilm-associated infections caused by A. baumannii are catheter-related blood stream infections and ventilator-associated pneumonia The first pathogen on the critical priority list of diseases for innovative antibiotics to turn into a “red-alert” human pathogen belongs to the K.pneumonieae due to the rapid emergence of multidrug resistant (MDR) & extensively drug resistant (XDR) strains and the associated high mortality rate due to the diminished efficacy of available therapeutic choices  and chronic kidney disease (CKD) to name a few. Virulence factors are utilized by K. pneumonieae both during biofilm formation and for survival while evading the immune system. It has the ability to form a thick layer of extracellular biofilm that maintains the bacterial attachment to living or inactive surfaces, preventing antibiotic penetration and reducing its effects -one (ACNQ) with ciprofloxacin shows an efficacy rate of 80%. This review also covers more combinational antibiofilm therapies acting against Gram-positive and Gram-negative organisms (Escherichia coli O157 (93%), using Nitroxide Carboxy-TEMPO (20uM) in combination with ciprofloxacin concentration of 320ng/ml for pseudomonal strain and 20ng/ml for E. coli strain  for both Gram-positive and Gram-negative isolates were 127 mg/L and 117 mg/L, respectively. Gram-positive isolates showed the greatest synergy with ciprofloxacin, while Gram-negative isolates showed the greatest synergy with amikacin, gentamicin, and cefepime. Efficiency rate of curcumin was about 57%, curcumin being a natural component possess advantages and can be used in large amounts with minimal side effects  directly affecting the stability of biofilm structure. Synergy is obtained when Fmoc-F is combined with vancomycin and ampicillin antibiotics  and photothermal therapy (PTT). Light induced eradication has a 99-100% efficiency rate. Some of the light-induced therapies are PDT and Ampicillin used against MTCC 509 . An orig effects . This woisolates . The nexications . Potentiibiotics . A studyibiotics . The thec effect . The stucrobials .3The stable structure of biofilms causes obstacles in therapy specifically disabling drug delivery systems and reducing the efficacy of antibiotic agents. Individual antibiotic therapies are known to become less effective on biofilm forming pathogens, hence studies on combination therapies have gained importance and proven effective in enhancing the activity of existing antibiotic or destroying the biofilms. This review intended to give a brief account on currently established combinatorial therapies using natural as well as synthetic entity along with existing antimicrobials to enhance eradication of biofilms present in host cells and medical devices. It is important to identify the genetic markers that induce biofilm production in the bacterial cell and analyze individual component of EPS matrix. Analysis should also be focused on quorum sensing mechanisms of pathogens as a potential drug target to destroy biofilms completely. Combining a synthetic entity with antibiotics has disadvantages of disrupting normal cellular functioning whereas, combination of natural agent with an antibiotic could be a promising solution as natural components have lesser side effects and can be used in large concentration with lower concentrations of synthetic antimicrobial agents.AB and TB contributed to conception and design of the study, acquisition, analysis, interpretation of data, drafting and ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. AP and SR contributed to manuscript revision, read, and approved the submitted version. All authors contributed to the article and approved the submitted version.