YRNA1, YRNA3, YRNA4 and YRNA5. All YRNAs are relatively small, made up of about 100 nucleotides each. YRNAs are characterized by a stem-loop structure and each part of that structure carries a different function. YRNAs are transcribed in the nucleus by RNA polymerase III. Then, the YRNA molecule is bound to the polyuridine tail of the La protein responsible for both its nuclear retention and protection from degradation. They also bind to the Ro60 protein, making the molecule more stable. In turn, YRNA-derived small RNAs (YsRNAs) are a class of YRNAs produced in apoptotic cells as a result of YRNA degradation. This process is performed by caspase-3-dependent pathways that form two groups of YsRNAs, with lengths of either approximately 24 or 31 nucleotides. From all four YRNA transcripts, 75 well-described pseudogenes are generated as a result of the mutation. However, available data indicates the formation of up to 1000 pseudogenes. YRNAs and YRNA-derived small RNAs may play a role in carcinogenesis due to their altered expression in cancers and influence on cell proliferation and inflammation. Nevertheless, our knowledge is still limited, and more research is required. The main aim of this review is to describe the current state of knowledge about YRNAs, their function and contribution to carcinogenesis, as well as their potential role in cancer diagnostics. To confirm the promising potential of YRNAs and YRNA-derived fragments as biomarkers, their significant role in several tumor types was taken into consideration.YRNAs are a type of short, noncoding RNAs. A total of four different transcripts can be distinguished, which are  Most of the human genome (approximately 98%) is built of non-coding RNAs ,2,3. DesIn this review, current knowledge about YRNAs and YRNA-derived small RNAs, as well as their function in the carcinogenesis process and potential implication in cancer diagnosis is described.Bombyx mori, Anopheles gambiae) and prokaryotes  ..19].YRNA-derived fragments seem to lack gene silencing activity. Unfortunately, the role of YsRNAs is not fully defined yet, but they were found to be dysregulated in many diseases, including cancer . PreviouPseudogenes are copies of genes that have lost their function due to an accumulation of different mutations. These mutations may trigger promoters’ inactivation, which are significant frameshifts that cause premature stop codons  to appear. Additionally, the translocation of genes to inactive regions of the genome may also take place .YRNA1 pseudogenes , 16 YRNA3 pseudogenes (with a similarity range of 30–96%), 33 YRNA4 pseudogenes (with a similarity range of 73–97%) and 10 YRNA5 pseudogenes (with a similarity range of 63–89%). They are all located on different chromosomes, including X chromosome [Next generation sequencing and bioinformatics methods identified 15,000–18,000 human pseudogenes, and up to 10% of them are transcribed ,34. Amonromosome ,36. Howeromosome .Due to the fact that pseudogenes may play an important role in diseases including cancer, it is very important to take a closer look at this issue and dispel any doubts or discrepancies. Further studies should focus on whether YRNA pseudogenes possess functions like those of parental genes, or whether they acquire new biological roles or are transcribed without function.Although the exact role of YRNAs has not yet been fully discovered, short non-coding RNAs definitely play an important role in human tumorigenesis. Up till now, the dysregulated expression of YRNAs has been described in several tumor types including bladder cancer, clear cell renal cell carcinoma, prostate cancer, head and neck cancer, triple-negative breast cancer and lung cancer . It is iYRNA1, YRNA3 and YRNA4 was highly correlated, while the level of correlation of YRNA5 to the other YRNA types was significantly less prominent. What is more, Tolkach et al. correlated the expression of YRNAs with clinicopathological features and proved that the expression of three YRNAs  was strictly associated with advanced stage  and grade. Furthermore, YRNA1 and YRNA3 expression levels were a meaningful predictive indicator for cancer-specific and overall survival among BCA patients, with a clear trend for YRNA4. Therefore, in patients with urinary bladder urothelial carcinoma the expression of YRNAs is downregulated and expression changes are related to stage of disease, higher grade and metastasis. This may have prognostic value for overall and cancer-specific survival rates [The expression profiles of YRNAs in BCA (bladder cancer) and normal urothelial tissue were studied. This comparison revealed that expression levels of all four YRNAs were significantly downregulated and the mean expression levels were 2- to 4-fold lower in BCA than in normal tissue. Interestingly, the expression of YRNA3 and YRNA4 in ccRCC compared to normal renal tissue [YRNA4 expressions in patients with lymph node metastasis and in patients with advanced stage of ccRCC was observed. The functional relevance of YRNA4 in pathogenesis was confirmed due to a prominent tendency for poor overall survival following partial or radical nephrectomy in patients with low YRNA4 tissue levels [YRNAs also play a significant role in clear cell renal cell carcinoma (ccRCC) and may have prognostic value in this tumor type. Nientdiet et al. indicated increased expression levels of l tissue . Moreovee levels .YRNA4 and YRNA5 expressions were lower compared to BPH individuals. There was also a correlation between all YRNA expression levels. YRNA1, YRNA3 and YRNA4 expression was highly correlated, while YRNA5 level was had a weaker correlation with YRNA1, YRNA3 and YRNA4. YRNA levels were also shown to distinguish normal/BPH and PCA tissue. Concerning adverse clinicopathological parameters related to PCA patients, the association of the YRNAs expression with ISUP  grade group of the tumor was found. Significant differences in the expression levels between ISUP grade groups 1 and 2 were apparent for YRNA1, YRNA3 and YRNA4. What is more, the expression of YRNA5 was interrelated with biochemical recurrence-free survival of prostate adenocarcinoma patients. The study indicates a few possible diagnostic applications of YRNA expression, as well as different biological roles of YRNAs in various tissue and cancer types. In order to confirm the independent prognostic significance of YRNA, further investigations in a larger cohort are still required [In the study of YRNAs, expression levels in prostate cancer (PCA) patients and the possible diagnostic role of YRNAs was investigated. The expression profile of YRNAs was studied in both PCA and normal tissue, as well as in benign prostate hyperplasia (BPH) tissue. The expression levels of all four YRNAs in PCA tissue were 2- to 4-fold lower in comparison to normal prostate tissue. Additionally, in PCA patients, YRNA1 expression is downregulated in different HNSCC cell lines, as well as in patients’ tumor samples. However, expression of YRNA1 was found to be significantly upregulated in stage T4 tumors. It was also observed that higher expressions of YRNA1 are associated with longer DFS and OS in HNSCC patients. YRNA1 expression is associated with changes in the expression of many genes involved in carcinogenesis and its high sensitivity and specificity makes it a possible HNSCC biomarker [The role of YRNA-derived small RNAs was investigated in HNSCC. Martinez et al. revealed the presence of circulating YRNA-derived small RNAs that were remarkably dysregulated in the sera among patients with HNSCC compared to normal tissue samples . The attiomarker .In a study by Guo et al., several YRNA molecules were identified in the luminal androgen receptor (LAR) subtype of triple-negative breast cancer (TNBC) that may be hormone dependent. Similar to PSA for prostate cancer patients, YRNA fragments detected in the serum of breast cancer patients may provide an opportunity for noninvasive monitoring of the LAR subtype of TNBC in the course of treatment .YRNA-derived small RNAs were also found in non-small cell lung cancer patients (NSCLC). The high-throughput sequencing of small RNAs in plasma extracellular vesicles (EVs) from lung adenocarcinoma (ADC) and squamous cell carcinoma (SQCC) patients and healthy controls was performed, and the analysis revealed that YRNA4-derived fragments were significantly upregulated in plasma EVs among ADC and SQCC patients. Interestingly, YRNA4-derived fragments were downregulated in NSCLC cells and they significantly suppressed the proliferation of NSCLC cell A549. Thus, it all suggests that YRNA4-derived fragments play an important role in NSCLC development and may serve as promising biomarkers for NSCLC diagnosis .Kolenda et al. indicate that the expression of YRNAs could serve a diagnostic utility in the case of BRAF-mutant metastatic melanoma patients. However, there are no significantly different plasma YRNA levels in patients with BRAF-mutated unresectable stage III and IV cutaneous melanoma compared to healthy individuals. In spite of that, the estimation of YRNAs appears to be a prognostic marker for BRAF-mutated metastatic melanoma patients and helps to assess progression free survival and overall survival .In the case of glioma, there is only one study that was dedicated to YRNA expression. The researchers used material derived from cells (patients-derived tumorigenic glioblastoma multiforme cells (GBM)), free RNP and extracellular vesicles including micro-vesicles and exosomes. The control material was obtained from a transcriptome analysis of human and mouse astrocytes, endothelial cells, neurons and microglia. Interestingly, all four YRNAs and YRNA-derived fragments were found to be highly abundant in every examined material. What is more, it was found that in extracellular fractions, there was a significantly higher number of YRNA-derived fragments than YRNAs, especially in RNP, compared to cellular fractions of YRNAs and YsRNAs .YRNA1, YRNA3, YRNA4 and YRNA5 were discovered in cervix cancer cell cultures [YRNA1 showed a significant overexpression [YRNA5 fragments was significantly higher with poly(I:C) treatment [Furthermore, increased expression of cultures . Howeverpression . It was reatment .YRNA1 and YRNA3 [YRNA5 was found to be overexpressed in HCT116 cells during poly(I:C) treatment [YRNA4 was found to be highly expressed in multiple variants [There are also few studies considering YRNAs expression in colon cancer. In colon cancer cell cultures, all four YRNAs were significantly overexpressed, especially nd YRNA3 . In anotreatment . What isvariants .YRNA1 and YRNA3 were found to be significantly overexpressed [Finally, in FFPE samples obtained from pancreatic ductal adenocarcinoma (PDAC) patients, xpressed . What isxpressed .YRNA1 expression is changed in seven out of 17 tested cancer types. In two of them the expression of YRNA1 is upregulated compared to normal samples and in the other five YRNA1 expression is downregulated. YRNA4 was found to be dysregulated in four cancer types out of the 17 examined and in all cases the expression of YRNA4 was downregulated in comparison to normal samples. YRNA3 was not detected to have changed in any of the 17 examined cancer types, while for YRNA5 there are no studies available. What is more, neither YRNA1 nor YRNA4 showed differences in overall survival in any cancer disease tested [An analysis performed using StarBase v2.0  showed that YRNAs are dysregulated in many cancer diseases. e tested . This daSince the discovery of YRNAs in the early 1980s , they haAs demonstrated in this review, YRNAs and YRNA-derived fragments undeniably show a possible influence on carcinogenesis and have great potential in becoming biomarkers for different types of cancer. Thus, the presence of YRNAs and YRNA fragments as well as changes in their expression can be used in the diagnosis of cancer, especially because they are easy to obtain from blood, serum and plasma. However, it is very important to learn more about their exact role in both healthy cells and cancer cells, which means much more research is needed in this field. The questions that remain unanswered are: (i) how the expression of YRNAs and YRNA-derived fragments is regulated, (ii) how they regulate different types of RNA transcripts and (iii) how interactions among them are dysregulated in cancer. In the future, in vitro and in vivo studies should focus on their biological role and their implication in various important processes, such as proliferation, apoptosis and epithelial-to-mesenchymal transition (EMT) process, as well as the maintenance of cancer initiating cells.


Primary cilia protrude from most vertebrate cell bodies and act as specialized ‘signalling antennae’ that can substantially lengthen or retract in minutes to hours in response to specific stimuli. Here, we review the conditions and mechanisms responsible for regulating primary cilia length (PCL) in mammalian nonsensory neurons, and propose four models of how they could affect ciliary signalling and alter cell state and suggest experiments to distinguish between them. These models include (i) the passive indicator model, where changes in PCL have no consequence; (ii) the rheostat model, in which a longer cilium enhances signalling; (iii) the local concentration model, where ciliary shortening increases the local protein concentration to facilitate signalling; and (iv) the altered composition model where changes in PCL skew signalling. Higher levels of this widespread modification reflect higher internal cellular nutrient levels [Alterations in neuronal PCL are also seen in response to diverse physiological changes. Some of the most compelling evidence comes from studies of the hypothalamus, a brain structure containing neuron populations that regulate many fundamental homeostatic and behavioural processes. For example, a recent preprint suggested that neurons in the suprachiasmatic nucleus(SCN) of mice, which help regulate circadian rhythms, underwent cyclic changes in PCL, ranging in length from ~1 μm at the onset of the dark cycle to ~6 μm at the onset of the light cycle. The same group also suggested that the rhythmicity of these changes in PCL was disrupted by mutations in the core circadian clock genes, and perturbations in the cilia, in turn, disrupted the robustness of suprachiasmatic nucleus-generated circadian rhythms . Althougoduction   [2+levels   but is a 3 (AC3)  Figure2A2+levels  activateRho-associated kinase (ROCK) to promote the expansion of actin stress fibres [Actin stress fibres are likely to regulate PCL, since their destabilisation by pharmacological,45 or ges fibres , which, s fibres  to promos fibres ,45. Simis fibres , whereass fibres , 48 are thought to be released from the base of cilia where endosomes can also be absorbed, whereas emal tip ,68–71 in vivo did not interfere with normal development or behaviour in mice [Although the primary cilium plays an important role in signalling , changes in mice ,75.(ii)Cnsk2α [Alonger ‘sensory antenna’ with a correspondingly larger surface area could increase the number of receptors and downstream signalling mediators without altering their concentration , so thatCnsk2α , or pharCnsk2α . In the Cnsk2α since lepCnsk2α , leadingCnsk2α .(iii)There are a number of ways in which the local concentration of ciliary proteins could be altered. For example, if the rate of protein trafficking into and out of the cilium is slow relative to the rate of change in PCL, then the total number of ciliary membrane proteins would remain relatively constant, increasing their concentration and potentially increasing the number of intermolecular interactions as cilia shrink. This increased local concentration of ciliary proteins could potentiate or alter their signalling in at least three ways: (i) increasing the likelihood that receptors such as GPCRs will interact with downstream targets, (ii) increasing the concentration of second messengers due to the decreased ciliary volume, and (iii) increasing the formation of receptor homomers, heteromers, and higher-order oligomers which could, in turn, shape their responses to ligands . Indeed,(iv)Mechanisms that might alter PCL such as ectosome shedding could alter the composition of ciliary proteins and lipids to influence the subsequent signalling, as observed when GPR161 was removed upon SHH binding . The cilWe have summarized the mechanisms potentially involved in the control of PCL in mammalian neurons and proposed models of how these changes could affect ciliary signalling and, consequently, alter the cell’s state. Here, we propose a set of experiments to determine which model (or combination of models) might explain how PCL could affect signalling pathways, keeping in mind that results from one experimental system may not apply universally (see Outstanding questions). For example, one could use cilia-targeted cAMP or calcium sensors  to test


The intestinal tract encompasses the largest mucosal surface fortified with a fine layer of intestinal epithelial cells along with highly sophisticated network of the lamina propria immune cells that are indispensable to sustain gut homeostasis. However, it can be challenging to uphold homeostasis when these cells in the intestine are perpetually exposed to insults of both endogenous and exogenous origin. The complex networking and dynamic microenvironment in the intestine demand highly functional cells ultimately burdening the endoplasmic reticulum (ER) leading to ER stress. Unresolved ER stress is one of the primary contributors to the pathogenesis of inflammatory bowel diseases (IBD). Studies also suggest that ER stress can be the primary cause of inflammation and/or the consequence of inflammation. Therefore, understanding the patterns of expression of ER stress regulators and deciphering the intricate interplay between ER stress and inflammatory pathways in intestinal epithelial cells in association with lamina propria immune cells contribute toward the development of novel therapies to tackle IBD. This review provides imperative insights into the molecular markers involved in the pathogenesis of IBD by potentiating ER stress and inflammation and briefly describes the potential pharmacological intervention strategies to mitigate ER stress and IBD. In addition, genetic mutations in the biomarkers contributing to abnormalities in the ER stress signaling pathways further emphasizes the relevance of biomarkers in potential treatment for IBD. The intestine houses a plethora of innocuous microbes that establish a symbiotic relationship in the host. Additionally, constant exposure to the external factors makes it susceptible to invasion by exogenous pathogens ensuing persistent immune response in the gut. Therefore, the lamina propria immune cells must be functionally fine-tuned to differentiate and exhibit tolerance toward commensals and immunity to pathogens. At this juncture, the intestinal epithelial cells (IECs) lining the gut play two major roles: segregation and mediation, conserve gut homeostasis ; avoid uThe ER is the primary site for facilitating the appropriate folding of proteins and dispatches them to their respective functional destinations in the IEC with secretory function . HoweverUPR pathways function with unique mechanisms of signal transduction operating in parallel utilizing IRE1α, PERK, and ATF6 . In theiIRE1α is the most evolutionarily conserved transmembrane kinase with endoribonuclease activity  domain, unlike IRE1α and PERK. Once the BiP dislodges from ATF6, it is trafficked from the ER to Golgi. S1P and S2P cleaves ATF6 releasing the N-terminal cytosolic domain of ATF6 (N) that translocate to the nucleus and promotes the transcription of chaperones, ERAD complex and XBP1 to mitigate ER stress  . RCI-H358) . OverallCI-H358) . The phoCI-H358) . HoweverContinual exposure to a myriad of gut microflora, exogenous antigens, dietary metabolites, and toxins impede the functional ability of IECs. Although nature has bestowed with evolutionarily conserved and sophisticated mechanisms to overcome these impediments, disruption in any of these mechanisms can cause chronic inflammation in the gut. Accordingly, there are two ways to look at the cause for the collapse of these mechanisms: i) IECs are pushed to synthesize copious amounts of proteins, cytokines, and peptides; activating UPR. In this scenario, cells that are competent enough will survive and the rest will succumb to stress. ii) Genetic deficiency of the genes that are involved in UPR, autophagy, secretion, immune response and inflammation can have various impacts and confer a genetic predisposition to IBD owing to decreased protein folding capacity and heightened immune response. Mechanistic studies conducted on murine models deficient in these genes facilitated the understanding of phenotypic outcomes in correlation to IBD Table 1.Xbp1 is linked to IBD. Cell-specific loss of Xbp1 in intestinal epithelial cells (ΔIECXbp1) displayed amplified ER stress  to DSS-induced colitis mitigated ER stress-mediated inflammation not only by precluding immune cell infiltration, but also improved epithelial barrier integrity by regulating tight junction (TJ) proteins, mucus layer protecting proteins, and antimicrobial peptides .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ischemia-reperfusion injury (IRI), which is triggered by a transient reduction or cessation of blood flow followed by reperfusion, is a significant cause of acute kidney injury (AKI). IRI can lead to acute cell death, tissue injury, and even permanent organ dysfunction. In the clinic, IRI contributes to a higher morbidity and mortality and is associated with an unfavorable prognosis in AKI patients. Unfortunately, effective clinical drugs to protect patients against the imminent risk of renal IRI or treat already existing AKI are still lacking. Fibroblast growth factors (FGFs) are important regulators of key biological and pathological processes, such as embryonic development, metabolic homeostasis and tumorigenesis through the regulation of cell differentiation, migration, proliferation and survival. Accumulating evidence suggests that altered expression of endogenous FGFs is associated with IRI and could be instrumental in mediating the repair process. Therefore, FGFs have been proposed as potential biomarkers in the clinic. More importantly, exogenous FGF ligands have been reported to protect against renal IRI and display promising features for therapy. In this review, we summarize the evidence and mechanisms of AKI following IRI with a focus on the therapeutic capacity of several members of the FGF family to treat AKI after IRI. Acute kidney injury (AKI) is a syndrome and significant cause of death among hospitalized patients. AKI is characterized by a rapid decline in renal function and predisposes the transition of AKI patients to chronic kidney disease (CKD) and end-stage renal disease (ESRD) . IschemiThe disease mechanisms underlying the etiology and pathogenesis of AKI are complex and include mitochondrial dysfunction, reactive oxygen species (ROS), endoplasmic reticulum stress (ERS), autophagy, inflammation, apoptosis, and necrosis . FurtherIn the context of IRI, several growth factors have been reported to exhibit protective effects as well as therapeutic potential as they not only prevent the damages from occurring, but also improve functional recovery after the damages are done Table 1.Here, we will summarize the available data on the roles and known mechanisms of FGFs in the pathogenesis, prevention, and repair of AKI with a focus on IRI. The discussion will first provide a succinct overview of FGF/FGFR signalling specificity and function, followed by a detailed summary of the published work on the roles of FGFs/FGFRs in the AKI, impact of endogeneous and recombinant FGFs as prevention and therapeutic measures for the IRI, pathophysiological processes and conclude with a highlight for future research to better understand underlying mechanism and FGFs in AKI disease and provide viable strategies to prevent IRI or avert progression to CKD.In mammalians, the FGF system consists of 18 ligands signalling through their specific FGFRs Table 2.via the classical secretory pathway. FGFs have been described to act both in an autocrine and paracrine fashion. They signal through their specific trans-membrane receptors consisting of five members, FGFR1-5. FGFRs bind their ligands with high affinity and various degrees of specificity  and FGF2 (or basic FGF) are the prototypic members of the FGF family that have a similar broad range of biological activities. Both are FGF1 is an autocrine/paracrine regulator known to act on cells from various organs and tissues, including liver, vasculature, and skin. FGF1 exerts significant beneficial effects in different organs after IRI . The skevia the activation of the PI3K/AKT and ERK1/2 pathways. The expression of FGF2 is not observed in the adult kidney during homeostasis, but a strong induction is observed in the regeneration phase of AKI. FGF2 reduces the expression of renal damage markers such as ED-1 and α-smooth muscle actin and participates in the regeneration process after ischemic acute renal failure , which is an alternatively spliced RNA isoform containing a unique domain  in the third IgG-like loop . FGF7 anBoth FGF7 and FGF10 bind FGFR2b, but exert largely distinct physiological functions. FGF7 is implicated in both the induction of basal urothelial cell proliferation and the expansion of transitional epithelium. It is an efficient growth and differentiation factor during development and wound healing. FGF7 also has a significant effect on the kidney. Compared to wild type littermates, Fgf7 knockout mice display smaller kidneys with fewer ureteric buds and nephrons. Earlier reports showed that the expression of both FGF-7 and FGFR2b is induced and segregated between interstitial and epithelial cells in response to chemically-induced proximal tubular damage. The activation of this mesenchymal to epithelial paracrine signaling is implicated in the regulation of tubular repair process . Additiovia interaction with its high-affinity receptor FGFR2b. Deletion of Fgf10 in mice led to kidney dysgenesis characterized by fewer collecting ducts and nephrons. Overexpression of a soluble dominant-negative FGFR2b isoform in transgenic mice revealed more striking defects, including renal aplasia or severe dysplasia  and an atypical, kinase inactive FGFR5 or FGFRlL . Some ofFGFRs elicit different functions in mammalian development and diseases. Conditional knockout of Fgfr1 in ureteric bud and metanephric mesenchyme did not lead to any kidney development defects . AblatioAlthough IRI mainly affects renal tubular cells, IR-induced pathogenic process and its repair involve interactions between interstitial cells, infiltrated inflammatory cells and epithelial cells. Interestingly, a recent study using a mouse model with fibroblast-specific ablation of Fgfr2 gene indicated that FGFR2 expression in fibroblast may contribute to kidney fibrosis after IRI through promoting renal fibroblast activation and proliferation .via impacting several key pathophysiological mechanisms.IRI represents a frequent underlying cause for both ischemic heart disease and AKI. Compared to the numerous publications on a broad spectrum and relatively extensive research on FGFs in ischemic heart injury, only a limited number of reports are available on FGFs  in renal IRI and AKI. Additionally, most of FGF23-related studies focus on its role as a key metabolic regulator and an injury or prognosis biomarker. Consequently, FGF23 is a proinjury factor rather than a protective or therapeutic agent for AKI. Therefore, our following discussions will only focus on FGF2 and FGF10, both of which exhibit potent protection against IRI and therapeutic potential Acute cell death of proximal renal tubules and inflammatory response of both innate and adoptive nature are hallmark features of IRI. Apoptosis plays an important role in the IR-induced pathogenesis and is a validated parameter to evaluate the cellular damage induced by ischemia. The protective role of FGF2 against IRI is well documented for ischemic myocardial infarction. Studies in genetically engineered mouse models established the antiapoptotic effect of endogenous FGF2 toward ischemic cardiac injury. FGF2 also exerts a positive impact on the repair process that may involve its activation of both MAPK and PKC pathways . Furthervia different mechanisms. Besides apoptosis, other types of cell death such as necrosis, necroptosis, pyroptosis, ferroptosis, have been also been implicated to underlie the tubular cell damage, the potential role of FGFs on these additional pathways and the their interplay remain to be characterized  is a highly conserved nuclear protein that functions as an architectural chromatin-binding factor and regulator of gene transcription. HMGB1 assumes diverse roles as an immuno-modulator in the form of a cytokine molecule or as nuclear chromatin and transcription regulator. HMGB1 can be activated and gets released from damaged parenchymal cells as a sterile inflammatory molecule and a major damage-associated molecular pattern (DAMP). Translocation of HMGB1 from the nucleus to the cytoplasm and subsequent release to the extracellular milieu is reported to promote inflammatory response  of TLRs . HMGB1-T of TLRs .via inhibiting NF-κB-dependent neuroinﬂammation and activating PI3K/AKT survival signalling pathway (Recent efforts to explore the potential protective effect of exogenous FGFs on renal IRI illustrated that, besides promoting proliferation and inhibiting apoptosis, both FGF2 and FGF10 effectively inhibited IRI-induced release of HMGB1 from the nucleus to the extracellular domain, which is associated with a marked decrease in the expression of inflammatory cytokines such as TNF-α, IL-1β, and IL-6 following IRI . It has  pathway . WhetherExtensive research in the past years have established FGFs as vital regulators in tissue repair and regeneration, as well as in metabolic homeostasis. Along with advances in our understanding of FGF biology and their regulation of various pathophysiologic processes comes the inspiration of harnessing their power for potential disease therapies.In this review, we summarized the available data on the FGFs that have shown promising features related to IRI either as a preventative/therapeutic agent or a biomarker. We highlighted some functional and biological aspects that constitute the promising features of FGFs, particularly FGF2 and FGF10, in averting IR-induced tubular cell death and inhibition of overt inflammatory response, which may all contribute to their net beneficial effect on reducing the IRI and promoting recovery.Admittedly, the exploration of FGFs as preventative or therapeutic agents in the management of clinical IRI is still in its rudimentary stages. Most of the published work has been conducted in pre-clinical settings. These widely used animal models, including the bilateral IR that is considered to best resemble clinical AKI, still have certain limitations in fully mimicking human AKI, which can be caused by numerous and complicated clinical conditions. Additionally, the underlying pathophysiology of IRI and how FGFs impact key biological and pathophysiological processes, such as apoptosis, autophagy, ERS, and oxidative stress, remains poorly understood. Future research should encourage more clinical-based and patient-oriented studies. Moreover, the utilization of advanced genetic animal models will also be instrumental in elucidating the disease mechanisms. This will allow for the labeling and tracing of specific cell populations to gain deeper insights about the effects of FGF. Collectively, these approaches, combined with proteomic and genomic technologies, will better delineate FGF signalling targets and support the therapeutic potential of FGFs toward AKI.J-SZ and L-CD conceived the study. L-CD, TA, SB, and J-SZ drafted and revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The handling editor is currently organizing a Research Topic with several of the authors SB, J-SZ, and confirms the absence of any other collaboration.


Cerebellar ataxias are a heterogenous group of degenerative disorders for which we currently lack effective and disease-modifying interventions. The field of non-invasive brain stimulation has made much progress in the development of specific stimulation protocols to modulate cerebellar excitability and try to restore the physiological activity of the cerebellum in patients with ataxia. In light of limited evidence-based pharmacologic and non-pharmacologic treatment options for patients with ataxia, several different non-invasive brain stimulation protocols have emerged, particularly employing repetitive transcranial magnetic stimulation (rTMS) or transcranial direct current stimulation (tDCS) techniques. In this review, we summarize the most relevant rTMS and tDCS therapeutic trials and discuss their implications in the care of patients with degenerative ataxias. Cerebellar ataxias encompass a heterogenous group of acquired and hereditary disorders with diverse clinical presentations. They manifest clinically with a combination of signs and symptoms, such as balance and coordination disturbances, oculomotor deficits, dysarthria, dysmetria, and kinetic tremor . Both thMost of the ataxias with a genetic origin are called spinocerebellar ataxias (SCAs), with more than 40 genetically distinct subtypes already identified, emphasizing the involvement of both the cerebellum and spinal cord, especially in SCAs with a CAG repeat expansion that encodes polyglutamine (polyQ) . MoreoveOther genetic forms of ataxia include dentatorubral-pallidoluysian atrophy , episodiThe most frequent acquired degenerative ataxias include the cerebellar variant of multiple system atrophy (MSA-C) ,22 and, Currently, the majority of degenerative ataxias lack effective pharmacologic disease-modifying therapies and there is growing interest in finding innovative therapeutic approaches to improve clinical symptoms in patients with this spectrum of debilitating disorders. Recently, a report of the Guideline Development, Dissemination, and Implementation Subcommittee of the American Academy of Neurology has systematically reviewed the evidence regarding ataxia treatment, with only few studies emerging as promising for the treatment of only a subset of cerebellar ataxias . What emIn this view, the field of non-invasive brain stimulation has recently gained much attention in the scientific community, in particular because stimulation techniques are non-invasive, provide novel information on cerebellar physiology, may modulate neural plasticity irrespectively of the underlying disease ,31,32,33In this review, we focus on the principal studies implementing non-invasive brain stimulation techniques, particularly transcranial magnetic stimulation (TMS) and transcranial direct current stimulation (tDCS), in the treatment of patients with degenerative ataxias.We searched articles in English published between Jan 1, 1996 and Jan 31, 2020 on Medline (PubMed) using the terms “ataxia”, in combination with “transcranial direct current stimulation” OR “transcranial magnetic stimulation” OR “repetitive transcranial magnetic stimulation” OR “brain stimulation” OR “cerebellar stimulation”.TMS is a noninvasive form of brain stimulation that uses electromagnetic induction as a highly effective, practically painless way to generate currents in the brain capable of discharging targeted neuronal populations. Magnetic fields readily penetrate into the brain without attenuation by the scalp or skull and generate a current according to Faraday’s law of electromagnetic induction .Several TMS protocols, as single or paired-pulse stimulation techniques, have been used to assess the involvement of the corticospinal tract and motor cortex circuitry in hereditary cerebellar ataxias . These Contrary to single-pulse TMS, repetitive TMS (rTMS) is able to change and modulate cortical activity beyond the stimulation period as a potential method for treatment . rTMS caHowever, it should be noted that the mechanism of generating cerebellar plasticity may be very different from that observed in the motor cortex. NMDA-dependent LTP and LTD-like plasticity changes are based on experiments on hippocampal slices  and the The effects of low frequency rTMS were first evaluated by Shimizu and colleagues in four patients with SCA . They applied 10 TMS pulses at 100% of maximum stimulator output with an interpulse interval of more than 5 s for 21 consecutive days on the cerebellum and observed a reduction of the time required for a 10 m walk and in the number of steps in tandem gait in all patients. They also observed a significant increase in blood flow in the both cerebellar hemispheres, putamen, and pons with single photon emission computed tomography (SPECT) .In a subsequent double-blind, sham-controlled trial performed by the same group, 74 patients with sporadic or hereditary spinocerebellar degeneration underwent a similar protocol with 10 TMS pulses applied over both cerebellar hemispheres and the inion for 21 consecutive days. They observed a greater improvement in gait speed and standing capacities in the intervention group compared to the sham group, with an increase in mean regional brain blood flow in the cerebellum and pons in the intervention group. Although placebo or training effects were demonstrated in gait and standing capacities, the effects of active stimulation were far beyond those of sham stimulation. They also observed in some patients that if cerebellar rTMS was repeated one or twice a week, the improvement persisted for at least 6 months. On the contrary, patients receiving rTMS every 2 weeks quickly returned to baseline conditions .By applying a similar protocol for 8 weeks in 20 patients with spinocerebellar degeneration , Ihara and colleagues extended these results by observing a decrease in oxidative stress biomarkers after rTMS in most patients, which correlated with the improvement in clinical scores. They observed a significant decrease in ascorbate free radical concentrations in the cerebrospinal fluid of patients after rTMS compared to baseline. While before rTMS ascorbate free radical concentrations were significantly higher in patients compared to healthy subjects, after rTMS concentrations did not differ significantly between the two groups . OxidatiThe same protocol was applied by Farzan and co-workers in one patient with idiopathic late-onset cerebellar atrophy for 21 days. They observed an improvement in dysmetria and tremor in the finger to nose and finger-chase tests in standing postural control and gait, evaluated kinematically and electromyographically, which were maintained at 6-month follow-up. They also assessed changes in CBI, observing a significant increase in facilitation at interstimulus intervals of 5, 6, and 7 ms (between the cerebellar and opposite motor cortex stimuli) .By applying a slightly different protocol in a patient with Minamata disease, Nakamura and colleagues observed an improvement in gait and tremor only after real TMS but not after sham stimulation. They applied rTMS with the coil centered 4 cm lateral to the right of the inion (three trains), with the coil centered on the inion (four trains), and with the coil centered 4 cm lateral to the left of the inion (three trains). Each train consisted of 5 Hz stimulation for 10 s with a 50 s inter-train interval .Another single-subject observation was made in a patient with SCA 6 by applying TMS over both the motor cortex and cerebellum for 5 days a week for 2 weeks, with an identical course repeated after 2 weeks. A total of 20 single pulses at approximately 0.3 Hz were applied twice over the motor cortices , followed by 10 single pulses at 0.5 Hz applied twice over the cerebellum (10 with current flowing counter-clockwise and 10 clockwise). They observed a marked improvement in diplopia, particularly after stimulation of the motor cortices, accompanied by an improvement in limb ataxia, evaluated with the International Cooperative Ataxia Rating Scale (ICARS) .Very recently, Dang and co-workers applied a slightly different protocol in another patient with SCA 6. They delivered 1500 pulses over the inion in 1 s trains at 10 Hz, with a 10 s inter-train interval. Each session was applied daily for 5 days a week for 20 weeks. They observed a significant improvement in posture and gait, kinetic functions, and speech, whereas no improvement in oculomotor movements was identified .All of these observations paved the way for a randomized, double-blind, sham-controlled trial performed in 20 patients with genetically confirmed SCAs , which was recently published. Manor and colleagues applied a 4-week, 20-session rTMS intervention targeting the cerebellum, guided by individual brain anatomy using structural magnetic resonance imaging (MRI). Ten pulses were delivered in every region, for a total of 30 pulses in each session. rTMS, compared to sham, was associated with greater improvement in the Scale for the Assessment and Rating of Ataxia (SARA) scores, particularly within the “stance” sub-score. This functional change was accompanied by improvement in several objective metrics of postural sway during eyes-open and eyes-closed standing. rTMS did not influence performance in the nine-hole peg test, the timed up-and-go test, or gait kinematics .In summary, 122 patients have been assessed in 8 published studies using rTMS see . All pattDCS is a form of non-invasive brain stimulation that modulates neuronal excitability in a polarity-specific manner by delivering prolonged (10–20 min) but weak (1–2 mA) currents to brain tissues via electrodes placed on the scalp . The effSimilarly to what has been observed in the motor cortex, cerebellar tDCS has been shown to modulate cerebellar excitability in a polarity-specific manner, with anodal cerebellar tDCS increasing the excitability of the cerebellar cortex and thus CBI, whereas cathodal cerebellar tDCS decreased CBI . These pInitial studies evaluated the effects of cerebellar tDCS on neurophysiological measures in ataxic patients. Grimaldi and colleagues observed in nine patients with several types of acquired and degenerative ataxias  a decrease in long latency stretch reflexes after tDCS stimulation with the anode over the right cerebellar hemisphere and the cathode over the contralateral supraorbital area. In this study, short latency stretch responses and coordination tasks in the upper limbs were unaffected .The same group subsequently evaluated the effects of cerebello-cerebro tDCS in two patients with SCA 2. They observed that, after 20 min of stimulation with the anode over the right cerebellar hemisphere, immediately followed by 20 min of stimulation with the anode over the contralateral motor cortex and the cathode over the contralateral supraorbital area, tremor and hypermetria decreased in both patients. These results were accompanied by an improvement in SARA scores .Taking into account that the excitability of the motor cortex is depressed in patients with cerebellar disease , Pozzi aFollowing these promising results, several randomized, double-blind, sham-controlled clinical trials were performed in larger groups of patients with degenerative ataxia. One of the first was a randomized, double-blind, sham-controlled, crossover study in 19 patients with ataxia . By applying a single session of 2 mA tDCS with the anode over the cerebellum and the cathode over the right deltoid muscle for 20 min, researchers observed a significant improvement in SARA and ICARS scores , at the nine-hole peg test and at the 8 m walking time compared to the sham stimulation. They also observed a significant negative correlation between the improvement in clinical scores and the impairment on activities of daily living. These results were also confirmed when only SCA and MSA-C patients were considered separately .Considering that repeated sessions of stimulation could have cumulative effects on synaptic plasticity , the samTrying to increase the possible effects of tDCS in patients with ataxia, which frequently have an involvement of the spinal cord, the same group performed a randomized, double-blind, sham-controlled, crossover trial in 21 patients with neurodegenerative ataxia  by applying a concurrent stimulation with the anode over the cerebellum and the cathode over the spinal lumbar enlargement (2 cm under T11) for two consecutive weeks (Monday to Friday). Additionally in this case, cerebello-spinal tDCS showed a significant improvement in all performance scores , in motor cortex excitability, and CBI compared to sham stimulation ,83.A following double-blind, auto-matched clinical trial performed in seven patients with ataxia  showed a significant improvement of gait parameters and SARA scores after five sessions of tDCS with the anode targeting both motor cortices (20 min on the left followed by 20 min on the right motor cortex) and the cathode over the contralateral supraorbital area .An important step forward was achieved by Pilloni and co-workers, who evaluated the feasibility of long-term at-home treatment with cerebellar tDCS in a patient with cerebellar ataxia. They delivered 60 tDCS sessions, 59 of which were administered remotely, observing an improvement in gait speed and manual dexterity. The applied tDCS montage involved placing the anode over the cerebellum and the cathode over the right shoulder . These fOn the contrary, two studies performed by the same group did not observe any significant effects of cerebellar tDCS in patients with ataxia. Hulst and co-workers assessed in 20 patients with cerebellar degeneration  the effects of approximately 22 min of tDCS with the anode over the right cerebellar hemisphere and the cathode over the right buccinator muscle. After 1 week, patients received another session of tDCS with the anode over M1 and the cathode over the contralateral supraorbital region. They did not observe any significant effects after cerebellar or M1 stimulation in a standard reaching task with force-field perturbations . By applAnother relevant aspect that should be more systematically assessed in tDCS studies is the effect of sham tDCS, which is fundamental due to the placebo response observed in non-invasive brain stimulation trials . Sham-coIn summary, 116 patients have been assessed in 10 published studies using tDCS see . All patCerebellar ataxias are a very heterogenous group of degenerative disorders for which we currently lack effective and disease-modifying interventions. The field of non-invasive brain stimulation has made much progress in the development of specific stimulation protocols to modulate cerebellar excitability and try to restore the physiological activity of the cerebellum in patients with ataxia but also in other neurodegenerative disorders . SeveralNevertheless, several studies both in healthy controls and in patients with ataxia have suggested that the modulation of the cerebellum by non-invasive brain stimulation may enhance postural control, gait, and motor coordination. Although the effects of cerebellar stimulation on cognitive functions have been abundantly studied in healthy controls , there iDespite the promising findings, the precise mechanism of action of non-invasive cerebellar stimulation still remains largely unknown. Other than modulating the excitability of Purkinje cells and increasing their activity on the dentato-thalamo-cortical pathway, several other mechanisms could be involved, such as inactivation or activation of specific cellular processes, including gene expression, protein synthesis, channel pump regulation, and receptor or neurotransmitter modulation .Several questions remain unanswered and could provide novel targets for future studies. For example, several studies have now shown that the effects of iTBS might be even more relevant than those obtained after rTMS , and encFuture studies should try to assess several issues: the inclusion of etiologically homogenous group of patients, perhaps in multicenter studies; defining the optimal timing of follow-up stimulation sessions and the effects of repeated sessions over time; the effects on cognition and emotions; the feasibility of at-home remotely supervised stimulation in larger cohorts; and if these effects may be intensified by concurrent motor training interventions or pharmacologic therapies. Critically, evaluation of target engagement using imaging or physiologic biomarkers, as well as the assessment of “dose” by using modelling to calculate the induced currents in the brain to define individual stimulation parameters, would be essential. Moreover, the cerebellum can show significant structural changes in term of climbing fiber Purkinje cell and parallel fiber Purkinje cell wirings in cerebellar ataxia and other disorders ,105,106,In conclusion, particularly in light of limited evidence-based pharmacologic and non-pharmacologic treatment options for patients with ataxia, non-invasive brain stimulation techniques with rTMS or tDCS are potentially promising tools for therapeutic approaches, but further work is needed before it can be broadly offered in the clinical setting.


Heterozygous mutations of the GBA1 gene, encoding for lysosomal enzyme glucocerebrosidase (GCase), occur in a considerable percentage of all patients with sporadic Parkinson’s disease (PD), varying between 8% and 12% across the world. Genome wide association studies have confirmed the strong correlation between PD and GBA1 mutations, pointing to this element as a major risk factor for PD, possibly the most important one after age. The pathobiological mechanisms underlying the link between a defective function of GCase and the development of PD are still unknown and are currently the focus of intense investigation in the community of pre-clinical and clinical researchers in the PD field. A major controversy regards the fact that, despite the unequivocal correlation between the presence of GBA1 mutations and the risk of developing PD, only a minority of asymptomatic carriers with GBA1 mutations convert to PD in their lifetime. GBA1 mutations reduce the enzymatic function of GCase, impairing lysosomal efficiency and the cellular ability to dispose of pathological alpha-synuclein. Changes in the cellular lipidic content resulting from the accumulation of glycosphingolipids, triggered by lysosomal dysfunction, may contribute to the pathological modification of alpha-synuclein, due to its ability to interact with cell membrane lipids. Mutant GCase can impair mitochondrial function and cause endoplasmic reticulum stress, thereby impacting on cellular energy production and proteostasis. Importantly, reduced GCase activity is associated with clear activation of microglia, a major mediator of neuroinflammatory response within the brain parenchyma, which points to neuroinflammation as a major consequence of GCase dysfunction. In this present review article, we summarize the current knowledge on the role of GBA1 mutations in PD development and their phenotypic correlations. We also discuss the potential role of the GCase pathway in the search for PD biomarkers that may enable the development of disease modifying therapies. Answering these questions will aid clinicians in offering more appropriate counseling to the patients and their caregivers and provide future directions for PD preclinical research. Parkinson’s disease (PD) is the second most common neurodegenerative disorder after Alzheimer’s disease, with a 0.3% prevalence in the general population, increasing to 1% in people older than 60 years and to 3% in the over 80s , which may present with either neurological involvement (neuronopathic) or not (non-neuronopathic). Interest in GBA1, as a causative factor for PD, was initially sparkled by the observation, in the 1990s, that among GD patients and GBA1 mutation carriers, a higher proportion developed parkinsonian symptoms  found throughout the central nervous system of PD patients  patients without GBA1 mutation, suggesting a central, more widespread involvement of this enzyme in PD pathogenesis  is very similar to iPD, except for an earlier onset and higher prevalence of cognitive deterioration and non-motor features, manifesting before the development of the PD motor features  or “mild” , with a residual GCase activity of 13–24% and 32–38%, respectively . The reduction in GCase activity causes an accumulation of its substrates with subsequent perturbation of lipid homeostasis. This affects trafficking, processing, and clearance of alpha-synuclein, resulting in accumulation and aggregation of the protein  alpha-synuclein  promotes balanced cell proteostasis, degrading altered proteins through ubiquitination and proteosomal degradation , greater incidence of sleep disturbances, and earlier development of cognitive deficits  were significantly worse in GBA-PD patients with severe mutations than in those bearing mild mutations  the same group also demonstrated a significant presynaptic dysfunction in GBA-PD, with a severe reduction of striatal uptake in both putamen and caudate nucleus, whereas asymptomatic GBA carriers had increased caudate uptake with normal putamen uptake  employing several different radioisotopes  has been used to assess brain metabolism in GBA-PD. Kono et al.  first deFluorodopa PET or single-photon emission computerized tomography (SPECT) with dopamine-sensitive ligands in GD-PD patients showed asymmetric striatal dopaminergic neuronal loss with the highest reduction in the caudate and posterior putamen nuclei; this reduction was also observed in iPD brains, although less represented  from GD, GBA-PD, and also iPD patients  and specificity . Preliminary results showed that higher dose ambroxol are effective and safe in PDD .Despite the availability of successful treatments for systemic manifestations in GD patients, these approaches do not affect glycosphingolipid accumulation in the central nervous system, because they are not able to cross the blood-brain barrier. Current experimental approaches available in clinical trials  are founAnother approach is being tested in the Netherlands by Allergan in a phase I clinical trial with LTI-291, a non-inhibitory chaperone of GCase able to both increase its catalytic activity and extend its half-life .An alternative therapeutic strategy that is being explored is to reduce the accumulation of glucosylceramide  by targeting the enzyme glucosylceramide synthetase. The substrate reduction restored glycosphingolipid balance and reduced alpha-synuclein accumulation in neuronal cells of PD with and without GBA1 mutations . Based on the assumption that a decrease in GCase activity might favor alpha-synuclein accumulation and synucleinopathy development, AAV- mediated GBA1 delivery has been tested in mouse models of GD-related PD to assess its effects on GCase and alpha-synuclein modulation  Family history of PD; (2) early onset of PD (before 50 years old); or (3) individual among “population at risk,” such as AJ individuals.Genetic screening should be also performed to: (1) stratify patients into subtypes to better understand the disease process; and (2) provide patients with the opportunity to participate in clinical trials with experimental therapies designed for patients with specific genotypes (Payne et al., GBA1 mutations play a pivotal role in the molecular pathogenesis of PD and are considered one of the most frequent risk factors for the disease. Increasing evidence suggests that GCase deficiency might also have a role in iPD.The pathobiological mechanisms underlying the link between a defective function of GCase and the development of PD are still poorly understood, but interactions with alpha-synuclein, lysosomal dysfunction, ER stress, and neuroinflammation may all play a crucial role.Further studies on large GBA1-positive populations will be needed to precisely identify the biological factors that link GBA1 mutations to PD. This will improve the diagnostic and caring process, possibly leading to the discovery of new therapeutic targets to engage in the frame of an innovative, personalized approach to PD therapy.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The scale of the SARS-CoV-2 pandemic has thrust a spotlight on the sex-based differences in response to viral diseases; morbidity and mortality are greater in men than women. We outline the mechanisms by which being female offers a degree of protection from COVID19, that persists even when confounders such as comorbidities are considered. The physiological and immunological mechanisms are fascinating and range from incomplete X chromosome inactivation of immune genes, a crucial role for angiotensin converting enzyme 2 (ACE2), and regulation of both immune activity and ACE2 by sex steroids. From this flows understanding of why lung and other organs are more susceptible to COVID19 damage in men, and how their distinct immunological landscapes need to be acknowledged to guide prognosis and treatment. Pregnancy, menopause, and hormone replacement therapy bring changed hormonal environments and the need for better stratification in COVID19 studies. We end by noting clinical trials based on increasing estrogens or progesterone or anti-testosterone drugs; excellent examples of translational physiology. This short review focuses on how differences in the physiology of women and men affect the outcome and survival of patients with COVID19. We first review the evidence that outcomes for females are more favorable, before considering the mechanisms and relating them to viral infection. We use the binary terms “male” and “female” so we can correctly report data in published studies, which so far have not considered if COVID19 has particular effects on trans and non-binary people.https://globalhealth5050.org/the-sex-gender-and-covid-19-project/) is recommended. At the time of writing (October 2020), it is almost a year since the first reports of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus-2019 (COVID19), appeared. Since then, there has been concerted international effort to understand the virus, the disease it produces and develop strategies to combat it. From the earliest findings, it emerged that more men than women suffer severe COVID19 disease and die from it . This fiWith the increases in available data it is possible to interrogate the statistics further and ask whether differences such as infection rates, age and co-morbidities can explain the sex differences in the outcomes of COVID19, . It appeWhile future studies will further refine our knowledge concerning disease outcomes, sex makes a significant contribution to outcomes; in COVID19, women have a degree of protection compared to men. We are not saying that all the aspects of COVID19 can be attributed to sex differences, but rather, that benefits will follow from understanding the disease better, and biological sex is a part of this. We will briefly mention how gender may impact on these data, before a more detailed discussion of the physiological mechanisms of the reported sex differences.There are many ways that gender can impact on COVID19 statistics. Compared to men, women may be more concerned about COVID19 . This maWe first overview how SARS-CoV-2 infects humans as the basis for understanding how sex-based differences can arise. We then focus on the role of angiotensin converting enzyme 2 (ACE2) and infection, and then sex differences in immunological responses to infection.1 That this is essential for viral entry was shown using ACE2 knock out mice , is relatively recent;  see FiguACE2 is a zinc containing, carboxy peptidase that removes an amino acid and converts angiotensin 1 to angiotensin 1–9 and angiotensin 11 into the vasodilator, angiotensin(1-7), and may have additional substrates . Its catUntil menopause, women are relatively protected from a variety of cardiovascular risks, including high blood pressure . Part ofTMPRSS2 is also widely distributed and highly expressed in epithelial cells in lungs, small intestine, heart, liver, and prostate. No significant difference in TMPRSS2 expression between males and females in human lung were found  showed a positive association between COVID19 and menopausal status, and a negative association with combined oral contraceptive pill use , support2 facilitating viral fusion with host cell membranes  or oral progesterone in men (NCT04365127). In Mexico, a trial is investigating the effect of a combined estrogen and progesterone patch (NCT04539626) on clinical response and mortality in non-severe COVID19 patients. A trial in Iran is also testing the effect of injectable estradiol and testosterone on recovery in male and female COVID19 patients with respiratory, heart or kidney failure (IRCT20150716023235N15).Trials exploring anti-androgen therapies are also underway, including in Sweden (NCT04475601), the USA  and Brazil (NCT04446429) with a view to reducing disease severity in older (>50 years) male and female patients, or males presenting with male-pattern baldness, by inhibiting the expression of androgen regulated proteins, such as TMPRSS2. Other trials are investigating the effect of decreasing TMPRSS2 action using TMPRSS2 inhibitors see .In Italy, a Phase II randomized trial is planned to assess the efficacy of intravenous oxytocin in patients affected by COVID19 (NCT04386447). Oxytocin known for its role in augmenting uterine contractions in labor , has alsPassive antibody therapy for COVID19 has already been discussed . Many vaOur main conclusion is that the sex-based differences in outcomes of COVID19 infection, tentatively reported at the beginning of the pandemic, have been reinforced by all subsequent studies. In addition, our understanding of the possible contributors to this is increasing but it is likely more exciting discoveries remain to be made., especially around the intersection of physiology, immunology and environmental factors.We note that, generally, more men are enrolled in clinical trials and research in animals is often focused on males to avoid the cyclic fluctuations in hormones. This poses a significant barrier in understanding the sex-based differences in infection severity. The disparity in the effects of COVID19 observed between the sexes, and recent data in other physiological systems and pathologies, highlights the need to include both males and females in future research. There is clearly much more to be understood about sex-based differences. Understanding the mechanisms behind them may help to find appropriate and sex specific therapies for COVID19 and other sexually dimorphic pathologies .Biological sex affects areas of physiology and pathophysiology, beyond the obvious. Importantly these sex-based differences impact on both symptoms of disease and effectiveness of medication. In the light of the COVID19 pandemic, an appreciation of how the physiological and immunological differences between female and male responses to viral diseases is crucial, and it is to this that this review contributes. Compared to males, females mount stronger immune responses to combat and clear viral loads, but also have different immunological landscape during infection. Sex-dependent steroid hormones link mechanisms between sex and response to viral infection. For example, estrogens promote the production of anti-inflammatory cytokines, and having two X chromosomes can increase activity of immune genes carried on the chromosome, due to incomplete X-inactivation. The host viral receptor is angiotensin converting enzyme 2 (ACE2). Sex differences in ACE2’s expression in lungs and viral handling, are being actively investigated to better understand the underlying mechanisms. These differences have manifested worldwide in fewer deaths and severe COVID19 complication in females compared to males, despite roughly equal infection rates. They have also led to active clinical trials of using sex hormone treatments, such as estradiol patches, to help mitigate the effects of COVID19.SW conceived the study. SW and SA wrote and edited the article. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


However, despite widespread acceptance of this concept, diagnostic blood testing is an excellent example of clinical care that is not yet customized to the needs of children, especially newborns. Cumulative blood loss resulting from clinical testing does not typically impact critically ill adult patients, but can quickly escalate in children, leading to iatrogenic anemia and related comorbidities. Moreover, the tests prioritized for rapid, near-patient testing in adults are not always the most clinically relevant tests for children or newborns. This report describes the development of a digital microfluidic testing platform and associated clinical assays purposely curated to address current shortcomings in pediatric laboratory testing by using microliter volumes (<50 µL) of samples. The automated platform consists of a small instrument and single-use cartridges, which contain all reagents necessary to prepare the sample and perform the assay. Electrowetting technology is used to precisely manipulate nanoliter-sized droplets of samples and reagents inside the cartridge. To date, we have automated three disparate types of assays  on the platform and have developed over two dozen unique tests, each with important clinical application to newborns and pediatric patients. Cell lysis, plasma preparation, magnetic bead washing, thermocycling, incubation, and many other essential functions were all performed on the cartridge without any user intervention. The resulting assays demonstrate performance comparable to standard clinical laboratory assays and are economical due to the reduced hands-on effort required for each assay and lower overall reagent consumption. These capabilities allow a wide range of assays to be run simultaneously on the same cartridge using significantly reduced sample volumes with results in minutes. Clinical laboratory tests are an invaluable component of medical care that aid in both the diagnosis of disease and the evaluation of response to treatment. Unfortunately, for neonatal and pediatric patients undergoing intense medical interventions, significant challenges may arise from the diagnostic blood sampling process. Cumulative blood loss from diagnostic testing can escalate quickly in critically ill patients resulting in iatrogenic anemia and need for transfusions, which are associated with higher risk of infection, donor erythrocyte injury, and other cardiac, vascular, and hemorrhagic complications ,2,3,4. IThe dangers of iatrogenic anemia have gained particular attention for newborns in the neonatal intensive care unit (NICU) setting , as the While the clinical need for maximizing the diagnostic output from low blood volumes is recognized, there are only a limited number of such tests available that comprehensively address the testing needs of pediatric or newborn patients. A handful of FDA-cleared platforms have emerged as leaders in low volume testing, which include the i-STAT , Piccolo Xpress , and Gem Premier series . While these products have gained considerable traction for rapid, point-of-care blood testing of analytes important for blood chemistry and gases, cardiac care, and liver and kidney function, they only cover a subset of the ~1000 tests  used in In this article, we describe a nanoliter scale analyzer that leverages the highly flexible liquid handling architectures inherent to digital microfluidic (DMF) technology to perform laboratory-equivalent chemistry assays, immunoassays, and molecular assays essentially on the same platform. All assays are performed in single-use, disposable cartridges that contain all assay-specific reagents onboard, unless specified otherwise. The system supports testing for a wide range of samples, including whole blood, serum, urine, saliva, and meconium (newborn stool). A novel and unique capability of DMF described here is that whole blood samples are separated into plasma on the cartridge and, as required, both whole blood and plasma samples are utilized simultaneously for various assays within the same protocol. All assays under development described here are designed specifically for vulnerable newborn and pediatric patients for whom small volume testing is especially important. Multiple assays are demonstrated utilizing microliter sample volumes dispensed from a 50 µL sample loaded into the cartridge to provide a comprehensive profile of patient status within minutes. Though each assay is performed using < one microliter of sample, choice of sample collection devices for low volumes is limited, therefore a 50 µL collection device is used.Digital microfluidics by electrowetting allows programmable manipulation of droplets . The heaters are controlled and monitored from the instrument software and enable much faster thermal cycling than previously described aluminum heater bars, which were placed in the deck of the instrument . Numerous internal quality control (QC) checks are integrated in the system to ensure the validity of reported results. Algorithms utilize within-run optical, electrical, and thermal measurements to verify the integrity of the data generated. These algorithms can detect issues with sample and reagent loading, reagent integrity, fluidic anomalies, and plasma separation. For example, electrical impedance checks are performed on all loaded samples to ensure that sufficient sample volume is available for testing. Samples below acceptable volumes are flagged to the user as inadequate. Measurements of optical absorbance at specific wavelengths are also performed to determine whether samples have exceeded the hemolytic, lipemic, or icteric interference limits of particular assays. All QC checks are fully automated and do not require intervention or interpretation from the user.This section is divided into materials and methods for sample preparation, chemistry assays, immunoassays, and molecular assays with each of these sections providing further details on reagents, droplet protocols, and data analysis methods.A lectin-based agglutination agent was spotted directly onto the PCB and air dried. Droplets of diluent were dispensed to reconstitute the dried agglutinin and then combined with whole blood droplets dispensed from the sample input reservoir. As the whole blood mixes with agglutinin, the red blood cells (RBCs) agglutinate into a clump surrounded by liquid plasma. This plasma fraction of the mixture is manipulated by electrowetting to separate from the agglutinated cells to yield clear plasma see . SeparatRBCs in droplets of whole blood were lysed osmotically on the cartridge by serial dilution of the blood sample with diluent. Two droplets of diluent were mixed with a single drop of blood, and then split to derive 3 droplets at a 1:3 dilution. The volume of each droplet on the cartridge is approximately 1 µL. One of the droplets is then mixed with another two droplets of diluent and this process is repeated until the necessary dilution of whole blood is obtained. Efficiency of whole blood lysis was confirmed through the absence of scatter at 775 nm.2), diazonium salt, silicone oil, succinic acid, glucose, glucose oxidase, peroxidase-HRP, bilirubin, dyphylline, excipients, surfactants, preservatives and stabilizers were procured from MilliporeSigma . Bromocresol Green (BCG) was obtained from Affymetrix . Creatinine kits (2-step colorimetric readout) were purchased from Abcam . Urine calibrator samples were procured from CST Technologies . β-nicotinamide adenine dinucleotide phosphate (NADP+), maleimide, glucose-6-phosphate (G6P), magnesium chloride , and albumin assays were performed using reagents that were spotted on the PCB and air dried under controlled temperature and humidity.2, and other additives in 100 mM phosphate buffer, pH 7.3) induced production of NADPH. Kinetic fluorescence measurements were collected by shuttling the reaction droplet around a loop of electrodes where every 10 s the fluorescence is collected at the designated detection electrode for a period of approximately 4 min. The rate of NADPH production is then quantified from the increasing fluorescence, which is proportional to G6PD enzymatic activity. The reaction occurs in the presence of maleimide, which is used to improve the specificity of the assay by minimizing the production of NADPH from 6-phosphogluconate dehydrogenase. Hemoglobin present in the lysed droplet was measured through spectral absorbance and used to normalize G6PD enzymatic activity, resulting in a final reported value of units activity per gram hemoglobin (U/gHb). For determination of G6PD activity, red blood cells were first lysed osmotically in the cartridge by mixing whole blood with diluent as described above. The lysed sample droplet was then mixed with assay-specific reagent 1 . Addition of a second reagent  chemistries, respectively. The previously described biochemical technique for on-cartridge plasma separation was performed prior to each assay. To measure TBil, a droplet of the separated plasma was combined with dried reagent . It should be noted that plasma is directly used to reconstitute the dried reagent. Bilirubin in the plasma transforms into azobilirubin, which caused a proportional increase in absorbance. To measure albumin, a droplet of the separated plasma was combined with a droplet of 1.0 mM BCG, surfactants, and excipients in 100 mM succinate buffer (pH 3.6) to form a complex with albumin, the concentration of which was proportional to the absorbance intensity. 2O2 as one of the end products. A single droplet of bilirubin calibrant was combined with a droplet of the reaction mixture and transported to the detector, where the spectrophotometric characteristics of the un-oxidized bilirubin were followed over time to detect the time-dependent pattern of oxidation. The amount of unbound bilirubin is then correlated to the rate of oxidation. Unbound bilirubin (UB) was measured using liquid reagents according to the protocol by Shimabuku and Nakamura  in whichThe creatinine assay was performed using liquid reagents as follows. Undiluted urine calibrant samples or clinical samples were dispensed from the sample reservoir; a single droplet of sample was then combined with the reaction mix and incubated. The reaction droplet was then transported to the detection electrode where absorbance was measured. Superparamagnetic bead immunoassay kits for free thyroxine (T4) and insulin were procured from Beckman Coulter . Superparamagnetic beads for the neutrophil gelatinase-associated lipocalin (NGAL) immunoassay were conjugated in-house using Dynal MyOne goat anti-mouse beads  with NGAL capture and detection antibodies obtained from R&D Systems  and ThermoFisher. 4-methylumbelliferyl phosphate (4MUP) was procured from MilliporeSigma.All immunoassays were performed using liquid reagents, which were loaded into the cartridge prior to initiation of the automated immunoassay protocol. For the free T4 assay (a competitive immunoassay), one droplet (approximately 1 µL) of sample was combined with a droplet containing the T4 capture antibody (conjugated to magnetic beads), and an excess of biotinylated T4 analog. Endogenous T4 in the sample competes with the biotinylated T4 and the fluorescence output of the assay from 4-MU derived from biotin-bound-streptavidin ALP is inversely related to the T4 level. The insulin and NGAL assays were sandwich immunoassays in which capture and detection antibodies form a “sandwich” with the analyte of interest and the final fluorescence signal generated is directly proportional to the amount of analyte. With some minor modifications, protocols described in Sista et al.  were folThe real-time PCR mixture and polymerase were procured from Roche Custom Biotech , primers and probe  were obtained from Applied Biosystems  and ThermoFisher using the CMV sequences published by Boppana et al. , and cusTarget DNA sequences were amplified on the cartridge using heaters printed onto the PCB itself, which are rapidly thermocycled using the instrument software. Fluorescence output from amplification of the 6-FAM probe was captured using the instrument fluorimeter. Reaction droplets  were subjected to 50 cycles, with each cycle consisting of a 2-s denaturing stage 93 °C), followed by a 2-s extension stage (60 °C). The fluorescence output of each cycle was detected at the site of amplification such that the entire 50 cycle reaction, including on-cartridge operations such as droplet dispensing, reagent mixing, and reaction setup, was completed in 5 min. Note that nucleic acid extraction from more robust organisms or matrices  may require additional steps, which have been demonstrated on the DMF platform [3 °C, folSamples utilized for the method comparison studies were from adults, obtained from the following sources: G6PD clinical samples were purchased from BioIVT  as whole blood samples. Plasma samples for albumin and bilirubin were purchased from Discovery Life Sciences . Procurement of samples from neonates or pediatric patients was not feasible as blood collection volumes needed for comparator assays were often milliliters of blood.Serum samples utilized for insulin and free T4 studies were assayed adult plasma samples  obtained by collaborators at Duke University under an IRB approved protocol. Samples were stored at −80 °C for 3 to 36 months prior to testing. Assayed urine samples utilized for the NGAL and creatinine studies were obtained from the University of Alabama at Birmingham under IRB approved protocols (discarded and de-identified). For each of the method comparison studies, the DMF assays were compared to a currently validated assay as outlined in the following. For the albumin and TBil assays, human serum and heparinized plasma samples were procured from a research sample biobank  and added to washed human red blood cells  to target 50% hematocrit. Each whole blood sample was analyzed using three different lots of cartridges. The remaining prepared whole blood sample was centrifuged to obtain plasma, which was shipped to LabCorp  for measurement (Hepatic Function Panel) using standard laboratory equipment used for routine clinical reporting . Albumin and total bilirubin values obtained from LabCorp were used as comparator values. For the G6PD assay, heparinized adult whole blood (BioIVT) was obtained and analyzed using three different lots of cartridges. The remaining whole blood was sent to LabCorp for G6PD measurement using the Pointe Scientific reagent kit on the Roche Cobas analyzer. All biological material for the albumin, TBil, and G6DP method comparison studies were derived from adult donors, with no specification on gender, ethnicity, or age range.For the unbound bilirubin assay, human plasma samples that were obtained from Discovery Life Sciences values were acquired using an in vitro diagnostic clinical analyzer. Creatinine values were acquired using a mass spectrometry based method run in the University of Alabama at Birmingham clinical laboratory. NGAL values were acquired using a Kidney Injury Panel 5 Human kit (KIP-5) microtiter plate assay  . InsulinHere, we describe integration of some of the pre-analytical steps such as plasma preparation, red blood cell lysis, and sample quality assurance, which are all required sample preparation and processing steps to deliver the analyte to the analytical assay.Due to the inherent simplicity in use of DMF technology and flexibility in its operation, the platform is designed for use in multiple settings including a hospital laboratory, NICU, physician’s office, or other distributed settings without ancillary laboratory equipment. We developed integrated protocols for common sample preparation requirements including plasma separation from whole blood, RBC lysis, and DNA isolation on the same cartridge. For plasma separation, we utilized a biochemical method which is based on lectin-induced agglutination to facilitate clumping of the RBCs directly on the cartridge . As illuWe have also demonstrated the capability to perform red blood cell lysis from whole blood on the DMF cartridge. RBC lysis is performed using the osmotic method which incorporates serial dilution of the blood sample with water. To demonstrate the efficacy of on-board DMF hemolysate preparation, whole blood from a single donor was serially diluted within the cartridge and the optical scatter was measured at 775 nm, providing a surrogate measure of incompletely lysed red blood cells C. The saThe cartridge can accept a wide range in input sample volume variations from the sample transfer pipette. Our DMF platform is the first of its kind to combine chemistry assays, immunoassays, and molecular assays together on a single system. Over three dozen unique assays relevant for neonatal diagnostics utilizing multiple types of samples have been demonstrated on our DMF platform so far and some are summarized in Among the most ordered chemistry assays for neonates are TBil and albumin, which inform neonatal hyperbilirubinemia diagnosis and subsequent treatment based on American Academy of Pediatrics guidelines for hyperbilirubinemia . Standarfree) is oxidized and detected kinetically according to the rate of decreasing absorbance. The DMF unbound bilirubin assay successfully measures the loss of absorbance in calibrant samples and results of a preliminary method comparison study  enzyme activity, which identifies the relatively common deficiency of this enzyme . PatienHere, we describe the development of a fully automated quantitative sample-to-answer DMF G6PD enzyme assay from 50 μL of whole blood. Onboard controls ensure that the operational temperature of the cartridge is regulated tightly, an important feature given that G6PD activity is highly dependent on temperature fluctuations. An automated osmotic shock lysis procedure shown in  was alsoWhile low blood volumes are generally preferred in newborn and pediatric patients for the stated reasons, minimizing the sample volume for other physiological samples, such as urine, is also highly desirable in this population. To broaden the compatibility of different types of samples, we have demonstrated a DMF assay for creatinine, which is commonly tested in urine . Urine gImmunoassays are another commonly used method in clinical laboratory testing to measure antibodies, antigens , or a combination of the two. We previously published DMF immunoassay protocols for Troponin I, insulin, and IL-6 ,13 usingInsulin assays are routinely ordered for newborns and children suffering from persistent hypoglycemia and access to quick results, while still minimizing blood volume, is desirable under such conditions. As shown in Free thyroxine (T4) is one of the most commonly ordered tests for newborns to investigate potential congenital thyroid disorders such as neonatal Graves’ disease and congenital hypothyroidism (incidence of 1 in 3000 births). We have demonstrated a competitive immunoassay for free T4 on the DMF cartridge. Competitive immunoassays are often used for small molecules and are based on the competition of the native analyte with an alkaline phosphatase (ALP) labeled version of the analyte; the two analytes compete for binding by a primary capture antibody. Addition of 4MUP induces signal generation from the ALP-labeled FT4 analogue and the signal produced is inversely proportional to the amount of analyte in the sample. Results of our DMF competitive immunoassay for free T4 are shown in While many clinical immunoassays are performed on blood samples, other biological samples may be tested to discern various etiologies. Kidney function is a common concern for preterm newborns that often requires analysis of urine samples. We also demonstrated an immunoassay for neutrophil gelatinase-associated lipocalin (NGAL) on urine samples on the DMF cartridge shown in C. The DMMolecular diagnostic laboratory tests are commonly used for the detection of infectious disease and genetic markers of disease risk. Recently published DMF protocols for polymerase chain reaction (PCR) from human genomic DNA, bacterial genomic DNA, and fungal genomic DNA ,10 used t values were 32, 26, and 19 respectively for 10, 1500, and 225,000 copies/µL, which is generally in line with expected relative cycle threshold values. An internal control lambda (600 copies/µL) was also multiplexed along with CMV (data not shown). PCR amplification completed within 5 min. These results show promise for performing rapid PCR with further optimization of thermal cycling of on-board heaters, reagent composition, and the reaction conditions.Congenital CMV (cCMV) infection increases a child’s risk for long-term health problems including hearing loss, and newborn screening for cCMV has been advocated . The meaThe DMF platform described in this report is the realization of a technology that was envisioned over 10 years ago : a smallWhile quality control checks are routinely implemented in mature technologies, a number of measurements of optical, electrical, and thermal parameters have been uniquely developed for the droplet handling platform. Several algorithms have been developed to flag any issues with sample loading, reagent integrity, fluidic anomalies, and blood separation—all of which are novel and essential to mature the DMF platform to produce diagnostic quality results. Chemistry assays including sample preparation of whole blood  were demonstrated on a DMF cartridge spanning dye-binding colorimetric assays  to rate kinetic fluorimetric enzymatic assays (G6PD) with dried reagents onboard in around 15 min including the sample preparation pre-analytical steps. Another chemistry assay (creatinine) with urine samples has also been demonstrated, as well as feasibility of an unbound bilirubin assay from plasma. Utilizing the same DMF platform, representative data are shown for competitive immunoassays (free T4) and heterogeneous sandwich immunoassays (insulin and NGAL) from plasma and urine samples, respectively. Additionally, by integrating the heaters onto the same DMF cartridge, a rapid PCR assay (CMV) has been demonstrated within 5 min. Precise droplet control in a DMF cartridge, coupled with the novel features presented, enable results that are comparable to standard clinical laboratory assays, but using only 1 µL sample volume and with greater flexibility. Interference and other analytical validations of the DMF assays are outside of the scope of this publication, however this information will be included in follow-up publications. The DMF platform described in this report is demonstrated to possess all the features that are required to bring the advantages of low sample volume and rapid testing to neonatal and pediatric diagnostics.


All these mechanisms are required to cope with the different micro-environment signals and maintain cellular homeostasis in order to sustain the proper dynamics of migrating neuroblasts and their faithful arrival in the target regions. Neuroblasts in the postnatal brain not only migrate into the OB but may also deviate from their normal path to migrate to a site of injury induced by a stroke or by certain neurodegenerative disorders. In this review, we will focus on the intrinsic mechanisms that regulate long-distance neuroblast migration in the adult brain and on how these pathways may be modulated to control the recruitment of neuroblasts to damaged/diseased brain areas.Neuronal migration is a fundamental brain development process that allows cells to move from their birthplaces to their sites of integration. Although neuronal migration largely ceases during embryonic and early postnatal development, neuroblasts continue to be produced and to migrate to a few regions of the adult brain such as the dentate gyrus and the subventricular zone (SVZ). In the SVZ, a large number of neuroblasts migrate into the olfactory bulb (OB) along the rostral migratory stream (RMS). Neuroblasts migrate in chains in a tightly organized micro-environment composed of astrocytes that ensheath the chains of neuroblasts and regulate their migration; the blood vessels that are used by neuroblasts as a physical scaffold and a source of molecular factors; and axons that modulate neuronal migration. In addition to diverse sets of extrinsic micro-environmental cues, long-distance neuronal migration involves a number of intrinsic mechanisms, including membrane and cytoskeleton remodeling, Ca Neuroblasts are immature cells of neuronal lineage that are highly motile and migrate to target brain regions from their birthplaces to become neurons and integrate into neural networks. They are polarized cells, with a long leading and a short trailing process. During cell migration, neuroblasts do not migrate continuously and have saltatory movements with migratory periods intercalated by stationary phases  of the dentate gyrus in the hippocampus  than in superficial and deep layers of the EGL  and may react to netrin-1 expressed by mitral cells in the OB  promotes neuronal migration in the RMS  complex , Ras-related C3 botulinum toxin substrate 1 (Rac1), and cell division cycle 42 (Cdc42) are particularly important in controlling actin network remodeling during cell migration Luo, . The expMicrotubules are the second cytoskeleton component involved in regulating cell migration. Microtubules are hollow filaments composed of α-tubulin and β-tubulin heterodimers  are a large family of proteins involved in different cellular functions. These proteins are grouped into different subsets such as type III, which includes vimentin and glial fibrillary acidic protein (GFAP), type V, which includes laminin family proteins, and type VI, which includes nestin  or by tyrosine receptor kinase (TRK) activation. These receptors may activate phospholipase C (PLC), which cleaves phosphatidylinositol 4,5 bisphosphate (PIP2) into 1,4,5-inositol triphosphate (IP3) and diacyl-glycerol (DAG)  to the plasma membrane of the leading edge of migrating cells  and fusion (elongation)  during the ATP production process , which is phosphorylated when the ATP/ADP ratio decreases  clusters around blood vessels are found in the ischemic penumbra   that need to be integrated at the cellular level. These factors modulate cell behavior and trigger intracellular processes affecting cell migration. Cytoskeleton remodeling, mitochondria dynamics, ATP production, autophagy, and CaCB and AS wrote the review article. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.