Retinol binding protein 4 (RBP4) is the specific transport protein of the lipophilic vitamin A, retinol, in blood. Circulating RBP4 originates from the liver. It is secreted by hepatocytes after it has been loaded with retinol and binding to transthyretin (TTR). TTR association prevents renal filtration due to the formation of a higher molecular weight complex. In the circulation, RBP4 binds to specific membrane receptors, thereby delivering retinol to target cells, rendering liver-secreted RBP4 the major mechanism to distribute hepatic vitamin A stores to extrahepatic tissues. In particular, binding of RBP4 to ‘stimulated by retinoic acid 6’ (STRA6) is required to balance tissue retinoid responses in a highly homeostatic manner. Consequently, defects/mutations in RBP4 can cause a variety of conditions and diseases due to dysregulated retinoid homeostasis and cover embryonic development, vision, metabolism, and cardiovascular diseases. Aside from the effects related to retinol transport, non-canonical functions of RBP4 have also been reported. In this review, we summarize the current knowledge on the regulation and function of RBP4 in health and disease derived from murine models and human mutations.  Rbp4 gene ). Injecting radio-labeled retinol and subsequently extracting retinol that had been bound to a carrier protein from plasma led to the discovery of RBP4 [The term vitamin A refers to retinol and its bioactive derivatives, which are known to mediate a plethora of physiological functions. Essential for humans , vitamin of RBP4 . Human R of RBP4 ,8. The s of RBP4 . Within  of RBP4 ,11. WithThe major part of retinoids (~80%) is stored in the liver, whereas other organs usually contain some, but overall much lower amounts when compared to the liver . ConsistRBP4 gene that are accountable for the high hepatic transcription have been identified, although the exact characteristics of these regulators remain to be clarified [Rbp4 mRNA expression in murine hepatocytes [Rbp4 mRNA expression [Rbp4 mRNA expression upon fasting was also detectable in mice lacking one of the hepatic fasting master-regulators, peroxisome proliferator-activated receptor α (PPARα) [Genomic elements in the 5’ flanking region of the human larified ,21,22. Tlarified ,24. Notaatocytes . Anotherpression ,24. The  (PPARα) , indicat (PPARα) .trans retinoic acid (atRA) and 9-cis RA were found to be potent inducers of Rbp4 gene expression in vitro (Hepa 1–6 cells) and in vivo (murine liver) [Rbp4 in adipose tissue but not in the liver, while repressing hepatic RBP4 protein and inducing serum RBP4 levels [Changes in the dietary supply of vitamin A—in form of sufficiency, depletion, and repletion—were shown not to alter mRNA expression or protein translation of RBP4 . Notablye liver) . Another4 levels .Rbp4 mRNA was increased upon acute re-feeding [Rbp4 translation [Rbp4 have not been identified so far.Furthermore, the translation of -feeding . The samnslation . Other tHuman RBP4 was found to be shortened by one or both leucine amino acid residues at the C-terminus in patients suffering from chronic renal failure . In thisRbp4 raises the question which of these organs contribute, and to what extent, to circulating RBP4 concentrations and whether a tissue-specific origin could render RBP4 exhibiting distinct properties. Strikingly, it was found that hepatocyte-specific deletion of RBP4 in mice resulted in undetectable RBP4 levels in serum, suggesting that, at least in mice, hepatocytes are the sole origin for circulating RBP4 [RPB4 in humans circulates at 2–3 µM, while in the circulation of mice, it amounts to ~1 µM . Most ofing RBP4 . This aling RBP4  is due ting RBP4 ,38. Liveing RBP4 ,40, furting RBP4 . In facting RBP4 ,43. Anoting RBP4 . Consisting RBP4 ,35,45. Ting RBP4 ,47. ThisRBP4 is circulating not only within the bloodstream, but also in the cerebrospinal fluid ,15. HereRbp1 gene product) was shown to be required for adequate retinyl ester accumulation in stellate cells [All vitamin A is derived from nutritional uptake . The ingte cells .For storage, retinol is transacylated by lecithin retinol acyltransferase (LRAT) to form retinyl esters, with retinyl palmitate as the predominant product ,58,59,60When RBP4 was first discovered, it was already realized that circulating RBP4 is bound to another protein that was named prealbumin, later transthyretin (TTR), with a molecular weight of 55 kDa, when in its more stable tetrameric form ,66,67. ROnce secreted into the bloodstream, the complex comprising retinol, RBP4, and TTR is distributed to extrahepatic tissues. On the surface of target cells ‘stimulated by retinoic acid 6’ (STRA6) has been identified as RBP4’s main receptor to induce the uptake of retinol , as showSTRA6 displays a structural feature that allows the β-barrel core of RBP4 to access a specific loop in the receptor to facilitate retinol release . A cryst2+, extracellular binding of apo-RBP4 and retinol export in vitro [Interestingly, STRA6 can bind calmodulin, which has been shown to promote, in association with Cain vitro . The delin vitro ,82. Thusin vitro . The phyin vitro ,80,81,83To some surprise, the phenotypes of murine STRA6 loss-of-function models showed less severe phenotypes than what has been observed in humans with STRA6 mutations. These mutations are associated with multiple pathologies and referred to as the Matthew–Wood syndrome. Among the detected abnormalities of this syndrome are anophthalmia, congenital heart defects, diaphragmatic hernia, alveolar capillary dysplasia, lung hypoplasia, and intellectual disability ,85,86.In the liver, STRA6 is barely expressed . InsteadAfter delivering retinol to target cells, RBP4 remains in the bloodstream without being bound to either retinol or TTR. Having lost these partners, RBP4 is a target of renal filtration. Nevertheless, within the proximal renal tubule, the megalin-cubilin receptor complex initiates almost complete reabsorption of RBP4 and therefore its recycling, as concluded from a kidney-specific megalin knockout mouse model ,93. This3H-retinol replacement in the binding pocket of human RBP4, providing another hint to the fatty acid binding and possible transporting function of RBP4 [Ongoing research on RBP4 proposes that this protein can transport other molecules aside from retinol. In crystallographic studies, expression-host-derived oleic and linoleic acid were found in RBP4’s binding pocket . Both RB of RBP4 .The following chapter describes the most important findings of RBP4’s contributions to health and disease, and cis retinaldehyde in the eye as the light-sensitive chromophore of the rhodopsin complex [Some of the most prominent consequences of vitamin A deficiency are in regard to vision: night blindness and full blindness , as well complex  require Rbp4 locus [Interestingly, RBP4 deletion in mice on a mixed genetic 129xC57BL/6J background and fed a normal chow diet induced visual defects  only in the first four to five months after birth . Howeverp4 locus , were nop4 locus . Moreovep4 locus . These sp4 locus ,121.Mice lacking the RBP4 cell surface receptor STRA6 displayed an ocular phenotype, including changed morphology and anatomy of the eye, but lacked major anomalies in other organ systems ,122,123.RBP4 in humans are linked to impaired vision. The compound heterozygous mutations p.I59N and p.G93D (I41N and G74D in the cleaved RBP4 protein) were found to cause night blindness and modest retinal dystrophy without effects on growth [RBP4 led to a much less effective binding of its substrate retinol while the affinity of the mutated RBP4 to STRA6 was highly increased. This in turn resulted in a disturbance of vitamin A metabolism, as its receptor preferably bound to the mutated and therefore empty carrier and thus did not allow for retinol uptake [RBP4 [RBP4 mutation produces a truncated RBP4 protein due to the introduction of an early stop codon. The encoded protein is predicted to be non-functional and the mutation is linked to the development of retinitis pigmentosa [Most of the identified mutations of n growth ,115. Then growth . Autosomn growth . In thesl uptake . Althougl uptake . Anotherke [RBP4 . Here, agmentosa . RBP4 null mutations have been identified [Vitamin A is highly relevant for embryonic development, which is also reflected in the finding that expression of the RBP4 receptor STRA6 is strongly induced during certain stages of embryogenesis ,76. Untientified . This maentified  and withentified . Howeverentified . These pentified  and retientified ,128,129 entified , the priOverall, the sensitivity of rodents and humans to alterations in retinoid homeostasis and vitamin A availability may differ substantially, as humans are much more susceptible to retinoid toxicity because of uncontrolled retinoid diffusion , which mRbp4 expression is upregulated in adipose tissue of obese and diabetic mice and humans, while the insulin-sensitizing thiazolidinediones lowered elevated RBP4 serum levels [RBP4 mRNA expression, visceral fat mass, and insulin resistance in humans [RBP4 were reported to be associated with metabolic diseases and related risk factors, including the risk of hypertriglyceridemia in Chinese Hans [It was shown more than 20 years ago that type 2 diabetic patients exhibited increased levels of RBP4 in their blood ,133. In m levels . Importam levels . A hypotn humans . Anothern humans . Howevern humans . Thus, Rn humans . Regardiese Hans , increasese Hans , risk ofese Hans , higher ese Hans , elevateese Hans , and typese Hans ,142,143.+ T-cells in adipose tissue, thereby promoting an inflammatory response, impairing insulin signaling and eventually leading to insulin resistance [So far, a variety of different mechanisms for how elevated RBP4 in the circulation may interfere with insulin sensitivity have been reported . The inisistance ,145,146.sistance ,145, sumAnother group suggested an alternative mechanism linking elevated RBP4 to insulin resistance, which was dependent on STRA6 and retinol , as depiA very recent study provided evidence that the growth hormone receptor promoted RBP4 expression and secretion via a phosphorylation of STAT5 in liver . MoreoveStra6 expression is especially high in β-cells. It was found that holo-RBP4 inhibits insulin synthesis via a STRA6/JAK2/STAT1/insulin gene enhancer protein-1 cascade as the underlying cause for the age-dependent decrease in glucose-stimulated insulin secretion of C57BL/6J mice with CAG promoter-driven, transgenic overexpression of human RBP4 [RBP4 was also shown to induce β-cell dysfunction . The autman RBP4 .Rbp4 expression in the adipose tissue of obese patients reported by some studies [Despite the fact that elevated levels of circulating RBP4 in insulin-resistant and type 2 diabetic subjects was reproducibly found in most studies, whether or not RBP4 is indeed causative and the exact mechanisms are still heavily debated in the field . Some di studies  and the  studies ,153. Oth studies . Moreove studies ,40, suggRbp4 [Adipose tissue shows substantial expression of Rbp4 , the funRbp4 . As descRbp4 . The autRbp4 , is currRbp4 . In vitrRbp4 . MoreoveRbp4 . The herRbp4  via, forRBP4 levels in blood are associated with cardiovascular diseases such as hypertension ,158,159.RBP4 single-nucleotide polymorphisms were linked with cardiovascular risk factors [Coronary artery disease (CAD) was also found to be associated with increased RBP4 levels ,164,165. factors . It was  factors  and prom factors . Nonethe factors ,170. A r factors . In mice factors .Not unexpectedly, chronic kidney disease (CKD) can also increase RBP4 in the circulation . ExperimRbp4 in mouse liver but not white or brown adipose tissue and elevated retinol concentrations in blood, indicative of increased mobilization of hepatic retinoid stores. Moreover, cold-exposed mice that lacked RBP4 failed to induce the thermogenic gene expression profile in their inguinal adipose tissue and failed to maintain their body temperature [Murine and human circulating RBP4 levels increase upon exposure to cold , suggestperature . This waperature . Whetherperature , which mRbp4 gene expression was found in many brain regions [To date, only few studies implicate RBP4 in the physiology of the brain and the central nervous system. Although  regions ,15,16,17 regions . These f regions .RBP4 coordinates retinol transport and mobilization of hepatic retinoid stores. Although highly homeostatic, certain conditions, delineated in detail in the previous chapters, may indeed benefit from a pharmacological reduction of RBP4 levels in the circulation or diminished retinol delivery that may associate with reduced activation of RAR/RXR-dependent pathways.Fenretinide, a synthetic derivative of retinoic acid, has antitumoral potency due to its antiproliferative and apoptotic properties in some tumor cells . FenretiFenretinide was also investigated for its metabolic effects in mice. It was observed that mice fed a high-fat diet and treated for six weeks with this retinoic acid derivative normalized their circulating RBP4 levels to those of standard chow diet-fed littermates . Both inFenretinide was also successfully used in human trials for age-related macular degeneration, where its benefits are thought to be mediated by its anti-angiogenic properties . Other dAnother compound to target RBP4 is the non-retinoid ligand A1120. This molecule interrupts the binding of TTR to RBP4 and thereby reduces circulating levels of RBP4 and retinol . The admThe compound BPN-14136 is another a non-retinoid ligand of RBP4 and a potent suppressor of the RBP4/TTR interaction, decreasing RBP4 levels in the circulation of mice . The comIndirect strategies to target RBP4 are so-called ‘transthyretin tetramer kinetic stabilizers’. Instead of being ligands for RBP4, these small molecules bind TTR and stabilize its tetrameric assembly , therebyTaken together, the number of non-retinoid compounds that lower RBP4 in circulation is growing and may lead the way to treating some of the described pathologies. Since all of these compounds reduce the concentration of RBP4 in blood, they are also referred to as RBP4 antagonists.RBP4 is the key transport protein to distribute stored retinoids from the liver to extrahepatic tissues. Significant progress has been made in our understanding of its function by characterizing transgenic mouse models and human mutations. Although alternative delivery systems for retinoids can compensate for dysfunctional RBP4, eye development and vision appear particularly reliant on the supply of retinol via RBP4. Still, many questions remain unanswered about the tissue-specific function of RBP4, alternative non-retinoid ligands, and retinol-independent effects of RBP4 on inflammatory pathways, which are likely to be addressed in future studies. These insights will help shape the therapeutic applicability of RBP4-modifying compounds for some of the described pathologies.


To investigate the association between geographic, clinical, socioeconomic factors and delayed management of pediatric testicular torsion (TT) in West China.A retrospective study was conducted on TT at Children’s Hospital of Chongqing Medical University in West China from November 2004 to December 2020. Univariate analysis and logistic regression analysis were conducted to determine the association between these factors and delayed management of TT.A total of 301 cases were included in this study. The misdiagnosis rate of TT in primary, secondary healthcare units and tertiary hospitals was 93.8, 71.1, and 8.9%, respectively. Approximately 26.9% of TT boys received timely surgical management (within 12 h from symptoms inset to surgery). Logistic regression analyses suggested the following factors were associated with delayed repair of TT:P = 0.001), with a history of symptoms progress (P = 0.001) or former treatment (P <0.001), absence of other diagnosis (P = 0.011) and those boys living far away from the main city zones (P <0.001).age less than 6 years (Delayed surgical management for TT was more likely for boys with age less than 6 years, the absence of other diagnosis, with a history of former treatment or symptoms progress, and those living far away from the main city zone. To maximize the possibility of timely surgical management for TT, it is vital to strengthen the public awareness of TT and conduct continuously re-education and update physicians working at primary and secondary healthcare units. Testicular torsion (TT) is an urological emergency and needs prompt management. The incidence rate of TT in pediatrics is about 3.8–5.9 per 100,000, TT is not common but a time dependent urgent event , 5. An eA large number of studies reported that, worldwide, the delayed diagnosis and management of TT was quite common, and a relatively high proportion of TT patients proven to be delayed even in tertiary hospitals , 8. AaroThe aim of this retrospective study is therefore to investigate the geographic, clinical, and socioeconomic factors associated with delayed management of pediatric TT in West China. This study consecutively investigated all the surgically confirmed TT cases regarding clinical and socioeconomic factors and delayed surgical repair at the largest children’s medical center in West China from November 2004 to December 2020, and gave further evidence on the correlation between the surgical findings and the duration of symptoms onset to surgery.A retrospective review of the medical records on TT at the Children’s Hospital of Chongqing Medical University from November 2004 to December 2020 was conducted. Only surgically confirmed TTs and boys age more than six months were consecutively included in this study, these patients refused to surgery or surgically found not to be TT were not included.https://uroweb.org/guideline/paediatric-urology), surgical detorsion of TT not exceed 12 h was found to avoid infertility, and all TT cases should undergo urgent surgical exploration within 24 h from symptoms onset to diagnosis. We defined delayed management of TT as duration more than 12 h from symptoms onset to surgery, in the meanwhile, we also identified 24 h as an important duration interval.According to European Association of Urology guidelines on pediatric urology 2020 , school-age (6–12 years), and puberty (13–18 years).The extracted clinical data included the following: laterality, other diagnoses, whether having concomitant extra-scrotal symptoms, whether having symptoms progress, history of former treatment, and class of first-consultant medical establishment and diagnosis. Other concomitant diagnoses included hydrocele, cryptorchidism, inguinal hernia, and testicular microlithiasis. Concomitant extra-scrotal symptoms included abdominal pain, nausea, vomit, inguinal pain or masses, and fever. A history of former treatment defined as these patients had a first visit to a primary health care unit or their local hospital before transferring to our hospital. All clinical data were extracted or transferred from reliable medical records.http://www.cpad.gov.cn/art/2012/3/19/art_50_23706.html. The nine districts in Chongqing City were considered neighboring main city zone. Whether these patients had a medical insurance, who was their main guardians, and the education degree of guardians were asked when these patients checked in. All these messages were recorded from reliable medical records.Socioeconomic information included the following: medical insurance, whether living in poverty counties, whether living in neighboring main city zones around this largest children’s medical center, main guardians, and education degree of guardians. Poverty counties were defined based on the average annual income of the local population, as listed at In order to figure out the relationship between duration of symptoms onset to surgery and surgical findings of torsed testes, and to, further verify the duration would indicate poor clinical outcomes, we also summarized and compared the differences of surgical findings in different duration intervals. The noticed surgical findings included: direction of rotation, degree of testicular twist, type of torsion, blood supply, grade according to Arda, and outcome of detorsion and sufficient supportive treatments. Grade of Arda was defined as: grade I, sufficient bleeding, bleeding or oozing immediately; grade II, insufficient bleeding, no bleeding immediately after the incision but starting within 10 min; grade III, no bleeding within 10 min .First, after general anesthesia, the skin and tunica vaginalis of affected side were incised and the testis was reset without tension. Second, the affected testis was detorsed, 1% lignocaine was infiltrated within the spermatic cord. Third, a small incision was made in the tunica albuginea to check the blood supply, and in the meanwhile, a piece of moistened gauze with warm saline was used to cover the testicular tissue for 10–30 min. Forth, the torsed testis was re-examined for potential salvageability. A communication with the guardians during surgery was made to decide whether perform the orchidopexy or orchiectomy of affected testis and fixation of contralateral testis, if coagulative necrosis of the testicular parenchyma was observed. The abovementioned surgical findings were all noted in operation records.p-value less than 0.05 was considered statistically significant.All the statistical analyses were performed using the IBM SPSS Statistics for windows, version 25.0 . Categorical variables were presented as ratios (%) and continuous variables were presented by mean ± standard deviation (SD) and median (interquartile range). Comparisons were conducted by χ2 test or Fisher’s exact test for dichotomous outcomes and unordered multiple outcomes, by Kruskal-Wallis H test for ordered multiple outcomes. Variables that determined to be significant in the univariate analysis of timely and delayed repair of TT (≤12 h vs >12 h) were included in the logistic regression analysis. A Among the potential candidates in this 16-year period, 301 surgically confirmed TT children were included in this study. The number of TT children referred to our center greatly increased in recent 10 years, while the percentage of TT patients received timely management was not improved obviously.P = 0.093), medical insurance (P = 0.318), identification (P = 0.692) and education degree of main guardians (P = 0.146) were not different significantly among those TT children who received delayed or timely repair (Table P = 0.020), with symptoms progress (P <0.001), a history of former treatment (P <0.001), a first-treat in a primary or secondary health-care unit (P <0.001), living in a poverty county (P = 0.002), and far away from the main city zones (P <0.001) were significantly higher than those percentages among the children who received timely surgical repair of TT. In addition, age was significantly different among TT children with delayed or timely management (P = 0.030), and our data indicated that the older the age was, the lower risk of delayed management the boys suffered . From the univariate analysis of geographic, clinical, and socioeconomic factors associated with delayed surgical management of TT, laterality (2 h. FromP = 0.001), with a history of symptoms progress (P = 0.001) or former treatment (P <0.001) were significantly more likely to receive a delayed surgical management than a timely one. Patients without other diagnosis (P = 0.011) and those boys living far away from the main city zones (P <0.001) also associated with a delayed repair.All of the variables that determined to be statistically significant by the univariate analysis between TT children who received delayed or timely management were included in the logistic regression analysis Table . Boys wiP <0.001).In surgical findings, the direction of rotation and type of torsion were not significantly different between those boys who received a delayed repair or not Table . HoweverOur study investigated the possible geographic, clinical, and socioeconomic factors that may cause delayed surgical management of pediatric TT, which was rarely reported in Chinese population. This study concerned the correlation between age, identification and education degree level of guardians, former treatment, absence of extra-scrotal symptoms, and whether a progress of symptoms and the time of duration from symptoms onset to surgery, which was also rarely reported in literatures. The definition of delayed surgical management of TT was still not well established, 12 h and 24 h were the most two discussed boundary , and wheThe surgical findings in this study furtherly confirmed the delayed management of TT would lead to less activity, insufficient blood supply, and poor outcome of torsed testes after adequate detorsion and supportive treatment. These conditions were all directly associated with subsequent poor clinical outcomes and ability of testicular salvage, consistent with literature findings , 14.According to our study, the misdiagnosis rate of TT in primary, secondary and tertiary hospital was 93.8, 71.1, and 8.9%, respectively  may suffer less pain, reddness and swelling, and this may further delay the treatment. About 52.5% of children lived far away from the Chongqing main city zone, these boys were apt to have their first consultant in the local healthcare units, while the local hospitals were suffering lack of experience on the identification and diagnosis of TT, thus made the delayed surgical management of TT.Though this study had a relatively large participants in pediatric TT, there still existed several limitations. First, our results were limited by the retrospective observational design nature of the analysis. Some clinical and socioeconomic factors that may be related to delayed surgery, such as family history of TT, family economic conditions, guardians’ work, traffic conditions, were not available. Although our analysis was not full-scale, few studies have concerned these factors in such population and few studies have participants more than 300. Second, all data were drawn from the largest child specialist medical center in West China, some selective bias could not be avoided. In addition, the general public has a preference for treating in tertiary hospitals regardless of the severity of disease . Third, Only approximately 26.9% of TT children received timely surgical management (within 12 h from symptoms onset to surgery) in West China. Delayed surgical management for TT was more likely for boys with age less than 6 years, the absence of other diagnosis, with a history of former treatment or symptoms progress, and those living far away from the main city zone. The misdiagnosis rate of TT in primary and secondary healthcare units was very high. It is vital to strengthen the public awareness of TT and conduct continuously re-education and update physicians working at primary and secondary healthcare units to maximize the possibility of timely surgical management for TT.


Tribulus terrestris (TT) extracts by addressing various possible mechanisms of action in order to see whether the use of TT supplements in diabetes and diabetes complications is justified. Diabetic rats were divided into three groups: diabetic control group, TT extract with low protodioscin content group (TT-LPC) and TT extract with high protodioscin content group (TT-HPC). After twelve weeks of treatment, fasting blood glucose, insulin, LH, FSH and testosterone levels were measured. Both TT preparations reduced elevated blood glucose level. Insulin and luteinizing hormone levels were not significantly different compared with the control group; however, the FSH and testosterone levels were significantly higher in the TT-HPC group compared with the diabetic control group. The testosterone level is correlated in part with the protodioscin concentration in extracts and is probably mediated through an FSH-linked pathway.The present project aims to evaluate  Tribulus terrestris (TT) is one of the most controversial plants encountered in the literature. Several studies suggest that the administration of extracts obtained from the fruits could improve sexual performance, improve the symptoms of erectile dysfunction and increase libido in animals and/or humans [r humans ,2. On thr humans . The preData from the literature attributes supposed endogenous testosterone secretion to steroidal saponins based on favoring pulsatile LH release. However, effects like increasing testosterone levels in the body and the appearance of muscle hypertrophy, were observed mostly in preclinical studies and less in clinical ones: rodent experiments have shown rat performance increase in intense training and elevations in testosterone serum levels, but clinical trials conducted so far have had contradictory results ,5.Erectile dysfunction in diabetes has a multifactorial etiology, including metabolic, neurological, vascular and psychological components and, unfortunately, it is more resistant to treatment than erectile dysfunction of non-diabetic patients .The main purpose of this experiment was to observe the effect of TT extracts on reproductive parameters in diabetes. A positive result could have a favorable impact on male diabetics, with a decrease in libido, testosterone production and inhibition of spermatogenesis. Secondary, other mechanisms of actions that can improve diabetic complications were noted.The objectives of this study were to (i) quantify the protodioscin (PRD) content in the samples, (ii) to evaluate the antihyperglycemic effects of the extracts and (iii) to observe the effects of TT extracts on diabetic complications.Tribulus terrestris low protodioscin content extract (TT-LPC).In the first herbal drug (TT-1.2-17), the concentration of protodioscin was under the limit of quantification (<0.5 μg/g sample). This product was used in the experimental determination as the Tribulus terrestris high protodioscin content extract (TT-HPC).The second herbal drug contained 162.42 µg/g protodioscin and was used in the experimental procedure as the Forty-eight hours after STZ injection, rats exhibited diabetes-characteristic symptoms such as polydipsia and polyuria, notable for excessive water consumption and marked urination.In the healthy group, the blood glucose level remained constant for 12 weeks. Blood glucose level significantly raised in all groups after STZ injection (week 1). After the beginning of the treatment (week 2), significant differences were noticed in TT-HPC group compared with the first week. As a response to the treatment, a statistically significant lower glucose level in the TT-LPC group was noticed in the fourth week compared with the first week. In this data analysis, the values from week 0 were used as control in the same group .Post-hoc testing revealed a significant increase in body weight in the TT-HPC group starting from the fourth week . In the th week of the experiment. As seen in OGTT was performed after 16 h of fasting in the 8Tribulus terrestris extracts in alleviating germ cells destruction in diabetes should be considered. Modifications in semen morphology  were noticed in every group, with high frequency in the DC group . In the As shown in Testosterone levels were significantly lower from a statistical point of view in the diabetic control group and in the group treated with an extract with low protodioscin content compared with TT-HPC group . The norA stage 3 or 4 of opacity was not observed in any of the groups . No signDifferent studies on the efficiency of TT administration have highlighted the existence of major discrepancies in terms of treatment efficiency. It is largely documented how pedo-climatic conditions influence the chemical composition of herbal drugs  to a grePrior to our experiment, an evaluation of TT supplements found in pharmacies was conducted . For thiThe control of postprandial blood glucose concentrations is critical for the management of diabetes. Most of the diabetic complications are associated with uncontrolled glycemia through the activation of different pathways: polyol, advanced glycation end products (AGEs), protein kinase C, hexosamine pathway and enediol .The diabetogenic activity of STZ is caused by its preferential accumulation in β-cells through the GLUT2 transporter system resulting in cytotoxicity; therefore, STZ-induced diabetes is an animal model that reproduces type I diabetes and its complications, including the effects on the male reproductive system . Tribulus terrestris appears is unknown, but recent studies suggest that this action could be related to the ability of TT to inhibit α-glucosidase and α-amylase [The mechanism by which the antihyperglycemic effect of -amylase . AlthougA weight loss was observed after the first week in TT-LPC and TT-HPC groups, but we consider it to be normal, given that this experimental model is of type I diabetes.Tribulus terrestris extract in normalizing blood glucose, both basal and postprandial, has already been reported in diabetic rabbits. An improvement in insulinemia was also observed in these studies [Tribulus terrestris on glucose level may be the result of increased alpha-glucosidase and aldose-reductase activity, suggesting that the action is antihyperglycemic and not hypoglycemic. The antihyperglycemic effect has been attributed to saponins, especially to protodioscin [In the case of TT-HPC, there was a marked decrease in fasting blood glucose from the first week of treatment, as opposed to TT-LPC where the decrease was slow, the lowest value being recorded only after 8 weeks of treatment. The efficacy of  studies . The effodioscin .The marked decrease of fasting blood glucose in the case of TT-HPC compared with TT-LPC observed in our study, could be determined by the differences in the concentration of the protodioscin, but not by its antihyperglycemic action. Although protodioscin was below the limit of detection in the analytical method used, a decrease in fasting blood glucose was also observed in this case, the differences being significantly different between the values determined in the first week and those at the end of the experiment. However, the decrease was slower, only in the eighth week were the values lower than those found in the DC group. Because, in this case, the effect cannot be correlated with the activity of protodioscin, we intended to see if the effect is hypoglycemic rather than antihyperglycemic. This type of action has been reported in the scientific literature, being proposed as a mechanism for the proliferation of pancreatic beta cells, with the consecutive increase of insulin secretion and stimulation of peripheral glucose utilization ,14. ThisTribulus terrestris herbal drugs are attributed to their content in steroidal saponins, mainly furostanol saponins. Experimental diabetes has been described in the literature to produce changes in the male reproductive system [Administration of TT extracts is believed to be responsible for the increase of testosterone or testosterone precursors in the blood, which explains the increasing number of nutritional supplements available on the market. The anabolic actions of e system . It has e system . TestostThe positive effects of TT on testosterone and sexual dynamics have been attributed to steroidal saponins, but polyphenolic content must not be ignored because these compounds play an important role in chronic conditions due to their antioxidant capacity .T. terrestris extracts in improving the negative impact of diabetes on sperm morphology. Interestingly, an improvement in sperm morphology was observed in both groups treated with TT extracts. However, in the TT-HPC group, testosterone and FSH levels were significantly higher than those in the TT-LPC group. In addition, these results can be explained by the differences in the concentration of protodioscin, structurally similar to DHEA (dehydroepiandrosterone), a precursor of testosterone. Therefore, administration of a protodioscin-rich extract explains an increase in plasma testosterone levels observed in the TT-HPC group. Male infertility is associated with sperm DNA fragmentation as a result of an apoptotic process or the destruction of cells by ROS (reactive oxygen species). Impairment of the integrity of the DNA may occur in the testis, epididymis, deferential vessels or after ejaculation. In diabetes, this degradation of DNA was associated with increased ROS production in seminal plasma [Studies with animal models of STZ-induced diabetes have shown that both type 1 and type 2 DZ alter male fertility by modifying sperm specific parameters  ,19. Thesl plasma .Tribulus terrestris is not to be ignored [Oxidative stress occurs when the balance between reactive oxygen species and antioxidants is inclined towards the formation of free radicals. Secondary metabolites of TT are known to have antioxidant properties. Although the content in polyphenols is lower than in other herbal preparations, the antioxidant action of  ignored . The adv ignored . Polyphe ignored ,24.Although the antioxidant action of TT has already been studied and demonstrated, a more plausible explanation of the superior effect of TT-HPC in reducing the negative effects of diabetes on morphological parameters could be attributed to FSH growth. The direct anti-apoptotic action of FSH on germ cells and also the inhibition of DNA fragmentation has been demonstrated in both preclinical and clinical studies .The high differences noticed between the two herbal drugs are presumably related to the protodioscin content. Overall, the results obtained are in correlation with other recently published studies that have shown that TT administration in male rats has positive effects on sexual parameters but also on plasma levels of gonadotropins and testosterone . Our resAs shown in our previous study, STZ-induced diabetes can, in time, lead to the development and progression of cataract in experimental animals. However, in this study, no signs of early cataract development were observed in diabetic animals. The difference between our studies could be related to the gender dimorphism . In our Tribulus terrestris herbal drugs (fruits) were purchased from a local pharmacy and were identified at the Department of Pharmacognosy and Phytotherapy, Faculty of Pharmacy, George Emil Palade University of Medicine, Pharmacy, Science and Technology of Targu Mures, Romania. A voucher specimen of each sample (TT-1.2-17 and TT-2.2-17) was archived at the same department. Two different 13C3 from Sigma-Aldrich Corp. , testosterone from AppliChem. All the other used reagents were of analytical grade.Protodioscin was purchased from Cayman chemical, Testosterone-2,3,4-Both samples were extracted twice for 60 min, with 70% ethanol, in an ultrasonic water bath. The ethanol was evaporated in a Rotary evaporator and the extracts were concentrated until each had a concentration in total saponins of 20% . The exLC MS/MS analysis was performed using a QTOF 4600 (AB Sciex), UHPLC Flexar FX-10 (Perkin Elmer) following a previously described method . Chromatm/z 737.41, m/z 739.42 and m/z 755.42 resulted from m/z 1047.7 at a collision energy of 65V.Ionization of analytes was performed in electrospray negative mode, whereas protodioscin detection was performed by monitoring the sum of ions Prior approval was received from the ethical committee of the university and from the Sanitary Veterinary National Authority. All experiments were conducted in compliance with the Arrive guidelines and in accordance with the EU Directive 2010/63/EU for animal experiments. According to the Guidance Document on Acute Oral Toxicity Testing based on oral LD50 value which were recommended by the Organization for Economic Cooperation and Development, the extract of TT may be assigned to be class 4 (LD50 > 300–2000 mg/kg body weight).Tribulus terrestris extract with low protodioscin content group (TT–LPC)–25 mg extract/bw and Tribulus terrestris extract with high protodioscin content group (TT - HPC) – 25 mg extract/bw. An additional healthy group of ten rats was used in order to compare the evolution of healthy animals versus diabetic animals. This group was also used to obtain orientation values for different parameters measured under the study’s conditions , but these values were not included in the statistical analysis. The extracts (TT groups) or vehicle only  were administered through gavage. Glucose level, body weight and cataract development were monitored for twelve weeks. For this experiment, fifty male Wistar rats were used (between 5 and 7 months old). The animals were kept in standard conditions, in individual cages, with 12 h light/dark cycles. All animals were allowed one week of accommodation prior to the beginning of the study. During the experiment all animals received standard pellets and water ad libitum. Diabetes was induced by a single i.p. injection with 60 mg/kg streptozotocin (STZ). After 48 h, blood glucose was measured using a commercial glucometer (Accu-Check Active) and rats with blood glucose level higher that 250 mg/dL were considered diabetic and were included in the present study (ten rats were excluded from the present study). A homogenous suspension of the extracts (previously defrosted), in water, was prepared freshly every time prior to administration. Thirty animals were assigned following simple randomization procedures to three experimental groups of 10 animals each: diabetic control (DC), Oral glucose tolerance test (OGTT) was performed in the 8th week of the experiment after 16 h of fasting. Rats were orally dosed with a D-glucose solution (2.0 g/kg bw) and blood glucose concentrations were subsequently measured at 0 , 30, 60, 90 and 120 min after the oral dosing of glucose.g for 10 min at 4 °C. The serum was separated and stored at −70 °C until analysis.Specific complications of diabetes were monitored during the experiment. At the end of the study, the rats were sacrificed by isoflurane anesthesia with no chances of recovery. The blood was collected in separate vials for each determination and centrifuged at 280× After the sacrifice of the animals, the proximal caudal portion of epididymis was dissected and a sample of sperm was collected in order to evaluate the morphological characteristics .We analyzed 200 spermatozoa for each slide and changes in the shape of the head and tail were noted.The analysis was performed according to the WHO guidelines for human semen, adapted for the rat semen . The folRattus norvegicus were used  according to the manufacturer’s instructions.For both luteinizing hormone and follicle-stimulating hormone from serum, enzyme-linked Immunosorbent (ELISA) assay kits for 13C3) internal standard from the serum samples obtained from rats by a “high throughput” extraction method (protein precipitation), the resulting samples were analyzed by reverse phase liquid chromatography and detected following specific transitions in MRM MS/MS mode of the mass spectrometer after pre-ionization by a positive electrospray ionization source (ESI +). The calibration curve with testosterone was composed of 8 levels with concentrations of 50–10000 pg/mL. The calibration curve was plotted using a weighting factor of 1/y2 and the accuracy of each calibration standard was calculated. There were no standards excluded from the final curve due to the deviation from the theoretical value > 15%. Testosterone-2,3,4-13C3 was used as an internal standard.An LC-MS/MS method was used to test testosterone from rat serum samples based on a modified method previously validated (manuscript under review). After extraction of the testosterone and testosterone  to an Eppendorf tube after which 150 μL of 6% perchloric acid solution in acetonitrile was added. The mixture was vortexed for 2 min at 2000 rpm and then centrifuged at 10,000 rpm for 10 min. The supernatant was transferred to a HPLC vial to be injected into the LC-MS/MS system. The lenses collected from each rat at the end of the experiment were analyzed immediately under the microscope. The lenses were analyzed according to the method described by Geraldine et al. and were classified into four grades: 1—no opacity, 2—minor opacity, 3—mild opacity and 4—severe opacity .TM for macOS . Normal distribution was confirmed by Kolmogorov–Smirnov test. One-way analysis of variance (ANOVA), followed by post-hoc Tukey test was used for the analysis of the results obtained at the insulin, testosterone, FSH and LH determinations. Two-way ANOVA followed by post-hoc Tukey test was used for the analysis of weight and glucose level variations during the study. Chi square test was used for the determination of association between groups and morphological changes of spermatozoa. Statistical analysis was undertaken only for studies where each group size was at least n = 8 , the group size being the number of independent values not technical replicates. The results were considered significant if p < 0.05. Values obtain from the healthy group were not included in the statistical analysis.Results were expressed as mean ± SD. Statistical analysis was performed using GraphPad PrismSeveral mechanisms of action have been put forward in order to explain the effects of TT extract on diabetic rats and our results indicate that TT extracts have the potential to reduce hyperglycemia in diabetic rats. The extract with a high protodioscin content can also alleviate testosterone levels.Pharmacokinetic data are still insufficient in order to properly evaluate the absorption, distribution, metabolism and elimination of the active compounds from TT extracts.As such, further studies are required to evaluate the bioavailability of steroidal saponins in the human body in order to outline the clinical relevance.


P < 0.001 for both comparisons). The CSM, control, and PD groups did not significantly differ in terms of CSF NSE levels. A receiver-operating characteristic curve analysis was performed to assess the diagnostic value of CSF NSE levels in distinguishing ALS from CSM. The area under the curve for CSF NSE levels was 0.86. The optimal cutoff value was 17.7 ng/mL, with a specificity of 87% and a sensitivity of 80%. Hence, CSF NSE levels are elevated in ALS and are effective in distinguishing ALS from CSM.The current study aimed to evaluate whether cerebrospinal fluid (CSF) neuron-specific enolase (NSE) levels are elevated in amyotrophic lateral sclerosis (ALS) and are effective in distinguishing ALS from cervical spondylotic myelopathy (CSM). We retrospectively evaluated 45 patients with ALS, 23 with CSM, 28 controls, and 10 with Parkinson’s disease (PD) who underwent analysis of CSF NSE levels. The control group comprised patients aged above 45 years who underwent lumbar puncture because of suspected neurological disorders that were ruled out after extensive investigations. CSF NSE levels were evaluated using the electro-chemiluminescent immunoassay. The ALS group had significantly higher CSF NSE levels than the CSM and control groups ( As there are no specific tests for ALS, a detailed set of diagnostic criteria has been established2. However, some patients with ALS do not fulfill the clinical criteria on ALS particularly at the early stage, and they are misdiagnosed with different neurological and medical disorders3. Importantly, a misdiagnosis of cervical spondylotic myelopathy (CSM) is an important problem. ALS is most commonly misdiagnosed as CSM5. Patients with ALS present with focal muscle weakness and atrophy without bulbar symptoms at the early stage of the disease6, which is similar to cervical spondylosis (CS). Among CS, ALS characterized by lower limb spasm but without radicular pain might be easy to distinguish from cervical spondylotic radiculopathy, but not from CSM. Further, a misdiagnosis of CSM may lead to unnecessary surgery8 and subsequently more rapid deterioration because some patients with ALS experience accelerated disease progression after operation10. A novel tool is required to differentiate ALS from CSM.Amyotrophic lateral sclerosis (ALS) is a progressive and fatal disease characterized by the neurodegeneration of both upper and lower motor neurons. The pathogenesis of the condition is unclear, and its diagnosis is made clinically11. The intraneuronal NSE is secreted into the extracellular space after substantial neuronal damage. However, NSE is not physically secreted. Therefore, an elevated CSF NSE level mainly reflects neuronal damage12. In fact, this phenomenon is observed in different conditions associated with central nervous system damage, such as traumatic brain injury13, traumatic spinal cord injury14, acute brain infarction16, Parkinson’s disease (PD)17, Alzheimer’s disease18 multiple system atrophy19, bacterial meningoencephalitis20, and Creutzfeldt–Jakob disease21. However, thus far, there have been no reports, at least those written in English, about CSF NSE levels in ALS.Neuron-specific enolase (NSE) is a glycolytic enzyme predominantly observed in neurons and endocrine cellsTherefore, this study investigated whether CSF NSE levels are elevated in ALS and whether they are a useful biomarker for distinguishing ALS from CSM.P < 0.001).The characteristics of the four groups at time of CSF sampling are shown in Table P < 0.001) and control  groups  and the control group . The CSF NSE levels did not differ between the CSM and control groups. In a subgroup analysis of women, the ALS group had significantly higher CSF NSE levels than the control group (P = 0.001). Moreover, the ALS group had higher CSF NSE levels than the CSM group (P = 0.133) although the results did not significantly differ possibly due to the small sample size. An ROC curve analysis was performed to assess the diagnostic value of CSF NSE levels in distinguishing ALS from CSM  had significantly higher CSF NSE levels than the CSM . Moreover, the CSF NSE levels were significantly higher in patients with ALS who had an ALSFRS-R score of > 36 (22.4 ± 5.3 ng/mL) than in those with an ALSFRS-R score of ≤ 36 . There were no significant differences in terms of CSF NSE levels between patients with and without bulbar symptoms; those who had a disease duration of ≤ 12 and > 12 months; and those with and without cervical cord compression on MRI. ROC curve analysis was performed to assess the diagnostic value of CSF NSE levels in distinguishing ALS with several features from CSM. The AUCs of CSF NSE levels were 0.91 in ALS without bulbar symptoms, 0.87 in ALS with cervical cord compression, and 0.91 in ALS that do not fulfill the criteria on definite, probable, or PLS.Figure 22. Moreover, this research showed elevated CSF NSE levels in ALS via the subgroup analyses of male and female patients and those aged < 70 and ≥ 70 years, which were performed to control the confounding effects of age and sex. To the best of our knowledge, this study, at least among those written in English, first showed elevated CSF NSE levels in ALS. Another main finding is that CSF NSE levels are higher in ALS than in CSM; therefore, they are useful in distinguishing ALS from CSM. In relation to the finding, the CSF NSE levels were not elevated in CSM. In the literature, the CSF NSE levels in CSM are controversial. That is, a previous report showed high CSF NSE levels in CSM23. Meanwhile, another revealed normal levels24. Taken together, CSF NSE levels in CSM may not be as elevated as those in ALS. Hence, they can be used to distinguish ALS from CSM.The primary finding of this study is that CSF NSE levels are elevated in ALS. Based on a previous study, they are influenced by age and sex12. First, the difference in CSF NSE levels may reflect different degrees of neural damage. A widespread and aggressive neural damage in ALS can result in significantly elevated CSF NSE levels. However, a limited and non-aggressive neural damage in CSM may not. Second, differences in CSF NSE levels can reflect varying affected areas. CSM patients have degenerations in the anterior horn cells of the spinal cord. On the other hand, ALS patients have degenerations not only in the anterior horn cells of the spinal cord but also the Betz cells of the brain cortex and the motor nuclei of the brainstem. Notably, the brain cortex, which could be involved in ALS but not in CSM, has been reported to have high NSE levels25. Third, differences in CSF NSE levels may reflect varying pathologic processes. Although NSE is expressed in neuronal cells and is secreted into the extracellular space after substantial damage of neuronal cells as mentioned previously, NSE has been reported to be included in not only neuronal cells but also microglia26, astrocytes27, and oligodendrocytes28. Moreover, NSE expression and activity are markedly increased in neuronal and glial cells under several pathologic processes. That is, NSE may not be a non-specific marker of neural damage, but may play a role in several pathologic processes in which not only neuronal cells but only glial cells may be involved, and the mechanism might occur in ALS. In fact, NSE has been increased and involved in pathologic processes such as neuroinflammation, particularly in the expression of pro-inflammatory cytokines and the proliferation of inflammatory glial cells29. In addition, the importance of neuroinflammation in ALS has been reported30. However, specific pathologic processes related to elevated CSF NSE levels in ALS is not addressed. Moreover, the pathologic process related to the elevation of CSF NSE levels might not be triggered by toxic factors in the CSF of patients with ALS. Askanas et al. treated cultured rat motor neurons with CSF from patients with ALS and measured the NSE levels in the neurons primarily as an index of neuronal health31. The authors found that treatment with CSF from patients with ALS did not alter NSE levels in cultured rat motor neurons; thus, they concluded that the study failed to demonstrate the presence of toxic factors in the CSF from patients with ALS that would influence rat motor neurons. We further speculate that they also failed to demonstrate the presence of toxic factors in the CSF from patients with ALS which would trigger pathologic changes related to increased NSE expression in rat motor neurons.There are several explanations why the ALS group had higher CSF NSE levels than not only the control but also CSM groups, even though NSE is generally a non-specific marker of neural damage32. Moreover, asymptomatic disk disease and spondylosis of the cervical spine in MRI imaging were reported to be frequent in individuals of middle of old age33. The findings are also consistent with our results that one-half of patients with ALS had cervical cord compression on MRI regardless of symptomatic or asymptomatic. Subsequently, the misdiagnosis of CSM has been reported to be frequent among patients with ALS5, and concomitant ALS might be missed in those with CSM. In terms of treatment approaches, a misdiagnosis of CSM in patients with ALS is crucial because it may lead to unnecessary surgery and subsequent more rapid deterioration as mentioned in introduction. Notably, overlooking concomitant ALS in patients with CSM might be also crucial. Surgical treatment should be carefully considered in patients with CSM and concomitant ALS, even in those with symptomatic CSM and concomitant ALS. Although surgery for CS results in temporary alleviation, the major cause of motor symptoms is usually attributed to ALS in those patients and the motor symptoms might deteriorate more rapidly in some cases32. Therefore, CSF NSE level is a useful marker to determine the presence of ALS without the potential influence of CSM.Spinal MRI is a useful but insufficient tool for distinguishing ALS from CSM. The reason is that ALS patients must frequently exhibit degenerative disk disease and spondylosis of the cervical spine in MRI imaging regardless of symptomatic or asymptomatic, because both ALS and CSM preferentially affect individuals of middle of old age. In fact, approximately half of the patients with ALS were reported to have concomitant CSDistinguishing ALS from CSM is challenging, particularly when patients with ALS present with cervical cord compression on MRI and they do not experience bulbar symptoms and do not fulfill the criteria on ALS. In this study, patients with ALS and such features had significantly higher CSF NSE levels than those with ALS without such features, or the CSF NSE levels of the former group was as high as those of the latter group. In detail, patients with ALS who do not fulfil the criteria on definite, probable, or PLS ALS had significantly higher CSF NSE levels than those who fulfilled the criteria. The CSF NSE levels of patients with ALS with cervical cord compression was as high as those of patients with ALS without compression. Moreover, the CSF NSE levels of patients with ALS without bulbar symptoms was as high as those of patients with ALS with the symptoms. Consequently, the diagnostic values of CSF NSE levels in distinguishing ALS with such features from CSM were higher or as high as those of CSF NSE levels in distinguishing whole ALS from CSM. In cases in which patients with ALS are challenging to distinguish from those with CSM, CSF NSE can be used. Hence, it may be an effective biomarker.The reason why patients with ALS who do not fulfil the criteria had significantly higher CSF NSE levels than those who fulfilled the criteria is uncertain. However, it could be explained by a hypothesis that CSF NSE levels might decrease with disease progression at a certain stage because it could be accompanied by a decreased number of motor neurons, which might be the source of CSF NSE. Notably, the hypothesis could also explain our findings that patients with mild ALS had higher CSF NSE levels than other patients.17. In this study, while mean CSF NSE levels were higher in PD group than in CSM and control groups and lower in PD groups than in ALS group, these differences did not reach statistical significance. Because these findings may be influenced by small sample size of the patients especially with PD, further studies are needed.CSF NSE levels have been reported to be elevated in PDOur study had several limitations. First, it had a small sample size and was retrospective in nature. Hence, further large prospective studies should be conducted. Second, the control group only comprised unhealthy patients who underwent lumbar puncture, which is an invasive test, because of suspected neurological disorders that were ruled out after extensive investigations.CSF NSE levels are elevated in ALS. Further, they can effectively distinguish ALS from CSM and prevent the misdiagnosis of CSM in patients with ALS. Thus, unnecessary surgery and subsequent rapid deterioration may be prevented. Notably, numerous physicians including those in general medical institutions can benefit from the use of this biomarker in daily clinical practice because NSE is a common tumor marker for diseases including small lung cancer and can be measured in general medical institutions. In addition, because elevated CSF NSE levels in ALS may reflect a specific pathologic process, this finding could provide new perspectives regarding the understanding of ALS pathogenesis and could facilitate the development of appropriate treatments.2. Since the criteria include EMG findings of fibrillation potentials, positive sharp waves, large motor unit potentials, reduced interference pattern, and unstable motor unit potentials, we performed EMG in all ALS patients and carefully check the presence or absence of these findings. The diagnosis of ALS based on the criteria requires the absence of electrophysiological or pathological evidence of other disease processes that might explain the signs of lower and/or upper motor neuron degeneration. Therefore, we performed nerve conduction study in all ALS patients. The diagnosis also requires the absence of neuroimaging evidence of other disease processes that might explain the observed clinical and electrophysiological signs, we performed brain MRI in all but one ALS patient in whom we performed brain CT at least, and cervical MRI in 21 of the 41 ALS patients. Those with ALS and concomitant CSM were classified under the ALS group. CSM was diagnosed based on the presence of myelopathic symptoms, such as limb numbness, problems with fine motor skills, and gait disturbance, and radiologic cervical cord compression in the stenotic canal, which is correlated with the patients’ symptoms. The control group comprised patients aged above 45 years who underwent lumbar puncture due to suspected neurological disorders that were ruled out after extensive investigations. As NSE was reported to be a biomarker of PD17, we included a group of 10 PD patients. Not only controls and patients diagnosed with ALS, CSM, and PD at the time of CSF sampling but also those diagnosed at a later time (up to February 2021) were included. Patients with other concomitant neurological or neuromuscular disorders were excluded.We retrospectively evaluated 45 patients with ALS, 23 with CSM, 10 with PD, and 28 controls who were admitted to Osaka Medical and Pharmaceutical University Hospital and who underwent lumbar puncture and subsequent analysis of CSF NSE levels from January 2014 to January 2021. Patients were diagnosed with definite, probable, or probable laboratory-supported (PLS) ALS according to the revised El Escorial criteria34.Information about age, sex, disease duration, neurological symptoms, disability, spinal MRI findings, and ALS categories according to the El Escorial criteria were collected at the time of CSF sampling. Based on the presence of a concave defect in the cervical cord caused by the impingement of the disc or osseous material on MRI regardless of defect degree and its symptoms, cervical cord compression was considered Fig. . DisabilThis study was conducted according to the 2013 Helsinki Declaration, and the Osaka Medical and Pharmaceutical University Ethics Committee approved the study protocol and the need for informed consent was waived because this was a retrospective study and the data were collected without individual patient identifiers .CSF samples were collected via lumbar puncture. Then, they were immediately brought to the laboratory for analysis. CSF NSE levels were evaluated using the electro-chemiluminescent immunoassay performed by SRL . The detection limit was 0.1 ng/mL.P value of < 0.05 was considered statistically significant. All analyses were performed using the JMP software version 15.0 .The Mann–Whitney U test was used to assess differences in continuous variables between two groups. The Kruskal–Wallis test, followed by the Dunn’s multiple comparison test, was utilized to evaluate differences between three or four groups. Meanwhile, the chi-square test was applied to examine categorical variables. To investigate the accuracy of biomarkers in differentiating ALS from CSM, a receiver operating characteristic (ROC) curve analysis was performed by calculating the area under the ROC curve (AUC). The optimal cutoff value was chosen using the maximized Youden index. The values were expressed as mean ± standard deviation, and a


This idea is a development of the already stablished experience with mechanical assistance devices. is a great contribution to the previous literature and suggests the anastomosis of the donor’s pulmonary trunk to the recipient’s right pulmonary artery, instead of the recipient’s pulmonary trunk, as suggested by Barnard and Losman, or to the right atrium, as Copeland published. It’s clear that this approach leaves the need of a conduit between both donor’s and recipient’s pulmonary trunks, but we reaffirm that this consists of both circulations in parallel, which has inherent risks as we wrote in our brief communication: “In Barnard’s technique for HHT, both circulations - right and left - are in parallel, which progressively turns the donor’s heart responsible for the entire blood flow and progressively reducing the activity of the recipient’s heart. This fact might lead to arrhythmias and other disastrous consequences, such as thrombus and embolus formation. Besides that, continuous dilation of the myocardium provides a favorable site for endocarditis. All these factors led to the discontinuation of Barnard’s technique.”.The paper entitled “Experience with Heterotopic Heart Transplantation in Patients with Elevated Pulmonary Vascular Resistance. Late Follow-up”Another point that should be clarified, we agree that the left ventricle is the main site of thrombus and we never stated the opposite, but we know that when both circulations are in parallel, the donated heart is responsible for both pulmonary and systemic circulations, which could lead to clot formation on the native heart as we described earlier, but not only, these could also lead to arrhythmias and infection, for example. Our intention when anastomosing the native superior vena cava to donor’s superior vena cava by an end-to-end fashion is to preserve the donor’s right ventricular function, considering that the right ventricle is a flow-dependent chamber and that the Copeland’s technique with an end-to-side anastomosis provided atrophy of this chamber, which was undesirable for our technique as in near future, after the first surgery, it will be responsible for pulmonary circulation when finished the second stage on orthotopic position, as we wrote: “Considering that the RV is a flow-dependent chamber, its function would be preserved, as it will receive all the flow from the total superior vena cava (SVC) venous return, contrasting the RV atrophy associated with the reduced flow from the side anastomosis of the original Copeland’s technique, and this benefit would be attained by the direct end-to-end anastomosis of the donor SVC to the recipient SVC close to the brachiocephalic veins , while closing the donor inferior vena cava (IVC) and the recipient SVC near to the right atrium taking care to avoid lesion of the sinoatrial node.”. suggests significant reduction of pulmonary pressure, and furthermore, your concern about coronary artery disease on the donor’s hearts has been taken in consideration since our initial idea, as some papers calls them “marginal” donor hearts, and it’s clear that those hearts promotes poor outcomes when used for heterotopic heart transplantation and the opposite occurs when using healthy hearts who does not fit in marginal criteria, as we wrote: “ The “marginal” donor hearts, which means hearts that suffered a long ischemia time and are at high risk of complications, for example: hearts who needed high inotropic support, previous cardiac arrest or arrhythmia, abnormal wall motion visualized on echocardiogram and/or an electrocardiogram suggestive of ischemia. It is worth mentioning that most donor hearts that present with those clinical scenarios are excluded for OHT donation. So, it is obvious to say that we will not use “marginal” hearts.”We read your comments about the need of sufficient decrease of pulmonary vascular resistance to perform an orthotopic transplantation and that is the core of our research, such as your own paperFinally, your concern about clinical indication and clinical practice of our proposal method, we briefly explain that it is the point of our research, and two hearts are two organs to control, which one is healthy and the other, even with regression of pulmonary pressure, is still an organ that suffered with pulmonary hypertension and its disfunction could be catastrophic. We hope to achieve satisfactory results so this method will create a window of opportunity to a select group of patients with contraindication for an orthotopic heart transplant around the world. Both history or concepts were not neglected or forgotten, but considering the brief communication format limits, all the significant contributions to this challenging pathology could not be brought forward.Thank you again for the letter and the opportunity to clarify such interesting concepts.Sincerely,


Pythium insidiosum in China.The objective of this study is to report typical clinical and laboratory characteristics of three cases of keratitis caused by  Pythium keratitis from 2017 to 2019 were included. One 45-year-old female and one 55-year-old male were exposed to river water, and one 51-year-old female was burned by ash in the eyes. All of them are of Han ethnicity. Upon slit-lamp examination, subepithelial and superficial stromal opacities were observed in a reticular pattern. After conventional treatment with antifungal agents, the clinical status worsened and therapeutic penetrating keratoplasty was performed. Unfortunately, enucleation was performed to remove all infected tissue and relieve pain. Pythium insidiosum was identified in culture and confirmed by internal transcribed spacer ribosomal RNA gene sequencing analysis. Following the systemic and local antibiotic regimens, the patients were cured ultimately and no regression of infection was observed.Three Chinese patients of Han nationality diagnosed withIt is significant for ophthalmologists and microbiologist to be alert to this eye-threatening infection, especially in patients who are resistant to antifungal treatments and with water-related exposure. Pythium insidiosum is a genus of aquatic fungal-like oomycete pathogen belonging to the kingdom Stramenopila [P. insidiosum is often found in tropical, subtropical, and temperate climates [P. insidiosum usually occurs in healthy people and poses a threat to the eyes [P. insidiosum keratitis include trauma, stagnant water exposure, and contact lens wear [P. insidiosum keratitis has been reported in several countries, including Thailand, India, China, Australia, Haiti, and Israel [P. insidiosum have been found to be under-recognized due to the rarity and limited knowledge of the disease.menopila . P. insiclimates . Human pclimates . Vasculaclimates , 3. Oculthe eyes , 4. Possens wear –8. P. ind Israel . Thailand Israel , 7. OculP. insidiosum keratitis is often misdiagnosed as fungus keratitis because they have similar clinical presentation and morphology in diagnostic scrapings. Accurate microbiological identification is based on zoospore induction. Nowadays, DNA sequencing can be used for its identification [P. insidiosum keratitis is recalcitrant to antifungal therapy because it lacks ergosterol in its cytoplasmic membrane [P. insidiosum keratitis, and combination of antibacterial medications has been proven to be an effective therapeutic regimen.fication . P. insimembrane . As the membrane . CurrentP. insidiosum keratitis have been reported worldwide, only one case of pythiosis originated from China, occurring in a 7-year-old child [P. insidiosum keratitis in adults from China that progressed to enucleation to control the infection despite intensive medical and surgical therapy.Although several cases of ld child . Here, win vivo were performed instantly. A mass of hyphae was found in the 10% KOH wet mount stained with lactophenol blue and examined via confocal microscopy. Fungal keratitis was identified, and right corneal keratectomy was performed. Empirical antifungal and antibacterial therapy was initiated including topical and systemic fluconazole, levofloxacin, and cefminox sodium. After treatment of 2 weeks, the ulcer and symptoms did not improve and the patient was admitted to our hospital. Conjunctival congestion persisted, and a central corneal ulcer with a diameter of around 6 mm that reaches deep into the stromal layer can be seen  gene sequencing with panfungal primers (ITS1/ITS4), which matched 99.23% with the P. insidiosum strain (GU137348.1). Then, the patient was adjusted with combined antibacterial treatment consisting of linezolid and azithromycin, and no recurrence of infection was observed on follow-up.A 45-year-old female of Han nationality presented to a cirneal clinic in August, 2017 with a history of pain, redness, and decreased vision in her right eye 1 week after being exposed to river water. Corneal scrapings and confocal microscopy P. insidiosum.. Three obvious protein peaks of the strain were found by MALDI-TOF-MS, which are 2094.01, 4834.62, and 9682.51 (× 103 m/z), respectively  was initiated and voriconazole was added. Twelve days after the surgery, corneal opacity was rescraped and infiltration extended deeply into the anterior chamber. A second penetrating keratoplasty, virtually limbus to limbus, and intracameral amphotericin B injection were performed sequentially. However, 10 days after the second operation, the infection still spread to the adjacent sclera and progressed to endophthalmitis. Enucleation eventually had to be done. Subsequently, the corneal cultures growing on PDA plate were identified as P. insidiosum by MALDI-TOF-MS, confirming the diagnosis of pythiosis.A 55-year-old male of Han nationality presented with irritation, pain, and hyposia in the left eye for 3 days after facial washing with contaminated river water. He went to a local ophthalmic clinic and was diagnosed with viral keratitis. After 4 days of antiviral therapy, the symptoms were not improved, so the patient was admitted to our hospital. Slit-lamp examination showed conjunctival hypertrophy and infiltrated growth into cornea about 2 mm from the nasal limbus. In the left eye, microvascular tissue hypertrophy of about 2.5 mm was seen across the corneal limbus, and hyperemia grayish-white infiltrate of cornea with a diameter of about 5 mm was observed (Fig. P. insidiosum, oomycete, is a fungal-like organism that develops hyphae similar to those found in true fungi and is found predominantly in aquatic environments. Pythium keratitis is a rare but destructive infectious disease, with most cases reported from Thailand [Thailand . RecentlThailand .P. insidiosum has been reported in other countries, only one case of P. insidiosum keratitis in a 7-year-old child was reported from China [Pythium keratitis [Pythium keratitis, but both reported cases also had a history of tap water contamination [Pythium keratitis.Although systemic infection with om China . In thiseratitis , 12, 13.mination , 14. ThePythium have some limitations. Culture identification with zoospore induction is used to confirm the pathogen [Pythium hyphae in a smear have a size of 3–10 μm, sparse septation, and perpendicular lateral branches [Pythium keratitis. However, confocal microscopy, KOH, and histopathology cannot discriminate Pythium from true fungal agents. PDA plate is a commonly available medium in most clinics and was used in our cases. The culture has been demonstrated as colorless to white with finely radiated colonies. Serological methods, including immunodiffusion and enzyme-linked immunosorbent assay, developed to detect antimicrobials to P. insidiosum have also been used to diagnose pythiosis. The immunological method have been reported to have high specificity, but the sensitivity is low with only 40% [P. insidiosum infection. In the present report, we have created a reference spectrum of P. insidiosum by MALDI-TOF-MS after ITRNS rA gene sequencing and added it to the Bruker database, which could be used to facilitate diagnosis ofP. insidiosum infection in the future. Moreover, we have compared the reference spectrum of P. insidiosum in our laboratory with the reported one, which showed high similarity [Currently, the diagnostic tests for pathogen . Howeverpathogen . KOH exabranches , 12. Hismilarity . TherefoPythium is not a fungus, antifungal reagents are not useful. In vitro susceptibility of P. insidiosum to antibacterials, including minocycline, tetracyclines, macrolides, and linezolid, has been reported and supports the use of antibacterial medications for this infection [P. insidiosum keratitis [P. insidiosum [P. insidiosum keratitis was made after enucleation surgery. Therefore, the patients were misdiagnosed and missed the best time for treatment. To our knowledge, this is the second report of P. insidiosum keratitis in Chinese and the first one originating from Chinese adults. Due to the rare occurrence of P. insidiosum keratitis, practitioners should be alert to this disease in patients exposed to river water and showing no improvement with antifungal therapy.The standard protocol for medical management of human pythiosis has not been reported. As nfection , 19. Comeratitis . One newsidiosum . In our P. insidiosum keratitis is often misdiagnosed as typical mycotic keratitis, and the use of antifungal therapy is ineffective. Therefore, this etiologic agent should be considered in cases of suspected keratomycosis with water-related exposure, particularly if the infection is resistant to antifungal treatments. The wide application of DNA sequencing and MALDI-TOF-MS methods can facilitate the early diagnosis of the disease. Moreover, pythiosis was not limited to tropical and subtropical areas and might occur widely. Furthermore, new antimicrobials need to be explored to eradicate this intriguing organism in ophthalmic practice.