The cerebellum represents a brain compartment that first appeared in gnathostomes (jawed vertebrates). Besides the addition of cell numbers, its development, cytoarchitecture, circuitry, physiology, and function have been highly conserved throughout avian and mammalian species. While cerebellar research in avian and mammals is extensive, systematic investigations on this brain compartment in zebrafish as a teleostian model organism started only about two decades ago, but has provided considerable insight into cerebellar development, physiology, and function since then. Zebrafish are genetically tractable with nearly transparent small-sized embryos, in which cerebellar development occurs within a few days. Therefore, genetic investigations accompanied with non-invasive high-resolution in vivo time-lapse imaging represents a powerful combination for interrogating the behavior and function of cerebellar cells in their complex native environment. The cerebellum—meaning the small brain in Latin language—forms an own and anatomically easily distinguishable compartment of the brain in jawed vertebrates (gnathostomes). Although small in size, it contains about half of the neurons of the entire brain, and these neurons are organized in a paracrystalline manner found in three separated layers . These lThe differentiated zebrafish cerebellum can be subdivided into three compartments Fig.  3]. The. The3]. orthodenticle 2 (otx2) and gastrulation brain homeobox 1 and 2  is juxtaposed mediated by pou domain homeobox gene 2 (pou2) [wnt family member 1 (wnt1) and a posterior half of fibroblast growth factor 8 (fgf8) expression , ezrin/radixin/moesin proteins (erm), polyoma enhancer activator 3 (pea3) or sprouty4 (spry4), and members of the engrailed (eng) transcription factor family among others. Among the signal transduction molecules, the role of FGF8 in zebrafish is well understood. Lack of its expression in the acerebellar mutant leads to the absence of a cerebellar primordium [homeobox protein a2 gene (hoxa2) in the anterior hindbrain and to delimit the expression of otx2 to the midbrain [hox gene expression, enabling its development into cerebellar tissue from the dorsal half. As a dorsal neural tube structure, patterning of the cerebellar anlage occurs in parallel from the roof plate involving signaling via bone morphogenetic proteins (BMPs) and the expression of transcription factors of the paired homeobox gene 2, 5 and 8  family. For example, injection of a function-inhibiting antibody against Pax2 impairs the maintenance of its own expression and compromises the expression of cerebellar primordium key genes wnt1 and engrailed2 resulting in failure of the embryo to establish a proper cerebellar primordium [The cerebellum is located in the anterior-most hindbrain and originates from the dorsal part of rhombomere 1 . Like in2 (pou2) –8. The oimordium . This phimordium . Thus, imidbrain . This suimordium .Fig. 2MoIn parallel to these molecular processes, morphogenetic changes occur by the inflation of the 4th ventricle during mid-somitogenesis stages  gene defines the ventricular zone [atonal homolog 1 (atoh1) delineates the rhombic lip [atoh1 paralogous genes with partially overlapping but also distinct spatio-temporal expression patterns [Importantly, the ventricular proliferation region is subdivided by the early genetic patterning events described above into two germinal zones: the ventricular zone (VZ) positioned in the ventral alar plate and the upper rhombic lip (URL) in the dorsal alar plate region Fig. b. Both olar zone , while embic lip , 17, andmbic lip . Becausepatterns –22.ptf1a expression in the VZ is prominent in neuronal progenitors that emanate from the ventral alar plate and soon delaminate from the neurogenic zone to migrate dorsally over short distances, during which they become postmitotic [ptf1a-expressing progenitors differentiate into the inhibitory neurons of the cerebellum, all of which use gamma-aminobutyric acid (GABA) as neurotransmitter namely Purkinje cells (PCs), stellate cells (StCs), and Golgi cells (GoCs) [Proliferation of neural cells in the VZ is regulated by hepatocyte growth factor (HGF) through Krüppel-like factor 8 (Klf8)-regulated expression of its receptor Met , 24. At tmitotic , 22, 25.s (GoCs) –28.ptf1a-expressing VZ, while a minor contribution from the URL to the EC population has been suggested as well [oligodendrocyte transcription factor 2 (olig2), which distinguishes them from PCs , the equivalent of deep cerebellar nuclei neurons in mammals, are also mostly derived from the  as well . These nPCs Fig. . The sizg. ptf1a , 25. Proand GoCs .Fig. 4Sceomesodermin homolog a (eomesa), RAR-related orphan receptor beta (RORβ), carbonic anhydrase 8 (ca8), voltage-gated potassium channel protein Kcnc3a, parvalbumin7 (parv7) [ca8 has been functionally investigated suggesting that it mediates survival of differentiating PCs [From 3dpf onwards, PCs together with ECs, oligodendrocytes, and astrocytic Bergman glia start to form a continuous layer of cells of roughly one cell thickness across the dorsal region of the differentiating cerebellum, with ECs being positioned ventrally at the border of this layer and reaching occasionally into the granule cell layer (GCL). To this medial layer, PCs are continuously added until about 6dpf, when a plateau with about 400 PCs is reached and the cerebellum matures , 31. Onl (parv7) , 34. Yetting PCs . Mature ting PCs . Also, oting PCs . In the ting PCs . Identifzic1 and atonal homolog 1 (atoh1) atoh1a, atoh1b or atoh1c in a partially overlapping manner starting around 18hpf [atoh1c expression in the URL, these signaling mechanisms seem to be conserved in the cerebellar primordium [wnt1 and atoh1a emigrate from the URL toward the MHB where they turn ventrally to leave the cerebellum and to populate the ventral hindbrain tegmentum [atoh1c around 24hpf leave the cerebellar primordium and differentiate into neurons of unclear identity near the locus coeruleus in the tegmental hindbrain. Yet, the early expression domain of atoh1c is not confined to the URL but located near the isthmus suggesting a function in arousal pathways [atoh1 paralogs give rise to extracerebellar structures and form tegmental neurons of different fate.Neuronal progenitors emanating from the URL express paralogs of nd 18hpf , 21, 22.nd 18hpf . As inhiegmentum . Here, tegmentum . Similaratoh1a-expressing neuronal progenitors in combination with pharmacological and genetic experiments has provided some insights into the cell biological mechanisms underlying this early URL-derived neuronal migration. The polysialylation of the neural cell adhesion molecule (NCAM) seems to be required for advancing migration, as removal of this posttranslational modification results in cease of migration close to the URL [2+ transients triggered by a pattern of neurotransmitter activity across the cerebellar primordium, which either promote or slow down migration with acetylcholine and glycine exerting a prominent role [2+ transients appear to act on microtubule trafficking to regulate intracellular cargo distribution for controlling migration [In vivo time-lapse imaging of  the URL . The speent role , 43. Intigration .neuroD1 that is often used to identify the granule cell lineage of the cerebellum [atoh1c-expressing progenitors [atoh1a but not atoh1c localizes only to the corpus cerebellum, but whether these different granule cell populations serve different functions remains to be clarified. Excitatory unipolar brush cells as URL-derived cerebellar neurons in mice have not been identified and characterized in teleosts so far [Starting at around 48hpf, the URL continues to release migratory neuronal progenitors, and their exit from proliferation is now mediated by craniofacial development protein 1 (Cfdp1) . When grrebellum , 22. Therebellum . Here threbellum . Studiesgenitors . Interess so far .Migration of these granule cells occurs glia-independent in contact with each other in chains that are held together by the adhesion molecule Cadherin2 (Cdh2). Besides regulating the coherence of this tangential migration, Cdh2 also controls the directionality of granule cell migration in a cell autonomous manner. Interestingly, Cdh2 is relocated within the membrane of migrating granule cells from the rear to the front during their forward movement likely to align the microtubule cytoskeleton and to reposition the centrosome . When reTo elucidate the functions of the zebrafish cerebellum, it is essential to understand the detailed connections of the teleostian cerebellar neurons. In zebrafish, most of the key cerebellar neurons found in mammals have been identified. Also the neuronal morphologies and overall neuroanatomical organization of the zebrafish cerebellum are reminiscent to mammals . This haThe cerebellar afferents provide the cerebellar neurons with input from neurons outside the cerebellum known as precerebellar neurons. The two types of afferent fibers to the cerebellum are the mossy fibers (MFs) and the climbing fibers (CFs) Fig. . The MFsMonosynaptic retrograde tracing with recombinant rabies viruses detected the inferior olive nuclei (ION) located in the caudo-ventral hindbrain as cerebellar presynaptic neurons . In addiIntracerebellar circuits are mainly formed by granule cells (GCs), Purkinje cells (PCs), Golgi cells (GoCs), and stellate cells (SCs) Fig. a. The glThe ML consists of PFs, PC dendrites, EC dendrites, and SCs. The ML is the layer in which one of the most prominent cerebellar synapses is formed, the PF-PC synapse, which provides excitatory input to PCs. In addition, imaging studies suggest that PFs provide input to SCs, which extend axons parallel to the PFs and in mice form inhibitory synapses with PC dendrites to modulate PF input . PFs andAs detailed above, some PC and GC subpopulation provide efferent connections to specific structures in the anterior hindbrain. Yet, the main cerebellar efferents are formed by the ECs that are located at the intersection between GCL and PCL. ECs receive input from PCs and probably GCs and project efferents beyond the cerebellum. Studies in which the genetically encoded transsynaptic anterograde tracer wheat germ agglutinin (WGA) was expressed specifically in PCs were able to identify the efferent cerebellar system in zebrafish by anti-WGA immunohistochemistry. The cerebellar efferent structures included the thalamus, the optic tectum, the octaval nuclei such as MON, DON, the reticular formation, the red nucleus, the nucleus of the medial longitudinal fascicle (nMLF), and others  Fig. 6)6). Compaolig2 and calbindin2b  in the cerebellum are expressed in specific regions, where medial ECs express olig2 and lateral ECs express calb2b [In addition, tracing of individual axonal projections of ECs revealed a regionalization of EC efferents. For example, most of the medial ECs of the cerebellum were found to project to the thalamus through tectal neuropil, whereas medio-lateral ECs projected only to the optic tectum, with axon terminals constrained to deep tectal nuclei . Among ts calb2b , 29. This calb2b . By thess calb2b , 67.Projections of ECs from the caudolateral and caudomedial cerebellum were identified to connect to the octaval nuclei in the ventral hindbrain, the projections are mostly ipsilateral and only few transmit information to the contralateral side. The ECs in the rostromedial part of the cerebellum have long ipsilateral and contralateral projections to the nMLF, to the reticular formation and only contralateral projections to the red nucleus in the midbrain tegmentum , 68. HowAlthough it may seem a paradox, the cerebellum is curiously one of the brain structures, most conserved and with highest variability throughout evolution. It emerged during the agnathan–gnathostome transition; as it is present in all jawed vertebrates, from the phylogenetically oldest or most ancient  to the most recent  living animals with an actual layered cerebellum –71. All Despite the high degree of evolutionary conservation with respect to a layered structure, the distribution and shape into three layers  differ among species. The distribution from external or marginal to internal side of the cerebellar cortex in the zebrafish is akin to that in mammals with granule cells underneath the PC layer, but a clear white matter area as in mammals is not present in teleosts . On the The Purkinje cells (PCs) in zebrafish are considered, as in all groups of gnathostomes, the main cerebellar neurons and represent the major processing information center of the cerebellum. However, this neuronal population, instead of displaying a compartmentalization of a zebra-like pattern as in mammalians , all Purkinje cells (PCs) are Zebrin-II (or Aldolase-C) immunoreactive , the zebrafish has eurydendroid cells, a scattered cell population spread across the cerebellar cortex, laying in the vicinity of PCs. These particular PC efferences in teleost fishes are considered homologous to the DCN of amniotes, since they implement the same main functions, receiving the output from PCs and sending connections to extracerebellar structures. Certainly, they could correspond to a modified version by evolutionary divergence from the original or ancestral cerebellar nucleus. Indeed, going back in the evolutionary history, cartilaginous fishes (the most ancient extant organisms with cerebellum) show a well-defined cerebellar nucleus with subdivisions , 78. Themmalians . Thus, tmmalians .Pholidapus dybowskii—[Pholidapus dibowskii [Regarding other cerebellar cell types, there are some interneurons in the amniote cerebellum that are generally lacking in fish Fig. . Basket ybowskii—, in whicibowskii .The cerebellar glial cells are also quite evolutionary conserved, as the Bergmann glia  are present in zebrafish like in the cerebellum of other vertebrate groups, and express specific markers, such as the brain lipid-binding protein (BLBP) or fatty acid binding protein 7 (FABP7), and glial fibrillary acidic protein (GFAP) . Other gThe basic circuitry of cerebellar cells is also highly preserved throughout evolution. In all groups of animals, including zebrafish, the cerebellar information is mostly processed by the PCs, the main neurons that receive directly or indirectly input from intracerebellar interneurons, and from extracerebellar or precerebellar nuclei Fig. .Fig. 8EvThe intracerebellar connections within the zebrafish cerebellar cortex are akin that in other groups, as for PCs receiving their main input from granule and stellate cells, and sending their output to the efferent cells, with the exception of a group of PCs located in the corpus cerebelli that directly project to the vestibular nuclei . InteresWith respect to the extracerebellar input information, the two main types of afferents (mossy and climbing fibers) in zebrafish are comparable to all groups of animals with a cerebellum Fig. . A thirdThe majority of afferent inputs ends on the granule cells as mossy fibers; and whose information is then indirectly transmitted to PCs. Even though, most of the precerebellar nuclei (origin of the mossy fibers) correspond to those in other cerebellated animals, two additional precerebellar nuclei present in zebrafish but not in other groups are: the nucleus valvula lateralis and the nucleus paracommissuralis. Moreover, the exceptional large size of the cerebellum in some animals implies further cerebellar connections, such as the pontine nuclei in mammals and birds, due to the lateral expansion of the cerebellum in these animals .Regarding the well-known direct connection between climbing fibers and PCs, while in mammalians, the climbing fibers reach distal branches of the PCs dendritic tree, in teleosts, they extend only until the basal dendrites. On the other hand, in basal bony fishes and in cartilaginous fishes, climbing fibers only reach the soma of PCs, reminiscent of the early developmental stages of climbing fibers in mammalians . WhetherOverall, the zebrafish cerebellum shows some particularities specific of ray-finned fishes , likely due to a partial evolutionary divergence or secondarily derived features in this group. However, most of its cerebellar scaffold, such as main cell types and their connectivity, is highly conserved compared to that in other vertebrate groups, thus prone to form part of the fundamental network of the vertebrate cerebellum. Because of the numerous genetic tools and state of the art techniques available in zebrafish, the research on the cerebellum of this model organism is valuable for addressing functional studies, to obtain a deeper understanding of the intricate operational or physiological mechanisms of the circuitry in the cerebellar system.The zebrafish cerebellum is considered to regulate body posture, to coordinate directed movements, to mediate motor learning, and to serve higher socio-emotional functions such as exploration or anxiety, analogous to conserved cerebellar functions in other vertebrates. The conserved circuitry is well designed for exerting these functions. Therefore, on the electrophysiological level, a well-functioning, fine-tuned but still highly plastic cerebellum is essential for the zebrafish, because sensorimotor behaviors need to be permanently adjusted during development, experience, environmental changes, social interactivity, and learning so that the organism can optimally adapt to changes from outside as well as self-induced changes , 100–102The main responsible neurons, which are required for the correct processing of neuronal activity within the cerebellum, are the highly conserved Purkinje cells , 105. ThThe first form is called simple spikes (SS) and they make up the majority of PC activity. Simple spikes are derived by synaptic input from excitatory granule cells at the PF-PC synapses formed at the PC-dendrites Fig. a, b or cKCNC3) result in a unique neurodegenerative disease termed spinocerebellar ataxia type 13 (SCA13) in mammals (including humans), a disease that can be genetically modeled in zebrafish [The second form is called complex spikes (CS) and they arise from synaptic input from climbing fibers Fig. b and conebrafish –123. ThiThe cerebellar layers and synaptic contacts onto Purkinje neurons are observed as early as 4 dpf . CalciumThe visuomotor processing of PCs might seem abstract, but in simplification, it compares the function in the broadest sense to a parking sensor in a modern car. Both obtain numerous complex information of two main sources but generate only a single and simple form of output. Like mentioned above, the first strong input source is represented by the granule cells (GC) and their axons—the parallel fibers—which are responsible for encoding velocity, orientation and direction of the fish, muscle activity and other motor-related context. The second source of input is the climbing fibers, which originate from the inferior olive (IO) and convey mostly sensory and visual information, which are permanently updating the brain about transient changes in motion, while hunting prey, exploring or escape maneuvers and have, therefore, strong ethological relevance. The same applies to a parking sensor which processes the direction and velocity of the car and the visual input from the camera, which recognizes objects in front of it. Like the parking sensor, healthy PCs also show constant tonic firing activity at a low frequency to show that the “system” is working and if something changes in the visual field, the frequency rises and eventually becomes a permanent high-frequency bursting activity to signal either danger or attention. Therefore, like in a parking sensor, two diverse and complex input sources with many different variables/information are pruned down and therefore simplified by PCs and reduced to a single output signal and therefore makes fast decisions possible, which can decide about life and death of the fish in dangerous situations but also about success when hunting prey or during mating.Z-direction.The localization of a PC is also important for the way how information is processed, because there exists some region and stimulus specific processing, localization and clustering across the cerebellum. This functionally defined regionalization is based on complex spike responses to specific sensory information, affecting the associated motor-related information about body and eye movements transmitted via simple spikes . The senEven though a lot of information about PCs in the zebrafish cerebellum were gathered over the last decades, they are still up for new surprising discoveries. It was commonly believed that zebrafish PCs are a homologous group of neurons, which all share the same morphology regarding their soma size and dendritic arbors, and therefore also information processing was believed to occur in all PCs in a similar way. But recently it was shown that this is not the case and that even though all PCs in the zebrafish are Zebrin-II positive and indistinguishable by genetic markers known so far , 36, theAnother electrophysiological characteristic shared between mammalian and zebrafish PCs is that both show functionally relevant bistability, that is driven by olivary input (= climbing fibers/complex spikes). Bistability means that these neurons can be found either in the tonic or bursting mode Fig. a, and thBut vice versa, it is also possible for simple spikes to actively change the impact of complex spikes during elevated motor-activity periods. Like mentioned above, during non-locomotor periods, CS activity can consistently increase or pause simple spiking for several hundreds of milliseconds in PCs. However, in PCs which fire with high simple spike rates during a motor-related task, a CS is only able to reset SS activity for a short time window (< 50 ms) before simple spikes return to their previous high frequency. The narrowing of this temporal window may serve to make finer adjustments of motor activity through very acute perturbations in network activity . BistabiCurrently, although much electrophysiological information was gathered for PCs, other cerebellar neurons of zebrafish are by far understudied. Yet their ease of access should provide valuable insight into the function of cerebellar circuitry in the future and represents a rewarding field of study, because in zebrafish molecular analysis, physiological and behavioral function can be easily combined for establishing true molecule to physiology and function relationships directly in vivo.Compared to cerebellar research in rodents and birds, investigations on the zebrafish cerebellum are still immature and knowledge about teleostian cerebellar development, physiology, and function is fragmented. Yet, the past years of zebrafish cerebellar research have laid a solid foundation for further studies. Moreover, the powerful combination of molecular manipulation with high-resolution in vivo imaging, electrophysiology and behavioral analysis possible in this model allows for contributing significant and unique insights into cerebellar properties and functions. Of note, the contributions of the cerebellum to vertebrate brain function and information processing are far from being understood and the traditional view about the cerebellum mainly controlling the smooth execution of movements is under revision since some time, ascribing the cerebellum a much larger portfolio of responsibilities in the central nervous system than previously thought. Studies on the zebrafish cerebellum could not only complement understanding of cerebellar development and function but also serve to model cerebellar diseases, to elucidate disease-causing cell biological mechanisms, and provide genetic in vivo models for developing therapeutic strategies by means of genetic interference or pharmacological compound testing and validation. Therefore, cerebellar research in zebrafish is a promising research field for new generations of neurobiologists.


The baseline Cobb degree negatively correlated (p = 0.0459) with the preoperative SST-LI. The plasma SST-LI significantly increased in fraction 3 compared to the baseline (p < 0.05), and significantly decreased thereafter (p < 0.001). In contrast, in herniated disc operations no SST-LI changes were observed in either group. The VAS decreased after surgery both in the traditional  and microdiscectomy groups . More extensive and destructive scoliosis surgery might cause greater tissue damage with greater pain (inflammation), which results in a significant SST release into the plasma from the sensory nerves. SST is suggested to be involved in an endogenous postoperative analgesic (anti-inflammatory) mechanism.Somatostatin (SST) released from capsaicin-sensitive sensory nerves in response to stimulation exerts systemic anti-inflammatory, analgesic actions. Its elevation correlates with the extent of tissue injury. We measured plasma SST alterations during spine operations (scoliosis and herniated disc) to determine whether its release might be a general protective mechanism during painful conditions. Sampling timepoints were baseline (1), after: soft tissue retraction (2), osteotomy (3), skin closure (4), the following morning (5). Plasma SST-like immunoreactivity (SST-LI) determined by radioimmunoassay was correlated with pain intensity and the correction angle (Cobb angle). In scoliosis surgery, postoperative pain intensity (VAS 2.) 1 day after surgery significantly increased (from 1.44 SEM ± 0.68 to 6.77 SEM ± 0.82,  Sensory neuropeptides released from the capsaicin-sensitive peptidergic sensory nerves play crucial roles in regulating various physiological functions, including tissue homeostasis, immune responses, endocrine signalling, and neural processes associated with pain and inflammation. These sensory fibres do not only transmit sensory input and pain signals to the central nervous system (afferent functions), but they also have important local and systemic efferent actions ,3,4,5,6.1–sst5) [1 and sst4 have been identified as key targets in mediating the anti-inflammatory and analgesic effects of SST [Proinflammatory neuropeptides, such as tachykinins  and calcitonin gene-related peptide (CGRP) mediate vasodilation, plasma protein extravasation, and immune cell activation in the innervated area collectively called neurogenic inflammation ,3. Moreo1–sst5) ,9,10. Ams of SST . These rWe have shown earlier both in animal models and humans that SST is released from the capsaicin-sensitive afferents in response to inflammatory processes  and tissue damage  ,13. DataIn light of the dense innervation of the spinal column and intervertebral joints by peptidergic nociceptive fibres, our study aimed to assess changes in plasma SST-like immunoreactivity (SST-LI) during scoliosis orthopaedic surgery and disc hernia neurosurgical interventions, both conducted under general anaesthesia. During these operations, we monitored the levels of SST-LI in the bloodstream to understand the dynamics of SST release in response to the surgical procedure and pain. The objectives were to establish the correlation between the plasma SST levels and the intensity of pain, as well as the extent of the tissue damage incurred during the procedures and to investigate whether the release of SST could potentially serve as a general protective mechanism during painful conditions and surgical procedures.Altogether 30 spine surgery patients undergoing orthopaedic and neurosurgical interventions were enrolled in our study, which lasted for 8 weeks. Patient numbers in the different groups and their mean ages are summarized in Orthopaedic surgery offers a treatment approach for scoliosis through the utilization of segmental fixation of the spine with Cotrel–Dubousset instrumentation. This technique provides patients with the ability to swiftly return to their normal lives and activities following the procedure. To assess the severity of spinal deformities in scoliosis, the Cobb angle is commonly employed. This measurement involves determining the sum of the tilt angles of the upper and lower end vertebrae, providing a quantitative evaluation of the magnitude of the deformity.In neurosurgery, disc hernia operations aim to alleviate nerve compression by removing small fragments of the intervertebral disc, bone, and ligaments. The primary goal of these procedures is to free the affected nerve root by eliminating any disc fragments and degenerated disc material that may be causing compression. Traditional discectomy techniques typically involve larger incisions and significant muscle retraction. However, the advent of microdiscectomy has revolutionized the treatment of herniated discs, offering a minimally invasive alternative. This procedure requires only a small incision and employs surgical glasses or a microscope to magnify the operative site, facilitating the meticulous manipulation of the nerve root. By utilizing microsurgical tools, the surgeon can operate within the confined space of the spine. This approach allows for the precise removal of the disc material causing the compression while minimizing trauma to the surrounding tissues.We assessed pain intensity using a standardized visual analogue scale (VAS). VAS is a commonly used tool for clinicians to assess the pain intensity of patients after surgery. VAS is a simple method to use, it is a straight line with two endpoints: it starts from a low intensity value  and ends with an extreme level of pain (unbearable pain sensation: 10). There is a numerical scale between the two endpoints, and the patient has to assess a value according to the pain sensation. A higher measurable value illustrates higher pain intensity, while a lower value points out less pain sensation.v/v; Abbott Laboratories, Wiesbaden, Germany). During the surgical procedure, the major opioid analgesic fentanyl was administered intravenously at doses ranging from 25 to 50 μg according to the patient’s physiological parameters.Prior to the surgical procedure, premedication was administered to the patients, involving the use of midazolam torrex  and atropine . The induction of general anaesthesia was initiated through the intravenous administration of 1% propofol  and fentanyl . Following intubation, the patients were placed on mechanical ventilation to maintain adequate oxygenation. A balanced inhalation anaesthesia approach was employed, utilizing a mixture of oxygen and medical air in a 1:2 volume ratio, along with sevoflurane at a concentration of 1.6–2%  during induction, and a maintenance dose of 10 mg every 20 min was administered intravenously. Towards the end of the operation, the effect of the muscle relaxant was reversed by administering a combination of neostigmine and atropine intravenously .For effective postoperative pain management, intravenous morphine  was administered as needed. Furthermore, intravenous doses of Tramadol , Diclofenac , and Paracetamol  were given according to pharmaceutical instructions to address pain as required after the operation.The procedures were conducted under the administration of general anaesthesia, which was augmented with the use of parenteral opioids for pain management. Prior to the surgical procedures, patients adhered to our hospital’s established surgical protocols, which involved fasting in accordance with the “nil by mouth” guideline starting from midnight on the day preceding the scheduled surgery. In both groups of orthopaedic and neurosurgical patients, blood samples were collected on five separate occasions to measure the levels of SST-like immunoreactivity (SST-LI) in the plasma. The first sample was obtained at the outset, before any surgical intervention took place (sample 1). Subsequently, additional samples were taken after the retraction of muscles, ligaments, and soft tissues (sample 2), following osteotomy or herniotomy (sample 3), at the time of closing the skin (sample 4), and the next morning at 8 o’clock (sample 5). To prevent clotting, two separate 5 mL blood samples were immediately collected in Vacutainers containing EDTA (18 mg REF 367525 and 143 I.U. REF 367674) that were maintained at a low, ice-cold temperature. Furthermore, to inhibit enzymatic degradation, 200 µL of the peptidase inhibitor aprotinin  was promptly added to the sample intended for somatostatin measurement. Following centrifugation at 1000 rpm for 5 min and subsequently at 4000 rpm for 10 min, the plasma was frozen and stored at −70 °C until further analysis. All samples were analysed under standardized conditions at the end of the study.It should be noted that previous investigations have indicated that a precise determination of SST-LI using the radioimmunoassay (RIA) requires 10 mL of blood and the subsequent acquisition of 6 mL of plasma. In cases where smaller volumes are used, the radioactivity measurements may not reliably align with the standard curve of the assay.The RIA method has been broadly used as a laboratory technique in clinical practice and research to precisely measure the concentration of different peptides and proteins. Its specificity and sensitivity allow this method to determine low concentrations of substances in biological samples . A highly specific antibody binds to the molecule, a radioactive tracer (labelled radioactive isotope) helps to measure the formed antigen-antibody complexes. After a certain incubation time, the antibodies compete with the radioactive antigen for the binding sites. Unbound antigens have to be removed from the complexes and the radioactivity of the separated complexes is measured. The higher the concentration of the substances, the lower the quantity of the radioactive labelled antigen that will be bound to the specific antibodies. After a calibration method the actual concentration of the substance can be measured. Plasma SST-LI was determined with a specific and sensitive RIA technique developed and validated by us as described earlier in detail ,13,26. Wt-test. Correlations between the plasma SST levels and other laboratory parameters were assessed by linear regression and Pearson correlation. Plasma SST-LI data were tested for outlier values using the ROUT method in all groups in an identical manner. For all statistical analyses p < 0.05 was accepted as significant.Statistical analysis was performed using the GraphPad Prism 6.0  software. Results are expressed as means ± standard errors of the mean (SEM). Comparisons between the SST levels measured at different time points within the same group were performed by repeated measures one-way ANOVA, followed by Tukey’s multiple comparison test. The VAS changes within groups were analysed by paired samples We made an assessment individually for each patient regarding the initial pain intensity (VAS 1.) and the level of pain after scoliosis surgery on day 1 (VAS 2.) using the VAS method. VAS 2. significantly increased in patients after scoliosis surgery A on day As we measured the level of plasma SST-LI activity during the operation on five different occasions, the plasma SST-LI activity significantly increased in fraction 3 compared to fraction 1. Plasma SST-LI activity peaked in fraction 4, followed by a significant drop in fraction 5, A. PlasmaIn disc hernia operations, initial pain intensity (VAS 1.) was high in both the traditional and microdiscectomy surgical groups, which is basically attributed to the nature of the original disease in these patients. Pain after the operation (VAS 2.) significantly and similarly decreased after both types of interventions A,B.Plasma SST-LI did not alter in disc herniated patients regardless of the type of the operation A,B. No sThe present pilot clinical study provides the first findings for the significant increase in plasma SST-LI during the orthopaedic scoliosis operation. However, a remarkable change in the plasma SST-LI was not detected after either a traditional or microdiscectomy type of disc hernia neurosurgical intervention performed under general anaesthesia. It is well-established that SST released from the peptidergic nociceptive fibers exhibits analgesic and anti-inflammatory effects ,7,27,28.We suggest that SST is released from the activated peptidergic capsaicin-sensitive fibres in response to tissue injury and/or inflammatory mediators and enters into the bloodstream, inducing systemic analgesic/anti-inflammatory effects. Although we have no direct evidence for this theory by our descriptive results, a broad range of earlier animal experimental data in inflammation and pain models might provide a sufficient explanation ,7,11,29.During scoliosis surgery, plasma SST-LI significantly increased, presumably in response to tissue damage throughout the whole operative procedure, but no increase was observed in disc hernia operations. Scoliosis surgery caused more extensive tissue damage and remarkable pain, as shown by the significantly increased VAS 2. values during the first postoperative day. The greater the correction degree of the spinal column (Cobb angle), the higher the pain intensity, which is explained by the more extensive and stressful surgical procedure resulting in greater damage on skeletal muscles, joints, and tendons. This triggers inflammatory processes with consequent sensory nerve activation and SST release into the circulation. The neurosurgical interventions to remove the ruptured and herniated intervertebral discs represent remarkably smaller tissue damage and sensory nerve stimulation . FurtherThe plasma SST-LI level negatively correlates with the initial Cobb angle (Cobb 1): the greater the deformity of the spinal column, the lower the plasma SST-LI level of the patients. SST might be depleted from the sensory nerves in patients with a greater deformity of the spinal column in response to continuous activation and ongoing chronic pain. Moreover, scoliosis is likely to be due to genetic defects and environmental factors , which mSST inhibits activity-dependent nociceptors, and reduces pain sensation and perception following intrathecal and epidural administration , therefoThe role of SST in pain and inflammation is complex, involving multiple signalling molecules and pathways in neuronal and immune cells. Identifying the specific role and effects of SST within the intricate network can be very challenging. When tissue damage or an inflammatory stimulus occurs, immune cells, such as macrophages and lymphocytes, are activated and release various signalling molecules including cytokines and chemokines, as well as further neuropeptides. These molecules modulate pain and inflammation by acting on immune cells at the site of injury/surgical site. Moreover, SST reduces the release of inflammatory mediators such as cytokines and histamine from immune cells ,37, and A limitation of the present research is the relatively small sample size involved in the study, which might not be well representative and conclusive for a larger population. Another limitation is that it did not thoroughly address the specific types of pain medication, including various analgesic drugs that patients had been using prior to the surgery. Therefore, it leaves a gap in the understanding and analysing the potential influence or interactions between pre-existing medication regimens and postoperative pain management strategies. Nevertheless, in the future, we aim to increase the number of patients and carry out further investigations including pharmacotherapy-based subgroup analysis.In our study we could establish a link between SST levels and the intensity of patients’ pain, as well as the extent of tissue damage resulting from medical procedures. Further data could help to understand the multifaceted role of SST in inflammation and pave the way for its potential clinical implementation as a valuable intervention in the field of postoperative pain management.SST release into the systemic circulation presumably from the sensory nerves is likely to be a general protective mechanism during spinal surgical procedures: scoliosis orthopedic surgery and disc hernia neurosurgical interventions with extensive tissue injury, pain, and inflammation. Tissue damage and pain sensation might increase plasma SST levels in patients during surgery.The results of this study have the potential to enhance our understanding of the neurophysiological mechanisms underlying pain and tissue response during spinal operations. By elucidating the role of SST, we can potentially explore novel therapeutic approaches that target the modulation of neuropeptide signaling to optimize pain management and improve patient outcomes in the context of surgical interventions.


The aim of our study is to compare the incidence of veno-venous shunts in male varicocele and evaluate the possibility to exclude them with manual compression or/and scrotal ligation in order to carry out the procedure of retrograde sclero-embolization.In our retrospective study, all patients undergone retrograde sclerotherapy for varicocele in our Interventional Radiology Unit in the last four years were evaluated. Collaterals toward other venous shunts were identified and how many and which patients would be able to complete the procedure safely were considered.i.e., 24.17%) patients presented anatomical variants, consisting on shunting into left iliac vein (9 [9.89%]), lumbar left veins (3 [3.29%]), right iliac vein (1 [1.09%]), both iliac veins (1 [1.09%]), left femoral vein (1 [1.09%]) or a more proximal portion of the ISV itself without shunting (3 [3.29%]). Patients with duplication could benefit from a more distal injection in order to prevent back-flow; of the 19 left, nine successfully underwent sclerotherapy with manual compression or/and scrotal ligation, whereas in 10 flow through the collaterals could not be interrupted and patients were demanded for surgery.Of the 91 patients, as many as 22  may as well undergo retrograde sclerotherapy safely if compression/ligation is applied.No large previous study highlighted the impact of veno-venous shunts in technical feasibility of retrograde sclerotherapy of varicocele. Most cases occur on the left and are secondary to valvular lack or malfunctioning, although other minor causes  are advocated. The main indication for treatment is the presence of pain (at any age), infertility in adult patients, and testicular hypotrophy under 18 years of age (since a spermiogram is not obtainable in youngsters). From literature data, it is not shown as an absolute superiority between surgical and non-surgery or endovascular percutaneous treatment. Retrograde sclerotherapy represents the most available percutaneous approach, consisting in the catheterization of the internal spermatic vein (ISV) and injection of a sclerosing agent (SA) under fluoroscopy. Manual compression of the external inguinal ring or/and scrotal ligation is mandatory to avoid penetrance of the embolic agent into the pampiniform plexus, inducing inflammation and thrombosis, especially in those subjects with aberrant communicating veins from the ISV to iliac, lumbar or other venous collectors; Valsalva maneuver is mandatory as well to prevent renal reflux. Currently, no clear and univocal guidelines about the most appropriate management of patients with veno-venous shunts are available. The objective of our study is to assess the incidence of veno-venous shunts in male varicocele and evaluate the possibility of excluding them with manual compression or/and scrotal ligation, in order to perform retrograde sclero-embolization safely.Varicocele represents a common condition within the male population, arising at the adolescent age and becoming evident in the adult,We present a retrospective study on 95 patients  subjected to percutaneous retrograde sclerotherapy of varicocele within the last four years in our Interventional Radiology Department. Clinical grading of varicocele before treatment  was determined on color Doppler ultrasound according to Sarteschi classification . Indications for treatment included testicular pain and discomfort, infertility, testicular atrophy, testicular or inguinal swelling, and prophylactic treatment. For patient under the age of 16–17, spermiogram was not obtained and indication to treatment were the presence of symptoms and a grade of varicocele superior to II according to Sarteschi classification. Patient’s characteristics and demographics date, imaging with venographic anatomy details, complications, and technical aspects were recorded in Percutaneous access was chosen based on patient comfort . Ultrasound-guided percutaneous access was performed, under local anesthesia, with a 4 Fr sheath; with an angled hydrophilic guide wire  and a 4-Fr catheter (Ber or Cobra C2) through the left renal vein, the internal spermatic vein was selectively catheterized under fluoroscopic guidance. Diagnostic phlebography was performed before and during Valsalva maneuver, in order to detect any potential collateral toward other venous collectors. If no contraindications were present, a mixture (foam) of 3% atoxysclerol 4 ml (ranging from 2 to 10 ml) and air (ratio 2:8) was injected and followed by a few mL of normal saline solution (to pull the remaining sclerosant out of the catheter lumen). Injection was performed during Valsalva maneuver with patient cooperation and manual compression of the external inguinal ring (for about two-three minutes), in order to extend action of the foam in the specific site and avoid non-target embolization. Varicocele anatomy was determined according to the classification by Bahren et al and the prevalence of each variant compared with prior studies in the literature. Patients were discharged the same day and a one-month, 6 months, and 1-year follow-up with Doppler ultrasonography was scheduled.Of 95 patients, three patients were excluded from our analysis for a right-side disease with previous treatment of left varicocele; one more patient was excluded for varicocele and impaired renal drainage due to “nutcracker phenomena”.i.e., 24.17%) presented collateral vessels arising from the internal spermatic vein and shunting into the left iliac vein (9 [9.89%]), lumbar left veins (3 [3.29%]), right iliac vein (1 [1.09%]), both iliac veins (1 [1.09%]), left femoral vein (1 [1.09%]) or a more proximal portion of the ISV itself without shunting (3 [3.29%]).From a final population consisting of 91 patients with left-side-primitive disease, 22 . Of the 19 left, in nine patients sclerotherapy proved feasible with successful treatment using manual compression or/and scrotal ligation ; in the other 10 patients, the same maneuvers did not manage to stop flow through the collaterals , thus the procedure was interrupted and the patients were referred to surgical treatment. Notably, in subjects with shunts toward homolateral or/and controlateral iliac vein/veins, the above-mentioned maneuvers were often enough to stop or adequately reduce blood flow through the collaterals (9 out of 12 [75%]); this may be related to the possibility to perform a strong compression at the level of external inguinal ring (due to the low vessels-skin distance) or appropriate ligation . To the knowledge, within our cohort (22) no subjects from the treated subgroup (12) showed recurrence of homolateral varicocele at 1-month, 6-month, or 1-year follow-up ultrasound; in three patients a minimum reflus was reported during Valsalva maneuver.The incidence of collateral vessels and anatomic variants based on Bahren’s classification was established  and a co The prevalence raises from pediatric (<1% under 10 years) to adolescent subjects (15%), supporting the progressive nature of the disease; spontaneous regression is possible but infrequent. It is usually left-sided  due to the perpendicular drainage of the ISV into the homolateral renal or suprarenal vein, together with an overall longer course and greater differences in pressure. However, an analogous fashion for the right ISV  is possible. Primary varicocele, due to valvular absence or insufficiency (found in 75% of venography studies) or aberrant communicating vessels draining into alternative venous collectors, must be distinguished from secondary forms caused by external compression, as in the so-called “nutcracker phenomenon”, where the left renal vein is squeezed between the aorta and the superior mesenteric artery. Other potential mechanisms include transient increases in arterial blood blow overcoming venous capacity of the PP, involution of collateral draining veins (“ontogenetic etiology”), a higher concentration of nitric oxide (vasodilator), altered motility of the fasciomuscular tube around ISV or atrophy of collagen within the wall of the spermatic cord. Most of the varicoceles are asymptomatic, with only scrotal or inguinal discomfort or pain, worsened after long periods of standing, with an incidence of 2–10% in adults, whereas adolescents are usually diagnosed incidentally. The main issue is actually infertility, reported in 5 to 20% of the male population and most evident in adult age. Reduced sperm count, motility, morphology, and alternative forms  are described as a result of testicular hypotrophy and altered spermatogenesis. Many mechanisms have been advocated, from increased scrotal temperature (due to stasis and reflux of warm blood) to excessive production of reactive oxygen species (ROS), chronic arteriolar vasoconstriction , high concentrations of adrenal cortical hormones (with arteriolar vasoconstriction) and FSH or low concentrations of testosterone  or inhibin B (correlated with testicular volume and Sertoli cell activity), toxic substances in the refluxing blood  and autoimmunity (antisperm antibodies due to the presence of breaches in the blood-testis barrier). Dilatation of the periprostatic venous plexus (DPVP) seems to reduce sperm motility and increase seminal fluid viscosity. Physical examination (sensitivity 50–70%) is followed by scrotal ultrasound : anechoic enlarged veins (>3 mm) with reflux lasting more than one second after Valsalva maneuver or showing spontaneously are present. Indications for treatment include discomfort or pain or male infertility in case of a clinically palpable varicocele, female regular fertility or correctable infertility, or one or more semen abnormalities; correction actually improves both sperm count and motility. When dealing with adolescents, since fertility is not well predictable yet, one must consider presence of the variable symptomatic expressions of disease and the presence of testicular hypotrophy (difference in a testicular volume greater than 2 ml or 20%) and, of lesser importance, abnormal semen parameters (when available). When considering the rare right varicocele, another indication is represented by a persistent or recurrent left varicocele in the absence of evident collaterals.By the term varicocele, we mean varicosity and tortuosity of the pampiniform plexus (PP), located in the spermatic cord around the testis, secondary to retrograde blood flow from the internal spermatic vein (ISV). Retrograde sclerotherapy represents nowadays the most used, effective, and safe percutaneous technique in the treatment of male varicocele and a valid alternative to surgery. Via a common femoral, internal jugular, or humeral venous access, the spermatic vein is catheterized and its distal portion (lower edge of the ischiopubic ramus) is injected with a sclerosing agent (SA). The patient is asked to perform Valsalva (or pressure on his hypochondrium is applied) in order to avoid the reflux of the SA and a distal barrage near the external inguinal ring  is placed to prevent the scrotal veins from phlebitis. When dealing with large SV or patients with bidirectional flow due to augmented cardiac output, a temporary (about 10 min) proximal occluding balloon  can replace Valsalva maneuver, favoring prolonged contact of the SA with the vessel wall, not influenced by vessel caliber or flow or the patient’s ability to keep Valsalva  and a reduced risk of venous rupture. We employed a mixture of 2–6 ml aetoxisclerol  mixed with air to form a foam which may easily distribute throughout ISV and potential collaterals. Sodium tetradecyl sulfate (STS) and, less likely, hydroxy-polyethoxy-docanol and sodium morrhuate, represent valid alternatives to aetoxisclerol. In case of persisting reflux at post-sclerotherapy phlebography coils or glue should be used. Embolization through these alternative techniques is widely validated but possible complications must be well known by the operator: coil may migrate or cause venous dissection or perforation and detectable coils are preferred; cyanoacrylate glue may also be employed in case of post-surgical recurrences but frequent complications are fixation of the delivery catheter, migration into the pulmonary circulation or phlebitis. However, some Operators even use primarily glue and, to a lesser extent, coils or/and vascular plugs (up- and downstream [“sandwich technique”] in order to prevent both thrombophlebitis of the pampiniform plexus and reflux) for the lower recurrence rate. No significant difference in terms of pain/discomfort or complications has been reported, although glue grants lower costs, radiation exposure, and recurrence rates, being effective also in the presence of collateral vessels and without risks of migration or need for large sheaths. Again, no substantial difference was observed using different types of mechanical agents like pushable hydrogel-coated and non-coated platinum and detachable fibered coils. Sclerotherapy’s advantages over surgery are the lower risks of hydrocele and testicular atrophy (in 3–33 and 1% of varicocelectomies), associated with injuries of lymphatic and arterial vessels, respectively; its main risks are venous thromboembolism, thrombophlebitis and allergic reactions.The options available for treatment of varicocele are surgery  or endovascular scleroembolization (antegrade or retrograde sclerotherapy). and conservative therapy (NSAD and cryotherapy) usually suffices. To date, there is no evidence demonstrating the absolute superiority of one technique over the other for varicocele treatment, since technical success is around 100% in both. When undergoing sclerotherapy, however, the patient remains in observation for 4 h only, and the only recommendation is not to perform any strong effort for the first 10–15 days.Postoperative pain at 24–48 h may occur secondary to the inflammatory reaction associated with the SA (acting for 45–95 days); in general, duplications are described in 74% of cases. In particular, Bahren’s venographic classification distinguishes five types, depending on the absence of reflux (0), reflux into a single gonadal vein (I), a single gonadal vein communicating with accessory gonadal, lumbar, iliac vein or vena cava (II), a gonadal vein duplicated caudally but with a single trunk for the renal vein (III), a renal hilar/capsular vein communicating with the gonadal vein (IV) and a gonadal vein draining into a circumaortic renal vein (V). We have already reported the distribution of our patients within these categories; as previously noticed, no substantial differences are seen when compared with other studies from the literature, even considering the high variability of the number of patients included in each cohort. The presence of these aberrant communications may contraindicate sclerotherapy due to the risk of SA reflux, with these patients being often demanded to surgery.As previously described, varicocele might be caused by collateral veins draining into the iliac (3–5%), lumbar (3%), distal or capsular renal (5%), colonic vessels, or the contralateral scrotum (rarely)The presence of abnormal communicating vessels from the internal spermatic vein into other venous collectors represents a relative contraindication to retrograde sclerotherapy, due to the risk of reflux of the sclerosing agent. Manual compression or/and scrotal ligation can often reduce or stop blood flow through these collaterals. Despite the small cohort of patients included in our study , we demonstrated how shunts towards the iliac veins are the most susceptible to our maneuvers, probably due to the proximity of the anatomical region and the possibility for ligation. Thus, most of these patients may safely complete the procedure, without requiring surgery. The main limitation of the study was the small cohort of patients involved  in comparison with other cohorts (Table 3); thus, larger studies should be carried out in order to support Our findings and suggest appropriate guidelines when dealing with veno-venous shunts.


Background: To estimate associations of sulfur-containing amino acids (SAAs) in the early trimester of pregnancy and gestational diabetes mellitus (GDM) and estimate associations of maternal SAAs with adverse growth patterns in offspring. Methods: We established a 1:1 matched case-control study (n = 486) from our cohort of pregnant women, and 401 children were followed up at ages 1 to 8 years. We conducted binary conditional logistic regression to estimate the risk associations of serum SAAs with GDM. Multinomial logistic regression was implemented to explore associations of maternal SAAs with adverse growth patterns in the offspring. Results: High serum methionine and cystine were independently associated with increased GDM risk . Conversely, a low level of serum taurine was independently associated with increased GDM risk . Maternal high cystine and low taurine were also associated with an increased risk of persistent obesity growth pattern (POGP) in offspring  and the effect was largely independent of GDM. Conclusions: High serum methionine, cystine and low serum taurine in the early trimester of pregnancy were associated with a greatly increased risk of GDM. Maternal high cystine and low taurine were associated with elevated risk of offspring POGP, largely independent of GDM. Gestational diabetes mellitus (GDM) is one of the most common complications of pregnancy and has affected approximately 14.0% of pregnant women globally in 2021, according to an estimate by the International Diabetes Federation ,4,5 and Sulfur-containing amino acids (SAAs), including methionine, cysteine, cystine and taurine, play major parts in the transmethylation and transsulfuration pathways ,10. DysrChildhood obesity is one of the concerns in GDM. Undue insulin resistance, hyperglycemia during pregnancy and/or DNA methylation in the fetus are potential risk factors for obesity during childhood. A small study including 64 prepubertal children (5–9 years old) showed that the level of plasma cystine was elevated in healthy obese children while plasma cysteine was not different compared to healthy lean children . The stuWe established a case-control study from our cohort of pregnant women and its follow-up data to examine (1) associations between the serum SAAs concentrations in early trimester of pregnancy and subsequent GDM risk; (2) the associations between maternal SAAs levels in early trimester of pregnancy with obesity-related growth patterns if any, in offspring at 1 to 8 years of age; and (3) whether GDM mediates the risk associations between maternal SAAs concentrations in early trimester of pregnancy and adverse growth patterns in offspring.The design and methods of this study have been reported previously ,22. In sAt 24–28 weeks of pregnancy, all pregnant women took a 50 g 1 h glucose challenge test (GCT). Thereafter, pregnant women with positive GCT were transferred to the GDM center, where they underwent a standard 75 g 2 h oral glucose tolerance test (OGTT) for diagnosis of GDM. GDM was diagnosed based on the International Association of Diabetes and Pregnancy Study Group’s (IADPSG) criteria .During the fieldwork, 2991 pregnant women out of 22,302 participants were invited and agreed with the provision of overnight fasting blood samples in the first trimester of pregnancy. Among them, we excluded 227 pregnant women who did not have GCT results or whose GCT results were positive but did not have OGTT results. Finally, we included 243 women with GDM and 243 women free of GDM matched by maternal age (±1 year) in the current analysis.At postpartum, 486 children of the included women were asked to attend the follow-up study and were offered health examinations, including measurement of body height and weight, each year from 1 to 8 years. Finally, 401 children (a response rate of 82.5%) participated in the postnatal follow-up and completed body weight and height measurements at 1 to 8 years of age.The nurses or obstetricians with uniform training measured maternal weight, height, and systolic/diastolic blood pressure (SBP/DBP) at the first antenatal appointment and offspring height and weight at each follow-up visit aged 1 to 8 years. We also collected other demographic information on maternal age, parity, ethnicity, family history of diabetes, the habits of smoking and drinking, and child gender. We calculate BMI by dividing weight (kg) by squared height (m).Measurement of serum sulfur-containing amino acids was previously described . Brieflyt-test if normally distributed or a Wilcoxon signed-rank test otherwise. Categorical data were evaluated by the McNemar test or Fisher’s exact test.We performed all statistical analyses using SAS version 9.4 . Quantitative data were expressed as mean ± standard deviation (SD) or median (interquartile range) where appropriate. Differences in quantitative data between the two groups were tested using a paired Student p < 0.05 at entry and exit).Binary conditional logistic regression was conducted to obtain odds ratios (OR) and their 95% confidence intervals (CI) of serum SAAs  for GDM. Restricted cubic spline (RCS) analysis was employed to check the linearity of the associations between SAAs concentrations and GDM risk as before ,26. We sA group-based trajectory modelling method applied to the longitudinal BMI data was performed to identify distinct BMI growth patterns from 1 to 8 years of age . The metThe baseline characteristics of the participating pregnant women are shown in Serum methionine and cystine in the early trimester of pregnancy are positively associated with GDM risk in nonlinear manners, while serum cysteine and taurine are negatively associated with GDM risk in nonlinear manners . Serum mBased on the group-based trajectory modelling approach, four distinct BMI growth patterns were observed in 401 children who participated in the follow-up visits. The four BMI growth patterns can be described as (1) persistent lean growth pattern (PLGP) characterized by a persistent low BMI over time; (2) normal growth pattern (NGP) characterized by middle and “normal” BMI over time; (3) persistent obesity growth pattern (POGP) characterized by a high and persistent increased BMI over time; and (4) late obesity growth pattern (LOGP) characterized by a normal BMI before 5 years of age and rapidly increased BMI thereafter . CompareMaternal cystine ≥ 150 nmol/mL  is associated with markedly increased risk of POGP in the offspring in univariate and multivariate analyses . Further adjustment for GDM slightly attenuates the OR of high cystine for POGP, but its statistical significance persists . However, maternal high cystine is not significantly associated with LOGP risk in offspring. Similarly, maternal taurine ≤ 21.9 nmol/mL  is associated with an elevated risk of POGP, while low taurine is not associated with LOGP risk in offspring . Further adjustment for GDM slightly attenuated the OR of low taurine for POGP, but the statistical significance remained . On the other hand, maternal methionine and cysteine are not significantly associated with the risks of POGP and LOGP in offspring .Our study explored and confirmed that (1) serum methionine and cystine in early pregnancy were positively associated with GDM risk, while serum taurine was negatively associated with GDM risk in the Chinese population; (2) maternal high serum cystine and low serum taurine in the early trimester of pregnancy were also associated with increased POGP risk in offspring, largely independent of the occurrence of GDM.In recent years, a few animal and human studies have surveyed the risk associations of SAAs concentrations with insulin resistance and T2D, but their findings were inconsistent. For instance, an animal study found that plasma levels of methionine and cysteine were markedly decreased, and hepatic methionine, cysteine and taurine were also decreased in Zucker diabetic fatty rats . A cohorBiological links between SAAs and GDM remain unclear. SAAs play a major part in the transsulfuration pathway, which can protect against uncontrolled oxidative stress and inflammation through sulfur’s role in redox biochemistry ,31. One In recent years, much research has surveyed the BMI growth patterns in children. A large longitudinal cohort study implemented in the USA identified three distinct BMI growth patterns in children aged 2–6 years using the group-based trajectory modelling method . The thrThe associations of maternal SAAs and offspring growth patterns had not been explored. A small research implemented in the USA showed that the concentration of plasma cystine was elevated in healthy obese children while plasma cysteine was not different compared to healthy lean children . The uteIt is biologically plausible that maternal high serum cystine and low serum taurine were associated with markedly increased POGP risk in offspring. There is growing evidence that epigenetic changes have been a potential mechanistic link between exposure to the uterine environment and childhood obesity in the offspring . The intGDM can increase the risks of maternal diabetes and cardiovascular disease in later life and childhood obesity risk in offspring ,5,6, wheThere were some limitations in our study. First, the findings of this study were obtained from a case-control study, which was from a single cohort of pregnant women and their offspring in Tianjin. These findings, therefore, need to be verified in other cohorts. Second, the levels of serum SAAs were related to dietary intake, but we did not obtain detailed diet information from the mothers. Third, homocysteine is an important sulfur-containing amino acid and may play a part in GDM. However, in the targeted LC-MS/MS assay, homocysteine was not specifically included in the measurement scheme. We cannot exclude the possibility that it may have confounded associations between other sulfur-containing amino acids and GDM in this report. Fourth, we noticed that the sample size of the POGP group was small, and the 95%CI of the risk association between maternal low taurine and offspring POGP was large. Further replications by other cohorts are certainly needed to reduce the large type 2 error.In summary, our study found (1) high concentrations of serum methionine and cystine and low concentrations of serum taurine in the early trimester of pregnancy were independently associated with greatly increased GDM risk, and (2) maternal high concentrations of serum cystine and low concentrations of serum taurine were also associated with greatly elevated POGP risk in offspring, largely independent of GDM. Further studies are warranted to verify our observations, and biological mechanisms underlying the intriguing findings also need exploring for better comprehension of the pathophysiology of GDM and biological links from early life SAAs exposure to adverse childhood growth patterns.


Cardiovascular disease is a leading cause of death worldwide. Loss of cardiomyocytes that occurs during many types of damage to the heart such as ischemic injury and stress caused by pressure overload, diminishes cardiac function due to their limited regenerative capacity and promotes remodeling, which further damages the heart. Cardiomyocyte death occurs through two primary mechanisms, necrosis and apoptosis. Apoptosis is a highly regulated form of cell death that can occur through intrinsic  or extrinsic (receptor mediated) pathways. Extrinsic apoptosis occurs through a subset of Tumor Necrosis Receptor (TNF) family receptors termed “Death Receptors.” While some ligands for death receptors have been extensively studied in the heart, such as TNF-α, others have been virtually unstudied. One poorly characterized cardiac TNF related ligand is TNF-Related Apoptosis Inducing Ligand (TRAIL). TRAIL binds to two apoptosis-inducing receptors, Death Receptor (DR) 4 and DR5. There are also three decoy TRAIL receptors, Decoy Receptor (DcR) 1, DcR2 and osteoprotegerin (OPG). While TRAIL has been extensively studied in the cancer field due to its ability to selectively induce apoptosis in transformed cell types, emerging clinical evidence points towards a role for TRAIL and its receptors in cardiac pathology. This article will highlight our current understanding of TRAIL and its receptors in normal and pathological conditions in the heart. Cardiovascular disease is a significant healthcare problem and leading cause of death worldwide . Loss ofApoptosis occurs through two primary mechanisms, the intrinsic or mitochondrial and extrinsic or receptor-mediated pathways . The intIn vivo studies have not been performed and in vitro studies have been controversial with MMP inhibitors being found to both prevent cleavage and have no impact on cleavage depending on the study . TRAIL was first discovered for its homology to other TNF family members and its ability to induce apoptosis in cancer cells without affecting non-transformed cell populations . TRAIL ihe study . Both sohe study . Membranhe study . Additiohe study . The abiIn humans, TRAIL binds to two death inducing receptors, Death Receptor (DR) 4  and DR5  . Like alFor receptor binding, TRAIL forms a homotrimer that binds to high affinity death-inducing receptors, DR4 and DR5, resulting in trimerization of the receptor, causing the formation of a death-inducing signaling complex (DISC), that can then recruit the adapter protein Fas-associated death domain (FADD), which acts as an intermediate between the receptor and the pro-domain of the initiator caspase, caspase 8  Figure . DimerizSince TRAIL was first discovered for its ability to induce apoptosis in cancer cells without affecting non-transformed cell populations , TRAIL rIt is widely appreciated that activation of DR4 and DR5 in the majority of healthy cells does not result in the induction of apoptosis and emerging evidence suggests it can lead to pro-survival pathway activation such as Extracellular-Regulated Kinase (ERK) 1/2, Akt and Nuclear Factor (NF)-κB  Figure . The mecWhile TRAIL and its receptors have been implicated in cancer for decades, emerging evidence suggests a role in a multitude of different diseases including viral infection, arthritis, diabetes and cardiovascular disease . Mice laWhile TRAIL and its receptors have been implicated in cancer for decades, emerging evidence suggests a role in a multitude of different diseases including arthritis, diabetes and cardiovascular disease . TRAIL aThere have been several human studies investigating the role of TRAIL and its receptors in specific types of cardiac disease. However, many of these studies have a low number and often homogenous population of patients. Nevertheless, TRAIL, DR5 and OPG are emerging as potential predictive markers, biomarkers and relevant in the study of cardiac diseases.Arrhythmias arise from disrupted electrical conduction in the heart. Changes in conduction promote apoptosis, fibrosis and remodeling in the heart, which potentiates the disease and can lead to heart failure development  This remAtherosclerosis develops in the walls of arteries causing narrowing of blood vessels and limiting blood supply to parts of the body. This is particularly important in the heart, where CAD limits oxygen and nutrient supply to the heart and can result in myocardial infarction. In a group of diabetic patients with early-stage atherosclerosis, TRAIL was not associated with carotid or femoral intima-media thickness . In patiMyocardial infarction arises when blood flow is limited or stops in the coronary artery of the heart leading to damaged heart muscle. There have been several studies associating TRAIL and its receptors with acute myocardial infarction (AMI). High soluble DR5 and TRAIL in atherosclerotic plaques have been found to be associated with increased risk of future cardiovascular events . SeveralInterestingly, TRAIL may be a powerful biomarker for myocardial infarction patients. In a study investigating 92 biomarkers linked to cardiovascular disease or inflammation to determine which biomarkers predicted long-term all-cause mortality in patients with AMI, growth differentiation factor (GDF)-15 and soluble DR5 were identified as the most powerful biomarkers in predicting long-term all-cause mortality . In thisHeart failure can arise from several different causes including myocardial infarction, high blood pressure, arrythmias and infection and culminates in the heart not pumping enough blood to meet the needs of the body. Proteomic profiling of 80 proteins associated with cardiovascular pathology in elderly males looking at predictors of heart failure development over an 11-year span identified OPG and soluble DR5 as two of eighteen proteins as being associated with heart failure incidence . In thisThe extrinsic pathway of apoptosis is known to play an important role in the development of myocarditis-induced cardiac dysfunction. Myocarditis arises as a result of viral, bacterial, toxin, autoimmune and other causes of inflammation. As with other forms of heart failure, myocarditis can result in cardiomyocytes death through multiple forms of cell death including the extrinsic pathway of apoptosis . Very feTaken together, these clinical studies have identified changes in TRAIL or its receptors in a number of cardiac pathologies including arrhythmias, coronary artery disease, myocardial infarction, heart failure and myocarditis. However, the findings from these studies are often contradictory with some studies showing a positive association with TRAIL and its receptors with disease severity while others show an inverse correlation. Many of these studies have a limited sample size and often lack a diverse patient population. Large scale, carefully controlled studies will be necessary to decisively identify the role of TRAIL and its receptors in cardiac disease. Additionally, it is important to consider that the contribution of TRAIL and its receptors may differ depending on the type of cardiac disease. In some instances, TRAIL may serve a pro-apoptotic function in cardiomyocytes and exacerbate cardiac dysfunction whereas it in other cases it may be limiting inflammation and beneficial to disease pathology. Future mechanistic studies will be necessary to determine the role of the TRAIL receptor system in various cardiac pathologies.With the growing abundance of human evidence suggesting the involvement of TRAIL and its receptors in multiple forms of cardiovascular disease and heart failure from several etiologies, it is surprising that our understanding of the role of TRAIL and its receptors in the heart is limited. The majority of human studies have investigated circulating factors such as TRAIL, soluble DR5 or OPG, with little direct evidence of a localized role for TRAIL signaling in the heart. However, there is emerging evidence that supports direct role in regulating cardiac function and pathophysiology.DR4 and DR5 have been shown to be expressed in the healthy heart . HoweverThe majority of studies investigating the role of TRAIL and its receptors in the heart have taken a systemic approach however, it is widely appreciated that the heart is composed of many cell types . While tInformation pertaining to the cell type specific roles of TRAIL and its receptors in the heart is limited. Multiple studies have identified TRAIL receptor expression on cardiomyocytes. Membrane localization of DR5 has been shown in rodent cardiomyocytes  and mechThough studies suggest higher DR5 expression in cardiomyocytes compared with non-myocyte cell populations , cardiacThe role of TRAIL in inducing immune cell death has been extensively characterized . In partThe role of TRAIL and its receptors in endothelial cells is not well described. Human endothelial cells express DR4 and DR5 . ReportsPowerful evidence from multiple studies have suggested that TRAIL or soluble DR5 could be a biomarker for the development or progression of cardiac disease. Of particular note, in several prospective observational studies, TRAIL or its receptors were identified as being amongst the most predictive biomarkers identified . This inStudies therapeutically targeting TRAIL/DR5 in the heart are limited. Human data is confounding with some studies suggesting a protective and other pointed towards a detrimental role for TRAIL signaling in the heart. However, administration of soluble DR5 in rats with AMI to prevent TRAIL from binding to functional DRs resulted in reduced infarct size and improved markers of cardiac damage such as serum cardiac troponin I and creatine kinase-MB (CK-MB) . In a sein vivo pharmacokinetic properties have been poorly defined at this time (As mentioned, DR5 has been an attractive therapeutic target due to the long-held belief that activation of DR5 specifically induced apoptosis only in transformed cells. Over the past 30 years, numerous approaches have been taken to activate DR5 in the setting of cancer . This inhis time . Should his time . In larghis time . Should In the past decade, TRAIL, DR5 and OPG have emerged as important indicators of cardiac diseases. Current understanding of the impact of TRAIL in the heart is limited. While DR4 and DR5 are classically thought to be death-inducing receptors, in non-transformed cell populations, TRAIL often has a non-death-inducing function. Mechanistic studies have not fully defined the role of TRAIL receptor activation and function in relevant cells of the heart. Studies from rodent models show both a pro-apoptotic and beneficial role for DR5 in cardiomyocytes and studies on other cardiac relevant cell populations is limited. Additionally, more is known about the role of OPG in cardiac diseases. However, the affinity of TRAIL for OPG is lower than DR4/5 and it is currently unknown of OPG is playing a role in TRAIL signaling in the heart. Many of these studies focus on the role of OPG in RANKL signaling and fail to consider TRAIL. Furthermore, many studies concerning TRAIL focus on a single receptor and neglect to consider other receptors. Clinical correlations suggest both a protective and detrimental role depending on the study and disease. This may occur in a unique manner depending on the type of cardiovascular disease being investigated. However, it is clear that TRAIL signaling is associated with cardiac disease making further investigation warranted. Better understanding the role of TRAIL and its receptors in cardiovascular disease and how they regulate cardiovascular protection or pathology may offer new diagnostic or therapeutic approaches. Indeed, a limited number of studies have shown a promising role for TRAIL neutralization in rodent, pig and monkey models of myocardial infarction. More careful consideration of how TRAIL is regulating cardiac physiology under normal conditions and during pathophysiology and further mechanistic studies will provide insight into the potential of using TRAIL or its receptors as a prognostic or therapeutic tool in the future.


Rice is a major food crop in the world. Owing to the shortage of rural labor and the development of agricultural mechanization, direct seeding has become the main method of rice cultivation. At present, the main problems faced by direct seeding of rice are low whole seedling rate, serious weeds, and easy lodging of rice in the middle and late stages of growth. Along with the rapid development of functional genomics, the functions of a large number of genes have been confirmed, including seed vigor, low-temperature tolerance germination, low oxygen tolerance growth, early seedling vigor, early root vigor, resistance to lodging, and other functional genes related to the direct seeding of rice. A review of the related functional genes has not yet been reported. In this study, the genes related to direct seeding of rice are summarized to comprehensively understand the genetic basis and mechanism of action in direct seeding of rice and to lay the foundation for further basic theoretical research and breeding application research in direct seeding of rice. Oryza sativa L.) is a major food crop for more than half of the world’s population  during seed germination , downregulate the expression of catabolism-related genes (OsABA8ox3), and significantly increase endogenous ABA content, but also regulate water uptake by affecting the hydropore protein OsPIP1;1 during seed germination and regulation of rice seed vigor  cycle. Mutations in OsOMT reduce the levels of amino acids involved in energy production during seed germination, which may reduce energy availability and lead to low seed vigor in Osomt mutants  is a major abiotic stress that affects rice growth and ultimately decreases grain yield  is a complex trait that significantly varies among rice cultivars. To date, more than 200 QTLs have been mapped in rice . OsSAP16 encodes a stress-associated protein containing two AN1-C2H2 zinc-finger domains. After GWAS analysis of LTG using 187 accessions, OsSAP16 was identified as a candidate QTL gene. Further functional analysis indicates that a higher expression of OsSAP16 can increase LTG in rice , a class of enzymes that hydrolyzes glycosidic bonds and plays an important role in the hydrolysis and synthesis of sugars and glycoconjugates in living organisms. Glycosidic hydrolases have been reported to be involved in the degradation of cell wall polysaccharides and in controlling the loosening of plant cell walls and thus regulating germination, growth and development, fruit ripening, abscission and cell adhesion  through the IAA pathway, thereby promoting endosperm elongation  and ethylene insensitive like (EIL) genes (Kazan EIN/EIL protein binds to the promoter region of the ethylene response factor (ERF) gene to initiate expression of ERF family genes  and SNORKEL2 (SK2), play key roles in the adaptation of deep-water rice to flooded environments. SK1 and SK2 encode ethylene response factors, and ethylene activates the ethylene signaling pathway when water submergence causes an increase in ethylene content. EIN3 binds to the promoters of SK1 and SK2 to promote the expression of both, activating the gibberellin response required for stem elongation and regulating stem elongation in deep-water rice under submergence  gene encodes GA20ox, a key enzyme in gibberellin synthesis. The Sd1 protein drives the increased synthesis of gibberellins, mainly GA4, and promotes internode elongation. Sd1 expression allows rapid elongation of rice internodes after the 10-leaf stage in deep water, helping rice leaves emerge quickly and obtain oxygen  interacting protein kinase 15(CIPK15), together with a CBL Ca2+ sensor, contributes to the decoding of Ca2+ signaling  so that the seeds have sufficient carbohydrates and energy to germinate in water. In this cascade, SnRK1 kinase is the key regulator of rice energy balance  in rice. OsTPP7 encodes alglucose-6-phosphate phosphatase, which plays an important role in the metabolism of alglucose-6-phosphate. It has been shown that the OsTPP7 gene ensures an energy source by regulating the source pool balance and increasing the sugar content delivered to the embryo as well as the germinal sheath, thus enabling rice germination survival under flooding stress  suppressors SLR1 and SLRL1. SUB1A can inhibit ethylene synthesis and reduce the rate of chlorophyll degradation in plants through the jasmonic acid (JA) pathway for adaptation to flooded environments. SUB1A can induce the expression of GA2ox, which degrades GA in rice and leads to lower energy usage by suppressing rice growth , the first key enzyme to formally enter the gibberellin biosynthetic pathway, which catalyzes the conversion of geranylgeranyl diphosphate (GGDP) to the bicyclic intermediate ent-copalyl diphosphate (CDP). After the mutation of OsCPS1, the plants were unable to produce any GA. Rice seedlings exhibits reduced vigor and even die  that control mesocotyl elongation in rice, and a superior haplotype combination of OsML1 and OsML2 increased mesocotyl length by 4 cm, resulting in 85% high seedling emergence in soils with 10 cm soil cover.Mesocotyl length is another important indicator of early seedling vigor, and is the main driver of the rapid emergence of rice seedlings from the soil  biosynthetic pathway. Gy1 mutations result in the inhibition of PLA1 activity, which inhibits jasmonic acid biosynthesis and promotes elongation of the mesocotyl and radicle.Xiong et al.  cloned GOsGSK2, a key component of the Brassinosteroids (BRs) signaling pathway, regulated mesocotyl length by GWAS analysis of rice mesocotyl length. BR inhibits phosphorylation of the U-type cell cycle protein CYCU2 by OsGSK2 and ultimately promotes mesocotyl elongation.Sun et al.  demonstrOsP60-1 and OsP60-2 promote rice mesocotyl elongation by promoting microtubule protein depolymerization and stimulating an increase in endogenous GA content  is another functional gene that controls rice mesocotyl elongation. Knockdown of the OsPAO5 gene results in a significant increase in mesocotyl length and neater emergence at the early seedling growth stage, as well as an increase in yield traits, such as grain weight and grain number. OsPAO5 is a target gene with the potential to improve direct seeding of rice performance  and thus increasing amylase activity and soluble sugar content under deep seeding conditions.Rice In addition to seedling vigor, root vigor, which refers to the rapid establishment of a root system that can supply additional nutrients and water to the seedling, is also important for the success of direct seeding  for early root growth traits have been identified in rice, including those related to maximum length, thickness, number, and mass  play important roles in root development in rice. In addition to the functional genes related to root growth traits in rice, the mechanisms underlying hormones that participate in root development have also been investigated. For example, a rice mutant of the IAA biosynthesis gene Lodging is related to stem bending, breakage, and root lodging in plants (Mulsanti et al. The short stature of plants is generally believed to be the most relevant to lodging resistance in rice. For example, the short stature of rice is the main target for improving lodging resistance, as well as the harvest index in rice, which is one of the most important landmark achievements in rice improvement over the past 50 years (Keller et al. Culm diameter and thickness are other major factors contributing to lodging resistance in rice (Ookawa and Ishihara Cultivation techniques are also important factors influencing lodging resistance in direct-seeded rice (Shrestha et al. sd1, SBI, APO1, Gn1a/OsCKX2, and so on. (Asano et al. As discussed above, lodging resistance is a complex quantitative trait that is affected by many traits in rice, including culm morphology, culm diameter and length, and cellulose content. Therefore, moderate plant height, large stem diameter, thick stem walls, and high lignin deposition have been recommended as the preferred traits to improve lodging resistance in direct-seeded rice (Yadav et al. sd1 gene, which confers semi-dwarfism to rice varieties, was the first gene utilized in several breeding programs to decrease plant height and substantially improve lodging in rice (Asano et al. SBI gene could convert active gibberellins into inactive gibberellin compounds. The highly active SBI locus resulted in a significant reduction in active gibberellin content in the basal nodes of rice stalks, thereby inhibiting elongation of the basal internodes. A series of new rice varieties with a reasonable stalk node length structure and high resistance to lodging has been produced using SBI allelic mutations.The APO1 encodes an F-box protein consisting of 429 amino acids, and is the first gene cloned to improve stalk strength. In addition, APO1 can increase the number of grains per spike; therefore, APO1 can be used to improve both yield and lodging resistance in rice (Ookawa et al. Gn1a/OsCKX2 led to cytokinin accumulation in the crown root tip and accelerated the development of adventitious roots (Fig. However, lodging still occurs in semi-dwarf varieties. This has prompted many studies to dissect genetic factors other than plant stature that confer lodging resistance in rice. For instance, the composition of the secondary cell wall in the culm is an important factor in determining lodging resistance because it is a rigid, thickened structure that determines the mechanical strength of the plant body (Zhang et al. ots Fig. .Fig. 7GeThis review focused on three main problems facing direct seeding of rice and six related important traits. Functional genes and their mechanisms of action were reviewed and organized. This review is expected to lay the foundation for more in-depth basic theoretical research and breeding application research on direct-seeded rice.However, many modern high-yielding and high-quality varieties are selected based on the cultivation method of transplanting, resulting in a large number of excellent functional genes for direct seeding-related traits that have been lost in modern varieties. Direct seeding-related traits are complex traits controlled by multiple genes, the effect of individual genes is small, and their expression is easily influenced by environmental and gene interactions. Therefore, mining related genes and trait improvements is very difficult. Researchers need to use germplasm resources with richer diversity, including wild rice resources, and modern technologies such as functional genomics, histology, and genome-wide association analysis to clone superior functional genes and dissect their molecular mechanisms. Simultaneously, using efficient and accurate modern molecular breeding techniques, the identified functional genes can be applied to the genetic improvement of direct-seeded rice. The process of selecting and breeding new rice varieties suitable for direct seeding will accelerate meeting the demand for direct seeding in rice production as soon as possible.


The early mortality rate after a medical procedure or treatment has been a historical measure of quality of care delivery. It was originally developed to assess the risk of surgeries or other procedures and later adopted across medical practices to evaluate differential risks of early mortality with similar therapies to define favorable or unfavorable outliers, and to improve delivery of care. Its subsequent adoption in cancer care had broad applications in areas with widely divergent environments and influencing factors. Some of the variables include the kind of tumor ; the type of surgery (extremes of Whipple procedure or lymph node biopsy); the stage (early or advanced); the treatment intensity ; and the risk of mortality with intensive chemotherapy (compromised bone marrow in leukemia or intact bone marrow reserve in solid tumors) , 2.In cancer, the general measure has been the rate of early mortality following some form of treatment, early mortality being defined in some analyses as death within 4 weeks of receiving chemotherapy, as well as receiving chemotherapy within 2 weeks of death in patients with advanced cancers. Because of the broad range of tumors, patients, treatments and environment factors influencing this early mortality, its value as a measure of quality of care has been questioned Table  3, 4]. , 4. 3, 4In leukemia, the mortality rate has been used in two almost diametrically opposed situations, with different interpretations, to measure the quality of care: (1) 4-week mortality after frontline induction chemotherapy in patients with newly diagnosed acute leukemia; and (2) death within 2 (or 4 weeks) weeks of therapy in “terminally ill”  patients with advanced or previously treated leukemia.In the first situation, death within 4 weeks of induction therapy can reflect a treatment that is too toxic. With similar treatments administered in different locations, the 4-week mortality may delineate relative expertise and supportive care levels. For example, in patients with newly diagnosed acute myeloid leukemia (AML) undergoing standard intensive induction chemotherapy , the 4-week mortality rate was 15% in academic centers versus 29% in non-academic centers; it was 12% in NCI-designated cancer centers versus 24% in non-NCI-designated cancer centers . At MD AIn the second situation, therapy given “at the end of life” is frequently viewed negatively by supportive care experts and is interpreted to reflect inappropriate care/poor judgment, where the patients might have been unnecessarily harmed by toxic therapy and died in an environment not to their preference. The argument goes that, if well-advised about their treatment options and realistic outcomes, patients might choose to forgo therapy and die at home or in hospice rather than in the hospital. Hence, the delivery of therapy within 2 weeks of death to such patients may negatively impact the quality of care score in a particular environment or hospital.But the view that withholding therapy in the last 2 weeks of life is a reliable indicator of the quality of care may not consider several important factors: (1) Novel, more effective targeted therapies and immunotherapies with favorable toxicity profiles are now producing startlingly positive results and potential long-term responses in previously incurable patients with leukemia, lymphoma and myeloma. For example, patients who are in second salvage or later can still get significant benefit from treatment with bi-specific T-cell engagers (BiTEs), chimeric antigen receptor T cellular therapy (CART), and other drugs targeting molecular abnormalities –17. (2) Next, we will discuss in more detail the two opposing situations.In frontline therapy, healthcare experts agree that the 4-week mortality is a reliable indicator of quality of care but emphasize that it is crucial to consider the population mix. A general hospital with fewer than 20% of patients with cancer and 1% of patients with leukemia will score on average a lower total 4-week mortality than a dedicated cancer hospital. A cancer center hospital with 5% of beds occupied by leukemia patients will score on average a lower 4-week mortality rate than one with 25% of beds with leukemia patients . A cancer hospital that serves as a referral center and offers investigational options will have a higher proportion of patients on salvage versus frontline treatment, which results in a higher 4-week mortality rate since patients on salvage therapy tend to be sicker and more likely to die. Such patients treated at referral centers are often self-selected in that they have the social and financial resources to travel and seek more therapies regardless of the odds. They may often reject the advice of the cancer and supportive care experts who recommend hospice or terminal care. Quality healthcare experts argue that such differences in mortality rates  would be nullified once all patient-associated co-morbidities were accounted for. However, this did not turn out to be correct because of patient- and tumor-associated latent (unmeasured) variables that are not factored in such analyses , 25. WhiHow about therapy given within 2 weeks of death in a terminally ill cancer patient? Here, the distinction between solid tumors and leukemia, and recently other hematologic cancers such as lymphoma and multiple myeloma, is important. In solid cancers, the tumor is already advanced and/or metastatic, the poor prognosis well-defined, and poor quality of life irreversible . Thus, the administration of chemotherapy in the last 2–4 weeks of life may not be of value. Leukemia is a different situation, where, for example, patients with acute lymphoblastic leukemia (ALL) in first or second salvage and beyond may still be rescued with effective combinations of chemotherapy and novel targeted or immune therapies, followed by allogeneic stem cell transplantation or CAR T-cells . The potAn important recent issue is the blurring of the line between active chemotherapy used as a treatment and as a supportive care measure. In solid tumors, the separation is clear: chemotherapy to shrink the tumor; narcotics as supportive care to control tumor-associated pain. In leukemia, pain is rare and usually alleviated with a short course of steroids, but supportive care involves addressing three common components: red cell transfusions to improve anemia and fatigue/poor quality of life; platelet transfusions for thrombocytopenia and bleeding; and antibiotics to prevent or treat infections associated with immunosuppression. But leukemia also has an added dimension not observed in solid tumors: a rise in peripheral blood blasts that compromises organ function, worsens quality of life, causes pain and shortens survival by, at times, several months. This can be ameliorated with agents classified as chemotherapy but that also can be used to control the peripheral blast counts as a supportive measure: hydroxyurea, cytarabine, hypomethylating agents . We believe that including such uses in the quality indication if patients receive them within 2 weeks of death may be counterproductive and potentially harmful if treating physicians feel pressured to withhold a measure that might benefit the patient.With the ongoing therapeutic revolution involving novel small-molecule targeted therapies and immunotherapies  , we haveIn summary, the 4-week mortality is a good quality indicator in frontline acute leukemia therapy. In leukemia salvage, such intensive chemotherapy with or without other novel therapies followed by allogeneic SCT may be curative but results in higher 4-week mortality rates that should be incorporated into the quality indicators. Finally, several therapies classified as “chemotherapy” but sometimes used as supportive care should be allowed without penalty and considered as favorable quality-improving measures even in the last 2–4 weeks of life.