Plasma cell-free DNA 5-hydroxymethylcytosine and whole-genome sequencing signatures for early detection of esophageal cancer.

Esophageal cancer is a highly incidence and deadly disease with a poor prognosis, especially in developing countries. Owing to the lack of specific symptoms and early diagnostic biomarkers, most patients are diagnosed with advanced disease, leading to a 5-year survival rate of less than 15%. Early (n = 50) and middle-advanced (n = 50) esophageal squamous cell carcinoma (ESCC) patients, as well as 71 healthy individuals, underwent 5-hydroxymethylcytosine (5hmC) sequencing on their plasma cell-free DNA (cfDNA). A Northern Chinese cohort of cfDNA 5hmC dataset of 150 ESCC patients and 183 healthy individuals were downloaded for validation. A diagnostic model was developed using cfDNA 5hmC signatures and then improved by low-pass whole genome sequencing (WGS) features of cfDNA. Conserved cfDNA 5hmC modification motifs were observed in the two independent ESCC cohorts. The diagnostic model with 5hmC features achieved an AUC of 0.810 and 0.862 in the Southern and Northern cohorts, respectively, with sensitivities of 69.3-74.3% and specificities of 82.4-90.7%. The performance was well maintained in Stage I to Stage IV, with accuracy of 70-100%, but low in Stage 0, 33.3%. Low-pass WGS of cfDNA improved the AUC to 0.934 with a sensitivity of 82.4%, a specificity of 88.2%, and an accuracy of 84.3%, particularly significantly in Stage 0, with an accuracy up to 80%. 5hmC and WGS could efficiently differentiate very early ESCC from healthy individuals. These findings imply a non-invasive and convenient method for ESCC detection when clinical treatments are available and may eventually prolong survival.

Photic sensitization is mediated by cortico-accumbens pathway in rats with trigeminal neuropathic pain.

Exposure to light stimuli may trigger or exacerbate perception of pain, also known as a common yet debilitating symptom of photophobia in patient with chronic orofacial pain. Mechanism underlying this phenomenon of photic sensitization in neuropathic condition remains elusive. Here, we found that rats developed hypersensitivity to normal light illumination after establishment of chronic constriction injury of infraorbital nerve (ION-CCI) model, which can be attenuated by blocking the exposure of photic stimulation. Additionally, this behavioral phenotype of light-sensitivity impairment was associated with overexpression of anterior cingulate cortex (ACC) c-fos positive neurons, enhancement of neural excitability in the ACC neurons and its excitatory synaptic transmission between nucleus accumbens (NAc). Optogenetic and chemogenic silencing of ACC-NAc pathway improved trigeminal sensitization in responses to light stimuli by decreasing spontaneous pain-like episodes in ION-CCI animals. In contrast, selective activation of ACC-to-NAc circuits enhanced photic hypersensitivity in dark environment. Thus, our data provided novel role of ACC and its projection to NAc in bidirectional modulation of photic sensation, which may contribute to the understanding of photic allodynia in trigeminal neuropathic pain status.

Establishing a new model of cancer neuropathic pain in rats.

To guide the treatment of malignant neuropathic pain (MNP) in clinical practice, by inoculating MADB-106 breast cancer cells into the right L4 nerve root in Sprague-Dawley rats, a rat model of MNP was established, providing basic conditions for the study of neuropathic pain and development and application of therapeutic drugs. As the tumor grew over time, it pressed the nerve roots, causing nerve damage. The spinal nerve ligation (SNL) model, which is a neuropathic pain model widely used in rats, was compared with the L4 nerve root SNL model, and histologic examination of the nerve tissue of both models was performed by electron microscopy. In addition to the infiltration and erosion of the L4 nerve by tumor cells, the tumor tissue gradually grew and compressed the L4 nerve roots, resulting in hyperalgesia of the rat's posterior foot on the operative side. Some spontaneous pain phenomena were also observed, such as constant lifting or licking of the posterior foot on the operative side under quiet conditions. Electron microscopy images showed that nerve injury was due to progressive compression by the tumor, cells of which were visualized, but the injury was lighter than that in SNL rats. Imaging showed a paravertebral tumor near the L4 nerve root in the carcinomatous neuropathic pain model rat. These results suggest that progressive compression of the nerve by a malignant tumor leads to nerve damage similar to the behavioral changes associated with chronic compression injury resulting from a loose ligature of the nerve. The cancer neuropathologic pain model at the L4 nerve root was successfully established in Sprague-Dawley rats.

A novel implantable device for sensory and affective assessment of orofacial pain in rats.

Background and objective: Orofacial pain, in particular, chronic orofacial pain remains a great challenge in clinical practice. To better understand the underlying mechanism of disease, it is essential to apply a feasible and stable preclinical measurement of facial pain. Here, we introduced a novel electrical noxious stimulator in freely behavioral rodents and examined its validation in both naïve and chronic orofacial pain animals. Methods: One subcutaneous device of electrical stimulator was implanted in the facial region for delivery of the nociceptive input. The sensory component of orofacial pain was assessed by response scoring tool, and conditioned place aversion (CPA) paradigm for pain affect respectively. To confirm its usage in chronic pain state, the chronic constriction injury of the infraorbital nerve (ION-CCI) model was then applied. Results: We found that responsive scores increased with stimulation intensity, and acted in a dosage-dependent manner, which can be attenuated by the administration of morphine intraperitoneally. Naïve rats displayed significant aversive reaction to the noxious electrical stimulation (25V) in the CPA testing. In addition, an obvious sensory hypersensitivity to electrical stimulation was confirmed by the increased response scores in ION-CCI rats. Furthermore, ION-CCI animal showed significant avoidance to electrical stimulation at relatively low intensity (10V), which was innoxious to naïve rats. Conclusion: Our findings may provide an alternative pre-clinical measurement of orofacial pain, to quantitively assess both sensory and affective component of orofacial pain.

Repair Effects of Bone Marrow Mesenchymal Stem Cells on Demyelination of Trigeminal Ganglion in Rats with Trigeminal Neuralgia.

OBJECTIVE: The current study investigated the effects of bone marrow mesenchymal stem cells (BMSCs) on pain behavior in rats with trigeminal neuralgia induced by infraorbital nerve chronic constriction injury (ION-CCI), and the repair effects of BMSCs on pathological changes in trigeminal ganglion demyelination. METHODS: BMSCs or phosphate-buffered saline (PBS) alone were injected around trigeminal ganglion in ION-CCI rats via a rat brain stereotaxic apparatus. Mechanical pain threshold (von Frey test) and face grooming behavior were measured in each group. Recovery of demyelination of trigeminal ganglion was observed via electron microscopy 2 weeks later, and BMSC differentiation was observed via immunofluorescence. RESULTS: Rats in the BMSC group exhibited significant improvements in mechanical pain threshold and face grooming behavior compared with the PBS group. BMSCs could repair demyelinating changes in trigeminal ganglion in ION-CCI rats. Only cells expressing GFAP, S-100, and p75 were observed via immunofluorescence, and no PKH67-labeled BMSCs were observed in the trigeminal ganglion. No BMSC differentiation was observed in the trigeminal ganglion. CONCLUSION: Injection of BMSCs around the trigeminal ganglion could relieve trigeminal neuralgia effectively and repair trigeminal ganglion demyelination. No differentiation of BMSCs injected around the trigeminal ganglion into Schwann cells was observed. The mechanism of trigeminal neuralgia demyelination repair requires further investigation.

A Patient With a Foreign Body in Mediastinum That Penetrated Into the Bronchus.

We report the rare case of a patient who had a foreign body in the mediastinum and trachea caused by trauma due to epileptic seizures. A 52-year-old man had an epileptic seizure 3 months before visiting our hospital and had an injury to his neck caused by a broken glass cabinet. Computed tomography scan revealed a foreign body in the mediastinum and trachea. After a detailed discussion among members of the multidisciplinary team, surgery was successfully performed to remove the foreign body. This rare case may help provide a reference for diagnosing and treating a mediastinal and tracheobronchial foreign body.

Climate warming promotes deterministic assembly of arbuscular mycorrhizal fungal communities.

Arbuscular mycorrhizal fungi (AMF) significantly contribute to plant resource acquisition and play important roles in mediating plant interactions and soil carbon (C) dynamics. However, it remains unclear how AMF communities respond to climate change. We assessed impacts of warming and precipitation alterations (30% increase or decrease) on soil AMF communities, and examined major ecological processes shaping the AMF community assemblage in a Tibetan alpine meadow. Our results showed that warming significantly increased root biomass, and available nitrogen (N) and phosphorus (P) in soil. While precipitation alterations increased AMF abundances, they did not significantly affect the composition or diversity of AMF communities. In contrast, warming altered the composition of AMF communities and reduced their Shannon-Wiener index and Pielou's evenness. In particular, warming shifted the AMF community composition in favor of Diversisporaceae over Glomeraceae, likely through its impact on soil N and P availability. In addition, AMF communities were phylogenetically random in the unwarmed control but clustered in warming plots, implying more deterministic community assembly under climate warming. Warming enhancement of root growth, N and P availability likely reduced plant C-allocation to AMF, imposing stronger environmental filtering on AMF communities. We further proposed a conceptual framework that integrates biological and geochemical processes into a mechanistic understanding of warming and precipitation changes' effects on AMF. Taken together, these results suggest that soil AMF communities may be more sensitive to warming than expected, highlighting the need to monitor their community structure and associated functional consequences on plant communities and soil C dynamics under the future warmer climate.

Clinical Outcome of Pulsed-Radiofrequency Combined With Transforaminal Epidural Steroid Injection for Lumbosacral Radicular Pain Caused by Distinct Etiology.

BACKGROUND: Lumbosacral radicular pain (LSRP) can be caused by disc herniation, spinal stenosis, and failed back surgery syndrome. The clinical effect of pulsed-radiofrequency (PRF) combined with transforaminal epidural steroid injection (TESI) for radiating pain in different population remains unclear. METHODS: We retrospectively reviewed the medical recordings of patients with LSRP caused by different etiologies, who underwent PRF and TESI treatment. The primary clinical outcome was assessed by a 10-point Visual Analog Scale (VAS) pre- and post-treatment. RESULTS: A total of 34 LSRP patients were identified and classified into 3 subgroups (disc herniation, spinal stenosis, and failed back surgery syndrome). The overall immediate pain reduction was 4.4 ± 1.1 after procedure. After a median follow-up of 9.5 months, the VAS decreased from 6.5 ± 1.0 to 2.4 ± 1.9 at the last follow-up. CONCLUSION: PRF combined with TESI is an effective approach to treat persistent LSRP in distinct population.

Beyond resource limitation: an expanded test of the niche dimension hypothesis for multiple types of niche axes.

The niche dimension hypothesis predicts that more species can coexist given a greater number of niche axes along which they partition the environment. Although this hypothesis has been broadly supported by nutrient enrichment experiments, its applicability to other ecological factors, such as natural enemies and abiotic stresses, has not been vigorously tested. Here, we examined the generality of the niche dimension hypothesis by experimentally manipulating both resource and non-resource niche dimensions-nitrogen limitation, pathogens and low-temperature stress-in a Tibetan alpine meadow. We found that decreases in niche dimensions led to a significant reduction in species richness, consistent with results from nutrient addition studies. However, different niche variables uniquely affected the plant communities. While nitrogen had largest effects on both community biomass and species richness, pathogens and low-temperature stress, in combination with nitrogen, had synergistic effects on them. Our results provide direct evidence demonstrating that both resource and non-resource niche dimensions can influence species coexistence. These findings suggest that other non-resource factors need to be taken into consideration to better predict the community assembly and control over biodiversity, particularly under the future multifaceted global change scenarios.

Ceftriaxone Relieves Trigeminal Neuropathic Pain Through Suppression of Spatiotemporal Synaptic Plasticity via Restoration of Glutamate Transporter 1 in the Medullary Dorsal Horn.

Using a rat model of trigeminal neuropathic pain (TNP) produced by chronic compression of the infraorbital nerve (CCI-ION), we investigated the analgesic effect and the underlying mechanisms of ceftriaxone (Cef), a ß-lactam antibiotic, that is thought to be a potent stimulator of glutamate transporter 1 (GLT-1). First, repeated intraperitoneal (i.p.) injections of Cef (200 mg/kg) for 5-days since Day 1 of CCI-ION could significantly relieve both mechanical and thermal pain hypersensitivity from day 10 after drug administration. Western blot and immunofluorescent results demonstrated that 5-days administration of Cef resulted in the restoration of GLT-1 expression to a level equivalent to the sham control which was dramatically lost under the TNP condition. Moreover, multi-electrode (8 × 8) array recordings of network field excitatory postsynaptic potentials (fEPSPs) were performed on the acutely dissociated medullary dorsal horn slice evoked by electrical stimulation of the trigeminal spinal tract. The results showed that the increased number of fEPSPs, induction rate, and maintenance of long-term potentiation caused by CCI-ION were significantly suppressed by 5-days administration of Cef. Taken together, the results indicate that Cef can relieve TNP through suppression of spatiotemporal synaptic plasticity via GLT-1 restoration in the medullary dorsal horn of the trigeminal nerve.

Soil acidification alters root morphology, increases root biomass but reduces root decomposition in an alpine grassland.

Soil acidification has been expanding in many areas of Asia due to increasing reactive nitrogen (N) inputs and industrial activities. While the detrimental effects of acidification on forests have been extensively studied, less attention has been paid to grasslands, particularly alpine grasslands. In a soil pH manipulation experiment in the Qinghai-Tibet Plateau, we examined the effects of soil acidification on plant roots, which account for the major part of alpine plants. After three years of manipulation, soil pH decreased from 6.0 to 4.7 with the acid-addition gradient, accompanied by significant changes in the availability of soil nitrogen, phosphorus and cations. Plant composition shifted with the soil acidity, with graminoids replacing forbs. Differing from findings in forests, soil acidification in the alpine grassland increased root biomass by increasing the fraction of coarse roots and the production of fine roots, corresponding to enhanced sedge and grass biomass, respectively. In addition, litter decomposability decreased with altered root morphological and chemical traits, and soil acidification slowed root decomposition by reducing soil microbial activity and litter quality. Our results showed that acidification effect on root dynamics in our alpine grassland was significantly different from that in forests, and supported similar results obtained in limited studies in other grassland ecosystems. These results suggest an important role of root morphology in mediating root dynamics, and imply that soil acidification may lead to transient increase in soil carbon stock as root standing biomass and undecomposed root litter. These changes may reduce nutrient cycling and further constrain ecosystem productivity in nutrient-limiting alpine systems.

Simulated warming enhances the responses of microbial N transformations to reactive N input in a Tibetan alpine meadow.

Alpine ecosystems worldwide are characterized with high soil organic carbon (C) and low mineral nitrogen (N). Climate warming has been predicted to stimulate microbial decomposition and N mineralization in these systems. However, experimental results are highly variable, and the underlying mechanisms remain unclear. We examined the effects of warming, N input, and their combination on soil N pools and N-cycling microbes in a field manipulation experiment. Special attention was directed to the ammonia-oxidizing bacteria and archaea, and their mediated N-cycling processes (transformation rates and N2O emissions) in the third plant growing season after the treatments were initiated. Nitrogen input (12 g m-2 y-1) alone significantly increased soil mineral N pools and plant N uptake, and stimulated the growth of AOB and N2O emissions in the late growing season. While warming (by 1.4 °C air temperature) alone did not have significant effects on most parameters, it amplified the effects of N input on soil N concentrations and AOB abundance, eliciting a chain reaction that increased nitrification potential (+83%), soil NO3--N (+200%), and N2O emissions (+412%) across the whole season. Also, N input reduced AOB diversity but increased the dominance of genus Nitrosospira within the AOB community, corresponding to the increased N2O emissions. These results showed that a small temperature increase in soil may significantly enhance N losses through NO3- leaching and N2O emissions when mineral N becomes available. These findings suggest that interactions among global change factors may predominantly affect ammonia-oxidizing microbes and their mediated N-cycling processes in alpine ecosystems under future climate change scenarios.

Combination of warming and N inputs increases the temperature sensitivity of soil N2O emission in a Tibetan alpine meadow.

Many high-elevation alpine ecosystems have been experiencing significant increases in air temperature and, to a lesser extent, nitrogen (N) deposition. These changes may affect N-cycling microbes and enhance emissions of nitrous oxide (N2O, a potent greenhouse gas) from soil. However, few studies have investigated whether and how the resulting changes in N-cycling microbes may affect the temperature sensitivity (Q10) of N2O emission and in turn feed back to N2O emissions. We conducted two incubation experiments to examine N2O emissions and their temperature sensitivities in soils that had experienced 3-yr field treatments of warming, N inputs and their combination in a Tibetan alpine meadow. Our results showed that neither N inputs nor warming alone affected the rate or Q10 of soil N2O emission, but combining the two significantly increased both parameters. Also, combined N and warming significantly increased the abundance of ammonia-oxidizing bacteria (AOB), corresponding with high soil N2O emission. In addition, N2O emission from nitrification accounted for 60-80% of total emissions in all soils, indicating that nitrifying microbes dominated the N2O production and its temperature sensitivity. Using random forest (RF) and structural equation model (SEM) analyses, we further evaluated the effects of various soil characteristics on soil N2O emissions and Q10. We identified soil moisture, pH, N mineralization and AOB abundance as the main predictors of the Q10 of N2O emissions. Together, these findings suggest that alterations in soil moisture, pH and ammonia-oxidizing bacteria induced by long-term N inputs and warming may increase temperature sensitivity of soil N2O emissions, leading to a positive climate feedback in this high-altitude alpine ecosystem.

Machine Learning Models to Predict Primary Sites of Metastatic Cervical Carcinoma From Unknown Primary.

Metastatic cervical carcinoma from unknown primary (MCCUP) accounts for 1-4% of all head and neck tumors, and identifying the primary site in MCCUP is challenging. The most common histopathological type of MCCUP is squamous cell carcinoma (SCC), and it remains difficult to identify the primary site pathologically. Therefore, it seems necessary and urgent to develop novel and effective methods to determine the primary site in MCCUP. In the present study, the RNA sequencing data of four types of SCC and Pan-Cancer from the cancer genome atlas (TCGA) were obtained. And after data pre-processing, their differentially expressed genes (DEGs) were identified, respectively. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that these significantly changed genes of four types of SCC share lots of similar molecular functions and histological features. Then three machine learning models, [Random Forest (RF), support vector machine (SVM), and neural network (NN)] which consisted of ten genes to distinguish these four types of SCC were developed. Among the three models with prediction tests, the RF model worked best in the external validation set, with an overall predictive accuracy of 88.2%, sensitivity of 88.71%, and specificity of 95.42%. The NN model is the second in efficacy, with an overall accuracy of 82.02%, sensitivity of 81.23%, and specificity of 93.04%. The SVM model is the last, with an overall accuracy of 76.69%, sensitivity of 74.81%, and specificity of 90.84%. The present analysis of similarities and differences among the four types of SCC, and novel models developments for distinguishing four types of SCC with informatics methods shed lights on precision MCCUP diagnosis in the future.

Chemoselective syntheses of spirodihydrofuryl and spirocyclopropyl barbiturates via cascade reactions of barbiturate-based olefins and acetylacetone.

The Michael addition initiated ring closure reaction of barbiturate-based olefins and acetylacetone with NBS has been explored. The efficient and chemoselective approach for the synthesis of barbiturate-fused spirocycles was established. Spirodihydrofuryl barbiturates and spirocyclopropyl barbiturates were synthesized selectively via cascade reactions under different basic conditions in moderate to excellent yields. The structure of 2-(4-chlorophenyl)-1,1-diacetyl-5,7-dimethyl-5,7-diazaspiro[2,5]octane-4,6,8-trione was confirmed by single crystal X-ray diffraction analysis.

Facile chemoenzymatic synthesis of Lewis a (Lea) antigen in gram-scale and sialyl Lewis a (sLea) antigens containing diverse sialic acid forms.

An efficient streamlined chemoenzymatic approach has been developed for gram-scale synthesis of Lewis a angtigen (LeaßProN3) and a library of sialyl Lewis a antigens (sLeaßProN3) containing different sialic acid forms. Intially, commercially available inexpensive N-acetylglucosamine (GlcNAc) was converted to its N'-glycosyl p-toluenesulfonohydrazide in one step. Followed by chemical glycosylation, GlcNAcßProN3 was synthesized using this protecting group-free method in high yield (82%). Sequential one-pot multienzyme (OPME) ß1-3-galactosylation of GlcNAcßProN3 followed by OPME α1-4-fucosylation reactions produced target LeaßProN3 in gram-scale. Structurally diverse sialic acid forms was successfully introduced using a OPME sialylation reation containing a CMP-sialic acid synthetase and Pasteurella multocida α2-3-sialyltransferase 1 (PmST1) mutant PmST1 M144D with or without a sialic acid aldolase to form sLeaßProN3 containing naturally occurring or non-natural sialic acid forms in preparative scales.

Reconciling multiple impacts of nitrogen enrichment on soil carbon: plant, microbial and geochemical controls.

Impacts of reactive nitrogen (N) inputs on ecosystem carbon (C) dynamics are highly variable, and the underlying mechanisms remain unclear. Here, we proposed a new conceptual framework that integrates plant, microbial and geochemical mechanisms to reconcile diverse and contrasting impacts of N on soil C. This framework was tested using long-term N enrichment and acid addition experiments in a Mongolian steppe grassland. Distinct mechanisms could explain effects of N on particulate and mineral-associated soil C pools, potentially explaining discrepancies among previous N addition studies. While plant production predominated particulate C changes, N-induced soil acidification strongly affected mineral-associated C through decreased microbial growth and pH-sensitive associations between iron and aluminium minerals and C. Our findings suggest that effects of N-induced acidification on microbial respiration and geochemical properties should be included in Earth system models that predict ecosystem C budgets under future N deposition/input scenarios.

Long-term N and P additions alter the scaling of plant nitrogen to phosphorus in a Tibetan alpine meadow.

Nitrogen and phosphorus are two important nutrient elements for plants. The current paradigm suggests that the scaling of plant tissue N to P is conserved across environments and plant taxa because these two elements are coupled and coordinately change with each other following a constant allometric trajectory. However, this assumption has not been vigorously examined, particularly in changing N and P environments. We propose that changes in relative availability of N and P in soil alter the N to P relationship in plants. Taking advantage of a 4-yr N and P addition experiment in a Tibetan alpine meadow, we examined changes in plant N and P concentrations of 14 common species. Our results showed that while the scaling of N to P under N additions was similar to the previously reported pattern with a uniform 2/3 slope of the regression between log N and log P, it was significantly different under P additions with a smaller slope. Also, graminoids had different responses from forbs. These results indicate that the relative availability of soil N and P is an important determinant regulating the N and P concentrations in plants. These findings suggest that alterations in the N to P relationships may not only alter plant photosynthate allocation to vegetative or reproductive organs, but also regulate the metabolic and growth rate of plant and promote shifts in plant community composition in a changing nutrient loading environment.

Complete structure and variation of the chloroplast genome of Agropyron cristatum (L.) Gaertn.

Agropyron cristatum (L.) Gaertner, a perennial grass in the tribe Triticeae (Poaceae), is a wild relative of cereal crops that is suitable for genetic improvement. In this study, we first sequenced the complete chloroplast (cp) genome of Ag. cristatum using Hiseq4000 PE150. The Ag. cristatum chloroplast genome is 135,554bp in length, has a typical quadripartite structure and contains 76 protein-coding genes, 29 tRNA genes and four rRNA genes. The cp genome of Ag. cristatum was used for comparison with other seven Triticeae species. One large variable region (800bp), which primarily contained the rpl23 (non-reciprocally translocated from IRs) and accD genes, was detected between rbcL gene and psaI gene within LSC region. The deletion of the accD and translocated rpl23 genes in Ag. cristatum indicated an independent gene-loss events or an additional divergence in Triticeae. Analyses of the dn/ds ratio and K2-P's genetic distance for 76 protein-coding genes showed that genes with evolutionary divergence might suffer from the effect of sequence regional constraints or gene functional constraints in Triticeae species. Our research will generally contribute to the knowledge of plastid genome evolution in Triticeae.

Enzymatic and Chemoenzymatic Syntheses of Disialyl Glycans and Their Necrotizing Enterocolitis Preventing Effects.

Necrotizing enterocolitis (NEC) is one of the most common and devastating intestinal disorders in preterm infants. Therapies to meet the clinical needs for this special and highly vulnerable population are extremely limited. A specific human milk oligosaccharide (HMO), disialyllacto-N-tetraose (DSLNT), was shown to contribute to the beneficial effects of breastfeeding as it prevented NEC in a neonatal rat model and was associated with lower NEC risk in a human clinical cohort study. Herein, gram-scale synthesis of two DSLNT analogs previously shown to have NEC preventing effect is described. In addition, four novel disialyl glycans have been designed and synthesized by enzymatic or chemoenzymatic methods. Noticeably, two disialyl tetraoses have been produced by enzymatic sialylation of chemically synthesized thioethyl ß-disaccharides followed by removal of the thioethyl aglycon. Dose-dependent and single-dose comparison studies showed varying NEC-preventing effects of the disialyl glycans in neonatal rats. This study helps to refine the structure requirement of the NEC-preventing effect of disialyl glycans and provides important dose-dependent information for using DSLNT analogs as potential therapeutics for NEC prevention in preterm infants.