Bacillus anthracis (B. anthracis), Brucella spp. (Brucella) and Coxiella burnetii (Coxiella). The analysis identified DNA signatures of B. anthracis (0.48%), Brucella (0.9%), and Coxiella (0.66%) in a total of 77 samples. Highest prevalence rates of B. anthracis, Brucella, and Coxiella were observed in wildebeest (56%), dik-dik (50%), and impala (24%), respectively. Fresh samples, those collected during the rainy season, and samples from Selous or Serengeti had a greater relative risk of being positive. Microbiome characterization identified Firmicutes and Proteobacteria as the most abundant phyla. The results highlight and define potential risks of exposure to endemic wildlife diseases from bushmeat and the need for future investigations to address the public health and emerging infectious disease risks associated with bushmeat harvesting, trade, and consumption.Meat from wildlife species (bushmeat) represents a major source of dietary protein in low- and middle-income countries where humans and wildlife live in close proximity. Despite the occurrence of zoonotic pathogens in wildlife, their prevalence in bushmeat remains unknown. To assess the risk of exposure to major pathogens in bushmeat, a total of 3784 samples, both fresh and processed, were collected from three major regions in Tanzania during both rainy and dry seasons, and were screened by real-time PCR for the presence of DNA signatures of  Bushmeat is not only a source of food and income for the poachers, their families, and communities, but is also used in many cultural, social, and religious practices2. A recent survey in the regions surrounding the Greater Serengeti ecosystem revealed that between 2 and 5 meals per week in a household contained bushmeat3. While the majority of bushmeat is consumed locally, a considerable amount is smuggled illegally into the United States and Western Europe. Reports suggest that up to 5 tons of bushmeat is smuggled via a single airport in Western Europe each week4. Bushmeat smuggling from Africa and elsewhere could act as a conduit for pathogen spread as suggested by the recent discovery of retroviruses and herpesviruses in bushmeat illegally imported to the US5. This is of particular relevance in light of the current global pandemic of SARS-CoV-2, and the increasing risk of newly emerging infectious diseases of zoonotic origin6 . It is widely recognized that 60% of emerging infectious diseases are zoonotic in origin, and of these 70% originate in wildlife7. In this study, we shall examine the prevalence of endemic Bacillus anthracis (B. anthracis), Brucella spp. (Brucella), and Coxiella burnetii (Coxiella) in bushmeat samples collected within Tanzania.“Bushmeat”, the meat and organs derived from harvested wildlife species, is a common source of animal protein throughout the tropics of the Americas, Asia, Australia, and Africa. In Tanzania, although bushmeat hunting is unlawful without permits, illegal wildlife poaching is a common practice year-roundBacillus anthracis is a spore-forming, Gram-positive bacterium and the causative agent of anthrax, a zoonotic disease that mostly affects grazing livestock and wild ungulates and poses a threat to human health. Studies have estimated that 20,000 to 100,000 human cases of anthrax occur worldwide annually9. The disease is endemic in poor rural areas, especially where there are extensive interactions between human, livestock and wildlife9. Outbreaks are regularly reported with an early event in 1962, causing mortality in 1200 impalas in Tanzania10. More recently, an outbreak in 2016–2017 in Northern Tanzania recorded positive cases in 131 wildlife and 39 livestock carcasses that were also used for consumption10. The recurrence of outbreaks in Tanzania are mainly the result of interactions between humans, wildlife, and livestock at specific “hotspots”, where both domestic and wild animals are potential sources of infections to humans11. People become infected when handling and consuming infected livestock and also bushmeat. Periods of drought facilitate anthrax outbreaks since they lead to aggregations of animals close to water sources, heavy overgrazing that exposes soil, and atypical migration patterns12.Brucella, a genus of Gram-negative bacteria, is the causative agent of a number of zoonotic infections including brucellosis. Infection passes to humans through direct contact with infected animals, by the consumption of unpasteurized dairy products (often raw milk), or via occupational contact with infected animals as well as by the inhalation of airborne agents13. In sub-Saharan Africa, including Tanzania, studies have indicated the presence of Brucella in wildlife and that livestock to humans is considered an endemic problem causing devastating economic losses and challenges for public health17. A recent study in Kenya on Brucella suggested that transmission to humans increased as a consequence of close contact between livestock and wildlife18. Another study from Tanzania identified geographical “hotspots” with high anti-Brucella antibodies in humans, cattle, goats, and buffalos14. Other studies have emphasized that transmission of Brucella to humans was caused by the preparation and consumption of bushmeat21.Coxiella burnetii, a Gram-negative bacterial CDC select agent that causes Q fever is also transmitted to livestock, wildlife, and humans via byproducts and the direct contact with infected animals. There are few studies on the prevalence of Q fever in Tanzania. One study on humans, livestock and wildlife found that the incidence of Q fever in cattle and wildlife was associated with arthropod vectors22. Another cross sectional sero-epidemiological study conducted in South Africa concluded higher risk of zoonotic transmission and economic loss was associated with the livestock-wildlife interface23 and this has been supported by a number of studies across Africa25.21, and suggest that characterization of the microbiomes of bushmeat samples with evidence of DNA signatures for the three select agents may help better assess the overall microbial burden and potential public health risks associated with bushmeat harvest, handling, and consumption.Metagenomics has transformed microbiology by leading the way for a cultivation-independent determination of microbial profiles in samples, especially in samples similar to bushmeat that are subjected to a supply chain before consumption. Recent microbiome analyses have provided preliminary evidence for the presence of select agents in bushmeat samplesB. anthracis, Brucella spp., and Coxiella. Taken together, the results highlight the presence of select agent signatures and help define potential risks of exposure to endemic wildlife diseases from bushmeat in the country.Thus, to better assess the prevalence of select agents and risks associated with the handling and consumption of bushmeat, we screened a total of 3784 fresh and processed bushmeat samples representing 42 animal species from the Serengeti, Ruaha, and Selous regions in Tanzania collected during rainy and dry seasons over a 2-year period for the presence of DNA signatures of 2 Serengeti ecosystem is located in northern Tanzania, bordering southern parts of Kenya26. It consists of several protected areas including the Serengeti National Park  and adjacent Ngorongoro Conservation Area, Game Reserves and Game Controlled Areas. Serengeti typically has a dry season (June-October) and a wet season (November–May). The Ruaha ecosystem (7° 30′ S 35° 00′ E), located in the southern region of Tanzania and at 20,226 km2, is comprised of Ruaha National Park and surrounding Game Reserves and Wildlife Management Areas. Ruaha typically has a dry season (May–October) and a wet season (November–April). The Selous Game Reserve (9° 0′ S 37° 24′ E) 50,000 km2  is another protected area in Southern Tanzania. It is contiguous with the Mikumi National Park and Udzungwa Mountains National Park and contains one third of the abundant wildlife of Tanzania. Selous typically has a dry season (June–October) and a wet season (November–May). Typically, Tanzania has a short dry season (January–February) and a long dry season (June-October) and a short rainy season (November–December) and a long rainy season (March–May), although variations are observed in different regions30. For the purpose of this study, we considered the dry season from June-October while the rainy season was considered from the months of November–May for all the regions in the study.The sampling was carried out at three study savannah dominant sites in Tanzania: the Serengeti National Park (Serengeti), the Ruaha National Park (Ruaha), and the Selous Game Reserve (Selous). The ~ 25,000 km31. The samples were collected by teams of veterinarians, anti-poaching units, and local enumerator networks from villages surrounding the protected areas, where bushmeat is sold32. All samples were collected through incidental sampling after obtaining requisite permits from national authorities and in consultation with village leaders. No wildlife species were harmed or directly surveyed as part of this investigation.Sample collection occurred over a two-year period between July 2016 and July 2018, from both dry and rainy seasons. Samples were identified as either “fresh” or “processed”. Fresh samples were those that appeared to be recently harvested while the processed samples included those that were either heavily salted, sun or air-dried, semi-boiled, or a combination of methods that resulted in dehydrated meat samplesBushmeat samples (~ 250–500 g) were collected in double zip lock freezer bags containing sterile non-toxic Silica Gel desiccant moisture absorbers/dehumidifiers. The samples were stored in − 20 °C vehicle freezers prior to transferring to − 20 °C solar freezers in the field. The samples were then transported to the Nelson Mandela African Institution of Science and Technology (NM-AIST) in Arusha via vehicle freezers, where they were stored at − 80 °C until processing. Cold chain maintenance was ensured through temperature trackers included with the samples throughout the entire process. Sample identification sheets were completed for all collected samples and the metadata, including information regarding sample condition, wildlife species, the collection site (using GPS coordinates), and seasonality.To avoid potential issues associated with surface contamination, three replicates of ~ 120 mg each, were obtained from the muscle tissue using sterile, disposable Rapid Core punch  and sterile disposable safety scalpels . The samples were placed in the MagMAX™ Lysis/Binding Solution Concentrate buffer . The homogenization protocol was optimized and the settings with the best DNA yield for both fresh and processed samples can be found in the Nucleic acid extractions were performed using MagMAX™ 96 DNA Multi-Sample Kit  and KingFisher Flex automated DNA purification system  per manufacturer’s instructions with minor modifications. If the yield and purity of extractions were not adequate for a specific sample, such as the DNA concentration was less than 4 ng/µl and/or if the quality of the amplicons analyzed by the Bioanalyzer did not pass the quality control, DNA was extracted manually using DNeasy PowerSoil Kit  per manufacturer’s instructions. Extracted DNA was quantified using Qubit™ 3.0 Fluorometer . To ensure purified DNA was of high-quality, DNA was also visualized through agarose gel electrophoresis.B. anthracis, Brucella and Coxiella) were carried out in 20 μl reactions with both positive and negative controls , 2 μl of 5 uM of each forward and reverse primer  encoded by plasmid pX01 and Capsule B (CAPB2) encoded by plasmid pX02  was the targeted gene to detect Brucella species and to detect Coxiella burnetti, Insertion element IS1111 was used as the target gene. Supplemental Table Amplification reactions for the detection of all pathogens  Hub in Nairobi, Kenya for the microbiome sequencing. The V3-V4 hypervariable region of the 16S rRNA gene was sequenced on the Illumina MiSeq System generating 2 × 300 bp paired-end reads (see methods in 34. The EDGE Bioinformatics platform (v2.3.0) implementation is based on QIIME v1.9.1 and includes demultiplexing and quality filtering, operational taxonomic unit (OTU) clustering, taxonomic assignment, phylogenetic reconstruction, diversity analyses, and visualizations of each of these analyses34. QIIME started the pre-processing step with 43,095,486 paired-end reads (in FASTQ format) by removing reads with a PHRED quality score lower than Q20. Data were further filtered to remove reads with more than one ambiguous base. If less than 50% of a read contained consecutive high-quality bases, the read was discarded. The forward and reverse reads were merged before analysis. A 94% nucleotide identity sequence clustering threshold (approximately genus level) was used to generate OTUs. The representative sequence for each OTU was then queried against the Greengenes database35 for taxonomic classification assignment. The depth filter was set to 1000 reads—any barcoded sample that had less than 1000 assembled reads did not undergo taxonomic classification or further alpha- and beta-diversity analyses. Next, the integrated phylotype command generated the consensus taxonomy using phylotype-based approach, where taxonomy-linked sequences were assigned to OTUs based on similarity.To guarantee a high quality data set, microbiome analyses were performed using an open source platform, Empowering the Development of Genomics Expertise (EDGE)prop.test with continuity correction function in the ‘stats’ package36. The relative risk of a sample being positive for one or more of the select pathogens was calculated using the risk-ratio function of the ‘epitools’ package36. To investigate the additive effects of all three categories on the outcome of a sample being positive for any of the pathogens, a multivariate analysis was performed using logistic Generalized Linear Model (GLM with logit link) with region, season and meat condition included as categorical variables37. To examine the potential for sample size biases in the microbiota sequencing, a rarefaction analysis was conducted where the fraction of taxa was captured for each sample38. Results show that we were able to consistently identify all the detectable OTUs present in every sample with a minimum reading depth of 14,050 sequences per sample. This suggests that the variation of the microbiota among samples is not likely caused by low sequence coverage and that further sequencing would not detect additional genera  was performed using the Bray–Curtis Matrix at the phylum level using the package ‘Vegan’39. Groups were clustered graphing ellipses estimating the 95% confidence interval (CI) around the average value, for each variable.All the statistical analyses were performed using the software R , if not otherwise stated. The proportion of positive samples by season , sampling region  and meat condition  was estimated using n = 1688; 33 villages), Selous , and Serengeti  as shown in Fig. Connochaetes taurinus), zebra (Equus burchellii quagga), buffalo (Syncerus caffer), impala (Aepyceros melampus), dik-dik (Madoqua spp.), and other less common species, including eland (Tragelaphus oryx), topi , and the African savannah hare (Lepus microtis)  samples were identified as positive for at least one of the three tested pathogens, B. anthracis, Brucella, and Coxiella. There was an overall prevalence of 0.48%  of B. anthracis in the collected bushmeat samples, 0.9%  prevalence of Brucella, and 0.7%  prevalence of Coxiella , the highest percentage of Brucella in Ruaha at 53% , and the highest percentage of Coxiella was found in Selous at 76%   prevalence of the three select agents in the Serengeti, 1.4%  prevalence in Ruaha, and 2.7%  prevalence in Selous  and the samples positive for Brucella were mostly from dik-dik (n = 17)  Fig. . The sament Fig. . Remarkason Fig. . Also, ason Fig. . Only onson Fig. .The relative risk (RR) associated with a sample being positive for one or more of the select pathogens was determined Fig. . The relMultivariate analysis was undertaken to investigate the combined effects of all three categories on the outcome of a sample being positive for any of the pathogens. Results showed that samples collected during the rainy seasons increase the log odds of being positive by 0.70, compared to the dry season. On the contrary, processed samples decrease the log odds of being positive by 1.1, compared to fresh samples. Among the regions, analysis indicated that compared to Ruaha, samples collected from Selous increase the log odds of being positive by 0.70, and samples collected from Serengeti increase the log odds of being positive by 0.70 Table .Firmicutes and Proteobacteria  and Firmicutes (− 0.90) and the second principal coordinate by the relative abundance of Bacteroidetes (0.99)  contained six samples, of which five were Brucella-positive with the majority of the samples being freshly collected  included 12 B. anthracis-positive samples collected from Serengeti, of which eight were from fresh wildebeest samples collected during the rainy season. Cluster III (n = 24) included 16 Coxiella-positive samples, of which 15 were fresh, with thirteen of these collected from the Selous ecosystem. Samples in cluster IV (n = 24) included 17 Brucella-positive samples collected from Ruaha and Selous, four Coxiella-positive samples collected from Selous, and three B. anthracis-positive samples collected from the Serengeti ecosystem. The final cluster, cluster V (n = 5) was the most diverse and included three Brucella-positive and two Coxiella-positive samples , Enterobacteriaceae (22%), and Moraxellaceae (14%). The alpha diversity revealed differences according to pathogen, host species, and region  with diverse wildlife composition and density, and proximity to major urban centers  where the bushmeat is often sold.A goal of this study was to assess the potential for risk of human exposure to three select agents, Brucella and Coxiella often go unrecognized and remain under-reported despite the considerable risks of causing disease41.While the presence of DNA signatures does not imply the presence of a viable organism, given that bushmeat consumption is as high as 5 meals per week in some households, and with an overall pathogen prevalence of 2.03% (1.6–2.5%), this risk of exposure remains a concern and worthy of further investigation. This is especially problematic considering that the risks associated with endemic infections such as 42. Changes in weather conditions force the movement and interactions of animals where rainy seasons and/or droughts affect animals spatial grazing patterns44. Also, exposure to anthrax may be higher for migratory ungulates like wildebeest that are found in different parts of the regions throughout the year3. This may be one reason why there is a higher likelihood of positive samples being collected during the rainy season. Also, during the rainy season there may be a higher consumption of fresh samples, which is consistent with the higher risk of positives in fresh samples, probably associated with methods of processing being unavailable or unsuitable during this season, or simply a higher availability of fresh meat. High salt, UV exposure, and boiling of processed samples may reduce pathogen loads associated with the fresh meat samples46; however, UV light does not necessarily decrease the microbial content of meat48. Results also indicate that there is a lower risk of a sample being positive if it was collected from Ruaha. There are some substantial differences among the three ecosystems, both biotic and abiotic, and we have no evidence of a dominant factor affecting the samples, and suggest that multiple components might be contributing to the relative risks associated with each ecosystem50.Relative risk associated with positive DNA signatures was assessed for variables including season, sample condition, animal species, and regions (ecosystems) and revealed notable associations. There was a higher risk of a sample being positive if it was collected during a rainy season, or if the sample was fresh, in contrast there was a lower risk if the sample was collected from Ruaha, than Serengeti or Selous Fig. . StudiesFirmicutes and Proteobacteria, as demonstrated by the PCoA  of purported “bushmeat” samples from Selous that were instead speciated as being of cattle in origin had a combined prevalence of 28.7% of the three select agents, highlighting a potential public health risk associated with the endemic diseases in livestock production. This suggests that B. anthracis, Brucella, and Coxiella circulate throughout the year in all the ecosystems and confirms their endemicity and the recurrent risk for the nearby communities, including the larger markets where the meat is regularly sold.Samples that were collected in the same condition were generally available to be sold to the public, hence they represent microbial communities that are endogenous to the samples, as well as the microbiota that is acquired during harvesting, processing, and transporting. While the analysis suggests that fresh samples, samples collected during rainy season, and samples collected from the Serengeti and Selous had a higher likelihood of being positive for one of the three pathogens, positive samples carrying DNA signatures of the tested select agents were also collected during dry season, from other animal species, and ecosystems. Interestingly, the results also revealed that a subset  in the ecosystems, and the role of endemic and emerging strains in the microbial community, caused by sedentary and migrating animal species in the region are also warranted since the extent of an outbreak varies from year to year and place to place. In conclusion, the current investigations provide important insights and a rational basis for the formulation of future studies to assess and address the public health and emerging infectious disease risks associated with bushmeat harvesting, trade, and consumption in Tanzania and elsewhere.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.


We measured plasma and cerebrospinal fluid (CSF) metabolite concentrations in a 5-day porcine sepsis model of fecal peritonitis. The objectives were: (i) to verify whether the expected pathways that had emerged in previous studies pertain only to the early inflammatory response or persist for the subsequent days; (ii) to identify metabolic derangements that arise later; (iii) to verify whether CSF metabolite concentrations were altered and if these alterations were similar to those in the blood or delayed. We observed an early response to inflammation and cytokine storms with alterations in lipid and glucose metabolism. The arginine/asymmetric dimethylarginine (ADMA) and phenylalanine/tyrosine balances changed 24 h after resuscitation in plasma, and later in CSF. There was a rise in ammonia concentration, with altered concentrations of metabolites in the urea cycle. Whether persistent derangement of these pathways have a role not only on short-term outcomes but also on longer-term comorbidities, such as septic encephalopathy, should be addressed in further studies. In survivors, organ dysfunction, including dysfunction of the brain, may have long-term consequences on physical and mental health, and on quality of live4.Sepsis is a life-threatening disease caused by a dysregulated host response to infection. This condition can deteriorate to septic shock in which circulatory and cellular/metabolic abnormalities may substantially increase the mortality riskThe uncontrolled inflammatory response in sepsis and septic shock condition activates or alters several metabolic pathways related to complex biological communication between vital organs. However, the temporal evolution of sepsis is variable, and the phenotype dependent, among others, on source of sepsis, amount fluid losses and elapsed time until diagnosis. Accordingly, time course and magnitude of metabolic abnormalities in response to the septic insult are difficult to interpret in retrospect.The aim of the present study was to measure the metabolic derangements induced by sepsis by quantitative metabolomics analysis of plasma and cerebrospinal fluid (CSF) in a clinically relevant 5-day animal model. This allows assessment of metabolites’ concentrations at health, before sepsis occurs, and then repeatedly at exactly defined time points after the septic insult and from different compartments. With this information, precise determination and quantification of early and late metabolic pathways in sepsis is possible, and also whether the pathways are similar, different or just delayed in cerebrospinal fluid compared to blood. Future studies may then investigate, whether certain pathways can beneficially be modulated or specific compounds used as biomarkers for sepsis and or brain dysfunction evolution.5.Twenty anesthetized, mechanically ventilated pigs were randomized to fecal peritonitis or sham n = 10/group): Blood and cerebrospinal fluid (CSF) samples were collect at baseline, 8 h of observation after the insult without sepsis treatment, and sequentially within 24, 48 and 74 h after resuscitation, for a total of five time points. The protocol is illustrated in Fig. 0/group: 5. Briefly, after induction of fecal peritonitis, before resuscitation animals had low cardiac output and blood pressure. Hemoconcentration and low filling pressures suggested capillary leakage with fluid sequestration. Resuscitation then produced the typical hyperdynamic state with increased temperature, heart rate and cardiac output (CO). Septic animals received more Ringer’s lactate, norepinephrine and glucose to maintain hemodynamic stability and homeostasis, but at the same time, had lower urine output and higher positive fluid balance than sham pigs5. At T2/T3, platelets and leucocytes concentration were lower in septic than in sham animals, but band neutrophils percentage remained significantly higher in the septic group until the end of the experiment, although decreasing from T2 to T5  taking into consideration the T2/T1 and T5/T1 fold change to characterize the main differences in the metabolic pathways between septic and sham animals after the insult and at the end of the experiment. Figure These preliminary analyses indicated that lysophosphatidylcholines (LPC), phosphatidylcholines (PC) and the ratio between the amounts of LPCs and PC were reduced after the development of sepsis and did not recover after resuscitation Fig. . The heaWe then focused on specific pathways characterizing the acute phase of shock and the phases of resuscitation, and clearly emerged from the multivariate analyses.The inflammatory insult induced in septic animals produced, as expected, a significant increase in lactate in different circulatory districts from T2 Table , Fig. S4We then focused on specific metabolic species related to glucogenesis as the energy imbalance in sepsis is crucial from the early phases. Glucogenic amino acids  can give a picture of glucose production and its related pathways.Glutamine and alanine, two glucogenic metabolites, appeared in the first ranks of the VIP score of the model on T2/T1 fold-changes  to aromatic AAs , often used as an indicator of liver damage T2 Fig. . At the  T2 Fig. . Among t T2 Fig. . These bThe urea cycle converts ammonia to urea and is essential for the ammonia detoxification. In the multivariate analyses citrulline, glutamine, aspartate and asparagine were at the first ranks of the VIP scores for the models based on metabolite fold-changes Table , and theThis complex urea biochemical network led us to investigate arginine, asparagine, aspartate, citrulline, ornithine, glutamate, glutamine, glycine and the following ratios: citrulline/glutamine, arginine/citrulline, ornithine/arginine, ornithine/citrulline, arginine/(ornithine + citrulline), as measures of arginine bioavailability, glutamine/glutamate, glycine/glutamate. This information was paralleled with the ammonia values and the ratio of ammonia to urea cycle related metabolites.Ammonia was significantly higher from the end of the observation period after the insult (T2) until the end of the experiment. At T2 ammonia in septic pigs was more than three times that in of sham group Fig. .Figure 5Sham animal showed an early increase in glutamine at T2, then glycine was significantly lower from T3, and the glutamine/glutamate ratio dropped at T4 and T5. This suggests that the reactions of the glycine cleavage system shifted in the direction of ammonia production from glycine. The conversion of glutamate and ammonia into glutamine also seemed involved, as the ratio of glutamine to ammonia was significantly lower in septic animals at T2, T3 and T4.Compared to sham animals the concentration of aspartate was significantly higher while the concentration of arginine and the arginine/citrulline ratio were significantly lower after the insult. The bioavailability of arginine did not significantly differ in the two groups and was quite stable. Asparagine was significantly lower from T3. These results hint at less conversion of aspartate to produce asparagine, by utilizing ammonia Fig. .Multivariate analyses indicated that asymmetric dimethylarginine (ADMA) and phenylalanine were in the top of the VIP score for the models based on metabolites changes Table .In our experiment, the ratio of arginine to ADMA decreased in septic animals and was significantly lower than in sham group from T3 Fig. . This miPhenylalanine is a precursor for tyrosine, the monoamine neurotransmitters dopamine, which is the metabolic precursor of norepinephrine  and epinephrine . The phenylalanine/tyrosine ratio was significantly higher in septic animals from T3 and this might be due to a decrease in tyrosine and an increase in phenylalanine, as these values were significantly higher in the septic group than sham animals.In 14 of the 20 pigs (6 sham and 8 septic pigs) cerebrospinal fluid (CSF) was collected and analyzed using the same technological platform as for plasma metabolomics. Fifty-four (54) metabolites were quantified, out of 186 measurable.The metabolite concentrations in CSF varied widely, but with few significant differences between the two groups. However, there were similar trends for some of the biochemical pathways previously highlighted in plasma metabolome. For example, the phenylalanine/tyrosine ratio in the CSF increased and was significantly higher than in sham animals from T4 Fig. , while iLactate in CSF was significantly higher at T4 and T5, later than in the arterial vessels. However, lactate in the superior sagittal sinus (SSS) was significantly higher in septic animals at T2, T3 and T4 and decreased at T5, to levels similar to those in sham animals. The balance between glucose and lactate was altered only slightly as glucose was significantly higher at T4 and the glucose/lactate ratio was significantly lower at T3 and T4.The main feature of this experiment is the long observation time after sepsis that enabled us to determine magnitude and time course of a large number of metabolic changes in responses to the inflammatory insult and the subsequent standardized sepsis treatment.After induction of fecal peritonitis, before resuscitation, the animals had low cardiac output and blood pressure. Hemoconcentration and low filling pressures suggested capillary leakage with fluid sequestration. Resuscitation then produced the typical hyperdynamic state with increases in temperature, heart rate and cardiac output (CO) Figs. , S2. TheIn comparison to similarly instrumented and anesthetized control animals, we observed in septic animals early metabolic changes, which rapidly resolved , partly resolved , or were persistent .8, sepsis altered the overall lipidome  to aromatic amino acids (AAAs)—often decreased in patients with hepatic failure—dropped 24 h after resuscitation in the sepsis group and never rose in 74 h after resuscitation, suggesting that subclinical hepatic alterations16 probably started with the septic insult, and did not recover in the post-resuscitation time. The higher plasma alanine and glucogenesis AAs  is a master metabolic regulator controlling the conversion of pyruvate to acetyl-CoA in the mitochondria and its inactivation contributes to the metabolic reprogramming that occurs in immune cells in response to inflammation22.In this study we did not consider the ATP/ADP/AMP axis and tricarboxylic acid cycle (TCA) cycle intermediates, such as succinate, fumarate and citrate, which play a crucial role in the response to septic insults23. The loss of anabolic biosynthetic pathways develops within hours of the onset of inflammatory shock and TCA cycle shifts from intermediate cis-aconitate to itaconate. Itaconate directly inhibits succinate dehydrogenase (SDH), leading to disruption of oxidative phosphorylation and decreased ATP synthesis24. If this negative feedback loop of repressed anabolism persists, a life-threatening clinical syndrome of immunometabolic paralysis develops.Pyruvate dehydrogenase kinase (PDK) phosphorylates PDC and inhibits the conversion of pyruvate to acetyl-CoA. The decreased synthesis of acetyl-CoA limits TCA cycle-supported anabolism and converts the high energy demands to a tolerable, catabolic low-energy supply state21.Modulation of TCA cycle seems to affect urea cycle intermediates as well. In a monocyte culture model of severe acute inflammation that simulates sepsis reprogramming, the inhibition of PDK increased urea cycle activity, with elevated arginine, ornithine, citrulline, aspartate and possible repletion of the TCA cycle intermediate fumarate, while decreasing itaconate, and increasing anaplerotic catabolism of branched-chain amino acids (BCAAs)25. A low arginine/ADMA ratio can indicate reduced NO production due to reduced availability of L-arginine to NOS27. We observed also a decreasing of arginine/ADMA ratio in the CSF of sepsis animals later time after resuscitation (T4), compared to plasma (T3)  and a concomitant reduction of the arginine/ADMA ratio, starting from T3. ADMA is an endogenous inhibitor of eNOS and the arginine/ADMA ratio is considered an indicator of NO bioavailability3) Figs. , 7.28. The liver is the main site of phenylalanine metabolism; therefore, deterioration of liver function may reduce this metabolism, raising systemic phenylalanine levels. The biosynthesis of Phe to Tyr mediated by phenylalanine hydrolase and the transformation of Tyr to Dopamine (DOPA) by tyrosine hydroxylase requires the tetrahydrobiopterin (BH4) as cofactor. Inflammation and immune activation affect BH4 availability, and as a consequence, the biosynthesis of phenylalanine and tyrosine is altered29. Moreover, BH4 modulates specific metabolic changes in inflammatory macrophages by the regulation of NO production. These include regulating respiratory function by changes in levels of the important TCA cycle metabolites, and inflammatory mediators, citrate, succinate, and itaconate30.Furthermore, our results point to the dysregulation of the phenylalanine-tyrosine ratio (Phe-Tyr). This was a late response after the insult, and was also observed in the CSF—at an even later time point Figs. , 7. Arte31. Currently the precise mechanisms responsible for the neurological effects of phenylalanine accumulation are unclear.The impaired conversion of phenylalanine to tyrosine in the liver and the concomitant decrease in tyrosine affect the amino acids transport into the brain and its further conversion to its downstream metabolites dopamine, epinephrine and norepinephrineFinally, the interplay among different metabolic intermediates and cofactors as well as organ crosstalk, such as liver-brain axis, need further investigations.This study illustrates the timing of different pathways associated with the development of sepsis and resuscitation. Our results confirmed the early metabolic responses to inflammation and cytokine storms with alteration in lipid and glucose metabolism. Other pathways, which may have an impact on brain function, such as the phenylalanine/tyrosine balance, occurred after T3 in plasma and later in CSF. In addition, besides the increase in ammonia concentration, our results suggest an alteration in the urea cycle.5.The animal study was conducted according to the EU Directive 2010/63/EU for animal experiments and the ARRIVE guidelines for animal research, and was approved by the Animal Care Committee of the Canton of Bern, Switzerland (BE103/16). Details of the animals, anesthesia, instrumentation and experimental protocol were previously reported2, 5 cmH2O PEEP, 8 ml/kg tidal volume and 20 breaths/min respiratory rate. This was later adjusted with the goal to keep pH 7.35–7.45 and PaCO2 35–45 mmHg.Briefly, 20 mechanically ventilated, unconscious domestic pigs (weight: 39.8 ± 2.7 kg of both sex) were used. The pigs were sedated with ketamine (20 mg/kg) and xylazine (2 mg/kg) intramuscularly. General anesthesia was induced with midazolam (0.5–1.0 mg/kg) and atropine (0.02 mg/kg) intravenously, and maintained with propofol (4–10 mg/kg/h) and fentanyl (3–20 μg/kg/h). The depth of anesthesia was evaluated continuously with 1 derivation EEG, and observation of spontaneous movements, while nociception was checked hourly through toe pinching. After tracheal intubation, the pigs were mechanically ventilated in volume-controlled mode, initially with 30%, FiO2 saturation and flow. The pig was then turned into a left lateral position and craniotomy was performed to place a single-lumen oximetry catheter  in the superior sagittal sinus together with a pressure probe  to measure sagittal sinus and intracranial pressures, used for a separate study5. After midline laparotomy in dorsal recumbency, a catheter was inserted into the urinary bladder for drainage and measurement of urine output. Flows in the portal vein, hepatic artery and right kidney artery were measured by means of probes  positioned around the vessels.With the pigs in sternal recumbency, a spinal catheter was percutaneously inserted to collect cerebral spinal fluid. Then, with the pigs in dorsal recumbency, a midline neck incision was made to allow placement of an arterial catheter  in the left carotid artery, a pulmonary artery catheter  via left external jugular vein, a venous catheter in the left internal jugular vein  for volume infusion, a triple-lumen catheter  in the right internal jugular vein, and a flow probe  around the right carotid artery to measure blood pressures, O2) and hepatic venous pressure (Phv). Another pulmonary artery catheter was placed in the right renal vein through the right femoral vein to measure renal vein O2 saturation and pressure. Before abdominal closure, a large-bore drainage tube was placed intraperitoneally for induction of fecal peritonitis later5.A single-lumen catheter was positioned in the portal vein through a mesenteric vein to measure portal vein pressure . An 8.5 F introducer was placed in the hepatic vein for a pulmonary artery catheter  to measure hepatic vein O2 saturation . Continuous cardiac output  was measured with a Vigilance monitor , and O2 saturations were measured continuously with a Vigilance monitor or intermittently with a blood gas analyzer . Blood flows were measured with ultrasonic transit time perivascular flowmeters using double-channel TS 140 420 flowmeters . The signals of all the pressures and blood flows were recorded at 100 Hz by a data acquisition system .As previously reportedAfter surgical preparation, one-hour stabilization was allowed, followed by baseline blood and cerebrospinal fluid (CSF) sampling and data acquisition within 30 min (time point T1). Then, the pigs were randomized to fecal peritonitis (n = 10)/sham (n = 10) using sealed envelopes and to groups with/without abdominal vacuum, for a separate study.Fecal peritonitis was induced by peritoneal instillation of 2 g/kg of autologous feces dissolved in 250 mL warmed glucose 5% solution. After 8 h observation, before resuscitation another set of blood and CSF samples were collected (T2), then protocol-based resuscitation was started and continued for three days . Adequate resuscitation is important, and we used a strict protocol adhering to the current recommendations. Details about the resuscitation protocol can be found in the Supplemental Material.Further blood and CSF samplings were done just before RP + 24 h (T3), RP + 48 h (T4) and a couple of hours after RP + 72 h (T5). At the end of the experiment, anesthesia was deepened, and the animals were euthanized with 40 mmol potassium chloride.The protocol is illustrated in Fig. At each time point, arterial blood samples were collected, immediately refrigerated-centrifuged and EDTA-plasma was isolated for metabolomics and laboratory analyses. Then we measured hemoglobin concentration, hematocrit, lactate from blood sampling at different sites, concentrations of ammonia, alanine amino transferase (ALAT), aspartate amino transferase (ASAT), platelet count, prothrombin time, leukocytes, band neutrophils, creatinine, creatinine kinase and bilirubin on top of arterial and mixed-venous blood gasses. At each time point, CSF samples were collected for metabolomics and laboratory analyses . We didn’t measure Adenosine triphosphate (ATP) or its products adenosine diphosphate (ADP) and adenosine monophosphate (AMP), as they are related to mitochondrion energy metabolism, they provide a rather global picture, as they pertain to all possible organ cells and they are not so specific for the pathways we considered in our animal model.12. Measurable metabolites and their extended names and abbreviations are listed in Supplementary Table A targeted quantitative approach using combined direct flow injection and liquid chromatography (LC) tandem mass spectrometry (MS/MS)  was employed for the metabolomics analysis of plasma and CSF. The assay quantifies 186 metabolites from six analyte groups: acylcarnitines, amino acids, biogenic amines, hexoses (sum of hexoses), glycerophospholipids and sphingomyelins. The method combines derivatization and extraction of analytes with selective mass-spectrometric detection using multiple reaction monitoring (MRM) pairs. Methodological details and data pre-processing have been reported in our previous articles32 and De Noord and Theobald33. In the multilevel models, the variation between and within subjects are separated. The aim of these analyses is to separate the contribution of subject variability by taking into account the fact that the measures are repeated, i.e. from the same subject. The between-subject variation in multilevel data analysis uses the average of the observations, while the within-subject variation uses the net differences between paired observations. The initial step in the multilevel model is therefore to separate the between-subject from the within-subject variation. We report the key steps of these methods in the supplemental material.MultiLevel Simultaneous Component Analysis (MLSCA) and multilevel partial least square discriminant analysis (MLPLSDA) models were proposed by Westerhuis et al.I = 10 septic shock animals at the five time points (Ki = 5) using all metabolites concentrations as variables (J = 124), in order to have a global picture about the changes induced by the sepsis over the time of the experiment.In this study, we applied a MLSCA model on Then we considered the fold-change from T1 to T2 and from T1 to T5, i.e. from baseline to development of sepsis and from baseline to the end of the experiment, in order to characterize the main changes induced by sepsis in septic and sham animals. As the number of features (124 metabolite concentrations) is much higher than the number of observations (20 pigs) we filtered the variable so as to avoid redundancies and collinearities, and considered the binary classification as septic and sham.34. This algorithm sorts the features according to their relevance to the classification (maximum relevance criterion) and their redundancy (minimum redundancy criterion) in relation to the other variables. The ranking is based on the mutual information between the outcome and each feature and on the mutual information between each couple of features. The features distribution is discretized according to the interquartile range in order to apply the mRMR algorithm and the first 40 ranked features were selected for further analyses. The first 40 ranked features were used in a partial least square discriminant analysis (PLS-DA) model to separate sham and septic animals, and we ranked the features according to the variable importance in projection (VIP) scores, which represent the weights of each feature in the PLS-DA model. A feature with a VIP score higher than 1 is usually considered significant.We adopted the method proposed by Peng et al., which is minimal-redundancy-maximal-relevance (mRMR)The values of each variable are compared for the septic and sham groups at each time point using the Wilcoxon rank-sum test. To deal with the large number of comparisons for the metabolites, we also computed the false discovery rate (FDR). For significant comparisons p < 0.05 and FDR < 0.05 were considered.As regard metabolomics data, we focused our univariate analysis on specific pathways characterizing the acute phase of shock and the phases of resuscitation, and clearly emerged from the multivariate analyses. For each specific pathway we analyzed also specific ratios commonly adopted in literature to characterize the bioavailability of pathway substrates.Supplementary Information 1.Supplementary Information 2.


Supplemental Digital Content is available in the text 12 (VitB12) levels after bariatric surgery. This study reports the phenomenon and adverse reactions of serum VitB12 elevation 1 month after surgery and explores the possible etiologies.Few studies have reported an increase in vitamin B12 data for 112 patients from January 2018 to October 2019. Then, 87 patients were included between November 2019 and August 2020. They were divided into 2 groups according to the level of VitB12 after surgery, and the demographic and clinical data were analyzed. Then, LASSO regression model analysis and multiple logistic regression analysis were performed to explore the risk factors for VitB12 elevation after surgery.Retrospective analysis was performed on VitB12 level was significantly increased 1 month after surgery. Comparison of data between the 2 groups found that more patients also had diabetes in the nonelevated group. The postoperative folic acid and VitB12 levels of the elevated group were significantly higher than those of the nonelevated group. More patients had concurrent constipation in the elevated group than in the nonelevated group. Two meaningful variables in LASSO regression analysis were incorporated into the multivariate logistic regression analysis, and constipation was found to be an independent risk factor for the increase in VitB12 after surgery. Of the 199 patients in this study, 111 patients had elevated VitB12 levels after surgery. Among them, 7 patients had peripheral nerve symptoms.Retrospective data showed that the VitB12 levels after surgery. High levels of VitB12 may cause some peripheral nerve symptoms. Therefore, it is necessary to pay attention to patients with postoperative constipation, monitor their VitB12 level as soon as possible, and take measures to improve constipation to avoid some adverse reactions caused by elevated VitB12 levels.Constipation is an independent risk factor for increased VitB It increases the risk for developing multiple diseases, such as hypertension, diabetes, cardiovascular disease, several types of cancers, and poor mental health. All of these affect quality of life and work efficiency and increase medical costs. It has been proven that weight loss can effectively prolong life and reduce medical economic burden. In the treatment of obesity, physical therapy, such as moderate exercise, diet adjustment, and oral or injection drugs are inefficient, making it easy to regain weight, while surgery can achieve sustained and effective weight loss and is considered to be the most effective and lasting method for the treatment of obesity.Obesity is a complex multifactorial disease. The worldwide prevalence of overweight and obesity has doubled since 1980 to the extent that nearly one-third of the world's population is now classified as overweight or obese. Besides, in China, more and more LSG + jejunal bypass (JJB) used to treat patients who are heavier. LSG + JJB is on the basis of Sleeve, idling a section of the small intestine further reduces absorption to achieve better weight loss. Clinical studies have shown that although bariatric surgery can effectively reduce the weight of obese patients, postoperative malnutrition caused by bariatric surgery deserves our attention, and vitamin B12 (VitB12) is the most commonly affected nutrient.,9,10 VitB12 is mainly absorbed at the end of the ileum and requires internal factors secreted by gastric parietal cells. The amount of VitB12 stored in the body is approximately 2000 μg, and only 2 μg is needed on average every day. Despite such large reserves, VitB12 deficiency is common after bariatric surgery. Malabsorption and inadequate oral intake are the 2 main causes of VitB12 deficiency in patients undergoing bariatric surgery. Other causes of VitB12 deficiency include postoperative food intolerance and bacterial overgrowth syndrome.Currently, the most commonly used method of bariatric surgery is laparoscopic sleeve gastrectomy (LSG) and laparoscopic Roux-en-Y gastric bypass (RYGB).12 levels after bariatric surgery. High serum levels of VitB12 after surgery may lead to some peripheral nerve symptoms, such as limb numbness, fatigue, pain, facial edema, and whole body stiffness. In addition, 1 study has reported that high serum levels of VitB12 can increase the incidence of unstable angina pectoris. Therefore, increased serum VitB12 levels after bariatric surgery need our attention, as this condition may affect the prognosis of patients. The purpose of this paper is to analyze the related risk factors of an increase in serum VitB12 after bariatric surgery through a case-control study and to obtain the theoretical basis for the increase in serum VitB12 levels after bariatric surgery, providing some ideas for the prevention of postoperative VitB12 imbalance.However, there are few reports of elevated VitB212 data before and at 1, 3, and 6 months after surgery for 112 obese patients  who underwent bariatric surgery from January 2018 to October 2019 at Third People's Hospital of Chengdu. Later, the analysis included 87 obese patients  who underwent bariatric surgery between November 2019 and August 2020 at Third People's Hospital of Chengdu. A total of 53 patients with elevated serum VitB12 levels (>771 pg/mL) were divided into the case group, and a total of 34 patients with nonelevated serum VitB12 levels (≤771 pg/mL) 1 month after surgery were divided into the control group. The general medical data of all patients were collected and recorded, including sex, age, body mass index (BMI), mode of surgery, complications , hematological indexes . All patients completed the surgery successfully, and a clear liquid diet was required within 1 month after surgery, with 60 g protein powder and 2 multivitamin tablets supplemented daily. The clinical data of BMI, serum VitB12 and folic acid were reviewed and recorded 1 month after surgery. Patients were asked if they took multivitamin tablets as prescribed by the doctor and whether there was constipation and/or acid regurgitation. The Ethics Review Committee of Chengdu Third People's Hospital approved the study. All the subjects provided and signed a written informed consent. This study has benn registered in Chinese Trial Registry. Clinical trial registration numbers and date: ChiCTR2000038232, on September 15, 2020. All methods were performed in accordance with the regulations.Retrospective analysis was performed for serum VitB2 or BMI ≥ 27.5 kg/m2 with high-risk comorbid conditions, such as cardiopulmonary problems , uncontrolled type 2 diabetes, or obesity-induced physical problems interfering with lifestyle, hyperuricemia, renal insufficiency, or a history of primary hyperparathyroidism; patients undergoing bariatric surgery in our hospital; patients with normal serum VitB12 levels before surgery; and patients who understood and volunteered for the study. Exclusion criteria included: secondary obesity caused by endocrine disorders; history of malignant tumor; severe liver disease or renal failure; preoperative use of corticosteroids and other drugs; patients who needed to take antiepileptic drugs, metformin, histamine H2 receptor inhibitors and other drugs that affect VitB12 levels after surgery; patients deemed to have uncontrollable mental illness; patients whose comprehension and cognitive abilities were not adequate enough to complete the follow-up; other reasons affecting the metabolism of VitB12; and patients who failed to follow-up after surgery. The demographic and clinical data of 87 patients are presented in Table Inclusion criteria included: BMI ≥ 32.5 kg/mt test or one-way ANOVA was used for comparisons. If they did not obey a normal distribution, the rank sum test was used, and the chi-square test or Fisher exact probability method was used for counting data. Statistical analyses were performed with SPSS . The LASSO regression model was used to screen out meaningful risk factors, and R software  was used to incorporate the selected risk factors into multivariate logistic regression analysis. P < .05 was the level of significance.Descriptive statistics consisting of means, standard deviations, and percentages were calculated for variables. When the measurement data between the 2 groups obeyed a normal distribution and the variance was uniform, a paired 312 level of patients was significantly increased 1 month after surgery (P < .001); the serum VitB12 level of the patients improved 3 months after surgery; and there was no significant difference in serum VitB12 level 6 months after surgery (P = .945). The retrospective data are presented in Table The retrospective data showed that, compared with that before surgery, the serum VitB12 group and nonelevated serum VitB12 group revealed some interesting findings. There was no significant difference in demographic data between the 2 groups. More patients in the nonelevated serum VitB12 group had diabetes than in the elevated serum VitB12 group (P = .015). The postoperative serum folic acid level of the elevated serum VitB12 group was significantly higher than that of the nonelevated serum VitB12 group (P = .012). The postoperative serum VitB12 level of the elevated serum VitB12 group was significantly higher than that of the nonelevated serum VitB12 group (P < .001). The comparison of demographic and clinical data between the 2 groups is presented in Table A comparison of the case data between the elevated serum VitB12 group than in the nonelevated serum VitB12 group . The comparison of postoperative-related factors between the 2 groups is presented in Table When comparing the postoperative-related factors between the 2 groups, more patients had concurrent constipation in the elevated serum VitBUsing the LASSO binary regression model to select related risk factors in the clinical data of patients, 18 factors were reduced to 2 potential risk factors with a nonzero coefficient in the LASSO regression model. The results of LASSO regression model analysis are presented in Figure P = .002, hazard ratio = 5.135, 95% confidence interval: 1.861–15.934). The results of the multivariate logistic regression analysis are presented in Figure 12 after surgery are presented in Supplemental Digital Content 2, http://links.lww.com/MD2/A788.These factors included preoperative diabetes mellitus and postoperative constipation. These 2 factors were incorporated into multivariate logistic regression analysis, and according to the analysis, postoperative concurrent constipation was an independent risk factor for the increase in serum VitB12 1 month after surgery  patients had higher-than-normal serum VitB412, also referred to as cobalamin, is a water-soluble vitamin. VitB12 in nature is synthesized by microorganisms, but higher animals and plants cannot produce VitB12, which can only be supplemented by exogenous VitB12. The liver is the main place of VitB12 storage. VitB12 is mainly excreted through bile and reabsorbed in the ileum. This rarely lost hepatointestinal circulation allows VitB12 to be completely retained, so it takes many years, for even strict vegetarians, to develop VitB12 deficiency. Bariatric surgery removes about two-thirds of the stomach, mainly the fundus and body of the stomach. The decrease in gastric parietal cells and the secretion of gastric acid and internal factors after the operation coupled with the use of H2-receptor inhibitors and proton pump inhibitors can lead to postoperative malabsorption of VitB12. However, due to the slow consumption of VitB12 stored in the liver, VitB12 deficiency is usually not detected in the early stage after the operation (within 6 months after the operation). Similarly, none of the patients in our study had serum VitB12 deficiency 1 month after surgery. Since VitB12 is mainly absorbed in the duodenum and proximal jejunum, VitB12 deficiency is more common after laparoscopic RYGB than after other surgical methods. To prevent postoperative micronutrient deficiency, perioperative support guidelines for weight loss patients recommend taking 1 or 2 chewable multivitamin and mineral supplements every day. According to the recommendations of the guidelines, patients are required to consume a transparent liquid diet within 1 month after operation, supplemented with 60 g of protein powder and 2 multivitamin tablets daily.VitB12 deficiency after bariatric surgery. However, there are few reports on the phenomenon and causes of an abnormal increase in VitB12 after bariatric surgery. B vitamins are currently considered to be safe, and there are no tolerable upper levels that have been set, mainly due to the lack of systematic research on adverse reactions. In this study, we found that some patients undergoing bariatric surgery had increased serum VitB12 1 month after surgery, which returned to a normal level within 3 to 6 months after surgery. During this period, some of the patients even had adverse reactions, such as limb numbness, fatigue, and tingling. Their serum VitB12 levels were all more than 1500 pg/mL, and through analysis, it was found that postoperative constipation was an independent risk factor for increased serum VitB12 within 1 month after bariatric surgery. Constipation is one of the common short-term complications after bariatric surgery. In this study, most of the patients suffered constipation within 1 month after surgery. It has been reported that patients with malabsorption surgery, such as biliopancreatic diversion and RYGB have a higher frequency of diarrhea and fecal incontinence than before the operation. Of the patients treated with B biliopancreatic diversion PD and RYGB, only 5% and 7% reported constipation after surgery, respectively. On the other hand, restrictive surgery, such as the gastric band or LSG, may make patients prone to constipation.,26 In this case-control study, the majority of patients were treated with LSG or LSG + JJB.There have been many studies on the phenomenon and mechanism of VitB At the same time, increased postprandial levels of glucagon-like peptide-1 and peptide-YY after bariatric surgery can also delay intestinal transport. All these may lead to postoperative constipation. In addition, dietary fiber intake plays an important role in determining stool volume and intestinal transport time. Our patients require a clear and liquid diet within 1 month after bariatric surgery, and their dietary fiber intake is significantly reduced. It can also increase the incidence of postoperative constipation. What's more, it is reported that the symptoms of constipation, indigestion and vomiting in obese patients may be caused by thiamine deficiency. After RYGB and LSG treatment, 18% and 25.7% of the patients developed thiamine deficiency, respectively. Combined with the existing information, we postulate that since VitB12 is largely reabsorbed through the enterohepatic circulation, constipation leads to an increase in enterohepatic circulation, resulting in an increase in serum VitB12. In addition, 1 study pointed out that supplementation of B vitamins for 3 months after bariatric surgery can rapidly increase the level of serum VitB12. However, in this study, it was found that multivitamin supplementation within 1 month after surgery was not a risk factor for increased serum VitB12. Multivitamin supplementation may have a synergistic effect with constipation. Folic acid is a water-soluble B vitamin, which is mainly stored in the liver. Folic acid deficiency can lead to many side effects, but high folic acid levels have no obvious side effects. Allergic reactions and gastrointestinal symptoms, such as nausea and abdominal distension, are occasionally seen. Some researchers worry that excessive folic acid may mask the lack of VitB12. In this study, folic acid increased at the same time 1 month after surgery, but it did not exceed the normal level, which may be similar to the increase in VitB12.LSG changes the shape of the stomach, reduces its volume, and alters its vagal innervation and quantity of secretory cells. These may increase intestinal transport.Several strengths and limitations of this study should be noted. The same surgical team performed all the bariatric surgeries. The patient's postoperative management and follow-up data were also recorded by the same person. Limitations of the study include a small sample size and using single-center data, as well as having only short-term follow-up data and a lack of prospective studies to verify these etiological hypotheses.512 after bariatric surgery is still an important clinical problem. Constipation is a risk factor for the increase in serum VitB12 in patients after this operation. To avoid postoperative vitamin-related complications, it is necessary to monitor the status of vitamins as soon as possible and solve problems such as constipation. High levels of VitB12 may cause peripheral nerve symptoms, such as limb numbness, fatigue, pain, facial edema, and whole body stiffness. Whether there are other toxic problems that require longer-term observation and study has not been elucidated. We should consider increasing the intake of dietary fiber to prevent and improve constipation after the operation and appropriately reducing VitB12 supplementation after the operation or formulating a multivitamin supplement program by stages after the operation to prevent excessive levels of VitB12. This topic is very important to improve the prognosis of patients after bariatric surgery.In summary, the level of serum VitBThe authors would like to thank the efforts of the medical staff who followed the patients in The Third People's Hospital of Chengdu.Data curation: Jinhua Zhou, Rui Mao.Formal analysis: Pengsen Guo, Huawu Yang, Jinhua Zhou, Dafang Zhan.Funding acquisition: Pengsen Guo, Huawu Yang, Tongtong Zhang.Investigation: Huawu Yang, Jinhua Zhou, Rui Mao, Dafang Zhan, Tongtong Zhang.Methodology: Pengsen Guo, Rui Mao, Dafang Zhan.Resources: Yanxi Ou.Validation: Yanxi Ou, Yanjun Liu.Visualization: Yanjun Liu.Writing – original draft: Pengsen Guo, Jiang Yuan.Writing – review & editing: Pengsen Guo.


Several well-known inflammatory markers increase in FMF. However, there is still a need for diagnostic tests for specifying FMF and monitoring inflammatory activity. CXCL16 is a chemokine produced by inflammatory cells that demonstrate efficacy in the acute phase response. In this study, we aimed to investigate the relationship between CXCL16 levels and FMF disease and to evaluate CXCL16 levels as a novel biomarker for FMF.Familial Mediterranean feverFifty-three male patients diagnosed with FMF and sixty healthy individuals were included in this cross-sectional study. Blood samples were taken in the first 24 h of the attack periods. Serum soluble CXCL16 was evaluated by enzyme-linked immunosorbent assay (ELISA) method.CXCL16 (P < 0.001), erythrocyte sedimentation rate (P < 0.001), C-reactive protein (P < 0.001), and fibrinogen (P = 0.005) were significantly higher in FMF group than in control group. Receiver operating characteristic (ROC) curve analysis revealed a cut off value of CXCL16 as 2.68 ng/ml with 83% sensitivity and 68% specificity (P < 0.001). Logistic regression analysis indicated that high CXCL16 and erythrocyte sedimentation rate levels were predictive parameters for FMF disease  . There was no correlation between CXCL16 levels and attack frequency and disease duration .To the best of our knowledge, this is the first study evaluating serum soluble CXCL16 levels as a biomarker for FMF. CXCL16 levels were significantly higher and were predictive for monitoring inflammatory activity in patients with FMF. CXCL16 may be a promising biomarker for FMF diagnosis. Familial Mediterranean Fever (FMF) is a monogenic autoinflammatory disease characterized by recurrent fever and pain episodes mainly secondary to polyserositis, e.g., in the peritoneum, pleura, or joints. Although FMF has a distinct ethnic distribution, patients have been reported from many countries in the world. FMF is commonly detected in Sephardic Jews, Armenians, Turks, Greeks, Arabs, and Italians in the Mediterranean region . FMF is a genetic disease resulting from MEFV gene variations . This gene forms the protein of pyrin, which plays an important role in inflammation and apoptosis [2–4]. However, exact mechanism of pyrin pathway is not clear yet. One speculation for MEFV mutation is that pyrin and antiinflammatory protein production decreases and inflammatory conditions cannot be suppressed, thereby resulting in fever and inflammation in certain areas of the body.There is currently no definitive diagnosis of FMF and it is diagnosed by clinical findings according to the Tel Hashomer criteria [1–5]. Ethnicity, family history, and the mutation in MEFV (Mediterranean fever) gene supports the diagnosis in patients with clinical findings although it is not a requirement. Other findings that contribute to the diagnosis are the high blood levels of acute phase reactants during attack periods of FMF. The major mechanism of pathogenesis for clinical manifestations includes overactivation of cytokine cascades . Abnormal pyrin protein, which is proposed as a result of MEFV gene mutations, was presented to precipitate ineffective suppression of inflammation and was responsible for the inflammatory process [3].Difficulties lead to delays in diagnosis of FMF. The main problems are atypical clinical presentations, which do not fully meet the diagnostic criteria and overlap diseases [6]. Considering that the average delay in diagnosis of FMF is 7.3 years; one can speculate that new, reliable, fast parameters are required for FMF diagnosis [7]. CXCL16 is a chemokine produced by inflammatory cells which are a member of the chemotactic cytokine superfamily and have an important role in inflammatory processes such as rheumatoid arthritis, inflammatory bowel disease, SLE, Behçet’s disease, and gout disease, as well as tissue damage and fibrosis [8–13]. CXCL16 activates CXC motif-receptor 6, which is synthesized in several cells, especially leukocytes [14]. Unlike the common chemokines, CXCL16 is expressed in two forms; on the surface as transmembrane protein and soluble CXCL16. sCXCL16 increases the migration of CXCR6 + cells  to secondary lymphoid organs and inflammation sites . Thus, an inflammatory cascade is initiated via sCXCL16. In this study, our aim was to investigate the serum CXCL16 levels of patients in the FMF attack compared to healthy individuals and to investigate the role of CXCL16 on the diagnosis and activity of FMF disease.Fifty-three male patients diagnosed with FMF and sixty healthy age-matched male individuals were enrolled in this cross-sectional study. Local ethics committee approved the study. Informed consents were obtained from all participants.The FMF attack period was diagnosed by Tel Hashomer criteria and combining high levels of acute phase reactants (APR) with positive physical examination. All patients had peritonitis in terms of phenotype characteristics. All findings of the patients were recorded for further evaluation and classification. Blood samples were obtained, and physical examination was performed to the patients who referred with FMF attack.We performed a strict exclusion criteria. Patients using nonsteroidal antiinflammatory drugs, steroids, immunosuppressants, or immune regulatory drugs, and patients with cardiovascular disease, acute or chronic liver, or kidney disease, chronic obstructive pulmonary disease, rheumatological disease, malignancy, thyroid disease, immune deficiency, hypertension, diabetes mellitus, acute/chronic infection, bleeding disease, and those who did not meet the study criteria were excluded from the study. Blood samples were obtained in the first 24 h of the attack period. Blood samples were centrifuged at 5000 rpm for 5 min and 2 cc serum was separated into eppendorf tubes for CXCL16 tests and stored at –80°C. After a sufficient sample size was reached, all samples were tested in the biochemistry laboratory. CXCL16 was analyzed by ELISA  according to the manufacturer’s recommendations.SPSS 20.0  was used for statistical analysis. Descriptive statistics were performed and Kolmogorov–Smirnov test was used to check the data for normal distribution. Comparisons of the groups were made by student T test for the data with normal distribution, and Mann–Whitney U test for the data without normal distribution. Categorical data were assessed by Chi-square test. Receiver operating characteristic (ROC) analysis was used to evaluate the sensitivity and specificity of CXCL16 in FMF patients. Logistic regression analysis was performed to determine the parameters predicting FMF attack. Pearson and Spearman analyses were used for the correlation analysis. The P value <0.05 was considered statistically significant.Demographic and clinical data of patient groups are presented in Table 1. CXCL16 (P < 0.001), CRP (P < 0.001), fibrinogen (P = 0.005), and ESR (P < 0.001) were significantly higher in the FMF group compared to the control group, while BMI (P = 0.011) was higher in healthy individuals. ROC curve analysis revealed a cut off value of CXCL16 as 2.68 ng/mL with 83% sensitivity and 68% specificity (P < 0.001) . Logistic regression analysis indicated that high CXCL16 and sedimentation levels were predictive parameters for FMF disease   (Table 3). There was no correlation between CXCL16 levels and annual attack frequency and disease duration of FMF patients .To the best of our knowledge, this is the first study evaluating serum soluble CXCL16 levels as a biomarker for FMF. In this study, we showed that patients with FMF attacks had higher serum CXCL16 levels compared to healthy individuals and CXCL16 had a predictive role in the diagnosis of FMF disease.Higher ESR, CRP, fibrinogen, white blood cell count, and serum amyloid A (SAA) are expected results in FMF disease compared to the attack-free period . However, in a systematic review investigating acute phase reactants used for FMF diagnosis, Erer et al. reported that there was no effective acute phase reactant to diagnose FMF disease [18]. Actually, maybe as one of the limitations in our study, we could not test the SAA between these parameters because SAA was not among our routine tests in our biochemistry lab. Nevertheless, Çakan et al. recently reported that SAA is a sensitive but nonspecific marker to show inflammation in FMF [19]. The authors demonstrated that SAA levels increase significantly in febrile upper respiratory infections [19]. Innate immune system in FMF patients is proposed to be disrupted and the disease progresses with episodes of systemic inflammation [20]. Innate immune system activates the adaptive immune system by antigen presenting cells. Thus, B and T cells respond and result as disease symptoms [20]. IL-1β, IL-1α, TNF α, TNF β and IL-6 play an important role in these mechanisms [5]. CXCL16 has been reported to play an activating role in the pathophysiology of these inflammatory cascades [14]. In addition, Funk T et al. noted that dendritic cells have critical roles in FMF pathogenesis and disease activation [21]. CXCL16 is also known to be expressed in dentritic cells and is effective in triggering inflammation [14]. Inflammatory cytokines, such as TNF α and interferon-gamma, which play an important role in the pathogenesis of FMF, have also been shown to increase CXCL16 production [22]. In the light of these mechanisms, our findings suggest that high CXCL16 levels can be a relevant diagnostic marker for FMF disease.Literature states an increase of CXCL16 in several inflammatory diseases . In our study, logistic regression analysis indicated that high CXCL16 and ESR levels were predictive parameters for FMF disease. We think that the major advantage of CXCL16 over common widely available conventional APRs would be its OR of 8.31 for predicting FMF while ESR’s OR was 1.27. In addition to that, ROC curve analysis revealed satisfactory scores of sensitivity and specificity for CXCL16. Eventually, there is need for further prospective studies to evaluate CXCL16 in different periods of FMF disease and its role at differential diagnosis.As an interesting result of our study, there was not any correlation between CXCL16 levels and attack frequency and disease duration of FMF patients. This result may indicate that CXCL16 is effective especially in attack period of FMF pathogenesis. On the other hand, colchicine treatment can cause this result as it has antiinflammatory effects and can cause a decrease in proinflammatory cytokine levels [2–4]. Another reason may be the cross-sectional design of the study that the samples reflect only one time period. Another finding in our study was that BMI (body mass index) values were significantly higher in control group than in FMF patients. A relationship has been proposed between obesity and CXCL16 [23]. In this regard, Lopes et al. reported that the overweight increases CXCL16 levels in young adults (18–30 years old) [23]. We consider that this finding of BMI values, which were significantly lower in patients with FMF than healthy controls, may consolidate the diagnostic value of CXCL16 in patients with FMF.There are other new candidate biomarkers for FMF in the literature . Pentraxin-3, omentin, fetuin, calprotectin, serum amyloid A, CD144+, and CD146+ as circulating endothelial microparticles, endocan, chitotriosidase, serum matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1, S10012A and resolvin D1 has been investigated and had promising results. However, there is still need for prospective large cohort studies to use these novel biomarkers in daily practice. This study has some limitations. First, the study population was small. Second, all study participants were male because the study was conducted at a tertiary military center. However, we anticipate that this limitation will not weaken our results due to the efficient homogenization between the groups. Third, CXCL was only determined during attack period and was not analyzed in between attacks. On the other hand, our study population is young and they have no comorbid situations. Thus our strict exclusion criteria render our research valuable.In conclusion, we showed that CXCL16 levels were significantly higher in patients with FMF than in healthy individuals. Serum soluble CXCL16 levels may be a novel biomarker for FMF disease. Prospective, randomized, large studies are needed for elucidating the role of CXCL16 in the pathogenesis of FMF.All participants provided informed consent. The approval from the ethics committee of GATA Haydarpaşa Training Hospital  was obtained for this study. The study was carried out in accordance the World Medical Association’s Declaration of Helsinki.


Early-onset neonatal sepsis (EONS), a bacterial infection that occurs within 72 h after birth, is associated with high likelihood of neonatal mortality. Latamoxef, a semi-synthetic oxacephem antibiotic developed in 1980s, has been brought back into empirical EONS treatment in recent years. In the preliminary work, we established a population pharmacokinetics (PPK) model for latamoxef in Chinese neonates. Moreover, in order to better guide clinical treatment, we conducted dose simulation and found that ascending administration frequency could improve the target rate of 70% of patients having a free antimicrobial drug concentration exceeding the MIC during 70% of the dosing interval (70% fT > MIC). Accordingly, this study is aimed to compare the 70% fT > MIC, efficacy and safety between conventional regimen and PPK model regimen for rational use of latamoxef in EONS treatment. A single-blind, multicenter randomized controlled trial (RCT) for latamoxef will be conducted in Chinese EONS patients. Neonates  admitted to the hospital with the diagnosis of EONS and fulfilling inclusion and exclusion criteria will be randomized (ratio of 1:1) to either a conventional regimen (30 mg/kg q12h) or model regimen (20 mg/kg q8h) latamoxef treatment group for at least 3 days. Primary outcome measure will be 70% fT > MIC and secondary outcome indicators will be the latamoxef treatment failure, duration of antibiotic therapy, changes of white blood cell count (WBC), C-reactive protein (CRP) and procalcitonin (PCT), blood culture results during administration and incidence of adverse event (AE)s. Assessments will be made at baseline, initial stage of latamoxef treatment (18–72 h) and before the end of latamoxef treatment. Ethical approval of our clinical trial has been granted by the ethics committee of the Beijing Children’s Hospital (ID: 2020-13-1). Written informed consent will be obtained from the parents of the participants. This trial is registered in the Chinese Clinical Trial Registry (ChiCTR 2000040064).It is hoped that our study will provide a clinical basis for the rational clinical use of latamoxef in EONS treatment. Neonatal sepsis, a leading cause of mortality in neonates worldwide, is divided into early-onset (EONS) and late-onset neonatal sepsis (LONS) . EONS ocTo assess the PK features of latamoxef in neonates, we performed a population pharmacokinetics (PPK) study of latamoxef in Chinese neonates and established a PPK model for them . CurrentThe proposed clinical trial will be a randomized, single-blind and multicenter intervention study for latamoxef in EONS treatment. This trial is registered in the Chinese Clinical Trial Registry (ChiCTR 2000040064) and is in full adherence to the principles of the Declaration of Helsinki and Good Clinical Practice (GCP) guidelines. This trial will be conducted at three hospitals in China , and 114Our research group will protect the rights and safety of participants by full compliance with the Declaration of Helsinki and Good Clinical Practice (GCP) guidelines. Ethical approval of this trial has been granted by the ethics committee of the Beijing Children’s Hospital (ID: 2020-13-1). Written informed consent will be obtained from the parents of the participants. Adequate measures will be taken to ensure confidentiality of data collected in this trial. Results of this trial will be disseminated to the public through relevant academic and professional journals, academic conferences and workshops.1) The suspected diagnosis of postnatal age in days (PNA) ≤3 contains any of the following: abnormal clinical manifestations, the mother has chorioamnionitis and premature rupture of membranes (PROM) ≥18 h. Sepsis can be ruled out if there are no abnormal clinical manifestations, including negative blood culture, and less than two consecutive non-specific blood tests  and procalcitonin (PCT), etc.) at 24 h intervals.2) The patient has been clinically diagnosed with clinical abnormalities  and met 3) When the patient has clinical manifestations , blood cThe diagnostic criteria of EONS will refer to expert Consensus on the Diagnosis and Management of Neonatal sepsis (version 2019) .1) The s1) PNA ≤ 3;2) Term infants and preterm infants with gestational age (GA) ≥ 32 weeks;3) The EONS diagnostic criteria;4) Suitable for latamoxef treatment;5) Written informed consent signed by the parent or legal guardian of the neonates.Patients will be recruited for this study if they meet all of the following criteria:1) Expected survival time shorter than the duration of the treatment cycle;2) Severe congenital malformations;3) Having the high risk of serious bleeding, such as disseminated intravascalar coagulation (DIC) and Vitamin K deficiency bleeding (VKDB) :9/L);A. Decreased platelet count ;C. Prolonged activated partial thromboplastin time ;D. INR ≥4 or a value >4 times the normal values in the presence of normal platelet count and fibrinogen level;4) Undergoing surgery within the first week of birth;5) Receiving other trial drug treatment;6) Having other factors that researchers believe are not suitable for inclusion.Patients with any of the following exclusion criteria shall not be admitted to this study:Inpatient neonates will be enrolled from Beijing Friendship Hospital, Beijing Obstetrics and Gynecology Hospital, and Children’s Hospital, Capital Institute of Pediatrics . We’ll uTo minimize selective bias, Beijing Six Yuan Space Information Technology Co., Ltd (Six Yuan) has been entrusted to conduct a stratified blocked randomization by using a computer random number generator in the system of Six Yuan. Participants will be randomly divided into the conventional treatment group and the model treatment group with a ratio of 1:1. The randomized code of the trial, derived from the random system of Six Yuan, will be the unique identification code of participants. After assignment of the randomization code, researchers are un-blinded to the treatment regimen of latamoxef. Considering that the drug concentration detection and data analysis will significantly affect the conclusion of this trial, all the research assistants in charge of drug concentration detection and statisticians will be blinded to treatment assignment until the trial is completed.After inclusion in the study, participants will be randomized into a conventional treatment group and a model treatment group. They will receive the following interventions at least 3 days. Conventional treatment group: latamoxef, 30 mg/kg q12h; model treatment group: latamoxef, 20 mg/kg, q8h. As steady-state plasma concentration of latamoxef will be reached after 18 h of dosing, the opportunistic blood sampling method will be used to collect samples for testing plasma drug concentration at initial stage of the treatment (18–72 h) .The primary outcome will be 70% fT > MIC, which is the target rate of 70% of patients having a free antimicrobial drug concentration exceeding the MIC during 70% of the dosing interval. 70% fT > MIC is appropriate to evaluate therapeutic efficacy of time dependent antibiotics in neonates . Based o1) Rate of latamoxef treatment failure: the symptoms and laboratory indicators of infection persist or worsen after latamoxef treatment. Neonatologists have to increase of the dose of latamoxef, or add other antibiotics to the treatment with latamoxef, or stop latamoxef treatment and switch to other antibiotics.2) Duration of antibiotic therapy: the length of antibiotic therapy for EONS;3) Changes of white blood cell count (WBC), CRP and PCT: comparison of the changes of WBC, CRP and PCT at baseline, initial stage of treatment (18–72 h) and before the end of treatment ;4) Blood culture results during administration: comparison of blood culture results at baseline, initial stage of treatment (18–72 h) and before the end of treatment ;5) Incidence of adverse events (AE)s: monitoring and recording AEs during latamoxef therapy .This trial has five secondary outcomes:In this trial, safety will be monitored, including AEs, serious AEs, and withdrawals due to AEs. The safety indexes are mainly composed of the routine blood, liver function, kidney function, coagulation function and vital signs. AEs of latamoxef mainly include anaphylaxis , gastrointestinal reactions , and other latamoxef associated AE .According to our preliminary PPK study and dose simulation for latamoxef in neonates , 60.2% oThe procedures and contents of data collection for this trial are detailed in p value < 0.05 will be considered statistically significant.Statisticians, independent of all the other processes, will conduct the statistical analyses by using SPSS version 15.0 for Windows . The statisticians will calculate the mean, standard deviation, median, minimum, maximum, lower quartile (Q1), upper quartile (Q3) for quantitative data and describe numbers and percentages for qualitative data . CompariIn order to ensure the quality of the trial, project director and researchers from each hospital have formulated detailed project implementation protocol and emergency plan at the beginning of this trial. During the clinical trial, researchers participating in this experiment shall undergo unified training to clarify and unify the recording methods and standards for data collection on the eCRF platform. Moreover, the clinical inspector, designated by the director, will make an inspection tour to each hospital periodically to ensure that the researchers strictly adhere to the clinical trial protocol and fill in the information correctly.In our proposed study, based on our preliminary PPK-PD analysis of latamoxef in neonates, we are aimed to conduct a single-blind, multicenter RCT and compare the 70%fT > MIC, efficacy and safety between conventional regimen and PPK model regimen for latamoxef in EONS treatment for the first time. Our study will provide PD data for optimizing latamoxef usage in EONS treatment.Group B Streptococcus (GBS) and Escherichia coli (E. coli) are most frequently bacteria; in China, E. coli and Coagulase-negative staphylococcus (CONS) were the leading pathogenic bacteria (Achromobacter xylosoxidans (A. xylosoxidans) and Klebsiella pneumoniae (K. pneumoniae). Therefore, broad-spectrum cephalosporins are often used as an important anti-infective drug in the Chinese EONS treatment, such as latamoxef. However, due to the limited PK and PD neonatal data, the dosing guidelines and optimal treatment regimen of latamoxef cannot be applied in EONS treatment.EONS, a serious threat for health of neonates, is mainly caused by bacterial pathogens transmitted vertically from mother to infant before or during delivery . The orgbacteria , followeTo ensure that the finding of this trial can comprehensively reflect PD characteristics of latamoxef in EONS treatment, 70% fT > MIC will be used as the primary outcome indicator, and efficacy and safety indexes will be employed as the secondary outcome indicators. Because there’s no clinical evaluation of latamoxef in EONS treatment, the trial design is derived from other clinical trials about cephalosporin treatment or therapeutic decision conducted in children or neonates . Rate ofE. coli and K. pneumoniae were 1 mg/L and 2 mg/L (According to the bacterial susceptibility data of latamoxef in relation to the bacteria commonly observed in Chinese EONS patients, the MIC90 values of d 2 mg/L . Thus, 7Our study had some limitations. First, since EONS patients often improve or recover at hospital discharge, follow-up is not designed in this trial. Hence our efficacy evaluation may miss some relapse situation after hospital discharge. Second, due to the few positive culture results and antimicrobial susceptibility results of bacteria in neonatal clinical practice, we chose 70% fT > MIC target with MIC of 2 mg/L in sample size calculation based on epidemiological microbiology data instead of individual microbiology. Finally, our study data are only from Chinese EONS patients in Beijing, limiting the generalizability to other populations and areas with different epidemiological microbiology data.We hoped that our study can provide reliable data to support rational use of latamoxef in EONS treatment.