Adequate knowledge about the safety of consumption of small-quantity lipid-based nutrient supplements (SQ-LNSs) is needed.We aimed to test the hypothesis that SQ-LNS consumption is noninferior to control with respect to child morbidityn = 1320) ≤20 wk pregnant were assigned to iron and folic acid until delivery with no supplementation for offspring; or multiple micronutrient supplements until 6 mo postpartum with no supplementation for offspring; or SQ-LNSs until 6 mo postpartum, and SQ-LNSs for offspring (6 mg Fe/d) from 6 to 18 mo of age [the lipid-based nutrient supplement (LNS) group]. We assessed noninferiority (margin ≤20%) between any 2 groups during 0-6 mo of age, and between the non-LNS and LNS groups during 6-18 mo of age for caregiver-reported acute respiratory infection, diarrhea, gastroenteritis, fever/suspected malaria, poor appetite, and “other illnesses.”Women  was3.3 episodes, overall mean prevalence (percentage of infant-days) was 19.3%, and the 95% CIs of the incidence rate ratio (IRR) and longitudinal prevalence rate ratio (LPRR) between any 2 groups were ≤1.20. During 6-18 mo, there were 240,097 infant-days for the non-LNS group and 118,698 for the LNS group. For all morbidity combined, group mean incidences were 4.3 and 4.3, respectively , and mean prevalences were 28.2% and 29.3%, respectively . Noninferiority was inconclusive for diarrhea, fever/suspected malaria, and poor appetite.J Nutr 2019;00:1-12.SQ-LNS consumption does not increase reported overall child morbidity in this population compared with the 2 other treatments. This trial was registered at clinicaltrials.gov as NCT00970866.  In low-income settings, the diets of many people are based predominantly on cereals and tubers and are of low nutrient quality . The conThe International Lipid-based Nutrient Supplements (iLiNS) Project developed small-quantity lipid-based nutrient supplements (SQ-LNSs) for enriching home-prepared foods for women and infants in low-income settings , as a poDespite the positive meta-analysis evidence so far reported , a possiThe iLiNS-DYAD trial in Ghana included both pre- and postnatal SQ-LNS consumption. In Ghana, child undernutrition has decreased in recent years , but micWe have described the iLiNS-DYAD Ghana trial (NCT00970866) in previous articles , 25. Wit1) the IFA group, assigned to consume 60 mg Fe/d + 400 µg folic acid/d supplements during pregnancy and 200 mg Ca/d as placebo during the first 6 mo postpartum, and no supplementation for offspring; 2) the MMN group, assigned to consume MMNs until 6 mo postpartum, and no supplementation for offspring; and 3) the LNS group, assigned to consume SQ-LNSs until 6 mo postpartum, and supplementation for the offspring with SQ-LNSs designed for infants from 6 to 18 mo of age. All supplements were intended for daily consumption.At enrollment, women were randomly assigned to the 3 groups as follows: The trial was approved by 3 ethics committees  and monitored by a Data and Safety Monitoring Board.As reported previously , 25, theThe daily dose of the SQ-LNS designed for infants (SQ-LNS-INF) contained the WHO, WHO/FAO, and FAO Recommended Nutrient Intakes (RNIs) for key micronutrients for infants 7-12 mo of age, with a few exceptions : the 6.0The IFA and MMN supplements as well as the calcium placebo were provided as capsules in blister packs of 10, whereas the SQ-LNS-P&L was provided in 20-g sachets, and the SQ-LNS-INF was provided in 10-g sachets. All supplements were meant for daily consumption.We determined women’s eligibility by using a screening questionnaire, and with the aid of information recorded from their antenatal cards. For eligible women agreeing to participate, we collected baseline information, including sociodemographic characteristics; gestational age ; anthropometric status (by using standard procedures); blood hemoglobin (Hb) concentration (HemoCue AG); zinc protoporphyrin concentration , and peripheral malaria parasitemia (Vision Biotech) .1) the study statistician at University of California, Davis, Janet M Peerson, developed the group assignments in blocks of 9 (SAS for Windows version 9.4); 2) the study data manager at the University of Ghana, who was not involved in enrollment, prepared opaque envelopes containing the assignments, which were numbered and stacked by block number; and 3) the study nurse at the field site performed the random assignment. At each enrollment, the study nurse shuffled 9 envelopes taken from the top of the stack and asked the participant to make a pick to reveal the group assignment. The nurse then returned the unused envelopes to the top of the stack. When there were <9 women left to be enrolled, the nurse shuffled whatever number of envelopes remained. Any allocation information was kept securely by the field supervisor in Ghana and the study statistician at University of California, Davis, only.Women were randomly assigned after the baseline assessments, as follows , 25: 1) At enrollment, the study nurse gave women a 2-wk supply of supplements, advice to take 1 capsule/d with water after a meal or one 20-g SQ-LNS-P&L sachet/d mixed with food, and a standard nutrition message: “Do not forget to eat meat, fish, eggs, fruits, and vegetables whenever you can; you still need these foods even as you take the supplements we have given you” , 25. We Field workers visited pregnant women in their homes biweekly to provide a fresh supply of supplements and monitor supplement intakes. All live-born singleton infants were enrolled into the study. In twin births, 1 infant was randomly selected during the first visit immediately after birth, and the selected infant was enrolled if he or she was born alive. After giving birth, women and their infants were visited weekly, but the women received their supplements or placebo and had intakes of these monitored biweekly as before.Women exited the study at 6 mo postpartum, but the weekly visits for the infants continued. At 6 mo of age, the study nurse gave the “minimum message” on complementary feeding to all mothers at the laboratory after the infants had participated in their first laboratory assessment: “Breastfeed your baby as you did before 6 mo of age; do not forget to feed your baby meat, fish, eggs, fruits, and vegetables whenever you can.” During the next usual weekly home visit, field workers delivered the first supply of SQ-LNS-INFs for infants in the LNS group, advised the mothers or caregivers on how to feed the supplements , and repeated the “minimum message” on complementary feeding given by the study nurse. For infants in the other 2 groups not assigned to any supplements, field workers only repeated the “minimum message” to the mothers or caregivers. Field workers delivered a fresh supply of infants’ SQ-LNSs during the usual weekly visits, but the “minimum message” was not repeated again thereafter. Infants exited the study at 18 mo of age.At each weekly home visit for study infants, field workers collected caregivers’ reports of common morbidity symptoms experienced by the infants, as well as the infants’ nonscheduled visits to any “treatment points” with the purpose to receive treatment of illness, during the period since the last visit or the last 10 d, if the current visit was delayed for >10 d. We defined “treatment points” to include hospitals, clinics, health posts, private physicians, pharmacists, nurses, midwives, drug stores not operated by pharmacists, and traditional healers, because residents of the study area typically seek treatment of illnesses from all such quarters, and therefore focusing on hospitals and clinics alone would underestimate treatments of morbidity. Whenever an infant was reportedly taken for a nonscheduled visit to a treatment point because of illness, the field worker asked about the specific illnesses that precipitated the nonscheduled visit.To facilitate data collection, field workers trained caregivers to use a calendar grid with pictures in its rows depicting the common morbidity symptoms  and a visit to a treatment point, to record information. The calendar grid had columns for recording information for each day ≤10 d. At each visit, field workers transferred the information recorded by the caregiver on the calendar grid onto a morbidity form after verification with the caregiver, and in addition, asked if the child had rapid breathing or difficult breathing on any day during the period. As much as possible, all morbidity data were collected using the daily calendar grid. As a complementary strategy, we asked fathers, older children, and other key members within the households to remind mothers about entering the children’s morbidity information on the calendar each day. With the intense weekly home visits by field workers, the filling out of the morbidity calendar soon became the normal daily routine for mothers and families. If a mother missed filling out the calendar grid, it was generally for a few days within the week and not the entire week or any longer period of time. If, during the weekly home visit, a field worker found that a mother had missed filling out the calendar on some days, the field worker probed the mother to determine if the child had any morbidity symptoms or was taken to any treatment centers on those days.Several trials across Africa have used a calendar grid to collect children’s morbidity data, including some in Malawi , 19 and 1) 0-6 mo of age [the period when the WHO recommends exclusive breastfeeding (1 ) the incidence and longitudinal prevalence of caregiver-reported common morbidity episodes, and 2) the incidence of caregiver-reported nonscheduled visits to a “treatment point” because of illness. The morbidity episodes were 1 ) acute respiratory infection (ARI), 2) diarrhea (caregiver-defined), 3) gastroenteritis, 4) fever or suspected malaria, 5) “other illnesses,” and 6) poor appetite. We separated caregivers’ diagnosis of diarrhea from gastroenteritis or the clinical definition of diarrhea  before any analyses began, we listed child morbidity as one of the secondary outcomes to be analyzed separately. An additional, more detailed SAP was developed specifically for this present analysis and finalized before data analysis began. We performed all analyses by using SAS version 9.4 (SAS Institute) and the complete case intention-to-treat principle  was 88.1%/85.7% for the IFA group, 87.0%/85.0% for the MMN group, and 83.7%/80.0% for the LNS group , and thaWe summarized background maternal and household variables by intervention groups according to the supplements women were assigned to at enrollment, as done previously , 25. ForFor the noninferiority testing, we predefined a noninferiority margin (δ) of no more than a 20% increase in an outcome for the intervention group compared with the control group. There is no standard noninferiority margin for assessing safety in micronutrient supplementation trials for children, but a similar margin has been used in several such contexts, including 2 trials in Bangladesh , 41 and In the analysis of outcomes during 0-6 mo of age, we compared infants in all 3 groups according to the supplements women were assigned to at enrollment, and for the analysis of outcomes during 618 mo of age, we compared infants in the LNS group (who consumed the SQ-LNSs) with those in the IFA and MMN groups combined (who did not consume any supplements). In the text, we have presented statistics on background characteristics as means ± SDs (continuous outcomes) or percentages (binary outcomes), and on the outcome variables as ratios and their 95% CIs.The iLiNS-DYAD-Ghana trial data were collected between December, 2009 and March, 2014. At enrollment, the women’s mean age was ~27 y, they had nearly 8 y of formal education, and were ~16 wk pregnant . Furthern = 1185) all of the infants enrolled completed the study at 18 mo of age, except 27 who died, 9 whose parents refused, and 7 who relocated. Apart from having a greater mean ± SD housing index , the dropouts did not differ in background characteristics from those who completed the study.A total of 1257 infants were born to the women in the study, of whom 1228 were enrolled; the remaining 29 not enrolled were stillborn . Nearly During the period 0-6 mo of age, there were 1197 infants in the study, who contributed >190,000 infant-days of follow- up for the 3 groups. During this period, the overall mean incidence and mean longitudinal prevalence of all morbidity events combined were 3.3 episodes per 100 infant-days and 19.3% of infant-days, respectively. In each of the pairwise comparisons, the 95% CIs of the IRR  or LPRR From 6 to 18 mo of age, the infants in the IFA and MMN groups combined contributed 240,097 infant-days of follow-up, whereas those in the LNS group contributed 118,698 infant- days of follow-up. During this period, the mean incidence and longitudinal prevalence of most of the morbidity events in the LNS group were noninferior to the corresponding mean incidence and longitudinal prevalence in the IFA + MMN groups (non-LNS group) combined . ResultsIn this semiurban setting in Ghana, infant morbidity during the first 6 mo of life was not increased by the provision of any of the maternal supplements , compared with infants whose mothers received either of the other 2 supplements. Similarly, child incidence and longitudinal prevalence of all morbidity events (combined) between 6 and 18 mo of age were not increased by the provision of SQ- LNSs to mothers and then to their offspring from 6 to 18 mo, compared with children who did not receive SQ-LNSs and whose mothers received IFA or MMNs, nor was the incidence of ARI, diarrhea, gastroenteritis, or fever/suspected malaria. We could not conclude noninferiority with regard to the prevalence of diarrhea and fever/suspected malaria, nor the incidence or prevalence of poor appetite, when comparing children provided with SQ-LNSs with those not provided with any supplements, but those results were close to the noninferiority margin threshold of 1.2.Our results that SQ-LNS consumption did not increase overall child morbidity agree with those from the iLiNS-DOSE trial in Malawi , in whicWith regard to caregiver-reported ARI, diarrhea, gastroenteritis, and fever/suspected malaria, our results are also generally comparable with those of other LNS supplementation trials. For ARI, our findings of noninferiority of the LNS group in incidence and prevalence during 0-6 mo of age and 6-18 mo of age agree with those from Ghana , Malawi For diarrhea, the pairwise comparisons of incidence and prevalence from birth to 6 mo of age suggest that the 3 groups were indistinguishable from each other with respect to those outcomes during that period. This is similar to results from Bangladesh , where dThere are several potential explanations for the differences in results across studies regarding LNS consumption and diarrheal morbidity or gastroenteritis in children. These include variability in the definition of diarrheal episodes across studies , possiblRegarding fever/suspected malaria, our results are generally consistent with previous reports from Ghana , Haiti , BangladThis study had several strengths and weaknesses. Strengths include a fully randomized design with active control groups, and intense weekly morbidity data collection along with caregivers’ use of a calendar grid to record infants’ morbidity and adherence to treatment. The study was originally designed as a superiority trial (for the primary outcomes) that maintained blinding to the group assignments to the maximum extent possible, and the noninferiority margin used in the present analysis was prespecified in our SAP. Such conditions are associated with less bias in results . The conIn conclusion, our data support the hypothesis that in this semiurban setting in Ghana, the provision of SQ-LNSs to women from early pregnancy to 6 mo postpartum, and to their infants from 6 to 18 mo of age, does not increase the infants’ reported overall morbidity from birth to 6 mo of age, or from 6 to 18 mo of age. Further research is needed to understand contextual differences in the effects of nutritional supplements on diarrhea and malaria between children in different study settings.


Saccharomyces cerevisiae antibodies (ASCA) and anti-neutrophil cytoplasmic antibodies (ANCA) and compare them with these same antibodies in controls to determine whether a correlation between LPS and barrier proteins could be found. We obtained 94 ASCA- and 94 ANCA-positive blood samples, as well as 188 blood samples from healthy controls. Samples were assessed for antibodies to LPS, zonulin+occludin, S100B, and aquaporin-4 (AQP4). Results show significant elevation in antibodies in about 30% of ASCA- and ANCA-positive sera and demonstrate positive linear relationships between these antibodies. The findings suggest that individuals positive for ASCA and ANCA have increased odds of developing intestinal and BBB permeability compared to healthy subjects. The levels of LPS antibodies in both ASCA- and ANCA-positive and negative specimens showed from low and moderate to high correlation with antibodies to barrier proteins. This study shows that LPS, by damaging the gut and BBBs, contribute to the extra-intestinal manifestation of IBD. We conclude that IBD patients should be screened for LPS antibodies in an effort to detect or prevent possible barrier damage at the earliest stage possible to abrogate disease symptoms in IBS and associated disorders.Individuals with intestinal barrier dysfunction are more prone to autoimmunity. Lipopolysaccharides (LPS) from gut bacteria have been shown to play a role in systemic inflammation, leading to the opening of the gut and blood-brain barrier (BBB). This study aims to measure antibodies against LPS and barrier proteins in samples positive for anti- Saccharomyces cerevisiae antibodies (ASCA) are associated with and used as biomarkers for CD, while anti-neutrophil cytoplasm antibodies (ANCA) are recognized as markers for UC [Inflammatory bowel disease (IBD) is a heterogeneous group of chronic inflammatory disorders of the gastrointestinal (GI) tract that has two main distinguishable forms, Crohn’s disease (CD) and ulcerative colitis (UC) . Accordis for UC . Saccharomyces cerevisiae, also known as baker’s or brewer’s yeast, is the most commonly detected fungi in human fecal samples and likely originates from food [S. cerevisiae; they are widely recognized as test markers for CD. For instance, in 2016 Huang et al. did a five-year follow-up study on 51 patients with CD compared to a control group. ASCA IgG had a sensitivity of 45.1%, a specificity of 100%, and a positive predictive value (PPV) of 100%, while ASCA IgA’s respective results were 35.3%, 91%, and 90% [rom food . ASCA arAnti-neutrophil cytoplasmic antibodies (ANCA) are a group of autoantibodies against antigens in the cytoplasm of neutrophil granulocytes and monocytes. ANCA are divided into cytoplasmic ANCA (c-ANCA), perinuclear ANCA (p-ANCA), and atypical ANCA (x- or a-ANCA) . c-ANCA IBD has been associated with a greater risk for increased permeability of the intestinal barriers ,11. ThisComorbidities of a neurological nature are not reported as frequently in conjunction with IBD , but anyEscherichia coli, Salmonella, Shigella, Pseudomonas, Helicobacter, Legionella, and Wolbachia. As an endotoxin, LPS increases the negative charge of the bacterial membrane and promotes the upregulation of pro-inflammatory cytokines, which contributes to gut dysbiosis [LPS is a component of the surface membrane of Gram-negative bacteria found in the gastrointestinal tract. Gram-negative bacteria include ysbiosis ,28,29,30ysbiosis . Evidencysbiosis . Systemiysbiosis ,37,38,39ysbiosis . Chelakkysbiosis .2 (about the size of a tennis court) [The single-cell epithelial lining in the intestines forms the single, largest interface between the human body and the external environment with a surface area of 300-ms court) . Compriss court) . An intas court) .The BBB is a physical barrier between the brain and the circulating blood, formed by the arrangement of endothelial cells and tight junctions that line the capillaries supplying blood to the brain. It is a highly selective barrier. The BBB naturally permits the passage of essential metabolites, oxygen, carbon-dioxide, hormones, cytokines, and other messengers. However, it restricts the movement of all soluble materials greater than 400 Da from the blood across to the brain. Acting like a filter, the BBB excludes many potentially toxic compounds, such as xenobiotics , viruses, food antigens and peptides present in the circulation . DespiteAstrocytes are characteristic star-shaped glial cells in the brain and spinal cord that protect the BBB. These cells secrete S100B, and their endfeet, which enwrap the BBB, abundantly express aquaporin-4 (AQP4). If the astrocyte is injured, S100B and AQP4 may be found in the bloodstream ,48. OnceBecause intact barriers support and maintain the health of the individual, a breach of one of these essential body barriers is an invitation for autoimmune reactivity to self-tissue, including the brain. Banks and colleagues  pinpointThus, the aim of this study is to compare antibodies against LPS and barrier proteins in ASCA- and ANCA-positive samples versus controls and to investigate the correlation between them.A total of 376 sera were assessed for levels of IgG, IgM, and IgA isotype antibodies against LPS, zonulin+occludin, the BBB protein S100B, and the water channel protein AQP4.n = 94), ANCA-positive subjects (n = 94), and healthy control subjects (n = 188). Each group had separate measurements of the three isotype antibodies. Based on ROC analysis, the percentages of samples with significant elevation in antibody levels were evaluated. Compared to healthy controls or ASCA-negative specimens, the ASCA-positive sera showed significant elevation in IgG, IgM and IgA antibodies against LPS. While the % elevation of LPS antibodies in controls was between 9% (8/94) and 12% (11/94), ASCA-positive samples showed significant levels of LPS antibodies in 30% (28/94) to 33% (31/94)  . p-valuep < 0.0001  to 31% (29/94) of ASCA-positive samples showed significant elevations in these antibodies, 5% (5/94), 12% (11/94), and 2% (2/94) of controls had elevations in IgG, IgM, and IgA antibodies against S100B, with inations .p < 0.0001 . The mean differences are illustrated in Overall, there were statistically significant differences in mean antibody levels for LPS, zonulin+occludin, S100B and in AQP4 in ASCA-positive subjects compared to healthy controls for IgG, IgM, and IgA antibodies  and intestinal barrier permeability (zonulin+occludin and LPS antibodies) were significantly higher for ASCA-positive subjects than the odds for healthy control subjects . The higp-value of 0.2 was the least significant for LPS IgG and S100B IgG (r 0.2) and for LPS IgG and AQP4 IgG (r 0.3).The correlation coefficients between antibodies to LPS and barrier proteins and between gut and BBB proteins in ASCA-positive subjects were calculated, with the highest being between AQP4 IgM and S100B IgM (r 0.9), followed by zonulin+occludin IgM and AQP4 IgM (r 0.8), LPS IgM and S100B IgM (r 0.8) and zonulin+occludin IgA with S100B IgA (r 0.8) . This cop < 0.0001  for each are shown in Setting the specificity at 31%, we found between 0%–9% of controls or ANCA-negative sera showed elevations in IgG, IgM, and IgA antibodies against LPS, zonulin+occludin, S100B and AQP4. This % elevation for all antibodies against all four antigens in ANCA-positive samples was 29%–33% with inations . The eleThe linear relationship between LPS antibody and gut and BBB proteins in relation to ANCA positivity was calculated. The data presented in The odds ratio for developing barrier protein antibodies with ANCA positivity was also calculated. The odds for developing blood-brain barrier permeability (S100B and AQP4 antibodies) and intestinal barrier permeability (zonulin+occludin and LPS antibodies) were significantly higher for ANCA-positive subjects than the odds for healthy control subjects . Similarp-values < 0.0001) and moderate-to-high r-values with intestinal barrier permeability and blood-brain barrier permeability for ANCA-positive subjects  causes a selective increase in intestinal tight junction proteins by inducing enterocyte membrane expression and localization of TLR4, without causing cell damage or cell death . But usiThis is why at high concentrations the inflammatory effect of systemic LPS has been linked to multiple disorders manifesting from gut to brain ,82,83,84A breach of the BBB allows for circulating autoantigens to enter the brain and nervous system and thus contribute to neuroautoimmunity. This may explain the moderate to high correlations that were found between IgA and IgM isotype antibodies against LPS, zonulin+occludin, S100B, and AQP4 in both ASCA- and ANCA-positive sera . The antibodies IgA and IgM in tandem have become the preferred biomarkers for inflammatory gastrointestinal disorders. Due to its polymeric quality, IgA, which is reflective of mucosal immunity, can be transported from the gut to the blood stream, making it a more specific biomarker for the gut. On the other hand, IgM is widely used to identify immune responses in the acute or inflammatory stage, making it an ideal inflammatory biomarker. In fact, our results showed a higher correlation coefficient with the IgA (0.5–0.9) and IgM (0.8–0.9) results. Correlation coefficients with IgG were less statistically significant (0.2–0.6). This shows that IgA and IgM reactivity to LPS puts a patient with IBD at a greater risk for damage to the gut barrier, including disruption of zonulin/occludin, which can result in consequences and manifestations beyond the gastrointestinal tract . We also examined the correlation between LPS, gut, and BBB protein antibodies in the control sera. We found a significant correlation between IgA and IgM antibodies against all four tested antigens in about 10% of control subjects who exhibited high levels of antibodies (data not shown). Because we did not have enough clinical information about the control sera, especially the 10% with the elevated antibody levels, we cannot exclude whether or not these individuals had any GI symptomatology or disorders.The question may be asked, why measure antibodies against LPS, zonulin+occludin, S100B, and AQP4 in blood? Why not directly measure the actual levels of these factors in the blood, instead of the levels of their antibodies?One answer to this question was given in our earlier research article about the fluctuation of zonulin levels in the blood versus the stability of antibodies . As we dWhile the half-life of zonulin in serum has still to be established, it is possible to examine the half-life of other similarly-sized proteins, such as LPS. LPS and zonulin are both involved in the induction of inflammation in type-2 diabetes. The half-life of LPS ranges from 2–4 min in blood . This giThis fluctuation is not unique to proteins such as LPS and zonulin. Take, for instance, the measurement of levels of U1 nuclear antigen in the blood. The detection of elevated blood levels of U1 antigen and circulating autoantibodies against U1 nuclear antigen are held to be the hallmark of systemic lupus erythematosus. However, although U1 antigen antibody levels were demonstrated to be highly stable, the level of U1 antigens fluctuated from day to day. This fluctuation in antigen level in sera may be connected to antibody-antigen complex formation in the circulation .The same caveat applies to measuring the blood level of S100B, which is currently regarded as a reliable biomarker only for acute cases of traumatic brain injury. Using kinetic modeling, researchers demonstrated that S100B concentration changes dramatically over timescales, and that the peak for its level was found to be at 27.2 h after traumatic brain injury . Thus, tSimilarly, caution should be applied in the utilization as a biomarker of direct levels of aquaporin-4 (AQP4). AQP4 is a water channel protein that conducts water through the cell membrane. It is found in many cells of the human body, including the brain, lung, stomach, and skeletal muscle , and is Additionally, since S100B and AQP4 is expressed not only in the BBB but also in the enteric nervous system, we cannot at this point determine whether the antibodies we detected against S100B and AQP4 originated from the brain or the enteric nerve system. Therefore, S100B or AQP4-mediated inflammatory effects are not limited to the brain, but their over-expression and the immune response mounted against them may also be associated with the onset and maintenance of inflammation in the gut ,103.The significant elevation of LPS antibody and its correlation with gut and BBB protein antibodies in ASCA- and ANCA-positive sera but not in healthy controls, as shown in this study, confirms that many patients with IBD indeed suffer from not only from gut inflammation but from systemic inflammation and enhanced gut and BBB permeability to macromolecules . It has been posited that intestinal permeability represents an early event that presages the actual onset of IBD. In 2019 Caviglia et al. measured serum as well as fecal zonulin in 118 IBD patients as biomarkers of intestinal permeability . In compVery recently, Michael Maes, in collaboration with our laboratory, reported increased plasma level of antibodies against Gram-negative bacteria, paracellular tight and adherens junction, transcellular cytoskeletal proteins, gut vascular barrier and blood brain barrier proteins in patients with deficit schizophrenia. Maes concluded that bacterial translocation and production of bacterial toxins is responsible for the upregulation and breakdown of tight and adherens junction in the gut, vascular barrier, and blood brain barrier in patients with schizophrenia ,59,60.We agree with Maes’s findings, and we therefore believe that gut dysbiosis, the release of LPS, and the production of LPS antibodies against them in a subgroup of patients with Crohn’s disease and ulcerative colitis should be detected and addressed at the earliest stage possible. Otherwise, the consequences of this breakdown in the barriers by bacterial toxins could be neuroinflammation, neuroautoimmunity, neurodegeneration, and neuropsychiatric disorders.We did not have any clinical information about our so-called healthy controls other than that they were screened based on a questionnaire and were negative for HIV and hepatitis-C antibodies. The additional information is important for the analysis of data from about 10% of controls who produced significant levels of antibodies against LPS as well as the barrier proteins.Likewise, other than their positivity or negativity when screened for ASCA or ANCA, we also did not have additional information about the clinical symptomatology of our ASCA- and ANCA-positive samples.We do not know whether the antibodies that we detected against AQP4 and S100B were made against these molecules in the brain, in the GI tract, or against both the gut and the brain glia and astrocytes.Due to our sample size and optical density variances, the confidence intervals were wide. This is due to the fact that only one-third of the ASCA- and ANCA-positive sera were highly reactive. However, there were still significant increased odds of 6- to 40-fold with the lowest tail of the confidence intervals.There are several limitations to our study:While we agree that these limitations should be addressed in future studies, our present findings suggest that individuals diagnosed with Crohn’s disease and ulcerative colitis with ASCA- and ANCA-positive antibodies have increased odds and correlations for both intestinal permeability and BBB permeability when compared to healthy subjects (ASCA- and ANCA-negative).Saccharomyces cerevisiae antibodies (ASCA) and anti-neutrophil cytoplasmic antibodies (ANCA) positivity or negativity. In this way we accumulated a total of 94 samples that were positive for ASCA and 94 samples that were positive for ANCA. We tested these samples using a kit procured from Inova, and compared them with sera from 188 healthy controls. Sera were assessed for serum immunoglobulin IgG, IgA, and IgM reactivities to LPS, as well as zonulin+occludin, human aquaporin (AQP4), and S100B. LPS from E. coli, Salmonella, Shigella, Klebsiella, and Pseudomonas were purchased from Sigma-Aldrich® . Occludin, zonulin, S100B and aquaporin-4 were purchased from Bio-Synthesis® . Commercially available sera of 24 patients with Crohn’s disease and 24 sera from patients with celiac disease were purchased from The Binding Site , Inova , Trina International , Diamedix , and Innovative Research . We also purchased from Innovative Research  additional blood samples obtained from donors who had been screened for anti-Antibodies against these proteins were measured using enzyme-linked immunosorbent assay (ELISA) as previously described .LPS, zonulin+occludin, S100B, and human AQP4 at a concentration of 1 mg/mL were dissolved in 0.01 M Tris buffer and diluted 1:50 in 0.1M carbonate buffer at pH 9.5. 100 μL of each diluted antigen was added to each well of the microtiter plate. Plates were incubated for 24 h at 4 °C and then washed three times with 200 μL 0.01 M PBS containing 0.05% Tween 20 at a pH of 7.4. After washing, 200 μL of 2% bovine serum albumin was added to each well to prevent non-specific binding of the antibody to the plate.Plates were washed, and then 100 μL of serum diluted 1:100 in serum diluent were added to duplicate wells coated with each antigen. Plates were incubated for an additional 1 h at room temperature. The plates were then washed five times with Tris-buffered saline (TBS)-Tween. Alkaline phosphatase-labeled anti-human IgG, IgM, or IgA antibodies were then added to all wells and incubated again for 1 h at room temperature. The enzyme reaction was started by adding 100 μL of substrate at a concentration of 1 mg/mL. The reaction was stopped by the addition of 50 μL of 1 N NaOH, and the samples were read by an ELISA reader; the optical densities were recorded. In addition to blank wells, several wells were coated with non-specific proteins such as human serum albumin (HSA) and rabbit serum. All reagents were added, and their ODs were recorded and used as controls for detecting the background and non-specific reaction.Statistical analysis was performed using GraphPad Prism 6.0 software . The diagnostic values of the indirect ELISA assays was evaluated by the receiver operating characteristic (ROC) curve. The optimal cutoff values were chosen according to ROC analysis, setting specificity at 0.31 since only about one third of the patients reacted strongly to LPS and barrier proteins. Additional data analysis was performed using STATA 14.2 software. Logistic regression, t-tests, and Pearson’s correlation coefficients were used to analyze the data. Bonferroni adjustments were performed to avoid a false discovery rate for multiple comparisons.This study was conducted in accordance with the Declaration of Helsinki, and the research received IRB approval from Partner’s Human Research Committee at Massachusetts General Hospital . Because bacterial toxins such as LPS produced by Gram-negative bacteria can damage the gut and blood-brain barriers, which are the two major gateways to autoimmune, neuroinflammatory, neurodegenerative, and neuropsychiatric disorders, we conclude that patients with IBD should be screened for LPS IgG, IgM, and IgA antibodies and their correlation with barrier protein antibodies in an effort to prevent BBB damage and its neurologic comorbidities. By regulating the levels of BBB and intestinal tight junction proteins, it is possible to abrogate disease symptoms, not only in Crohn’s disease and ulcerative colitis, but in associated disorders as well.However, much more remains to be done regarding understanding the major factors that are involved in the pathogenesis of IBD in order to successfully implement lifestyle modifications.


Gallus domesticus, Anas platyrhynchos domesticus, Ovis aries, Sus scrofa domesticus, Bos taurus, Equus caballus, Columba livia domestica, Rattus norvegicus, Mus musculus, and Canis lupus familiaris) and human beings by amplifying 22 short tandem repeat (STR) loci in a multiplex PCR using a set of five fluorescently labeled dyes. This novel 22-STR panel was validated by optimization of PCR conditions as well as species specificity, sensitivity, reproducibility, precision, DNA mixture, and tissue/organ consistency. The results of developmental validation showed that the 22-STR loci achieved high species specificity among 10 animal species and human beings, and the sensitivity of this panel was 0.09 ng. This 22-STR panel identified different meats in mixed samples, and the minimum detected mixture ratio in the current test was 10% (0.1 ng/1 ng). This sensitive, accurate, and specific 22-STR panel can be used for forensic species identification and the detection of meat fraud and adulteration.Species identification of unknown biological samples is of fundamental importance for forensic applications, especially in crime detection, poaching, and illegal trade of endangered animals as well as meat fraud. In this study, a novel panel was developed to simultaneously identify 10 different animal species ( Biological samples left behind at crime scenes always contain a great deal of valuable information that can provide helpful clues for the criminal investigations. In addition to human biological specimens, non-human biological samples acquired from a crime scene can also suggest certain directions for tracing the suspects. With the continuous progress of biotechnological achievements made in the field of forensic genetics, there has been much interest in the forensic identification of non-human species. For example, domestic pet hairs left at a crime scene could be evidence indicating that the suspect (pet owner) might have been present at the scene of the crime .Rhinoceros horn and tiger bone were two substances that were formerly used in traditional Chinese medicine. Although the production and import of these protective animal-derived traditional Chinese medicine are strictly prohibited in China, occasional illegal trade occurs. Species identification is then of great importance in the criminal investigation of poaching and illegal trade of endangered animals . MoreoveWith the growing size of the human population and increasing of social affluence in recent years, meat consumption has been increasing annually . Along wBased on the morphological and structural differences of cells and tissues in different species, the morphological observation was one of the most traditional techniques used for species identification a few decades ago . The serb and cytochrome oxidase subunit 1, which are located on mitochondrial genome. Although there have been more than five million DNA barcodes published in the Barcode of Life Data System (BOLD)1, scientists found a severe lack of adequate taxonomic coverage of some animal species within BOLD, which might be the results of anomalous or invalid identification  and the high level of sensitivity achieved in recent years . DNA barfication . Howeverfication . Hence, Appearing as a repeat unit of a 2–6 bp core sequence, the short tandem repeat (STR) loci, distributing widely in the human genome with high polymorphisms, have been widely used in the field of forensic genetics . In receIn this study, we selected 22-STR loci of 10 different animals as well as human beings, with two STR loci for each species, which enabled high species specificity among pig, cattle, goat, chicken, duck, rat, mouse, horse, pigeon, canine, and human samples. And then, we constructed a novel five-dye typing panel based on the CE platform. To evaluate the forensic efficiency of this 22-STR panel, we conducted a series of validation tests such as sensitivity, species specificity, DNA mixture, reproducibility studies and so on.Gallus domesticus), duck (Anas platyrhynchos domesticus), sheep (Ovis aries), pig (Sus scrofa domesticus), cattle (Bos taurus), horse , and pigeon (Columba livia domestica) were purchased in the local market. Samples of Sprague-Dawley (SD) rat (Rattus norvegicus), Kunming mouse (Mus musculus), and dog (Canis lupus familiaris) were acquired from the Medical Experimental Animal Center of Xi’an Jiaotong University. The environment of this center meets the standard for feeding practices conducted for experimental animals (GB 14925-2010). Human (Homo sapiens) blood stains were previously collected by our laboratory. SD rats and Kunming mice were anesthetized and euthanized by cervical dislocation. Euthanasia of the experimental animals, sample collections, and the following experimental processes were approved by the ethics committee of Xi’an Jiaotong University, Health Science Center.Samples of chicken . DNA was quantified with a NanoDrop 2000c spectrophotometer . If the concentration of extracted DNA was not greater than 1 ng/μl, the DNA was re-extracted.Twenty-two STR loci showing species specificity among 11 species were selected from published studies following the criteria: (1) primer sequences designed for each STR locus of one species did not share homologous sequences with other species; (2) priority was given to STR loci whose core sequences were tetranucleotide; and (3) priority was given to STR loci that had fewer alleles. Primer 5.0 software was used to design the STR primers. Oligo 7 software  was usedFor the 22-STR loci, 20 unrelated individuals of each species were collected to determine the variabilities of the alleles observed in each species. Moreover, variabilities of the alleles for some STR markers were screened from previously published studies. Allelic ladder was generated according to previous reports .® Polymer . Loading samples for CE contained 1 μl of PCR product, 0.3 μl of QD550 internal size standard, and 8.7 μl of Hi-Di Formamide. The alleles were genotyped using GeneMapper ID-X software v1.5 . Next, equal amounts of DNA from each species were mixed, and then the mixture was diluted to 1 ng/μl (DNA mix). The DNA mix was used as positive control DNA, and deionized water was used as the negative control.Unless stated otherwise, standard PCR amplification and genotyping procedures were as follows. We used a 10-μl reaction volume containing 1 μl of DNA template (1 ng/μl), 2 μl of Primer set , 4 μl of Master Mix I , and 3 μl of deionized water. The PCR was conducted using a GeneAmp PCR System 9700 Thermal Cycler  under the following conditions: 5 min of initial denaturation at 95°C, followed by 29 cycles of 94°C for 30 s, 59°C for 60 s, and 72°C for 60 s, with the final elongation at 60°C for 60 min. Electrophoresis was performed by an ABI 3500xL Genetic Analyzer  using 36-cm capillary arrays with POP-4To evaluate the specificity and amplification efficiency of a pair of primers, we amplified each STR locus according to the standard PCR components and reaction conditions.PCR cycling parameter studies were conducted on the 10-μl volume system. Total cycle numbers of 27, 28, 29, 30, and 31 cycles, and annealing temperatures at 57, 58, 59, 60, and 61°C were separately tested in order to choose the most optimal PCR parameters.Two different total reaction volumes, 10 and 25 μl, were adjusted to evaluate the performance of this 22-STR panel. The value of each reagent in the 25-μl PCR system was 2.5 times larger than that in the 10-μl system, and the PCR conditions were in accordance with those mentioned above. To evaluate the PCR performance in various PCR thermocyclers, we conducted the PCR in the Applied Biosystems Veriti Thermal Cycler, Applied Biosystems ProFlex Thermal Cycler, Applied Biosystems 9700 Thermal Cycler, and Applied Biosystems 2720 Thermal Cycler . The DNA mix was regarded as the DNA template, and the reaction conditions used were as mentioned above.® Company . Genomic DNA was extracted from the liver, heart, spleen, lung, kidney, and muscle of the same SD rat and Kunming mouse, respectively, which was used to evaluate the concordance of genotyping results of this panel in detecting different organs or tissue of the same individual.We performed a serial of concentrations of DNA mix to obtain 5 ng/μl, 2 ng/μl, 1 ng/μl, 0.5 ng/μl, 0.25 ng/μl, 125 pg/μl, and 62.5 pg/μl that were used to evaluate the sensitivity of this 22-STR panel. To evaluate the reproducibility of this panel, the DNA mix was genotyped three different times, comparing with the ladder profile. The mean sizes in base pairs and the standard deviations were calculated for each allele. We selected some samples of different species and sequenced the alleles of each STR locus using the Sanger sequencing method in order to verify the STR profile results of the CE platform. Sanger sequencing was conducted by Sangon BiotechWe genotyped DNA of each studied species based on this multiple STR panel to evaluate if the panel was capable of avoiding the genotype of other non-targeted species. Samples from practical cases were usually composed of more than one animal species, therefore, it was important to evaluate the reliability of this panel for the detection of mixture samples of different species. Mixture studies were also performed to evaluate the lowest detection limit  of this 22-STR panel. For this purpose, we set two types of DNA mixture patterns with different mix ratios, and these two mixture patterns are shown in TM card at room temperature for up to 7 years were used to represent common samples in the practical cases. We selected four unknown cooked meats to evaluate the efficiency of the detection of cooked meats. These four meats were named SE (braised), SY (roasted), SG (poached), and SN (stir-fried). The surface of all samples was rinsed with ultrapure water before DNA extraction to remove all inhibitory substances. All samples were amplified in a 10-μl volume, and the PCR conditions were the same as those mentioned above.Human blood stains preserved on an FTAIn this research, 22-STR loci with high-species specificity among 10 animal species and human were selected: D8UIA2, D0UIA21; PG5, PG6; FH2361, FH2100; GCT025, LEI0049; HMS6, HMS3; BT165, BT150; D3S3045, TPOX; NC000070, NC000084; APH14, CAUD056; MAF33, MCM164; SW742 and EF046 loci.Before the construction of this novel panel, we amplified each STR locus to evaluate the specificity and amplification efficiency of a pair of primers, and the results showed that specific peaks of each pair of primers were only detected at the corresponding locus for each species, and no peak was found in other species.A series of temperatures at 57, 58, 59, 60, and 61°C were used to determine the optimal primer annealing temperature. All loci could be detected at these five different annealing temperatures, and the amplification efficiencies at 58, 59, and 60°C were higher than those at 57 and 61°C. The highest value of average peak height was observed when the annealing temperature was 58°C, but more optimal peak height balance was found at 59°C. We finally chose 59°C as the most optimal annealing temperature. Related genotype profiles are shown in This 22-STR panel was tested over a range of 27, 28, 29, 30, and 31 total amplification cycles. With the increase in the number of cycles, the peak height increased obviously. All alleles could be detected, and more optimal allelic peak height balance was obtained when the number of amplification cycles was 29. We finally chose 29 as the optimal number of amplification cycles. Related genotype profiles are shown in We also evaluated the PCR efficiencies in the different reaction volume systems of 10 and 25 μl. As shown in We used a serial of input DNA concentrations of 5 ng/μl, 2 ng/μl, 1 ng/μl, 0.5 ng/μl, 0.25 ng/μl, 125 pg/μl, and 62.5 pg/μl of DNA mix to evaluate the sensitivity of this 22-STR panel. As shown in Genomic DNA extracted from the liver, heart, spleen, lung, kidney, and muscle of the same SD rat and Kunming mouse, respectively, were used to evaluate whether the STR genotyping profiles of different tissues or organs from the same individual showed exactly the same STR genotyping result. As shown in Genomic DNA templates of the studied species were amplified separately based on this multiplex STR panel so that we could evaluate the species specificity of this 22-STR panel, and the corresponding STR genotyping results are shown in We made two types of DNA mix models with different mixture ratios to evaluate the performance of this panel in the detection of each DNA mixture. In TM cards at room temperature to evaluate the performance of this novel panel on aged samples. The results showed that all samples could be successfully genotyped at D3S3045 and TPOX loci.We genotyped human blood stains preserved for up to 7 years on FTAWe also genotyped four types of cooked meats of unknown species origin to evaluate the efficiency of the detection of cooked meats, and the profiles are shown in With the increasing occurrence of illegal incidents such as meat fraud and adulteration or illegal trade of protected animals , it is fOver the past two decades, STR loci have been used extensively in population genetics, individual identification, and paternity tests for protected wild animals or domestic animals . DespiteCompared to human forensic genetics, research progress of non-human genetics has been more gradual, largely because no rich unified databases of wild animal or domestic animal were available. Besides, genetic markers used in the field of animal genetics have not been systematically validated by forensic medicine, which made them difficult to be used in the forensic genetics .Most kits have been developed based on RT-PCR, dPCR, or liquid chromatography-tandem mass spectrometry . AlthougThe purpose of this research is to develop a panel that could distinguish 10 animal species as well as human beings, which could then be used in the application of forensic species identification and the detection of meat fraud and adulteration. The choice of the studied species is fully considered based on actual adulteration cases. Pork, beef, mutton, and chicken are the most common meats found in China. Beef or mutton has been found to be adulterated with inexpensive meat such as duck, horse, and even mouse meat, and therefore, chicken, duck, sheep, pig, horse, cattle, rat, and mouse are selected for this study. Additionally, because canine and pigeon meat are also popular in some cities of China, these two animal species are chosen for this study.We selected 22-STR loci with high species specificity among 11 species and then constructed a novel five-dye multiplex amplification panel that could be analyzed using the CE platform. Before the validations, we evaluated the performance of different thermal cycling parameters. As anticipated, an increasing cycle number led to an apparent increase in overall allelic peak height. All loci could be detected in reasonable ranges of thermal cycling parameters. At 29 cycles, we observed a more balanced peak height.The annealing temperature affected the specificity of the PCR. In tests of different annealing temperatures, the amplification efficiencies at 58, 59, and 60°C were higher than those at 57 and 61°C. After we considered that low annealing temperature led to non-specific amplification , we finaIt is essential to evaluate the efficiency of a novel panel before it is used for casework. Here, we performed a series of developmental validations studies including sensitivity, reproducibility, precision, specificity, mixture, and tissue/organ consistency and so on. In forensic practice, we could not always acquire sufficient DNA amounts, and therefore, any potential panel should be capable of genotyping trace amounts of DNA template. In the current study, we evaluated the sensitivity of this 22-STR panel with serial input DNA amounts. According to the results, one dropped peak  was observed when the input DNA was 0.5 ng, indicating that the minimum input amount of DNA template should be more than 0.5 ng. Reproducibility and precision studies were performed to validate the reliability and accuracy of this 22-STR panel. The results of reproducibility studies showed that the STR profiles of three trials were consistent, and allele calling was consistent with their known amplicon sizes, which demonstrated that this panel could ensure proper allele detection.It was critical to ensure that this 22-STR panel exhibited no cross-reactivity between different species. The primer specificity of the STR loci was the key to the specificity of this panel. To ensure that no cross-reactivity occurred among the 11 species, we designed the primers according to the highly conserved region of each species’ genome and used BLAST to evaluate the specificity of each primer sequence. The present species specificity study showed that the specific peaks of the STRs were detected only at the corresponding loci for each species, and no allelic peaks were found in the STR loci of other species, indicating that all the primers in this panel exhibited no cross-reactivity between different species.In the ongoing investigations of meat fraud and adulteration, it is usually found that various inexpensive meats such as chicken or duck are often added to beef or mutton, which not only decreases food safety, but also disrupts market order. Illegal addition of animal-derived ingredients in feedstuff might spread infectious diseases such as bovine spongiform encephalopathy or scrapie . TherefoIn addition to meat fraud and adulteration, processed meat or animal tissues are also commonly investigated in forensic casework. Poached and roasted lamb, beef, and pork are popular in the Chinese diet. High temperatures and various condiments used during cooking could damage DNA. Therefore, the efficiency of the detection of cooked meats or animal tissues is also essential. In this research, we used four different cooked meats to evaluate the detection efficiency of the mixture of cooked meat. Full profiles were acquired for SG, SY, and SN meats, and one locus was detected in SE meat. The results indicated that this 22-STR panel could be used for the detection of individual species in cooked meat.In this research, we developed a novel 5-dye panel that could simultaneously identify 10 animal species and human being, and co-amplify 22-STR loci using one PCR system. This panel was validated by a series of tests including optimization of PCR conditions, sensitivity, reproducibility, precision, species specificity, DNA mixture, and tissue/organ consistency. In present results, this 22-STR panel achieved high species specificity among 11 species and a detection capacity for a mixture of meat samples. The results of the developmental validations demonstrated that this panel can be used for forensic species identification and the detection of meat fraud and adulteration.All datasets generated for this study are included in the article/The studies involving human participants were reviewed and approved by the Ethics committee of the Xi’an Jiaotong University, Health Science Center. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Ethics committee of the Xi’an Jiaotong University, Health Science Center.BZ designed and was responsible for this research. WC and XJ built up this 22-STR panel and prepared the preliminary data. WC, YG, and CC analyzed the data. WC wrote the draft manuscript. WZ, JL, YW, and BZ reviewed and revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This study will evaluate the effectiveness of different acupuncture courses for functional constipation (FC) through network meta-analysis.Eight database , China National Knowledge Infrastructure (CNKI), China Biomedical Literature Database (CBM) and Wanfang Database) will be searched from inception to October 2019. Only randomized controlled trials comparing different acupuncture courses or acupuncture versus sham acupuncture or placebo will be included. The outcomes involved weekly stool frequency, Bristol Fecal score, responder rate and safety evaluation. The risk of bias assessment and quality of evidence will be appraised using the Cochrane Risk of Bias Tool and the Grading of Recommendations, Assessment, Development and Evaluation guidelines. RevMan 5.3 software, STATA V.14.0 and GeMTC software will be used to perform the network meta-analysis.This work will compare and arrange the comparative efficacy of different acupuncture treatments for FC by summarizing the current evidences. The results will be submitted in the form of a journal publication.The results of this network meta-analysis may help doctors determine the best treatments for patients to manage FC.CRD42020153801. In addition to digestive system symptoms or diseases, it can also cause anorectal disease, anorexia, diverticulosis, pain and mental symptoms.–5 Although FC is not life-threatening, it makes a very significant adverse impact on the quality of life and economic costs of patients.,7 Most epidemiological studies report prevalence in the general population between 12% to 19%,–11 and female sex, reduced caloric intake, and increasing age are risk factors for FC.,13 These negative impacts and high rates make FC a major public health issue.According to the Rome IV criteria, supplementing the diet with fiber and exercising are recommended to improve bowel movement, but the strength of recommendation is weak. Medicines, such as osmotic and stimulant laxatives,,18 secretagogues, serotonin(5-HT) 4 agonists and peripherally acting mu-opioid receptor antagonists, are reported with definite efficacy, but the majority of medicines have the adverse effects, such as drug-dependent, diarrhea, bloating, gas, bowel cramps, and esophageal obstruction. Therefore, the search for effective complementary and alternative therapies with few side effects for FC has attracted the attention of both doctors and patients.The basic goal of FC treatment is to increase bowel frequency, alleviate the symptoms in patients with FC. Guidelines show management of constipation in stages. Commonly used to manage constipation manners include basic lifestyle and dietary modifications and pharmacological therapies. Increasing their fluid intake, Clinical and laboratory research have studied its application, along with electroacupuncture (EA), in constipation.–26 Several studies have found that acupuncture may treat FC by regulating the nervous system and peripheral gastrointestinal hormones content.–28 Moreover, some randomized controlled trials (RCTs) have proved that acupuncture can treat FC patients from multiple aspects, by increasing bowel movements, relieving symptoms, and improving quality of life.,25,29–31 Whereas, we have found similar effects of different acupuncture courses in these RCTs.Acupuncture is a treatment modality that originated in ancient China and has been used to treat various digestive diseases.,33 Thus, this study will summarize the current evidences and conduct network meta-analysis to appraise the effectiveness of acupuncture in different courses for patients with FC.However, there are no meta-analyses to compare 2 or more courses. Therefore, it is unclear what the optimal course of acupuncture is. Network meta-analysis can visualize large amounts of evidence, assess and arrange the comparative effectiveness of all intervention methods, despite lacking direct comparisons.2–36 The registration number is CRD42020153801 in the PROSPERO.The protocol will be performed on the basis of the PRISMA guidelines, the PRISMA network meta-analysis extension statement and the recommendations of the Cochrane Handbook.2.12.1.1The trials will include RCTs that used a 2-, or 3 -arm parallel design regardless of blinding. Other types of trials will be excluded, such as quasi-randomized trials, reviews, case reports, secondary analysis and animal researches.2.1.2Patients over the age of 18 years who were diagnosed with FC using the Rome IV/III/II criteria will be included regardless of race, sex, education status or severity of disease. Patients can be included if they meet other clinical research guidelines and have no specific pathological cause. Studies with participants that included special populations, such as pregnant women, lactating women, addicts, strokes or those diagnosed with constipation due to irritable bowel syndrome, will be excluded.2.1.32.1.3.1The experimental group should be treated by acupuncture or EA, without restrictions in terms of electrical stimulation intensity, acupuncture depth, the number of acupuncture points and treatment courses. However, the trials that the intervention group is acupuncture combines with other conventional therapy should be excluded.2.1.3.2Through preliminary research, we have not found RCTs that directly compare different acupuncture courses. Therefore, we will indirectly compare the effects of different acupuncture courses. And sham acupuncture or placebo will be classified into a category as inert control. And trials simply comparing different acupuncture prescriptions or acupuncture forms will be excluded.2.22.2.1The primary outcome is weekly stool frequency (including spontaneous bowel movement (SBM), and complete SBM (CSBM)) at the end of all sessions. Specifically, a SBM refers to defecation without any medication or other means in the past 24 hours. A CSBM was defined as a SBM with the sense of complete evacuation.2.2.2The secondary outcomes involved stool consistency assessed with Bristol Fecal score, responder rate and safety evaluation. Bristol Fecal Scale uses 7 scales to evaluate stool characteristics, with scores of 1-2 indicating Lumpy or hard stools, 6 to 7 indicating mushy or entirely liquid stools. Responder rate is defined as the number of participants having at least 3 CSBMs or Bristol score of 3 to 5 per week. Safety evaluation is defined as the ratio of the number of reported adverse events to the total number.2.3We will systematically search electronic database from inception to October 2019, including 4 English databases: PubMed, Embase, Web of Science, Cochrane Central Register of Controlled Trials , and 4 Chinese databases: China National Knowledge Infrastructure (CNKI), China Biomedical Literature Database (CBM) and Wanfang Database. According to patients, intervention and study type, the following search terms are used: constipation, FC, colonic Inertia, acupuncture, acupuncture therapy, acupuncture points, EA, randomized, randomized controlled trial and so on . In the process, any inconsistencies will be discussed between the 2 reviewers and decided by a third party. Missing information was obtained by contacting the correspondent authors via e-mail.2.5Two reviewers will evaluate the quality of included RCTs based on the Cochrane handbook V.5.1.0. The tool includes six domains: random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, selective reporting and other bias. They will be rated on 3 levels, “low risk”, “high risk” or “unclear”. Disagreements were resolved in consultation with the third reviewer. Moreover, we evaluated the quality of evidence for the outcomes of the included studies in our review using the Grading of Recommendations Assessment, Development and Evaluation guidelines, including 5 factors: study limitations, consistency of effects, indirectness, inaccuracy, and publication bias. Finally, the level of evidence for each outcome indicator can be divided into: high, moderate, low, and very low.2.6 The magnitude of heterogeneity was measured using the I2 statistic: when I2 < 50%, a fixed-effects model will be used for pooled data; and when I2≥50%, a random-effects model was used. A meta-analysis of indirect treatment comparisons will be retrieved from the available evidence if head-to-head comparisons are lacking.First of all, we will use RevMan 5.3 software to perform the direct meta-analysis. For continuous variables, such as CSBM, SBM and Bristol score, the mean difference or standard mean difference with 95% confidence interval was used for analysis. For dichotomous data, such as the rates of responder s and adverse events, the relative risk (RR) with 95% confidence interval was utilized for analysis. If there are any data issues, we will deal with them according to the method described in the Cochrane handbook.Then, we will use STATA V.14.0 to make a network diagram and inconsistency test if there are 3 or more courses of treatment. The network diagram is showed by dots and lines. Among them, the dot represents a course of acupuncture with the size represents the number of subjects. The lines between the points indicate whether there is a direct comparison with the thickness represents the number of studies. And we will use the methods described in the previous article to test consistency. We will use the Brooks-Gelman-Rubin method to assess convergence. Estimation and inference will be performed when the convergence state is stable. The GeMTC initial parameters will be set as follows: the number of chains is 4; the initial value is set to 2.5; the step size is 5; the number of simulation iterations is 100,000; the number of adjustment iteration is 20,000. Parameters can be adjusted appropriately according to specific conditions. With the potential scale reduced factor close to 1, we will determine whether the consistency of the model is reliable. Moreover, a ranking figure of all treatment sessions will be generated if multiple intervention times exist.Finally, The indirect meta-analysis will be showed in the GeMTC software by using the Markov chain Monte Carlo method.2.7I2>50%, subgroup or sensitivity analysis was performed to find the cause.Some factors may contribute to the heterogeneity, such as stimulation parameters, the severity of constipation, participants’ age or gender. When the 2.8When the number of included studies in each outcome is sufficient, we will use a funnel plot that evaluates the reported bias. (n>10)3,31,42 However, no systematic review on the comparison of different acupuncture courses has been published in the world. This systematic review will compare the curative effect of different acupuncture treatments on FC through network meta-analysis. We hope that this review will provide a reference for clinical decisions by acupuncturists.The factors affecting the efficacy of acupuncture in the treatment of FC are complex and diverse, of which the amount of acupuncture stimulation is the main factor, such as the selection of acupoints, acupuncture cycle, the depth of acupuncture, and the intensity of current.Conceptualization: Lu Wang.Data curation: Mingmin Xu, Ying Liu, Tinghui Hou.Formal analysis: Dong Wang, Wei Cao.Investigation: Qianhua Zheng.Methodology: Lu Wang, Mingmin Xu, Tinghui Hou.Project administration: Lu Wang, Ying Liu.Supervision: Ying Li.Writing – original draft: Lu Wang, Wei Cao.Writing – review & editing: Mingmin Xu, Ying Li.


The NME (Non-metastatic) family members, also known as NDPKs (nucleoside diphosphate kinases), were originally identified and studied for their nucleoside diphosphate kinase activities. This family of kinases is extremely well conserved through evolution, being found in prokaryotes and eukaryotes, but also diverges enough to create a range of complexity, with homologous members having distinct functions in cells. In addition to nucleoside diphosphate kinase activity, some family members are reported to possess protein-histidine kinase activity, which, because of the lability of phosphohistidine, has been difficult to study due to the experimental challenges and lack of molecular tools. However, over the past few years, new methods to investigate this unstable modification and histidine kinase activity have been reported and scientific interest in this area is growing rapidly. This review presents a global overview of our current knowledge of the NME family and histidine phosphorylation, highlighting the underappreciated protein-histidine kinase activity of NME family members, specifically in human cells. In parallel, information about the structural and functional aspects of the NME family, and the knowns and unknowns of histidine kinase involvement in cell signaling are summarized. Ubiquitous nucleoside diphosphate kinases (NDPKs) were identified early in the 1950s based on their ability to catalyze the transfer of a phosphate from nucleoside triphosphates (NTPs) to nucleoside diphosphates (NDPs) in yeast . A few yThis review will discuss the complexity and polyvalence of NDPK activities and the challenges faced in working on this unstable post-translational modification, and outline the progress that has been made in the past few years in understanding how protein-histidine phosphorylation is catalyzed by NME family members and used to regulate cellular functions . NME (for non-metastatic) genes, also called NM23 (for non-metastatic protein 23) genes, and are highly conserved through evolution with multiple synonyms gene names in different organisms, including AWD, NDK, YNK, Gip17/Guk, NmeGp1sd [NDPK enzymes are expressed from NmeGp1sd . This muNmeGp1sd . A wholeNmeGp1sd  that areNmeGp1sd ). There NmeGp1sd ,12 althoNmeGp1sd ,13. NME1NmeGp1sd .The sequence alignment reveals common features, particularly in the catalytic domain, but distinct features in loops and the N- and C-termini. As mentioned above, NME kinases were first associated with a nucleoside diphosphate kinase (NDPK) activity that catalyzed the transfer of the γ-phosphate from nucleoside triphosphates through a phosphohistidine enzyme intermediate to nucleoside diphosphates resulting in the synthesis of nucleoside triphosphates other than ATP ,16,17 F. But sevNME kinases are ubiquitous, but only a few NME partners and protein substrates are currently known. An additional complexity is that NME family members can interact with each other, raising the functional importance of NME oligomerization, that will be also addressed. NME1 was originally defined as the first metastasis suppressor gene, because its RNA level was higher in tumors with low metastatic potential [Disregarding the molecular mechanism, NME members are reportedly involved in multiple physiological and pathological conditions . Indeed,otential . Besidesotential . NME1 isotential , and knootential ,28.Taken together, the publications related to NME and the synonymous NM23 or NDPK, have grown significantly since 1990, with around 1823 publications according to PubMed. Recent progress in elucidating the functions of this intriguing protein family was discussed by experts in the field at the 11th International Congress on the NDPK/NME/AWD protein family in October 2019 in Annecy, France. Their presentations clearly illustrated the complexity mentioned here, with emerging importance for understudied members such as NME3/4/6/7. In this review, we focus on the histidine kinase activity of the NME family, and its role in physiological and pathological conditions related to NME family members.Phosphohistidine (pHis) belongs to the N-phosphate class of SONAtes  and is characterized by an acid-labile and thermosensitive phosphoramidate bond, in contrast to the O-phosphate category, which includes the conventional phosphoserine (pSer), phosphothreonine (pThr) and phosphotyrosine (pTyr) ,9,29. pHHistidine phosphorylation is particularly well-known in the two-component system (TCS) or multi-component phosphorylation system used in bacteria for cell signal transduction. In TCS signaling, a transmembrane histidine kinase, generally a sensor kinase, is autophosphorylated on a catalytic histidine, and this phosphate is then transferred onto an aspartate in a response regulator protein, which transmits the signal. Histidine phosphorylation is found not only in bacteria, but also in various other organisms such as fungi and plants and more recently has been associated with important mammalian cellular processes ,31.Despite its long history, the mammalian pHis proteome has been difficult to study because of the acid-lability and thermosensitivity of this modification, which is not compatible with most of the standard phosphoproteomic methods used for pSer, pThr and pTyr analysis. Indeed, this is one of the reasons why our knowledge about phosphorylating events that generate phosphoramidate bonds has progressed slowly. The higher free energy of these P-N bonds leads to rapid hydrolysis at low pH or upon exposure to high heat. Even exposure to mild heat and acidic conditions, such as pH = 6.0 and 60 °C for 30 min, as well as certain primary amines, effectively hydrolyzes pHis. It has also been reported that the 1-pHis isomer is less stable than the 3-pHis isomer ,33. Yet, over the last few years, several new tools have emerged and have led to a resurgence in the study of histidine phosphorylation, specifically thanks to the development of histidine phosphate analogs and pHis monoclonal antibodies ,35,36,37These recent studies have begun to highlight the potential of histidine phosphorylation in multicellular organisms as a reversible regulatory protein modification and support a possible role for histidine kinases in cell signaling. NM23 gene products, primarily NME1-4, is seemingly not necessary for all of their biological functions [The intrinsic nucleoside diphosphate kinase (NDPK) activity associated with the unctions . This stE. coli showed that NDP kinase can phosphorylate histidine protein kinases, such as EnvZ and CheA, which both catalyze pHis-pAsp phosphorelay signal transduction systems in bacterial TCS [Arabidopsis thaliana, one report suggested that NME1 phosphorylated the kinase suppressor of Ras (KSR) at Ser392, as well as Ser434 when Ser392 was mutated [Additionally, studies in rial TCS . Similarrial TCS ,53. Addi mutated .Several studies have shown that NME phosphorylates the histidine in the catalytic site of ATP-citrate lyase (ACLY) ,56,57 anInterestingly, NME2 forms complexes with βγ subunits of heterotrimeric G-proteins and contributes to G protein activation by increasing the high-energy phosphate transfer onto GDP via formation of a phospho-H266 intermediate in Gβ subunits . FurtherThese diverse observations are among several types of evidence that support the concept that NME acts as a histidine kinase. However, as will be discussed further, one issue with NME acting as a protein kinase is that structure of its active site is designed to bind nucleotide tri/diphosphates, which might be expected to restrict access of protein histidine residues. Nevertheless, the fact that histidine residues such as H266 of Gβ are exposed on the protein surface, and also the recent finding that the NME1 activator molecule (NMac1) induces NDPK activation through allosteric conformational changes, suggest possible ways in which NMEs could use proteins as substrates ,62.Newly developed tools, such as sequence-dependent phospho-specific NME antibodies and NME inhibitors ,63, willDuring the past 25 years, more than 10 structures of human NMEs have been solved, including crystal structures of NME1, NME2, NME3, NME4, and NME10 . The firHuman NME1-4 are highly conserved (sequence identity > 50%), but relatively distinct from human NME5–10. Since 2002, structures of other human NME subtypes have been solved, showing highly similar architectures compared to NME2 . NME1, 2Human NMEs have been found to be multifunctional enzymes, with activities including NDP kinase, protein kinase, and 3′-5′ exonuclease. Substrates of the NMEs acting as NDP kinases and 3′-5′ exonuclease (NDP and dinucleotide respectively) have been observed bound to the same site of the enzyme (PDB ID: 3BBB ). DespitH. sapiens, M. xanthus, and D. discoideum, etc. Both ADP and GDP substrates bind to the NDP kinases in a similar manner. The active site is located in a cleft formed by two helices (helix 44–52 and helix 60–70). The base of the nucleotide is near the protein surface, exposed to the solvent. More specifically, by using NME2 as an example, F60 of helix 60–70 and V112 of the Kpn loop (94 to 114) form a clamp, stabilizing the base of the substrate  and even extracellular localizations ,105,106.nhibitor . FurtherDrosophila, the equivalent histidine is H119 in the awd gene, which encodes the NME homolog. Loss of function of the awd gene can be rescued by expression of WT AWD but not with the mutated H119A form, showing the importance of the catalytic site in this gene function [NME1/2 null background, re-expression of NME2 H118N catalytic inactive mutant led to lower basal G protein activation compared to the WT form [To understand the biological effects related to NME’s function as a protein-histidine kinase, site-directed mutagenesis of the catalytic site has been performed. Mutations of H118 to prevent its autophosphorylation, which is essential for NME kinase function, have been explored in several models. In function . On an N WT form . This reDrosophila as the “killer of prune” mutation since it was lethal in combination with the prune gene mutation [Because H118 is important for both nucleotide and protein-histidine kinase activity, experiments carried out by mutating only this amino acid are unable to define which activity is responsible for the observed phenotypes. To distinguish between these two functions, the consequences of mutating Pro96 of NME1 has been investigated. Mutation of Pro96 to serine causes conformational changes in NME affecting its binding to protein substrates . This mumutation ,116,117.mutation . Recent mutation . InteresAnother recent paper from the Steeg group reported that P96S mutant NME1 was unable to promote endocytosis or inhibit EGFR activation , demonstWork using NME mutants has revealed how its histidine kinase function is important in cell migration and invasion, in cellular growth and development, endocytosis, and cell signal transduction. As a summary, some NME protein kinase functions are depicted in Histidine phosphorylation can be reversed by phosphohistidine phosphatases, but as is the case for histidine kinases, only a few are currently known. NME2 pH118 is dephosphorylated by Phosphoglycerate Mutase Family Member 5 (PGAM5) . PGAM5 hAnother recently discovered histidine phosphatase is phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP), which was first purified from swine brain tissue . AlthougOther phosphatases such as STS-1 and STS-2 are members of the PGAM family and share sequences with histidine phosphatases. Therefore, they are potential candidates to dephosphorylate histidine residues possibly targeted by the NME histidine kinase . Other pWhile many of the molecular functions of NME in the cell remain unknown, recent progress has been made by investigating NME binding partners. To a large extent, NME function and its partners have been studied in the context of cancer, an outgrowth of the fact that NME was first described as a metastasis suppressor gene. Nevertheless, studies in different organisms have revealed that NME is essential for biological activities related to development and other disorders in addition to cancer. Not a lot is known about NME protein kinase activity and cell signaling. Here, we discuss the few instances where it is known that NME regulates signal transduction through histidine phosphorylation. However, most of the following studies relate to NME function and signaling modulation in different models, independent of its protein histidine kinase activity, but it is interesting to reconsider some of them in the light of histidine phosphorylation. NME1 was first described as a metastasis suppressor in a study comparing RNA levels in mouse metastatic melanoma cell lines with non-metastatic cells, which showed that Nm23/NME1 expression was downregulated in the metastatic cells . NME1 isCaenorhabditis elegans Masoudi, et al. [NME has a cell proliferation inhibitory function. To achieve this, NME1 through its protein phosphotransfer function activates KSR by phosphorylation of S392 leading to altered subcellular localization . KSR is , et al. . Deletio, et al. ,152. pMA, et al. . Surpris, et al. ,153. A p, et al. . NeverthAnother signaling pathway where histidine phosphorylated NME might act is through the AKT signaling pathway. In a mouse hepatocellular carcinoma model, levels of activated pS473 AKT were increased in tumors expressing higher levels of NME and its histidine phosphorylated form compared to the normal control . AlthougThere are other important signaling pathways regulated by NME for metastasis inhibition. However, there is no direct link between histidine phosphorylation and NME function in these cases, and it remains to be determined whether histidine phosphorylation might be important in some of them. For instance, silencing of NME increases Rac1 signaling and MAPKs, including ERK, JNK, and PI3K/AKT pathway activation . NME1 alC. elegans, binding of microRNA mir-35 to NDK-1 modulates the activation of the MAPK pathway resulting in DNA damage-induced apoptosis of germ cells [C. elegans and in humans NME promotes the engulfment of apoptotic cells through its interaction with Dynamin1 and 2 [NME1 can also promote cell death or enhance the engulfing phagocytic capacity of cells . In C. erm cells . In a carm cells ,164. In n1 and 2 ,165,166.Is it possible that NME regulates MAPK/AKT signaling mediated cell proliferation, motility, and apoptosis by its histidine kinase activity? Because most of the previous studies did not focus on detecting pHis protein signals, this question cannot yet be answered, and future experiments, including with other NME family members, will be needed. Interestingly, ongoing work presented at the 11th International Conference on the NME gene family (NME 2019 conference) has highlighted a critical function for NME3 in maintaining genome stability and a possible role in breast tumor metastasis . Likewis2+ channel activity [NME1 and 2 and the catalytic H118 residue are highly conserved from yeast to humans ,72. NMEsactivity , which iactivity . Phosphoactivity . Moreoveactivity ,172,173.activity . The samDrosophila has demonstrated that the deletion of the NME homolog awd gene, causes defects in the formation of imaginal discs during the development of larvae to adults [awd plays a role in the migration of cells during tracheogenesis and border cell movement, with awd working through receptor endocytosis to modulate the activation of FGFR/VEGFR/PDGFR and JAK/STAT signaling [C. elegans NDK-1 plays an important role in gonadal development [Xenopus, the NME4 homolog, NM23-X4, is essential for the development of the retina [Work with o adults . In the ignaling . In C. eelopment . In zebrelopment . In Xenoe retina . In micee retina ,177, bute retina . Furthere retina . In mouse retina . Moreovee retina ,181,182.e retina . Furtherawd and shibire (dynamin homolog) in the Drosophila demonstrates that awd functions as a GTP supplier to dynamin to mediate endocytosis [Besides development and cancer, there are studies showing the importance of NME in other biological contexts. Work with ocytosis . This haocytosis ,102. NMEocytosis ,184. Morocytosis , and NMEocytosis . All theThe detection of histidine phosphorylated NME has been challenging given that, until five years ago, we lacked the appropriate tools to selectively detect 1-pHis and 3-pHis in proteins. This is mainly why most of the work done so far has not explored NME protein-histidine kinase activity by evaluating changes in pHis protein levels. This began to change with the development of stable phosphoryltriazolylalanine (Tza) analogs of 1-pHis and 3-pHis by the Muir and Webb groups ,187, whiThe development of sequence-specific and sensitive monoclonal antibodies to individual pHis-containing proteins, and pH118 NME1/2 in particular, will be important for our understanding of the significance of NME protein histidine kinase activity in diverse cellular activities. There is a need for additional molecular tools in this field of study and complementary approaches, such as the development of selective NME histidine kinase inhibitors, would be valuable. Given the rapidly increasing amount of data and the identification of new histidine phosphorylated proteins, there is no doubt anymore that the extent of histidine phosphorylation was underestimated for many years but is now being recognized as an important regulatory post-translational modification. Many of the signaling roles of histidine phosphorylation remain to be explored, and important questions include whether other undefined histidine kinases and phosphatases exist and in what ways the addition of a phosphate to a histidine residue can initiate downstream signaling.